The role of chloride intracellular channel 1 in immune/inflammatory responses

Kanin Salao A thesis submitted in fulfillment of the requirement for the degree of Doctor of Philosophy

School of Medicine The University of New South Wales

Sydney, Australia December 2016 PLEASE TYPE

THE UNIVERSITY OF NEW SOUTH WALES

Thesis/Dissertation Sheet

Surname or Family name: SALAO First name: KANIN Other name/s· Abbreviation for degree as given in the University calendar: PhD of Medicine

School: St Vincent's Clinical School Faculty: Medicine Title. The role of Intracellular chloride channel 1 in immune/inflammatory------responses-- ---

Intracellular chloride channel protein Abstract1 (CLIC1) 350 wordsparticipates maximum: in (PLEASE inflammatory TYPE) processes, at least in part by regulating macrophage phagosomal functions such as pH, proteolysis. Here, we sought to determine if CLIC1 can regulate immune/inflammatory responses by actions on dendritic cells (DC), the key professional antigen presenting cell.

To study role of CLIC1 in immune/inflammatory responses, I first investigated immune cell phenotype changes in various tissue compartments associated with germline deletion of the gene for CLIC1 and found that both normal physiological conditions and under inflammatory responses, the immune cell composition is altered significantly in CLIC 1·1· mice when compared CLIC 1.,. mice. To further investigate the role of CLIC1 in dendritic cells, I first generated bone marrow-derived DC (BMDCs) from germline CLIC1 gene deleted (CL1c1·'·) mice and examined the effect of CLIC1 gene deletion on dendritic cell migration from peripheral 1 footpad to secondary lymph nodes of mice. I found that more cuc1· · BMDCs migrated from the site of 1 injection in the footpad and homed to popliteal and to inguinal lymph nodes than CUC 1 • ·sMDCs in both 1 1 cuc1• • and cuc1··mice. Subsequently, I identified the subcellular localization of CLIC1 in BMDCs after phagocytosis and found that cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from cuc1·'· BMDCs displayed impaired acidification and 1 1 proteolysis, which could be reproduced in wild type cuc1· ·, but not cuc1· • cell were treated with IAA94, a CLIC family ion channel blocker. CLIC r'· BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) reduced MOG induced experimental autoimmune encephalomyelitis.

These data suggest that CLIC1 regulates immune/inflammatory responses by means of changing immune cell composition, altering dendritic cell migration, modulating dendritic cell phagosomal pH and facilitating optimal processing of antigen for presentation to antigen specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic tar et for inflammator diseases. ------, Declaration relating to disposition of project thesis/dissertation

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iii Acknowledgments

I would like to thanks to my supervisor, Prof Samuel N Breit and co-supervisor, Dr Lele Jiang and Prof David A Brown for their advice, guidance, encouragement, understanding, patience and support throughout the study and preparation of this thesis. I especially thanks Sam for always give me a second chance one after the other. I learned a lot from you and all of these will be with me for the rest of my career. For Lele, you are like my Mum who has never given up on me. I couldn’t thanks you more.

I would like to express my sincere thanks to the Royal Thai Government for the award of the scholarship and faculty of medicine UNSW for top up scholarship for the financial support and opportunity during my PhD study.

I also would like to thanks to our colleges Dr Mohammad Mohammad, Dr Vicky Tsai Dr Xu Wei Luo, Dr Yasmin Husaini, Hui Li, Michelle Lee, Tamara Kuffner, Hong Ping Zhang and other inflammation group members at St Vincent’s for Applied Medical Research (AMR) for their suggestion and assistance throughout these years.

My thanks to friends, Jennifer, Kuan, Jod, Yut, A, Nadine and Bird and Pan and other soccer mates for their friendship and support throughout these years. I especially would like to express my thanks to Reeni Donohoe for her English correction one draft after the other. I would like to also sincere thanks Dr Xu Wei Luo for his moral support and also English proof reading.

Big thanks to my Dad for his love and support for most of my difficult time. Finally very importantly, I thank Aoom, my wife to be, for her love, support, understanding and encouragement throughout these years.

For my Mum and beloved sister, I wish you two here with me to see me hand in this Thesis. Without you both, things were not easy. I just hope that you both realize how important your support were and are. Thank you for having me as brother and son.

v Table of contents 1 Literature review ...... 1 1.I Chloride Intracellular Channel PROTEIN (CLICs) ...... 1 1.I.A Introduction ...... 1 1.I.B Electrophysiology of CLICs ...... 3 1.I.C Biochemistry of CLICs ...... 10 1.I.D Structure of CLICs ...... 26 1.I.E Tissue and SubCellular distribution of CLICs ...... 34 1.I.F Biological functions of clics ...... 43 1.II Dendritic cells (DCs) ...... 68 1.II.A Introduction ...... 68 1.II.B DC Subsets ...... 69 1.II.C DC Development ...... 75 1.III Functions of cDCs ...... 82 1.III.A Introduction ...... 82 1.III.B Tissue surveillance ...... 84 1.III.C Antigen uptake ...... 88 1.III.D Antigen processing ...... 98 1.III.E Maturation ...... 106 1.III.F Migration ...... 109 1.III.G Antigen presentation ...... 112 2 Materials and methods ...... 119 2.I Materials ...... 119 2.I.A Chemicals and reagents ...... 119 2.I.B Antibodies used for flow cytometry ...... 122 2.I.C Antibodies used for immunohistochemistry ...... 123 2.I.D Particles ...... 124 2.I.E Cells ...... 124 2.I.F Mice ...... 128 2.II Methods ...... 129 2.II.A Immunostaining for Cell profiling ...... 129 2.II.B Dendritic cell migration experiment ...... 130 2.II.C CLIC1 cellular localisation ...... 133 2.II.D Intraphagosomal acidification measurement ...... 133 2.II.E Proteolyis assay ...... 134 2.II.F In vitro Antigen presentation Experiment...... 135 2.II.G In vivo antigen presentation experiment ...... 136 3 Results ...... 138 3.I Immune cell profiling ...... 138 3.I.A Introduction ...... 138 3.I.B Background ...... 138 3.I.C Results ...... 140 3.I.D Discussion ...... 157 3.II Dendritic cell migration ...... 160 3.II.A Introduction ...... 160 3.II.B Background ...... 160 3.II.C Results ...... 163 3.II.D Discussion ...... 179

vi 3.III CLIC1 localisation in Dendritic cells ...... 182 3.III.A Introduction ...... 182 3.III.B Background ...... 182 3.III.C Results ...... 184 3.III.D Discussion ...... 186 3.IV phagosomal acidification and proteolysis ...... 187 3.IV.A Introduction ...... 187 3.IV.B Background ...... 187 3.IV.C Results ...... 190 3.IV.D Discussion ...... 195 3.V Antigen presentation ...... 199 3.V.A Introduction ...... 199 3.V.B Background ...... 199 3.V.C Results ...... 202 3.V.D Discussion ...... 215 4 General Discussion ...... 219 5 Conclusion and future direction ...... 227 5.I Conclusion from this thesis ...... 227 5.II Future direction ...... 227 6 References ...... 229

vii List of figures

Figure 1 Multiple sequence alignment of human CLICs ...... 2 Figure 2 Crystal structure of CLIC1 ...... 31 Figure 3 Key transcription factors and important cytokines that regulate DC subset differentiation...... 78 Figure 4 An overview of cDC function as antigen presenting cells...... 83 Figure 5 Pattern recognition receptors (PRRs) used by cDCs to drive inflammation. ... 85 Figure 6 Three possible routes of extracellular antigen uptakes in cDCs ...... 89 Figure 7 Formation of phagocytic cup, phagocytosis and phagosome maturation ...... 93 Figure 8 The antigen processing and presentation pathways in cDCs...... 99 Figure 9 cDCs migration from peripheral tissue to lymph nodes via lymphatic vessel 110 Figure 10 T helper cell polarization...... 113 Figure 11 Schematic drawing of localization of lymphoid organs in mouse...... 139 Figure 12 Circulating blood from CLIC1-/- mice has more T cells but less monocytes and neutrophils...... 142 Figure 13 Spleen from CLIC1-/- mice has less T cells and macrophages...... 144 Figure 14 Cervical lymph nodes from CLIC1-/- mice have less macrophages...... 147 Figure 15 Inguinal lymph nodes of CLIC1-/- mice have less macrophages...... 149 Figure 16 Peritoneal cavities of CLIC1-/- mice contain less T and B cells but more macrophages...... 152 Figure 17 Gating strategy used to identify immune cell subsets from peritoneal cavity of mice stimulated with thioglycollate medium...... 155 Figure 18 CLIC1-/- mice recruit more monocytes and have more resident macrophages than CLIC1+/+ mice after thioglycollate stimulation...... 156 Figure 19 Lymph node drainage in mouse...... 161 Figure 20 Proportion of CLIC1+/+ and CLIC1-/- BMDCs in pooled sample prepared for footpad injection ...... 163 Figure 21 Gating strategy used to identify migrated BMDCs ...... 165 Figure 22 CLIC1-/- BMDCs migrated to popliteal lymph nodes more than CLIC1+/+ BMDCs when injected into CLIC1+/+ recipient mice...... 167 Figure 23 CLIC1-/- BMDCs also migrated to popliteal lymph nodes more than CLIC1+/+ BMDCs when injected into CLIC1-/- recipient mice...... 168 Figure 24 The presence or absence of CLIC1 in recipient mice has no influence on ratio of BMDCs found in popliteal lymph nodes...... 169 Figure 25 Toxicity from fluorescent dyes had no influence on BMDC migration as swopping fluorescent dyes on BMDCs gave consistent results...... 171 Figure 26 CLIC1-/- BMDCs migrated faster than CLIC1+/+ BMDCs from footpad to more distal inguinal lymph nodes of CLIC1+/+ mice...... 173 Figure 27 CLIC1-/- BMDCs migrated faster than CLIC1+/+ BMDCs from footpad to more distal inguinal lymph nodes of CLIC1-/- mice...... 175 Figure 28 Attenuated migration of CLIC1+/+ BMDCs is not due to cell death...... 178 Figure 29 Phagocytosis triggers CLIC1 translocation to BMDC phagosome membrane...... 185 Figure 30 pH Calibration curve for zFITC...... 191 Figure 31 CLIC1-/- BMDCs display impaired phagosomal acidification...... 192 Figure 32 CLIC1-/- BMDCs display impaired phagosomal proteolysis...... 194 Figure 33 Immunopathogenesis of EAE ...... 201 Figure 34 Gating strategy...... 203

viii Figure 35 129X1/SVJ and C57BL/6 cell mixing experiments...... 204 Figure 36 CLIC1-/- and CLIC1+/+ BMDCs differentially activate CD4+ T cells depending on whether they are pulsed with MOG35-55 or MOG1-125 peptide...... 205 Figure 37 Antigen pulsed CLIC1-/- BMDCs have a reduced capacity to activate CD4+ T- cells...... 206 + +/+ Figure 38 Chloroquine reduces CD4 T-cell activation by MOG1-125pulsed CLIC1 but not CLIC1-/- BMDCs...... 208 + +/+ Figure 39 IAA94 reduces CD4 T-cell activation by MOG1-125 pulsed CLIC1 but not CLIC1-/- BMDCs...... 210 Figure 40 CLIC1 has no effect on LPS induced BMDC activation cell surface molecules...... 211 Figure 41 EAE disease clinical scores of mice immunised with MOG1-125 pulsed BMDCs...... 214

ix List of tables

Table 1 Reported chloride ion channel activities of CLICs ...... 6 Table 2 Reported membrane insertion of CLICs ...... 14 Table 3 Reported interaction of cytoskeletal and scaffolding proteins with CLICs ...... 22 Table 4 Reported interaction of small GTPases with CLICs ...... 25 Table 5 Reported crystal structure of CLICs ...... 28 Table 6 Tissue and subcellular distribution of CLICs ...... 35 Table 7 Reported biological functions of CLICs ...... 48 Table 8 Summary of phenotypes of the major mouse DC subsets ...... 70 Table 9 Transcription factors and cytokines guiding DC development ...... 76 Table 10 Pattern recognition receptors (PRRs) of cDC subsets ...... 86 Table 11 Functional and phenotypical differences between immature and mature cDCs ...... 107 Table 12 Chemicals, reagents and materials used in this project ...... 119 Table 13 Antibodies used for flow cytometry ...... 122 Table 14 Antibodies used for immunohistochemistry ...... 123

x Abbreviations

°C Celcius Ab Antibody ADP Adenosine Diphosphate APCs Antigen Presenting Cells ATP Adenosine Triphosphate B cells B lymphocytes BM Bone Marrow BMDCs Bone Marrow derived Dendritic Cells BSA Bovine Serum Albumin Cat Cathepsin CD Cluster of Difference cDCs Classical Dendritic Cells cDNA Complementary DNA CFA Complete Fluid Adjuvant CHO-K1 Chinese Hamster Ovary K1 Cl- Chloride ion CLIC Chloride Intracellular Channel Protein CLIC1 Chloride Intracellular Channel Protein 1 CLIP Class II-associated Invariant chain Peptide CO2 Carbondioxide Cy2 Cyanine dye 2 Cy3 Cyanine dye 3 Cys Cysteine DCs Dendritic Cells EAE Experimental Autoimmune Encephalomyelitis ER Endoplasmic Reticulum FCS Fetal Calf Serum FITC Fluorescein Isothiocynate Flt3 FMS-like tyrosine kinase-3 Flt3L Fms-like tyrosine kinase3-ligand FoxP3 Forkhead box P3 FRET Förster Resonance Energy Transfer g Gravity force GFP Green Fluorescent Protein GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor GSH Glutathione GST Glutathione S-transferase GTP Guanosine-5'-triphosphate HLA-DM Human Leukocyte Antigen DM Hr Hour I-A/I-E Mouse MHC class II IAA94 Indanyloxyacetic acid 94 IFNγ Interferon gamma IgE Immunoglobulin E IgG Immunoglobulin G IL-4 Interluekin 4 IL17 Interleukin 17

xi infDCs Inflammatory Dendritic Cells kDa Kilo-Dalton KO Knock out LC Langherans Cells LN Lymph Node LPS Lipopolysaccharides M-CSF Macrophage Colony-Stimulating Factor mDCs Myeloid Dendritic Cells MHC I Major Histocompatibility Complex I MHC II Major Histocompatibility Complex II Min Minute MOG Myelin Oligodendrocyte Glycoprotein MyD88 Myeloid Differentiation primary response 88 N-terminus Amino-terminus NADPH Nicotinamide Adenine Dinucleotide Phosphate Oxidase ng Nano-gram NOX Nitrogen Oxide NS Not Significant p Probability PBS Phosphate Buffer Saline pDCs Plasmacytoid Dendritic Cells pH Potential of Hydrogen ion pKa Negative log of Ka pmoles Picomoles RhoA Ras homolog gene family, member A RM-ANOVA Repeat Measure ANOVA ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute (culture medium) RT Room Temperature RyR Ryanodine Receptor T cells T lymphocytes TCR T cell receptor TH T helper cells TLR Toll Like Receptor Treg Regulatory T cells Trp Tryptophan Val Valine VCAM-1 Vascular Cell Adhesion Molecule 1 WT Wild type Zy Zymosan κ Kappa ug Micro-gram ul Micro-litter uM Micro-molar

xii Publication and presentation

Publications

Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification.

Jiang L, Salao K, Li H, Rybicka JM, Yates RM, Luo XW, Shi XX, Kuffner T, Tsai

VW, Husaini Y, Wu L, Brown DA, Grewal T, Brown LJ, Curmi PM, Breit SN. J Cell

Sci. 2012 Nov 15;125(Pt 22):5479-88. doi: 10.1242/jcs.110072. Epub 2012 Sep 6.

(Published)

CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis.

Salao K, Jiang L, Shi XX, Tsai VW, Husaini Y, Wu L, Brown DA, Brown LJ, Curmi

PM, Breit SN. Biol Open. 2016 May 15;5(5):620-30. doi: 10.1242/bio.018119.

(Published)

Characterisation of immune cell profiling of lymphoid and nonlymphoid organs of

CLIC1-/- mice

Salao K, Jiang L, Shi XX, Tsai VW, Husaini Y, Wu L, Brown DA, Brown LJ, Curmi

PM, Breit SN (In preparation)

xiii Presentations

Kanin Salao, Lele Jiang, David Brown, Xin Xin Shi, Pual MG Curmil, Louis Brown, and Samuel N Breit (2010) Immune cell profiling of lymphoid organs from CLIC1-/- and synergenic WT mice. 19th St Vincents & Mater Health Sydney Research Symposium,

Sydney, Australia

Kanin Salao, Lele Jiang, David A Brown, Vicky W-W Tsai, Yasmin Husaini, Paul MG

Curmil, Louise Brown, and Samuel N Breit (2012) The role of intracellular chloride ion channel 1 CLIC1 in antigen processing and presentation. Inflammation Conference

2012 “Inflammation- the key to much pathology”, Melbourne, Australia

Kanin Salao, Lele Jiang, David A Brown, Vicky W-W Tsai, Yasmin Husaini, Paul MG

Curmil, Louise Brown, and Samuel N Breit (2013) CLIC1 function in antigen processing and presentation through modulation of phagosomal proteolysis and acidification. The 21th St Vincents & Mater Health Sydney Research Symposium,

Sydney, Australia

Lele Jiang, Kanin Salao, Louise Brown, Paul Curmi, David Brown, Samuel Breit.

(2015) CLIC1 regulates lysosomal pH and antigen processing and degradation in macrophages and dendritic cells. International workshop on CLIC protein, NIH, USA

xiv Abstract

Intracellular chloride channel protein 1 (CLIC1) participates in inflammatory processes, at least in part by regulating macrophage phagosomal functions such as pH, proteolysis.

Here, we sought to determine if CLIC1 can regulate immune/inflammatory responses by actions on dendritic cells (DC), the key professional antigen presenting cell.

To study role of CLIC1 in immune/inflammatory responses, I first investigated immune cell phenotype changes in various tissue compartments associated with germline deletion of the gene for CLIC1 and found that both normal physiological conditions and under inflammatory responses, the immune cell composition is altered significantly in

CLIC1-/- mice when compared CLIC1+/+ mice. To further investigate the role of CLIC1 in dendritic cells, I first generated bone marrow-derived DC (BMDCs) from germline

CLIC1 gene deleted (CLIC1-/-) mice and examined the effect of CLIC1 gene deletion on dendritic cell migration from peripheral footpad to secondary lymph nodes of mice. I found that more CLIC1-/- BMDCs migrated from the site of injection in the footpad and homed to popliteal and to inguinal lymph nodes than CLIC1+/+BMDCs in both

CLIC1+/+ and CLIC1-/- mice. Subsequently, I identified the subcellular localization of

CLIC1 in BMDCs after phagocytosis and found that cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis.

Phagosomes from CLIC1-/- BMDCs displayed impaired acidification and proteolysis, which could be reproduced in wild type CLIC1+/+, but not CLIC1-/- cell were treated with IAA94, a CLIC family ion channel blocker. CLIC1-/- BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) reduced MOG induced experimental autoimmune encephalomyelitis.

xv These data suggest that CLIC1 regulates immune/inflammatory responses by means of changing immune cell composition, altering dendritic cell migration, modulating dendritic cell phagosomal pH and facilitating optimal processing of antigen for presentation to antigen specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target for inflammatory diseases.

xvi 1 LITERATURE REVIEW 1.I CHLORIDE INTRACELLULAR CHANNEL PROTEIN (CLICs)

1.I.A INTRODUCTION

CLIC family proteins consist of six members, namely CLIC1, CLIC2, CLIC3, CLIC4,

CLIC5 and CLIC6. All CLICs contain a conserved structural module that consists of about 230 residues, with approximately 50-75% sequence identity among six human

CLICs (Figure 1). As depicted in Figure 1, all CLICs have a conserved cystein residue of either CPFS or CPxC motif close to the N-terminus. Although CLIC protein sequences share great deal of similarity, some of the CLICs such as CLIC5 and CLIC6 contain extra N terminal extensions. CLIC5 which has two isoforms (CLIC5A and

CLIC5B), generated by alternative splicing, both contain an additional 170 residues

(Shanks et al., 2002), while CLIC6 possess an additional 440 amino acid residues

(Friedli et al., 2003).

CLICs were initially thought to be ion channels; however, doubts have been raised about this as structural studies of the soluble form of CLICs indicate that they do not resemble to any conventional ion channel proteins. Instead they belong to the glutathione S-transferase (GST) fold superfamily of proteins. Multiple studies from several groups have demonstrated that CLICs display chloride ions conductance in cells and lipid bilayer systems. This has led to the debate as to whether CLICs function as an ion channel and/or have other non-ion channel functions.

1

Figure 1 Multiple sequence alignment of human CLICs Protein sequences six human CLICs were aligned using CLUSTALW (Larkin et al., 2007) and presented using ESPript (Gouet et al., 2003). The secondary structures belong to CLIC1 are shown on top of the alignment. Conserved residues are painted in red, while similar amino acid residues are shown in red fonts.

2 1.I.B ELECTROPHYSIOLOGY OF CLICs

Perhaps the most prominent characteristic of CLICs is their ability to form chloride ion channels. The first identified CLIC, originally called bovine p64 (CLIC5B) (discovered in the search for the cystic fibrosis transmembrane conductance regulator (CFTR) of bovine kidney cortex microsomes) displayed chloride ion channel activity in binding to the chloride ion channel inhibitor, indanyloxyacetic acid 94 (IAA94) (Landry et al.,

1993). Since then many studies have demonstrated that most CLICs display chloride ion channel activity and these studies are summarised in Table 1. All CLIC proteins that have been studied display ion channel activity but to date no ion channel conductance has been studied in CLIC3 and CLIC6.

CLIC1 has evidence of chloride ion channel activity in both cell systems and artificial planar lipid bilayers. In resting untransfected cells CLIC1 single channel currents on the plasma membrane are a rare event. However transfection of CLIC1 into CHO-K1 cells results in a markedly increased probability of detecting CLIC1 single channel events in either the nuclear or plasma membrane (Tonini et al., 2000). By using FLAG tagged

CLIC1 either on N or C terminus, when the N terminus is exposed to the anti-FLAG antibody in outside out, the channel activity is completely interrupted. In contrast, anti-

FLAG antibody has no effect using the inside out configuration. Conversely, anti-

FLAG antibody inhibit only the inside out carboxyl FLAG tagged CLIC1 but has no effect in the outside out configuration. Taken together, this indicates the epitope was on the same side of the membrane as the antibody. This demonstrated that CLIC1 is a transmembrane protein, whose N terminus projects outwardly while the carboxy- terminus projects inwards (Tonini et al., 2000). To date, the strongest evidence

3 supporting CLIC1’s role as an ion channel came from a study using point mutations on two positively charged residues putative transmembrane region (Averaimo et al., 2013).

In this study, when the only two positively charged residues (Lys37 and Arg29) on

CLIC1 putative transmembrane region were replaced by a neutral alanine, and transfected in Human embryonic kidney (HEK) 293 cells, the ion channel behaviour of these mutant transfected cells altered significantly when compared to wild type protein, as determined by both single cell and whole cell recording (Averaimo et al., 2014).

Although the mechanism by which these two residues may contribute is unclear, this study strongly suggests that both Lys37 and Arg29 are functionally important for

CLIC1 ion channel. Therefore, these results indicate that the observed chloride ion channel activity is a result of ion channel formation by CLIC1 expressed CHO-K1 cells.

CLIC1 has also been studied in artificial lipid bilayer systems where only purified recombinant CLIC1 protein is present. Purified recombinant soluble CLIC1 when reconstituted into artificial planar lipid bilayer, forms chloride ion channels and thus provides that the most direct evidence that CLIC1 itself is capable of forming a chloride channel in the absence of ancillary subunits or accessory proteins (Tulk et al., 2000).

The chloride ion channel formed by purified CLIC1 when reconstituted into artificial system is indistinguishable to that in CLIC1 transfected CHO cells (Harrop et al., 2001).

In absence of detergent, purified soluble CLIC1 can insert from aqueous phase into a preformed phospholipid membrane, increasing chloride permeability (Tulk et al., 2002).

Furthermore, forming of CLIC1 into the artificial planar lipid bilayer is pH dependent

(Warton et al., 2002) and redox controlled (Littler et al., 2004). Low and high pH favour the formation of a channel, while oxidised CLIC1 favours the channel formation.

Furthermore, electrophysiology studies of CLIC1 show multiple conductance states: a

4 small conductance with slow kinetic (SCSK), follow by a high conductance channel with fast kinetic (HCFK) (Warton et al., 2002), suggesting at least 4 subunits of CLIC1 are needed to form a channel. This hypothesis is supported by the FRET study, suggesting at least 6-8 subunits of CLIC1 are required to form selective ion channels(Goodchild et al., 2011). Taken together, these studies strongly indicate that

CLIC1 itself is able to form a chloride ion channel in the artificial lipid bilayer.

Despite convincing electrophysiological data, whether CLICs are ion channels has remained controversial, to a large extent because structural studies of soluble CLICs do not resemble any conventional ion channels but belong to the GST fold superfamily of proteins (Cromer et al., 2007b; Harrop et al., 2001; Littler et al., 2005; Littler et al.,

2010a). To date, there is no high resolution structure of the purified integral membrane form that might suggest a mechanism by which CLIC1 may conduct chloride ions.

5 Table 1 Reported chloride ion channel activities of CLICs CLICs Experimental System Detection Method Conductance Reference CLIC1 1) CLIC1 was transfected 1) Whole cell recording 22 pS (cell Valenzuela et al., 1997 into CHO-K1 cells 2) Single cell recording membrane) 2) Nuclei were isolated for 3) Nuclear attached patch clamp 33 pS (nuclear electrophysiology study membrane) CLIC1 CLIC1 was transfected into 1) Cell attached patch clamp 7.8 pS Valenzuela et al., 2000 CHO-K1 cells 2) Nuclear attached patch clamp CLIC1 CLIC1 was transfected into Patch clamp recording 8 pS Tonini et al., 2000 CHO-K1 cells -outside out recording 9 pS @ 20 mM Cl- -inside out recording 11 pS @ 50 mM Cl- 17 pS @ 140 mM Cl- CLIC1 Purified recombinant Chloride efflux assay and single 161pS @ 300 mM Tulk et al., 2000 CLIC1 was reconstituted in channel recording KCl 67 pS @ 150 vesicles that were then mM KCl added to planar lipid bilayer. CLIC1 1) Purified recombinant 1) Single channel recording 31 pS @ 140 mM Harrop et al., 2001 CLIC1 was added to planar 2) Inside out patch clamp KCl lipid bilayer. (single channel for 2) CLIC1 transfected CHO purified CLIC1) cells 29 pS @ 140 mM KCl (inside out of CLIC1 transfected CHO cells)

6 CLICs Experimental System Detection Method Conductance Reference CLIC1 1) CLIC1 expressing CHO 1) Patch clamp recording 7 pS @ 140 mM KCl Warton et al., 2002 cell line 2) Single channel recording & 2) Soluble recombinant 30 pS @ 140 mM CLIC1 was added to KCl phospholipid bilayer. CLIC1 Monomeric and dimeric Chloride efflux assay and single 28 ps @ 140 mM Littler et al., 2004 purified CLIC1 was directly channel recording KCl mixed with vesicles, or (monomeric CLIC1) added to planar lipid bilayer 28 ps @ 140 mM without adding any KCl detergent. (dimeric CLIC1) CLIC1 Aβ peptides treatment of Single channel recording 6.9 pS (BV-2) Novarino et al., 2004 1) Microglia cell line (BV2) 7.1 pS (Rat 2) Rat primary microglia microglia) cells CLIC1 Aβ peptides treatment of Whole cell patch clamp - Milton et al., 2008 1) Microglia cell line (BV2) 2) Rat primary microglia cells (PMG) CLIC1 Soluble recombinant CLIC1 Impedance spectroscopy - Valenzuela et al., 2013 was added to tethered lipid membrane with varying concentration of cholesterol

7 CLICs Experimental System Detection Method Conductance Reference CLIC1 1) Purified recombinant 1) Single cell recording 12 pS @ 140 mM Averaimo et al., 2013 mutant & WT CLIC1 was 2) Cell attached patch clamp KCl added to planar lipid 3) Whole cell patch clamp bilayer. 2) Mutant and WT CLIC1 was transfected HEK cells CLIC1 Forskolin stimulated mouse Single channel recording 9 pS Averaimo et al., 2014 retinal ganglion cells CLIC4 Microsomal membrane Single channel recording 43 pS @ 50 mM Duncan et al., 1997 vesicles (mainly Choline chloride endoplasmic reticulum) containing recombinant CLIC4 (p64H1) of transfected HEK-293 cells were reconstituted to planar lipid bilayer. CLIC4 Overexpressing CLIC4 Whole cell recording 1 pS -10 pS Proutski et al., 2002 (p64H1) plasma membrane Single channel recording of HEK-293 cells CLIC4 Soluble purified Chloride efflux assay and single 30 pS @ 140 mM Littler et al., 2005 recombinant CLIC4 was channel recording KCl directly mixed with vesicles, or added to planar lipid bilayer without adding any detergent.

8 CLICs Experimental System Detection Method Conductance References CLIC4 Soluble purified Single channel recording 15 pS (poorly Singh and Ashley, 2007 recombinant CLIC4 was selective) reconstituted to planar lipid bilayer p64 Purified border chloride Whole cell recording 15 pS @ 140 mM Schlesinger et al., 1997 (CLIC5B) channel was reconstituted Single channel recording KCl into asplectin vesicles, and then incorporated into planar lipid bilayer p64 Membranes of CLIC5 (p64) Single channel recording 42 pS @ 140 mM Edwards et al., 1998 (CLIC5B) transfected HeLa cells were KCl reconstituted into planar lipid bilayers.

9 1.I.C BIOCHEMISTRY OF CLICs

There are three biochemical/biophysical properties of CLICs that have been widely studied: 1) Ability to insert into plasma membrane, 2) Ability to interact with cytoskeletal and scaffolding proteins, and 3) Ability to interact with small GTPases.

Discussion of these characteristics forms the major part of this chapter. In this section, although the biochemical properties of all CLICs will be reviewed, CLIC1 will be the main focus.

1.I.C.i ABILITY TO INSERT INTO PLASMA MEMBRANE

An important biochemical/biophysical property of CLICs that distinguishes them from other ion channel proteins is that they do not have a leader peptide and are synthesised in the cytoplasm as soluble proteins. This triggers the insertion of CLICs into membranes. The studies that support this property of CLICs are summarised in Table 2.

All CLICs except CLIC3 and CLIC6 have been reported to insert into plasma membrane.

The first observation suggesting that CLICs can act as integral membrane proteins comes from studies of p64 (CLIC5B) expressed in Xenopus oocytes. Using cell fractionation experiments it showed that p64 incorporated into the microsomal membrane (Landry et al., 1993). From these studies, further experiments demonstrated that alkaline treatment failed to dissociate p64 related protein, p64H1, later identified as

CLIC4, from microsomal membrane vesicles (Duncan et al., 1997), CLIC4 was believed to insert into the microsomal membrane. Similar results were also observed in the alkaline treatment of human kidney cortex and medulla membrane where it failed to separate CLIC1 from the membrane. However CLIC1 associated alkaline washed

10 membrane was solubilized with Triton X-100, reinforcing CLIC1 presence in membrane (Tulk and Edwards, 1998). These all suggest that CLICs can exist in membrane integrated form.

Among CLICs, CLIC1 is the best studied CLICs for its ability to migrate from a soluble cytoplasmic protein into a membrane protein. CLIC1 has no a leader sequence required for synthesis in the endoplasmic reticulum, and under resting conditions, it exists almost entirely in cytosol. When purified soluble CLIC1 was incubated with artificial liposomes for different times between10 mins to 4 hrs, increasing amounts of CLIC1 became associated with the liposome fraction as determined by western blotting of the purified liposomes (Warton et al., 2002). When purified recombinant soluble CLIC1 is mixed with preformed artificial phospholipid vesicle, chloride permeability increased by 10 fold (Tulk et al., 2002). However, these studies failed to show the direct binding of soluble CLIC1 to the membrane. More recently, a study using sucrose-loaded vesicle sedimentation assay was able to separate soluble protein from membrane bound protein.

The study demonstrated that when reduced monomeric CLIC1 was added to the vesicle,

32 % of CLIC1 bound to the membrane, as confirmed by both sedimentation and amido-black protein assay (Goodchild et al., 2009). Furthermore, when an oxidative reagent was also included in the mix, the percentage of membrane bound CLIC1 was markedly increased to 75% (Goodchild et al., 2009). Thus, this study clearly indicates that CLIC1 can spontaneously insert into membrane, and thus promote membrane insertion. Likewise, a recent study used size-exclusion chromatography of mixture containing a 30- amino acid residue encompassing the putative CLIC1 transmembrane domain (TMD) peptide in sodium dodecyl sulphate (SDS) micelles or 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, and found that in absence of C

11 terminal domain of CLIC1, a total of 66% of the total peptide coelutes with sodium dodecyl sulphate (SDS) micelles, while almost all the total peptide coelutes with 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (Peter et al., 2013).

Thus, this study strongly indicates that in absence of C terminal domain, CLIC1 transmembrane domain (TMD) is necessary for efficient insertion into membranes.

While it is clear that, at least under some circumstances, CLICs associates with membranes, the process underlying this is less clear. Using fluorescence resonance energy transfer (FRET), it revealed that the integral membrane form of CLIC1 undergoes large scale unfolding of the N-terminal transmembrane domain away from the C-domain as it inserts into the bilayer (Goodchild et al., 2010). The same group also used FRET to show that there is a large-scale structural rearrangement in the formation of membrane CLIC1 which forms multimers in the artificial membrane. Modelling of this data suggests that it forms a large oligomer of 6–8 subunits upon membrane insertion (Goodchild et al., 2011). More recent studies using fluorescent spectroscopy were able to independently confirm this data (Peter et al., 2013). However, in vivo membrane insertion of CLIC1 may be much more complex and to date there is no high resolution structure of the membrane integrated form of any CLIC. Nevertheless, it is now well established that soluble CLICs from aqueous solution can insert into artificial bilayers in the absence of other cellular components in an artificial setting.

For CLICs to insert from aqueous solution to the membrane, significant structural rearrangement is required to separate the N and C terminal domains in order for the N terminal domain to refold in a configuration that can allow it to form a transmembrane domain (TMD). Whilst the mechanism is largely unknown, there is evidence available

12 to suggest that at least three possible environmental factors could facilitate this transition. These include oxidation, pH and cholesterol. The impact of environment factors on CLIC1 membrane binding/insertion will be reviewed in this section.

13 Table 2 Reported membrane insertion of CLICs CLICs Membrane type Experimental system Detection Method Conclusion Reference

p64 (CLIC5B) Microsomal p64 (CLIC5B) RNA transcripts Membrane p64 protein when Landry et al., membrane vesicle were injected into Xenopus fractionation and expressed in 1993 oocytes. immmunoblot Xenopus oocytes was incorporated into the microsomal membrane. p64H1(CLIC4) Microsomal Microsome was treated with Protein digestion CLIC4 is part of the Duncan et al., membrane vesicle alkaline solution. microsomal vesical 1997 membrane. p64 related Osteoblast antibody was tagged to 18 amino Immunogold CLIC4 spans through Schlesinger et al., protein membrane vesicle acid end of C terminus of p64 electron vesicle membrane 1997 related protein. microscopy using their 18 amino acid on C terminus. CLIC1 Kidney cortex and Membrane fraction of kidney Membrane CLIC1 is part of the Tulk and medulla membrane cortex and medulla was fractionation microsome vesicles. Edwards, 1998 extracted with alkaline solution. CLIC1 Phospholipid Soluble CLIC1 was added to Chloride efflux CLIC1 is Tulk et al., 2002 vesicles preformed phospholipid assay successfully inserted vesicles. into phospholipid vesicle and confer chloride efflux. CLIC1 Liposome Purified CLIC1 was added to Artificial liposome Low pH facilitates Warton et al., liposomes under various pH and biphasic CLIC1 the 2002 conditions. detergent interaction with fractionation liposomes.

14 CLICs Membrane type Experimental system Detection Method Conclusion Reference CLIC1 Liposome CLIC1 was added to sucrose Sucrose loaded 75% of soluble Goodchild et al., loaded vesicle. sedimentation CLIC1 was 2009 assay successfully loaded into vesicle. CLIC1 Liposome CLIC1 was added to sucrose Fluorescence CLIC1 forms Goodchild et al., loaded vesicle. resonance energy oligomers upon 2010) transfer (FRET)& oxidation in the Sucrose loaded presence of the sedimentation membrane. assay CLIC1 SDS micelles and CLIC1 was added to SDS Size-exclusion 66% of CLIC1 Peter et al., 2013 POPC liposomes micelles and POPC liposomes. chromatography interact with SDS micelle, while most of CLIC1 interact with POPC liposome.

15 1.I.C.i.a Oxidation promotes CLIC1 membrane insertion

CLIC1 adopts glutathione-S-transferase (GST) like structure that contains a glutathione- binding site containing Cys-x-x-Ser (CxxS) motif on its N domain (Harrop et al., 2001).

The putative transmembrane domain (TMD) of CLIC1 (residues 24-46), which crosses the membrane an odd number of time, also contains a Cysteine residue 24 (Cys-24)

(Harrop et al., 2001). This has led to the suggestion that biochemical properties and membrane insertion of CLIC1 may be under redox control.

In presence of the oxidising reagent, H2O2, monomeric CLIC1 forms intramolecular disulfide bonds between Cys24 and Cys59 exposing large hydrophobic patches on the protein that are the site for the subsequent non-covalent dimerization (Littler et al.,

2004). The crystal structure of this oxidised dimeric CLIC1 is changed radically from reduced monomeric CLIC1 (Littler et al., 2004). Studies using fluorescence quenching of endogenous Trp53 demonstrated that under reducing conditions monomeric CLIC1 does not enter an artificial liposome membrane (Goodchild et al., 2009). In contrast, under oxidising conditions there is maximal membrane association (Goodchild et al.,

2009). Furthermore, a study from the same group, using fluorescence resonance energy transfer (FRET) technique suggests that upon oxidation in the presence of membranes, monomer CLIC1 forms oligomers which may represent an intermediate state preceding membrane association (Goodchild et al., 2011; Goodchild et al., 2010). Thus, in addition to the previously discussed studies indicating increased ion channel activity of oxidised CLIC1, these strongly suggest that oxidation promotes CLIC1 membrane insertion.

16 1.I.C.i.b Low pH regulates CLIC1 membrane insertion

CLIC1 ion channel formation and activity is highly pH-dependent. When CLIC1 was added to artificial vesicles in buffers of varied pH (5.0, 6.0, 7.0, 8.0, 9.0), the chloride efflux was lowest at neutral pH, while greater rates of chloride efflux was observed at lower and higher pH (Tulk et al., 2002). This suggests that both protonation/deprotonation of some residues at low pH enhanced chloride efflux rate.

However, the result from this study is inconclusive because it is hard to pinpoint whether these pH effects are due to changes in protein residues or the membrane. When soluble purified recombinant CLIC1 was added to bath solution at various pH (5.0, 5.5

6.0, 6.5 7.0) together with artificial membrane, by reducing bath solution to pH 5, the time of the first ion channel opening after addition of CLIC1, is lessen compared to higher pH bath solution (6 or 7) (Warton et al., 2002). Further, using a single channel recording, when CLIC1 was in low pH (5.0 or 5.5) bath solution, the ion channel formed in low pH was characterised by increase occurrence of high conductance channel with fast kinetic (HCFK), while at high pH (6.5 or 7.0), the occurrence of

HCFK was decreased and replaced by increased occurrence of small conductance channel with slow kinetic (SCSK) (Warton et al., 2002). Thus, this study suggests that low pH favour the formation of aggregated ion channels. These all led to the suggestion that CLIC1 membrane insertion may be in part regulated by acidic pH.

1.I.C.i.c Cholesterol provides CLIC1 membrane initial docking site

Several studies have shown that CLICs membrane insertion into artificial lipids is highly dependent on membrane lipid composition (see Table 2). Studying the role of lipids on CLIC1 membrane insertion and activity showed that purified phospholipid alone tended to have significant endogenous permeability to chloride unless they are

17 supplemented with at least 10% cholesterol (Tulk et al., 2002). The same study also showed that increasing liposome cholesterol to 30% resulted in a decrease in CLIC1 ion channel activity as determined by chloride efflux rate (Tulk et al., 2002). Thus, this study indicates proper proportion of cholesterol is important for CLIC1 membrane insertion and activity. Later studies from independent groups reinforce this view and add that although CLIC1 appeared to insert into lipid bilayers regardless of lipid composition, the channel assembly and functioning in planar lipid bilayers require at least 1 molar cholesterol as a part of lipid mixture that contains 4 moles of neutral phosphatidylethanolamine (PE) phospholipid and 1 mole of net negative charge phosphatidylserine (PS) (Singh and Ashley, 2006). In absence of cholesterol, in a

Langmuir monolayer film, CLIC1 membrane insertion was reduced as measured by reduced membrane capacitance identified by impedance spectroscopy analysis.

However, in a film containing 16.7 mol% cholesterol, an increased amount of CLIC1 was inserted into the layer (Valenzuela et al., 2013).

It is unclear how cholesterol exerts its effect. Pre-incubation of CLIC1 with cholesterol prior to its addition to membrane, resulted in complete abrogation of CLIC1 membrane conductance, suggesting that cholesterol may act as a potential binding or docking site on the membrane (Valenzuela et al., 2013). Thus, it is proposed that the CLIC1

GXXXG motif which is adjacent to its putative transmembrane domain, may be the binding site for cholesterol, and this enhances assembly and oligomerisation that is necessary for ion channel activity (Valenzuela et al., 2013).

18 1.I.C.ii INTERACTION PARTNERS OF CLICS

1.I.C.ii.a Introduction

To date, the exact roles of CLICs within the cells are not fully understood. Although there is evidence to indicate that CLICs function as chloride channels, the biological functions of CLICs are not yet well described. Studies of CLIC interaction partners may aid in better understanding the biological functions of CLICs.

CLICs have been reported to interact with two main groups of proteins: cytoskeletal or scaffolding proteins and small GTPases. Evidence of CLICs interaction with cytoskeletal or scaffolding proteins as well as small GTPases will be reviewed in this section.

1.I.C.ii.b Cytoskeletal or scaffolding proteins

The cytoskeletal proteins comprise of a diverse group of intracellular proteins that provide cells both a scaffolding structure for static strength as well as flexibility to accommodate cell movement and help coordinate processes such as cellular differentiation, mitosis, cytokinesis, membrane trafficking and signalling.

CLICs interacts either directly or indirectly with cytoskeletal and scaffolding proteins.

Studies directly demonstrating interactions will be described immediately below, including data showing colocalisation of CLICs with elements of the cytoskeleton.

Studies of CLIC5 and CLIC4 illustrated that CLICs extensively interact with cytoskeletal and scaffolding proteins in different tissues. The interaction of CLICs with cytoskeletal and scaffolding proteins is recently reviewed extensively by (Jiang et al.,

2014) and is summarised in Table 3.

19 CLIC5 was first purified from placenta microvilli using affinity chromatography column containing Ezrin, a cytoskeletal protein (Berryman and Bretscher, 2000). Later pull down assay studies by the same group showed that when soluble recombinant

CLIC5A was added to a soluble microvillus extracts, CLIC5A formed complexes with actin, α-actinin, ezrin, gelsonin and IQ Motif Containing GTPase Activating Protein 1

(IQGAP1) (Berryman et al., 2004). Using yeast two hybrid screening and coimmunoprecipitation (Shanks et al., 2002), CLIC5B was also found to interact with A kinase anchoring protein 350 (AKAP350), a scaffolding protein.

CLIC4 has also been found to interact with cytoskeletal and scaffolding proteins. In brain tissue, a study using a gel overlay assay, showed that CLIC4 binds directly to brain dynamin I without any requirement of a linker or adaptor molecule, whereas, using the same protocol, the study found that CLIC4 binds indirectly with actin, tubulin and 14-3-3 isoform (Suginta et al., 2001). Furthermore, in cultured mammalian cell line,

CLIC4 co-localised with A kinase anchoring protein 350 (AKAP350) (Berryman and

Goldenring, 2003), a scaffolding protein. Thus, these all suggest that CLIC4 interact both directly and indirectly with cytoskeletal and scaffolding elements.

There is an emerging evidence of CLIC6 binding to scaffolding proteins to form multimeric complex necessary for downstream signalling pathway. Using yeast two hybrid system of rat brain cDNA library to screen for interaction partner to dopamine receptor D3R, CLIC6 was found to interact with scaffolding proteins such as multi postsynaptic density-95, disc-large, and zonulin-1 (PDZ) protein, Multi-PDZ-domain protein 1 (MUPP1) and Radixin (Griffon et al., 2003). However, the reason for this interaction is currently unknown.

20 Although there is no direct evidence to suggest that CLIC1 interact directly to cytoskeletal or scaffolding proteins, CLIC1 was found to co-localise with A kinase anchoring protein 350 (AKA350) scaffolding protein in confocal microscopy (Shanks et al., 2002).

Nevertheless, these all suggest that interaction of CLICs with cytoskeletal proteins could facilitate in some cellular signalling events associated with cytoskeletal and scaffolding proteins, possibly through the membrane remodelling mediated by the cytoskeleton network to place CLICs into a specific subcellular compartment to form an ion channel or to modulate channel activity. Therefore, further work is needed to validate this implication.

21 Table 3 Reported interaction of cytoskeletal and scaffolding proteins with CLICs CLICs Interaction Interaction Possible Reason Reported By Partner determined by for interaction Ezrin Affinity chromatography ? Berryman and Bretscher, 2000 Ezrin actin, Pulldown assay Assembly of F actin Berryman et al., 2004 α-actinin, Immunoprecipitation based structure at the cell CLIC5 gelsonin and Immunofluorescent cortex IQGAP1 microscopy Ezrin, Myosin Immunoprecipitation Stabilize membrane- Salles et al., 2014 VI, Radixin, Immunofluorescence cytoskeleton attachment AKA350 Yeast two-hybrid screen Scaffolding CLIC5 Shanks et al., 2002 Immunostaining to discrete location Actin, tubulin Affinity chromatography Cell membrane Suginta et al., 2001 14-3-3 isoform Iimmunoprecipitation remodelling, Control of cell shape CLIC4 Anion channel activity AKA350 Yeast two-hybrid screen Cytoskeletal organization Berryman and Goldenring, during the cell cycle 2003 CLIC6 Multi-PDZ Yeast two-hybrid screen ? Griffon et al., 2003 protein, MUPP1, Radixin CLIC1 AKA350 Yeast two-hybrid screen ? Shanks et al., 2002

22 1.I.C.ii.c Small GTPases

Another key interaction partners of CLICs are small GTPases such as Rho, Ran and

Rab family proteins. These small GTPases function as GDP/GTP-regulated molecular switches between active and inactive states to enable the modulation of complex cellular processes (Wennerberg et al., 2005). Small GTPases have high affinity to GDP and GTP. The cycling of GDP/GTP is controlled by two main classes of regulatory proteins GEF and GAP, with GEF promoting the formation of active GTPases (Schmidt and Hall, 2002), while GAP enhancing the formation of inactive GDP bound form

(Bernards and Settleman, 2004). In this section, the interaction of CLICs to small

GTPases is reviewed and summarised in Table 4.

CLICs proteins have been reported to associate with Rho and Rab GTPases

(Dozynkiewicz et al., 2012; Ponsioen et al., 2009). The most well studied CLICs is

CLIC4. CLIC4 was found to co-immunoprecipitates with Ran, a small GTPase, and was shown to facilitate CLIC4 translocation to nuclear compartment upon tumour necrosis factor (TNF)-α induction (Suh et al., 2004) or by S nitrosylation (Malik et al., 2010).

CLIC4 when artificially expressed in mouse neuroblastoma cells rapidly translocates to

RhoA-activating receptor complex consisting of scaffold proteins on cell membrane

(Ponsioen et al., 2009). Thus, this study suggests CLIC4 interacts directly with RhoA, which may facilitate in cellular events such as vesicle fusion, membrane expansion and actin dynamism (Ponsioen et al., 2009).

Studies of CLIC3 in ovarian cancer cell line showed that CLIC3 collaborate with Rab25 to direct endosomes containing active α5β1 integrin for recycling back to plasma membrane instead of directing to lysosome for degradation (Dozynkiewicz et al., 2012).

23 Consistent with this, in renal cell carcinoma, it has been demonstrated that the cells with human plasma membrane sialidase NEU3 (a key regulator of integrin recycling) silenced up-regulated Rab25 expression whilst down-regulating CLIC3 expression, which directed integrin to the lysosome for degradation (Tringali et al., 2012). Thus, these two studies indicate that CLIC3 is associated with Rab25 in some cancer cell line, however, whether the association is direct or indirectly still unclear.

For CLIC1, although confocal microscopy of murine peritoneal macrophages has shown that 5 minutes after phagocytosis, both CLIC1 and RhoA are localised to phagosomes but it was uncertain that these two proteins are indeed colocalised (Jiang et al., 2012).

Nevertheless, several CLIC studies strongly suggest that some CLICs especially CLIC4 and CLIC3 are associated with some of small GTPases in various cell types. This therefore merits further work to conclude whether CLIC interacts with small GTPases.

24 Table 4 Reported interaction of small GTPases with CLICs CLICs Interaction Interaction Detection Possible Reason Reported By Partner Determined by Method of interaction CLIC3 Rab25 Colocalisation Immunohistochemistry Recycling of integrin Dozynkiewicz et al., 2012 Confocal microscopy Ran Co-immunoprecipitation Western blot Importing CLIC4 to Suh et al., 2004 nuclear compartment RhoA Protein silencing reduce Pull down assay Mediating cell motility Spiekerkoetter et al., 2009 Rac1 small GTPases distribution. CLIC4 Ran Co-immunoprecipitation Immunoblot Importing CLIC4 to Malik et al., 2010 nuclear compartment RhoA Colocalisation Immunofluorescece Vesicle fusion Ponsioen et al., 2009 Mutational analysis membrane expansion Confocal microscopy actin dynamic CLIC1 RhoA Localisation to Immunofluorescence Unknown Jiang et al., 2012 Rac2 phagosomes Confocal microscopy

25 1.I.D STRUCTURE OF CLICs

In their soluble form CLICs adopt a glutathione-S-transferase (GST) fold structure.

CLICs contain two folds; N terminal folds containing β stranded mixed with 3 helices fold resembling thioredoxin like domain, and a C terminal domain consisting of all helices fold with foot loop between helix 5 and 6, which together resemble a glutathione-S-transferase (GST) like domain. To date, four of the vertebrate CLICs,

CLIC1 (Harrop et al., 2001), dimer CLIC1 (Littler et al., 2004), CLIC2 (Cromer et al.,

2007a; Cromer et al., 2007b), CLIC3 (Littler et al., 2010a), CLIC4 (Littler et al., 2005) and two invertebrate CLICs, EXC-4 in C elegans (Littler et al., 2008), DmCLIC in

Drosophila (Littler et al., 2008) crystal structures have been explained. However, there is no study on describing the structure of CLIC5 and CLIC6 so far. A brief overview of the crystal structures is summarised in Table 5. Whilst the structures of all CLICs will be reviewed, particular attention will be paid to CLIC1, which is the main topic of this thesis.

As depicted previously in Figure 1, N terminal domain of CLICs resembles thioredoxin like domain. Thioredoxin domain is best characterised as a four stranded β sheet sandwiched by three α helices in the order βαβα ββα and conserved Cys-x-x-Cys

(CxxC) or Cys-x-x-Ser (CxxS) motif for an active site (Qi and Grishin, 2005). In

CLICs, the N terminal domain is responsible for ~90 residues (except for CLIC5 and

CLIC6 where they possess an additional 170 and 440 residues respectively). CLICs adopt an thioredoxin domain in monomeric form (Cromer et al., 2007b; Harrop et al.,

2001; Littler et al., 2005; Littler et al., 2010a) whereas in their dimeric form, CLICs, for example CLIC1, the four stranded β sheets are replaced by extended helices (Littler et

26 al., 2004). Most of CLICs utilise a CxxS motif, but CLIC2 and CLIC3 are unique that they have a double cysteine motif and adopt Cys-x-x-Cys (CxxC) rather than Cys-x-x-

Ser (CxxS) motif for the active site (Cromer et al., 2007b; Littler et al., 2010a). Due to the presence of this motif, CLICs are believed to be controlled by redox reactions as previously discussed in section 1.I.C.i.a.

27 Table 5 Reported crystal structure of CLICs CLICs Crystal structure N terminal domain GSH binding site C terminal domain Reported By (Monomer) Soluble monomer form 4 of β strands of and 3 of CxxS motif of helix All 7 α helices (h4-h9) of Harrop et al., 2001 at 1.4-Å resolution α helices (h1-h3) of 90 1 via Cys24, Pro65 150 amino acids with more amino acids containing and Asp76 flexible foot loop GSH binding site CLIC1 Soluble dimeric form Significant changes - Slightly changes Littler et al., 2004 at 1.8-Å resolution 1) β strands disappears and replaced by extended helices 2) Forming of disulfide bond between Cys24 and Cys59 CLIC2 Soluble monomeric form 4 of β strands of and 3 of CxxC motif of helix All 7 α helices (h4-h9) of Cromer et al., at 1.8-Å resolution α helices (h1-h3) of 94 1, when oxidised 138 amino acid with the 2007b amino acids containing show intramolecular long flexible foot loop GSH binding site disulfide bond between h5 and h6 CLIC3 Soluble monomer form 4 of β strands of and 3 of CxxC motif of helix All 7 α helices (h4-h9) Littler et al., 2010 at 2-Å resolution" α helices (h1-h3) with 1 with more open with additional well additional 19 residues of and polar, when ordered helix F between hexahistidine tag oxidised show h5& h6 intramolecular disulfide bond CLIC4 Soluble monomer form 4 of β strands of and 3 of CxxS motif of helix All 7 α helices (h4-h9) of Littler et al., 2005 at 1.4-Å resolution" α helices (h1-h3) of 90 1 viaCys35 150 amino acid with amino acids containing disordered and flexible GSH binding site foot loop between h5 and h6 28 The C terminal domain of CLICs have ~150 residues, where all ten α helices resembles a glutathione-S-transferase (GST) fold like domain. The C terminal domain is connected to N terminal domain with a flexible proline rich foot loop at around helix 3.

The C terminal domain consists of seven helices (h4a, h4b, h5, h6, h7, h8 and h9) in both monomeric and dimeric forms (Littler et al., 2004). Between helix 5 and helix 6,

CLICs is inserted with 15 residues forming a foot loop that connect helix 5 and helix 6.

This foot loop possesses a highly negative charge and is usually flexible in most of

CLICs. The foot loop are particularly disordered in CLIC4 (Littler et al., 2005), but an ordered structure has been identified in helix F of CLIC3 (Littler et al., 2010a). This foot loop is believed to be a putative site for protein interactions in CLIC1 and perhaps to other CLICs.

Although all CLICs share most of their core structures that make them as a family, high-resolution crystal structures of individual CLIC reveals that there are indeed some structural differences that are unique to each class of CLIC and are perhaps responsible for the differences in behaviours and functions of individual CLIC. Individual crystal structure of all CLICs is far beyond the scope of this thesis, only CLIC1 crystal structure will be discussed in detail in the subsequent section.

29 1.I.D.i STRUCTURES OF CLIC1

1.I.D.i.a Introduction

Soluble and membrane integrated forms of CLIC1 are more likely to have their own structure. However to date, only soluble CLIC1 high-resolution crystallographic structure has been solved (Harrop et al., 2001). There are currently no reports on the crystal structure of the membrane-integrated form of CLIC1 due to the difficulty its isolation. In this thesis, the currently known structures of soluble CLIC1 will be discussed in details in this section.

1.I.D.i.b Structure of soluble CLIC1

Previous studies have illustrated that CLIC1, has a structure homologue to glutathione-

S- transferase (GST) superfamily protein, similar to other CLICs. For example, using

BLAST searches and sequence alignment, CLIC1 was found to share several similarities including the length and number of conserved active cysteine residues to omega class glutathione transferase GSTO1-1 (a class of GST superfamily protein)

(Dulhunty et al., 2001). Consistent with this, high resolution crystal structure of CLIC1 were achieved using crystal structure technique and confirmed that soluble monomeric

CLIC1 is indeed a structural homologue to GST superfamily (Harrop et al., 2001). The discovery of the CLIC1 crystal structure further revealed that the N domain has a thioredoxin fold that consists of a four stranded mixed β sheet sandwiched with 3 α helices, while the C domain is all helical, resembling omega class GST with two exceptions; 1) the insertion of a highly negatively charged loop region at the foot of the molecule and 2) the position of the carboxyl terminal helix h9 Figure 2A (Harrop et al.,

2001). The N and C domains are linked by the proline rich loop from Cys89-Asn100

30 A Monomeric CLIC1

B Dimeric CLIC1

Figure 2 Crystal structure of CLIC1 A: Crystal structure of monomeric CLIC1 showing GST folds that consists of two domains; thioredoxin folds on N terminal domain and all α helices fold (PDB ID: 1K0M). B: Crystal structure of dimeric CLIC1 showing N terminal domain undergoes tremendous conformation changes to all helices domain while only minor change was seen on C terminal domain (PDB ID: 1RK4).

31 which joins helices h3 to h4a. This interface is plastic and can be found in two conformations (Harrop et al., 2001), suggesting a candidate site responsible for structural alteration of CLIC1.

Within the N terminal domain, CLIC1 has one Cys24 which is located in the region corresponding to the typical glutathione-S-transferase (GST) active forming h1 and s2 within this active site and it is postulated that this active site could form a mixed disulphide with glutathione (GSH) and closely resemble a glutaredoxin protein (Harrop et al., 2001). The GSH binding properties of CLIC1 are much weaker than those observed for other GSTs, with less extensive interactions occurring between the CLIC1 active site and GSH (Harrop et al., 2001). There is a covalent complex between CLIC1 and oxidised glutathione (GSSG) and its structure has been crystallised. GSSG binding revealed only a fractional change in the protein structure with glutathione covalently attached to Cys24. Therefore, the presence of Cys24 residue on the GSH binding site suggest that CLIC1 may be under redox control.

To prove this, the crystal structure of oxidised soluble CLIC1 has been carried out.

High-resolution crystal structure demonstrated that upon treatment with an oxidative agent (H202), monomeric CLIC1 forms dimeric CLIC1 through intramolecular disulphide bond. This dimer is completely reversible by using a reductive agent (DTT)

(Littler et al., 2005). In comparison with monomeric CLIC1, dimeric CLIC1 comprises two third of the total proteins (Figure 2B). The C domain (residue 91-241) changes slightly, whereas the N domain undergoes significantly rearrangements. These N domain radical structural changes includes; 1) the formation of intermolecular

32 disulphide bond between Cys24 and Cys59, 2) the disappearance of the β-sheet and replaced by extended α helices (Littler et al., 2005).

1.I.D.i.c Structure of membrane integrated CLIC1

Although the crystal structure of the soluble form of CLIC1 has been resolved for years, less is known about its membrane integrated form as currently there is no crystal structure available. However, hypothetical structures have been proposed by various research groups.

33 1.I.E TISSUE AND SUBCELLULAR DISTRIBUTION OF CLICs

To date, there are several reports describing the distribution of CLICs in both human and mouse tissues as well as subcellular distribution of CLICs within different cell types. By searching on GeneCards database (www.genecards.org) that provides up to date information on tissue and subcellular distribution of the human gene of interest, the overall tissue and cellular distribution of human CLICs can be shown in Table 6.

According to the search for all CLIC homologues, CLIC1 and CLIC4 gene expression are widely distributed to almost all of the tissue examined (45 tissues for CLIC1 and 44 tissues for CLIC4). On the other hand, high expression of CLIC3 and CLIC6 gene are predominantly found in fewer tissues, for example high level expression of CLIC3 gene is found in placenta, whereas high level expression of CLIC6 is found in ganglia.

Likewise, CLIC3 and CLIC5 are highly expressed in placenta and spinal cord respectively.

For subcellular localisation, all of CLICs are found in plasma membrane, whereas most of CLICs except CLIC5 and CLIC6 are found in nucleus. While all CLICs can generally be found in cytosol and localise to plasma membrane, some CLICs localise to specific organelles and compartments. For instance, CLIC1 is localised to phagosome membrane. CLIC2 also localise to endoplasmic reticulum. CLIC3 is localised endosomes, lysosomes and vacuoles. CLIC4 is localised to cytoskeleton, mitochondrion and vacuoles. CLIC5 is localised to cytoskeleton and Golgi apparatus.

34 Table 6 Tissue and subcellular distribution of CLICs Tissues Normalized mRNA Subcellular localisation CLICs expression (%) salivary gland 8.46 plasma membrane pancreas 5.76 nucleus membrane stomach 5.65 nucleus ascites 5.11 cytoplasm skin 5.06 phagosome membrane blood 4.07 pharynx 3.76 parathyroid 3.75 bone 3.43 bone marrow 3.38 Lung, prostate,vacuolar, liver, Less than 3.00 cervix, mammalian gland, CLIC1 intestine, nerve, uterus, umbilical cord, ovary, lymph node, placenta, bladder, embryonic tissue, esophagus, connective tissue, eye, adrenal gland, ganglia, tonsil, thyroid, spleen, brain, mouth, pituitary gland, testis, thymus, muscle, trachea

35 Tissues Normalized mRNA Subcellular distribution CLICs expression (%) parathyroid 16.86 plasma membrane tonsil 14.38 cytoplasm endoplasmic reticulum blood 8.26 salivary gland 7.92 ganglia 6.09 trachea 6.06 esophagus 5.15 spleen 4.21 bladder 4.03 nerve 3.29 bone, kidney, muscle, pancreas, less than 2.2% CLIC2 heart, ovary, bone marrow, connective tissue, uterus, lung, thymus, lymph node, liver, placenta, prostate, brain, intestine, eye, skin

36 Tissues Normalized mRNA Subcellular localisation CLICs expression (%) placenta 34.53 plasma membrane lung 12.86 nucleus bladder 10.32 cytoplasm heart 7.51 endosome lysosome cervix 6.53 vacuole embryonic tissue 3.54 blood 3.25 pancreas 1.89 skin 1.50 uterus 1.37 brain 0.27 CLIC3

37 Tissues Normalized mRNA Subcellular localisation CLICs expression (%) vascular 12.14 plasma membrane nucleus trachea 10.58 cytoskeleton uterus 6.22 cytoplasm nerve 5.07 mitochrondria CLIC4 vacuole ganglia 4.69 thyroid 4.11 umbilical cord 3.33 connective tissue 3.32 lymph node 3.11 placenta 2.87 kidney, bone, muscle, bone less than 2.72 marrow, pineal gland, brain, heart, testis, ear, cervix, skin, parathyroid, embryonic tissue, tonsil, bladder, salivay gland, liver, lung, intestine, eye, stomach, ovary, spleen, mammalian gland, mouth, adrenal gland, ascites, prostate, pancreas, lymph, blood

38 Tissues Normalized mRNA Subcellular localisation CLICs expression (%) spinal cord 28.37 cytoskeleton golgi apparatus pharynx 14.90 cytoplasm placenta 7.15 plasma membrane ganglia 6.45 kidney 5.87 muscle 5.43 heart 4.66 spleen 4.47 ovary 4.09 intestine 2.90 CLIC5 connective tissue, adrenal gland, less than 2.18 eye, stomach, mammalian gland, ascites, lung, lymph node, brain, uterus, testis, pancreas, liver

39 Tissues Normalized mRNA Subcellular localisation CLICs expression (%) ganglia 30.90 plasma membrane cytoskeleton uterus 9.51 lung 8.19 eye 6.62 intestine 5.20 CLIC6 bone 3.73 blood 3.23 kidney 2.68 brain 2.65 connective tissue 2.61

40 1.I.E.i TISSUE AND CELLULAR DISTRIBUTION OF CLIC1

Several studies have revealed that CLIC1 is the most widely expressed CLIC because

CLIC1 mRNA is found in nearly all human and mouse tissues that were examined

(Berryman and Bretscher, 2000; Tulk and Edwards, 1998; Valenzuela et al., 2000). One study has shown that CLIC1 mRNA was absent in the brain (Berryman and Bretscher,

2000). In spite of the consistency of the CLIC1 tissue distribution from these studies, various lengths of CLIC1 mRNA transcripts have been reported, which no clear explanation is reported. Using Northern blot of whole tissues probed with NCC27 cDNA and actin cDNA as a control, the study identified two transcripts that are1.0 kb and 1.2 kb (Valenzuela et al., 2000). In contrast, the study that employed human tissues

300 bp of C terminal half of CLIC1 (576–871) of cDNA, identified 1.7 kb transcript in nearly all human tissues examined (Tulk and Edwards, 1998). Thus, it is likely that these variations may be due to the difference in the probes they used or the difference in experiment setting. Nevertheless, because of the abundance of CLIC1 in nearly to all tissues, CLIC1 is likely to participate in some basic biological functions common to most of cells.

Similar to Northern blot studies, a study using mouse tissues stained with polyclonal antibody against whole CLIC1, although CLIC1 protein is consistently present in most of tissues examined (Ulmasov et al., 2007), there was no staining of tissues that with previously mRNA expression such as spleen (Valenzuela et al., 2000). This raises an issue of a possibility of nonspecific staining from the polyclonal antibody. Similarly, many studies using polyclonal antibodies have reported that majority of CLIC1 localises in cytoplasm, intracellular organelles, plasma membrane and even nucleus. In fact, it is controversial and problematic to interpret because the reported cellular

41 distribution is varied and inconsistent. It is because these studies used polyclonal antibodies that raises against different part of CLIC1, which may be not specific to

CLIC1 at all, can bind to some other CLICs, thus non-specific to CLIC1.

Using polyclonal antibody against C terminal residue 228-241 of CLIC1, the majority of CLIC1 staining was found in the nucleus and nuclear membrane, whereas a small fraction of CLIC1 was found in cytoplasm and plasma membrane, determined by immunohistochemistry and western blotting (Tulk and Edwards, 1998). Similar results are reported when using polyclonal antibody against the whole CLIC1 protein (Ulmasov et al., 2007). Thus, to circumvent the issue of non-specificity, our group employed

CLIC1 knockout cells as a control and thus enable us to firmly identified cellular distribution of CLIC1 in macrophages, which CLIC1 localised in the cytoplasm and the phagosome membrane (Jiang et al., 2012). Similarly, a study using the same control and approach, CLIC1 was further confirmed to present on platelet membrane and cytoplasm

(Qiu et al., 2010). Considering high sequence homology between mammalian CLIC proteins, it is important to study tissue and cellular distribution by using monoclonal or anti-peptide antibodies specific to individual CLICs and using corresponding CLIC knockout cells/tissues as controls. Although it is uncertain whether and how CLIC1 tracking within the cytoplasm and intracellular organelle, the abundance of CLIC1 suggests its basic roles common to most of cells.

42 1.I.F BIOLOGICAL FUNCTIONS OF CLICs

1.I.F.i INTRODUCTION

Although the mechanism of action of CLICs is poorly understood, several studies have unravelled the roles played by individual CLICs in specific cellular processes. In this review, current findings on the biological roles from invertebrate and vertebrate CLICs will be discussed.

1.I.F.ii INVERTEBRATE CLICs

1.I.F.ii.a Introduction

Apart from the six vertebrate CLIC proteins, there are CLIC like proteins that have been studied in invertebrate species such as C. elegans and Drosophila. EXC-4 and EXL-1 are CLIC like proteins found in C. elegans, whereas DmCLIC is the only CLIC family protein found in Drosophila. Sequence alignment of EXC-4, EXL-1 and DmCLIC to human CLIC1, suggests that although these proteins are from very different organisms, they share significant similarity. Human CLIC1 shares a sequence identity of 25% with

EXC-4, 24% with EXL-1 and 30% with DmCLIC, while EXC-4 shares a sequence identity of 28% with EXL-1 and 39% with DmCLIC. EXL-1 shares 35% similarity to

DmCLIC. Phylogenetic analysis indicates that vertebrate and invertebrate CLICs diverged independently.

1.I.F.ii.b EXC-4 and EXL-1 in C. elegans

Functional studies of CLIC proteins have been extensively explored in vivo in C. elegans which only has 2 CLICs whilst vertebrate cells endogenously express multiple

CLICs. Thus, studies of this organism deal more effectively with the issue of possible redundancy between vertebrate CLICs. In C. elegans there are two genes that encode

43 CLIC like proteins, EXC-4 and EXL-1 (EXC-4 like 1) (Berry et al., 2003). C. elegans bearing its EXC-4 gene deletion display a distinct phenotype of enlarged cysts within a unicellular excretory canal cells (Berry et al., 2003). The same study using green fluorescent protein tagged EXC-4 in wild-type nematode, also showed that EXC-4 is localized to the luminal membrane of the excretory canal cell. Studies from the same group, using EXC-4 null mutant nematode, elegantly showed that introduction of wild type protein into the null mutant nematode rescued this cystic defect (Berry et al., 2003;

Berry and Hobert, 2006). Thus, these two studies emphasize an essential role of EXC-4 for the development of excretory canal perhaps through its membrane related functions.

Whilst the precise mechanism is unclear it indicates that Exc4 is involved in the process of tube formation.

The second and only other CLIC like protein in C. elegans is EXL-1. An EXL-1 construct is also localised to the membrane of lysosomes and Golgi apparatus of excretory cells but largely distinct from those associated with EXC-4 (Berry et al.,

2003). Using EXC-4 null mutant as an in vivo assay, the study showed that EXL-1 rescues the EXC-4 mutant phenotype, while human CLIC1 and CLIC4 failed to do so

(Berry and Hobert, 2006). Taken together, the results from these studies suggest that although invertebrate and vertebrate CLICs share some sequence homology, these two

CLICs have distinct functional characteristic when expressed in C elegans. This may be because the sequence difference is too great for the mammalian CLICs to rescue the

Exc4 phenotype or because the wrong mammalian CLICs were chosen for the attempted rescue.

Studies in C. elegans also provided the first insight into biophysical properties of CLIC protein in vivo. For example, interrupting the first 66 residues on N terminal domain of

44 EXC-4 and EXL-1 resulted in patchy membrane localisation and cytosolic partitioning, and failed to rescue the EXC-4 null phenotype (Berry et al., 2003). This suggests the 66 residues on N terminal end of EXC-4 and EXL-1 are indispensible for translocation of the protein from cytosol to the membrane. Further, both EXC-4 and EXL-1 are later confirmed as a putative transmembrane domain (PTM) that contained within the first 66 amino acid residues of their N terminal domain to attach to the membrane of the excretory canal luminal membrane or lysosomal membrane respectively (Berry and

Hobert, 2006).

The PTM region does not only direct invertebrate CLIC proteins to the membrane, it also plays key roles in functional specificity. Using chimeric protein (comprising of

PTM region of EXC-4 and other C terminal three quarters of CLIC1, GST-44 and GST-

36), the study illustrated that all CLICs were successfully targeted to the luminal membrane and rescued the mutant cystic phenotype (Berry and Hobert, 2006). Thus, this study suggests that C terminal three quarters of EXC-4, CLIC1, GST-44 and GST-

36 are functionally interchangeable, which the function is common to feature of GST fold and not specific to either omega class or to anion channel property of CLICs. The

PTM that direct EXC-4 to specific compartment, may therefore provide the functional specificity.

45 1.I.F.ii.c DmCLIC in Drosophila melanogaster

Less is known about the one CLIC-like proteins in Drosophilla called DmCLIC

DmCLIC is structurally closer to vertebrate CLICs than they are to any other GST fold family members (Littler et al., 2008), suggesting that they have a common evolutionary ancestor and may perhaps share similar molecular and cellular function in ion channels

(Littler et al., 2008).

46 1.I.F.iii VERTEBRATE CLICS

1.I.F.iii.a Introduction

Although the biological role and mechanism of action of CLIC proteins is poorly understood there are some studies that are informative and they are summarised in

Table 7. Section 1.I.F.iii.b-g provides an overview of the biological roles of CLICs such as in kidney function, angiogenesis, cell cycle and differentiation, inflammatory mediators production, intracellular vesicle trafficking, intracellular acidification and enzymes. Whilst all biological functions will be reviewed, particular attention will be paid to the functions of CLIC1, which is the main subject of this thesis.

47 Table 7 Reported biological functions of CLICs Biological Role CLICs Systems Possible function(s) Reported By Podocytes & Interaction with Ezrin Wegner et al., 2010 CLIC5A Glomeruli Podocytes Maintenance podocyte integrity Pierchala et al., 2010 Kidney function CLIC5A through interaction with Ezrin/radixin/moesin Maintenance of fetal kidney Edwards et al., 2014 CLIC4 Kidney morphology Drive conversion of fibroblast to Ronnov-Jessen et al., 2002 CLIC4 Fibroblast myofibroblast through TGFβ interaction Berryman and Goldenring, CLIC4 Epithelial cells Regulate cytoskeleton rearrangement during cell cycle 2003 Drive cell cycle arrest through CLIC4 Keratinocytes altering chloride concentration and pH of nucleus Suh et al., 2007 Regulate proliferation and Li et al., 2010 Cell division CLIC5 Mouse skeleton cell line differentiation of myoblasts into myotubes Drive cytokinesis at plasma Valenzuela et al., 2000 membrane by regulating CLIC1 CHO cells swelling of both cells and nucleus, leading to subsequent pinching off of two cells Setti et al., 2013 Glioblastoma cancer Drive Self-renewals and cell Gritti et al., 2014 CLIC1 stem cells proliferation through CLIC1 mediated chloride ion activity

48 Biological Functions CLICs Systems Possible function(s) Reported By Bone marrow Produce IL-6, IlL-12 He et al., 2011 CLIC4 derived macrophages and TNF Malik et al., 2012 Peritoneal macrophages Produce IL-6, IlL-12 CLIC4 and TNF Inflammatory mediator CLIC4 Peritoneal macrophages Produce iNOS Malik et al., 2012 production CLIC1 Microglial cells Produce TNF-α Novarino et al., 2004 CLIC1 Microglial cells Produce ROS Milton et al., 2008 CLIC1 Peritoneal macrophages Produce ROS Jiang et al., 2012 CLIC1 Cancer cell Produce ROS Wang et al., 2014 Ovarian and pancreatic Facilitate a recycling of integrin Dozynkiewicz et al., 2012 CLIC3 tumour cells α5β1 Facilitate a recycling of a Intracellular vesicle trafficking transmembrane CLIC3 Breast cancer cells matrix metalloproteinase Macpherson et al., 2014 HeLa Internalise integrin into CLIC4 MDA-MB-231 cells, endosomal system Argenzio et al., 2014 Mediate phagosome Kim et al., 2013 acidification after Listeria Macrophages cell line Monocytogenes uptake CLIC3 (THP-1) Mediate large vacuole Ulmasov et al., 2009 Intracellular acidification acidification and support CLIC4 Endothelial cells tubulogenesis Mediate chloride influx for electric shunting for proton influx in vesicular acidification CLIC5B Osteoclasts important for bone reabsorption Edwards et al., 2006

49 Biological Functions CLICs Systems Possible function(s) Reported By Mediate proton transportation through interaction with proton pump ATPases Intracellular acidification CLIC6 Parietal cells Shin et al., 2009 Mediate phagosome acidification through chloride CLIC1 Peritoneal macrophages channel activity Jiang et al., 2012

Platelet coagulation Regulate platelet activation via CLIC1 Platelets P2Y12 receptor Qiu et al., 2010 Enhance regulatory volume Cell volume regulation decrease via CLIC1 mediated CLIC1 Colon cancer cells chloride channel activity Wang et al., 2012

50 1.I.F.iii.b Kidney function

There is emerging interest in the function of CLICs in podocytes, specialised epithelial cells forming part of renal glomeruli of kidney (Edwards, 2010; Edwards et al., 2014;

Pierchala et al., 2010; Wegner et al., 2010). Since the first identified CLIC (p64;

CLIC5B) was isolated from a bovine kidney (Landry et al., 1993) several studies have tried to unravel the roles of CLICs in kidney function. Studies of jitterbug (jbg/jbg) mice that lack CLIC5 protein were found to have proteinuria (urine protein loss during glomerular filtration) (Pierchala et al., 2010; Wegner et al., 2010). Renal glomeruli podocytes cover the outer layer of glomerular basement membrane where they function to stabilise glomerular capillaries. The podocyte’s structural stability is crucial for glomerular barrier function and this is mainly achieved through interaction of the apical membrane of podocytes with the cytoskeleton network via ezrin, radixin and moesin

(ERM) proteins (Lowik et al., 2009). Failing to achieve this interaction, results in proteinuria (Asanuma et al., 2007). CLIC5A is localised to the apical plasma membrane of podocytes where it colocalises with ezrin (Pierchala et al., 2010; Wegner et al., 2010) and membrane associated protein such as podocalyxin (Pierchala et al., 2010). The absence of CLIC5 in jitterbug (jbg/jbg) mice, also leads to the disappearance of ezrin from the apical domain (Wegner et al., 2010). This suggests the role of CLIC5 in the podocytes may be to generate a stable cell structure tethering the podocyte apical membrane to the cells cytoskeleton through its interaction with ezrin, radixin and moesin (ERM) complex which in turn bind to the cytoskeleton network. Additionally in line with this view, CLIC5-/- podocytes are shorter than those of CLIC5+/+ mice, suggesting it has a role in the development and maintenance of proper podocyte architecture (Pierchala et al., 2010). Furthermore, a recent study which attempts to unravel the mechanism by which CLIC5A may interact with ezrin suggests that

51 CLIC5A may act by recruiting and clustering phosphatidylinositol (PI)-4,5- bisphosphate (PI(4,5)P2), a protein necessary for ezrin phosphorylation, to the apical plasma membrane of podocytes. This clustering phosphatidylinositol (PI)-4,5- bisphosphate (PI(4,5)P2) to the apical plasma membrane activates ezrin phosphorylation which in turn activates its association with cytoskeleton elements and thus stabilise podocyte integrity (Al-Momany et al., 2014).

CLIC4 have also been implicated in kidney biology. Using CLIC4 null mice a study compared the kidney to body mass ratio of CLIC4 null mice determined that both body and kidney of CLIC4 null mice are smaller than wild-type mice (Edwards et al., 2014).

This is consistent with the finding that CLIC4 null mice are smaller than littermate wild-type or CLIC4 heterozygote mice (Padmakumar et al., 2012). By counting number of glomeruli and identifying the density of peritubular capillary, the same study showed that CLIC4 null mice have less glomeruli and less dense peritubular capillary, suggesting impaired renal angiogenesis may be due to the absence of CLIC4 in CLIC4 null mice (Edwards et al., 2014). Because small kidney size is also associated with proteinuria, the study measured protein loss and confirmed that CLIC4 null mice have about 3.5 folds increased protein loss. Furthermore, in an acute kidney injury model,

CLIC4 null mice suffer from more severe acute kidney injury when compared to wild type mice, while the rate of recovery from the injury as determined by area of scar or fibrosis, is indistinguishable between gene deleted and wild type control mice (Edwards et al., 2014). In addition to actin, a recent study using CLIC4-/- mice and CLIC4+/+ control has demonstrated that new born CLIC4-/- mice have a defect in renal formation as demonstrated by less number of microvilli, while abnormally dilated proximal tubules were evident in the adult knockout mice. Because when CLIC4 is suppressed,

52 this study also showed that the delivery of Rab11 from early endosome tubule to recycling endosome is impaired, suggesting that CLIC4 function in renal tubule formation perhaps through apical delivery of endosome (Chou et al., 2016). These all suggest that CLIC4 play an important role in kidney. Whether CLIC4 plays direct or indirect role in other kidney function merits a future research.

Although there is no direct evidence for CLIC1’s role in kidney function, CLIC1 is expressed in the kidney especially in apical membrane of proximal tubule cells (Tulk and Edwards, 1998) and a proximal tubule mouse cell line (Ulmasov et al., 2007), suggesting a potential role for it in renal function mediated by CLIC1 in kidney similar to CLIC4-5. However, little is known about CLIC1 and kidney function.

1.I.F.iii.c Angiogenesis

Angiogenesis is the formation of new blood vessels from existing vasculature, which involves endothelial tubulogenesis and formation of capillaries network (Carmeliet,

2003). To date, several reports have suggested that that several CLICs such as CLIC4,

CLIC5 and CLIC1 have roles in angiogenesis.

The most convincing evidence of CLIC role in angiogenesis is from CLIC4 studies. It started from EXC-4 study that strongly suggested that this CLIC4 homolog plays a part in C elegans tube formation (Berry et al., 2003). Then a proteomic study reveal that

CLIC4 is one of the 120 proteins that changes in response to vascular endothelial growth factor type A (VEGF-A) driven endothelial tubulogenesis, providing an initial clue that CLIC4 may be a strong candidate for angiogenesis. In line with this, an in vitro silencing of CLIC4 in endothelial cells reduced capillary network formation and lumen

53 formation in vitro. Knocking out CLIC4 in mice resulted in fewer glomeruli and less dense capillary networks, suggesting impaired renal angiogenesis due to the absence of

CLIC4 (Edwards et al., 2014). Consistent with these results, a study performed using a matrigel angiogenesis assay on CLIC4-/- mice and showed that matrigel plugs from

CLIC4-/- mice displayed abnormal endothelial lumens with dilated cystic structure compared to well-formed lumen in wild-type mice. However, how CLIC4 facilitates angiogenesis is still unclear. One mechanism might be related to CLIC4 function as a chloride channel. Chloride channels help provide electric shunting which is needed to compensate for the proton entry necessary for vacuolar acidification necessary for vesicular fusion, A study that measured intracellular pH of vacuoles from endothelial cells of CLIC4-/- mice showed that large vacuoles of CLIC4-/- endothelial cells acidified less well than CLIC4+/+ vacuoles, resulting in an inhibition of endothelial tubulogenesis

(Ulmasov et al., 2009).

Unlike CLIC4, several studies have reported that CLIC1 may have both pro- angiogenetic and anti-angiogenetic properties. In vitro knockdown CLIC1 by short hairpin RNA (shRNA) in endothelial cell line (HUVEC) resulted in a reduction of endothelial cell growth, a decline in migration into the wound area and an inhibition of capillary-like network formation forming less dense, when compare to control cells

(Tung and Kitajewski, 2010). It has been showed that CLIC1 is co-localised with anti- angiogenic peptide (CTL1) at the cell margin of endothelial cells to mediate an internalization of CTL1 peptide to induce cytotoxicity and anti-tumour activity. Thus, this study indicates that apart of pro-angiogenetic effect, CLIC1 may exert anti- angiogenetic properties.

54 1.I.F.iii.d Cell division

Cell division is takes place in almost every cell. As some CLICs can be found in nucleus of cells and cell division may involve chloride ion channel activity, several studies have attempted to unravel their role in cell division. However, the precise mechanism of CLIC’s contribution to this function is still under investigation.

Several studies using different cell type have shown that CLIC4 is associated with different phases of cell division. Using immunofluorescence microscopy, CLIC4 was found in centrosome and midbody in cultured epithelial cells during mitosis, where

CLIC4 colocalised with A kinase anchoring protein 350 (AKAP350) (Berryman and

Goldenring, 2003), a scaffolding protein that is important for multiprotein clustering.

Given that CLIC4 was previously shown to interact with actin cytoskeleton (Suginta et al., 2001), the study thus proposed that CLIC4 may acts as adaptor protein that link scaffolding proteins to cytoskeleton network, fulfilling its role in cell division

(Berryman and Goldenring, 2003). This study is important because it introduce the additional function of CLIC4 independence of its chloride channel activity. Moreover, a study employed Ca2+ induced cell differentiation of CLIC4 knockdown, overexpressing and non-transfected keratinocytes, determined that CLIC4 is also required for cell cycle arrest and subsequent cell differentiation. Upon entering cell cycle arrest, CLIC4 was found abundantly in nuclear membrane where CLIC4 cause chloride flux into the nucleus, promoting cell differentiation. Additionally, However, this study confirmed their finding by showing that increased chloride flux of CLIC4 of over-expressing keratinocytes induced cell cycle arrest and promoted keratinocytes differentiation, while knocking down CLIC4 using either antisense adenovirus vector or RNA interference inhibited cell cycle arrest and prevented the cell differentiation (Suh et al., 2007). Thus,

55 the study supports the role of chloride flux mediated by CLIC4 in cell cycle arrest.

Taken together, these studies suggest that, at least in cell division, CLIC4 may function differently depend on the phase and cell type.

CLIC5 is highly expressed in skeletal muscle (Berryman and Bretscher, 2000). CLIC5 is also involved in cell cycle and cell differentiation processes (Li et al., 2010). When the mouse skeletal muscle cell line, C2C12, is starved of growth factors, the cells undergo cell differentiation and cell fusion to form multinucleated myotubes. A study examining total cell lysate from C2C12 cells after serum starvation, demonstrated that

CLIC5 protein level expression is markedly increased during this C2C12 cell differentiation. The study employed CLIC5 transfected cells and non-transfected control, and when these cells were grown in serum-free medium for 24 hrs, more

CLIC5 transfected cells were in G0/G1 phase than control cells. In contrast, the percentage of CLIC5 transfected cells in G2/M phase was lower than the cells in control group (Li et al., 2010). Thus, the results from this study indicates that CLIC5 may promotes cell cycle exist and exert cell differentiation. However, it is currently unknown whether chloride channel activity mediated CLIC5 plays a role in this function as previously reported from CLIC4 study (Suh et al., 2007).

Because CLIC1 distributes ubiquitously in most of mouse and human fetal and adult tissues (Valenzuela et al., 2000), intensive research has been investigated to determine whether CLIC1 has a role in various cellular biological functions—one of which is cell growth and cell cycle. Unlike CLIC4 (Suh et al., 2007), CLIC1 chloride conductance mediates cell mitosis rather than cell cycle arrest (Valenzuela et al., 2000). The study measured chloride conductance of CLIC1 transfected and non-transfected CHO-K1

56 during both G2-M phase and G1-S phase, and showed that, chloride conductance of

CLIC1 transfected cells was double in G2-M phase than in G1-S phase, in contrast, chloride conductance of non-transfected cells can only be found in G2/M phase while no current was detected in G1-S phase. Further, several CLIC ion channel blocker including indanyloxyacetic acid (IAA94), anthracene-9-carboxylic acid (A9C) and 4,4′- diisothiocyano-2,2′stilbene-disulfonic acid (DIDS) when used to treat CHO-K1 cells increased the percentages of cells in G2-M phase of the cell cycle compared to untreated cells. Thus, these results firmly indicate that CLIC1 chloride conductance is important for CHO-K1 cell mitosis.

1.I.F.iii.e Inflammatory mediators production

There is increasing evidence suggesting that CLICs, particularly CLIC4 (He et al.,

2011; Malik et al., 2012) and CLIC1 (Averaimo et al., 2010; Jiang et al., 2012; Milton et al., 2008; Novarino et al., 2004), play significant roles in early host defences particularly by producing inflammatory mediator products such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS) in innate immune cells such as macrophages and microglia. Recently, a study that examined CLIC4 expression in fetal and adult organs using green fluorescent protein knock-in mouse line, demonstrated that in spleen, high expression of CLIC4 was found in macrophages and myeloid origin cells such as monocytes, dendritic cells and B cells, while much lower expression of CLIC4 was evident in T cells (Padmakumar et al., 2014). Thus, this result indicates that CLIC4 may be an immune modulator.

Studies of CLIC4 have shown that it has dual roles in innate immunity by contributing to both pro-inflammatory (shortly after infection) (He et al., 2011) and anti-

57 inflammatory responses (Malik et al., 2012). Northern blotting of bone marrow derived macrophages (BMDM) treated with lipopolysaccharide (LPS) found that CLIC4 mRNA expression level was increased immediately in response to LPS (He et al., 2011), providing the first clue of CLIC4 role in early host defence. Further, CLIC4 transfected

RAW264.7 cells, when exposed to LPS, secrete increased amounts of several proinflammatory cytokines and chemokine including Interleukin (IL)-6, Interleukin

(IL)-12 and CCL5. Thus, this study indicates the association of increased CLIC4 with the enhanced macrophage pro-inflammatory responses in vitro. To validate the importance of CLIC4 in vivo, the study compared the early host response to LPS and

Listeria monocytogenes of CLIC4 null mice, and determined that CLIC4 null mice are protected from cytokine storm induced by LPS but have a higher burden of Listeria.

These results suggest that CLIC4 is important for early host innate immunity. In addition, CLIC4 has also been implicated in anti-inflammation responses especially for macrophage deactivation.

The study led by independent group has demonstrated that when peritoneal macrophages were exposed to LPS and INFγ, CLIC4 protein and induced nitric oxide synthase (iNOS) was upregulated to allow CLIC4 to undergo S-nitrosylation and translocates to nuclear membrane (Malik et al., 2012). Using real time PCR of several cytokine and chemokine transcripts of extracted RNA from CLIC4-/- macrophages and wild type control, the study showed that, upon exposure to LPS, there were sustained elevated levels of IL-1β mRNA for more than 24 hrs. Thus, the study suggests that

CLIC4 is needed to suppress IL-1β activity of proinflammatory macrophages. On the other hand, the results from this study indicate that nuclear localisation of S-nitrosylated

CLIC4 from iNOS, functions to decrease pro-inflammatory responses of macrophages

58 by suppression of IL-1β activity, thus deactivate macrophage function. Taken together,

These two studies firmly implicate that CLIC4 have roles in both promoting pro- inflammatory mediator production as well as suppressing pro-inflammatory responses depending on the microenvironment (Malik et al., 2012). However, the fact that the cells examined in these two experiments are from different sources i.e. bone marrow derived macrophages vs peritoneal macrophages. One might be aware that this may influence the inconsistent function of CLIC4 in innate immunity.

Of all the CLICs, CLIC1 provides the best data suggesting for a role in inflammatory mediators production. Since CLIC1 was first cloned after macrophage activation

(Valenzuela et al., 1997), CLIC1 is always thought to play an important role in macrophage function. The first piece of evidence directly supporting this view is the association of CLIC1 expression and the production of nitrogen intermediates and tumour necrosis factor-α (TNF-α) in microglial cells (macrophage like cells in the brain) treated with amyloid beta (Aβ) peptides that promote oxidative stress and cause neurodegenerative processes (Novarino et al., 2004). The study also showed that indanyloxyacetic acid 99 (IAA94) or small interfering RNA (siRNA) that block the chloride channel and knockdown CLIC1 respectively, prevented the release of amyloid beta (Aβ) peptides stimulated tumour necrosis factor-α (TNF-α). The same group of researchers later confirmed their findings by demonstrating that when using live cell imaging, amyloid beta (Aβ) peptides induced the rapid translocation of CLIC1 to the microglial plasma membrane to form a chloride conductance that is essential for ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Milton et al., 2008). These studies suggested that chloride influx mediated by CLIC1 may help

- in charge compensating O2 generated during the respiratory burst. Recently, our group

59 employed real time measurement of HRP enhanced luminol chemiluminescence and demonstrated that CLIC1-/- peritoneal macrophages produced 30% less ROS after phagocytosis of serum opsonized zymosan particles. When using indanyloxyacetic acid

99 (IAA94) as a CLIC inhibitor reduced the ROS production of CLIC1+/+ peritoneal macrophages to the level of CLIC1-/- peritoneal macrophages (Jiang et al., 2012) but had no effect on CLIC1-/- macrophages, suggesting deletion of CLIC1 impairs macrophages production of ROS. Supporting this, using K/BxN arthritis animal model which its pathogenesis is driven by ROS production by macrophages play significant role, revealed that CLIC1-/- mice are protected from K/BxN arthritis with lessen swelling when compare to CLIC1+/+ mice, emphasizing the important role of CLIC1 in

ROS production in vivo. Taken together, several studies consistently reported that

CLIC1 facilitates production inflammatory products such as oxidative stress and ROS especially in innate immune cells such as brain microglia cells and peritoneal macrophages.

1.I.F.iii.f Membrane vesicle trafficking

There are emerging studies that suggest roles for CLICs in membrane vesicle trafficking in some cells particularly in cancer cells. Direct evidence from CLIC3 studies supports this argument.

Using real time PCR, CLIC3 mRNA expression was increased 20 folds in human ovarian carcinoma cell line transfected with Rab25 and plated onto cell derived matrix- a thick, pliable matrix composed mainly of fibrilla collagen and fibronectin, compared to non-transfected cells (Dozynkiewicz et al., 2012). Thus, this study indicates that enhanced CLIC3 expression is associated with elevated Rab25. Further, the same study

60 expressed photoactivatable GFP-α5β1 integrin and Cherry-CLIC3 in the Rab25 expressing cells, and showed that activated α5β1 integrin was routed to CLIC3 positive late endosomes/lysosomes and rapidly recycle back to the plasma membrane, thus resulted in an increase in cell movement (Dozynkiewicz et al., 2012). Hence, this study proposed that the increased CLIC3 expression in Rab25 expressing cancer cells is associated with poor survival.

Similarly, a recent report from the same group, using microarray on breast cancer tissue, showed that CLIC3 mRNA expression is also enhanced in breast carcinomas when compared to adjacent normal tissue, and is associated with poor survival in a cohort study (Macpherson et al., 2014). However, in breast cancer cells that Rab25 expression is low, the role of CLIC3 in α5β1 integrin recycling was not affected by knockdown

CLIC3 gene. Instead, when CLIC3 was knockdown in breast cancer cell line, the rate of return of internalized transmembrane matrix metalloproteinase (MT1-MMP) to the plasma membrane was reduced (Macpherson et al., 2014). Using live cell imaging, this study showed that CLIC3 was colocalised with MT1-MMP in a subset of late endosome/lysosomes, suggesting a role for CLIC3 in facilitating recycling of MM1-

MMP from late endosome/lysosomes back to plasma membrane (Macpherson et al.,

2014).

61 1.I.F.iii.g Intracellular Acidification

Several studies from most CLICs except CLIC2, suggest that CLICs may play a part in intracellular acidification of various organelles. However, the mechanism by which

CLICs facilitate intracellular acidification is uncertain.

Recently, using yeast-two hybrid screening and coimmunoprecipitation against a complement receptor, C3b receptor, a receptor that recognise C3b on opsonized bacteria, CLIC3 has been found to interact and coimmunoprecipitated with cytoplasmic tails of C3b receptor on macrophages (Kim et al., 2013). Intracellular bacteria Listeria

Monocytogenes, are taken up by the phagosomes, and activate C3b signailing. In response to this C3b signaling, the phagosomes containing Listeria Monocytogenes, chloride influx was increased when measured with chloride sensitive fluorescent dyes using confocal microscopy. The phagosomes also acidified to as low as pH 4.5, identified by pH sensitive fluorophore probe using live cell imaging. Additionally, knockdown CLIC3 expression by siRNA in the CLIC3+/+ macrophages eliminates the difference of phagosomal acidification and chloride influx to a level similar to that in

CLIC3-/- macrophages (Kim et al., 2013). Because phagosomal acidification is known to facilitate their fusion with lysosomes, this study also demonstrated that the fusion did not take place in absence of CLIC3, whereas, successful fusion were evident in

CLIC3+/+ macrophages. Taken together, all of these results suggest that C3b-mediated killing of Listeria Monocytogenes in macrophages is facilitated by CLIC3 chloride influx in which mediate efficient phagosome acidification (Kim et al., 2013). This paper proposed that the chloride ion transportation mediated by CLIC3 may act as counterion of proton influx, important for fusion of phagosomes with lysosomes.

62 A study that measure intracellular pH of vacuoles from CLIC4-/- endothelial cells their wild type control, showed that large vacuoles (> 4 µm in diameter) of CLIC4-/- endothelial cells acidified less than CLIC4+/+ vacuoles (Ulmasov et al., 2009). However, no difference in intracellular pH of late endosome/lysosome or small vacuoles (< 2 µm in diameter) was observed between CLIC4-/- endothelial cells and wild-type control.

Thus, this study suggests that CLIC4 functions in angiogenesis by supporting vacuolar acidification along intracellular tubulogenic pathway.

CLIC5B is another member of CLICs that has been reported to play a part in intracellular acidification in cells particularly in osteoclasts (Edwards et al., 2006) and parietal cells (Bradford et al., 2010). In osteoclasts with CLIC5B gene knockdown by antisense nucleotide reduced cellular chloride transport when compared to untreated cells. The impaired chloride transport in these cells was associated with reduction in both rate and extent of vesicular acidification (Edwards et al., 2006). Because vesicular acidification is achieved by influx of proton which is mediated by a proton pump via V type ATPases (Toyomura et al., 2000), chloride influx is indispensible for electric shunting (Blair et al., 1991). Thus, the study indicate that at least in osteoclasts,

CLIC5B mediated chloride conductance determines vesicular acidification, important for their function in bone reabsorption (Edwards et al., 2006). Likewise similar to osteoclasts, in separate a study using CLIC5 mutant mice (jitterbug) consistently showed that the pH of stomach contents of CLIC5 mutant mice was higher than their wild-type littermates, providing further supporting evidence that CLIC5 in stomach parietal cells and bone osteoclasts may share common regulatory role in hydrochloric acid (HCl) secretion (Bradford et al., 2010). However, as CLIC5 is less abundant in the

63 stomach and parietal cells (Bradford et al., 2010), it is difficult to implicate CLIC5’s role as gastric chloride channels or gastric regulators.

Unlike CLIC5, CLIC6 is abundantly present in parietal cells of the stomach and was found to colocalise with proton pump ATPases (Bradford et al., 2010). However, whether the chloride channel activity mediated by CLIC6 is necessary for proton transportation during gastric secretion of parietal cells, is under investigation.

For CLIC1, a single report from our group has recently reported that peritoneal macrophages from CLIC1-/- mice when fed with pH sensitive fluorescent dye had impaired phagosomal acidification. This same study from our group has also shown that shortly after phagocytosis, CLIC1 moved to the phagosome membrane where it colocalised with NADPH oxidases. Using zymosan coated with pH sensitive dye and insensitive dye as a control, CLIC1-/- phagosomes of peritoneal macrophages acidify less (pH 4.13 ± 0.02) than CLIC1+/+ phagosomes (pH 3.92 ± 0.03) (Jiang et al., 2012).

Thus, these results indicate that movement of CLIC1 to phagosome membrane regulate phagosome acidification. However, the mechanism on how CLIC1 regulate acidification is still unsolved.

1.I.F.iii.h Enzymes

CLICs are a member of glutathione- S- transferase (GST) superfamily protein and contain active site on their N terminal domain, suggesting their potential role as an enzyme. A recent study using 2-hydroxyethyl disulfide (HEDS) as an substrate for assaying glutaredoxin enzymatic assay, has confirmed that CLIC1, CLIC2 and CLIC4 have glutaredoxin enzymatic activities (Al Khamici et al., 2015). Interestingly, among

64 these CLICs, CLIC1 and CLIC4 provided similar enzymatic activities while CLIC2 is less active, possibly due to the composition of the active site between CLIC2 and the other two CLICs.

1.I.F.iii.i Platelet coagulation

By using western blotting of lysate from platelets of CLIC1+/+ and CLIC1-/- mice, a study has shown that that human platelets contain a huge amount of CLIC1 proteins

(Qiu et al., 2010). The same study employed immunofluorescence confocal microscope, and showed that, upon activation, CLIC1 was predominantly stained along plasma membrane while this was absent during resting platelets. Thus, this high level of CLIC1 prompted this study to investigate platelet function in these animals Whilst CLIC1-/- mice appear normal and have no embryonic lethality, despite having 15% more platelet count, CLIC1-/- mice have twice prolonged bleeding time when compared to CLIC1+/+ mice, after stimulated with Adenosine diphosphate (ADP) (Qiu et al., 2010). Because stimulation of Adenosine diphosphate (ADP) leads to platelet aggregation and activation, this study also examined a defect in platelet aggregation from CLIC1-/- mice and found that adenosine diphosphate (ADP) stimulated platelet aggregation is 40% lower in CLIC1-/- when compared to CLIC1+/+ mice. By using flow cytometry to quantitate activated form of GPIIbIIIa, the study also demonstrated that CLIC1-/- platelets display attenuated activation when compare to CLIC1+/+ mice. Thus, these results both indicate abnormal platelet function in CLIC1-/- mice. Because adenosine diphosphate (ADP) can initiate platelet activation via P2Y1 receptor or P2Y12 receptor, this same study also sought out which pathway that CLIC1 may be involved in platelet function. By using antagonist to either P2Y1 receptor or P2Y12, they demonstrated that

+/+ only antagonist to the P2Y12 receptor reduced platelet activation from CLIC1 to the

65 same level of CLIC1-/- for the full range of all antagonist concentration, whereas, high antagonist concentration is require for P2Y1 receptor to eliminate the difference between CLIC1-/- and CLIC1+/+ mice. Thus, taken together, this study suggests that

CLIC1 may regulate platelet activation via P2Y12 receptor. However, the mechanism by which CLIC1 regulates platelet function via P2Y12 is currently unknown. As P2Y12 receptors need free thiol group at Cys17 and Cys270 to function optimally, it was postulated that CLIC1 is a redox protein that controls the local redox environment and in turn influence platelet activation (Essex, 2009).

1.I.F.iii.j Cell volume regulation

CLIC1 has been reported play a part in cell volume regulation which is important for cell survival, replication and migration. A study showed that CLIC1 mRNA relative expression is most highly upregulated of other chloride channel genes such as ClC3,

ClC2, Best1, Best2 and Best3, when microglia are placed in a hypotonic solution

(Ducharme et al., 2007). Thus, in response to hypotonic environment, this study suggests that the chloride channel activity mediated by CLIC1 is activated and the efflux of chloride ion can lead to a decrease in cell volume or regulatory volume decrease (RVD). Similarly, a recent study using colon cancer cell lines with high

(LOVO) and low (HT29) metastatic potency has showed that, in response to hypotonic solution, the rate of regulatory volume decrease (RVD) of LOVO cells was higher than

HT29 cells, suggesting that RVD capacity is correlated with the metastatic potential of cancer (Wang et al., 2012). When RT-PCR was used to measure CLIC1 mRNA expression from these two cell lines, the study showed that CLIC1 mRNA was 1.85 fold higher in LOVO cells compared with H29 cells. Further, when these two cell lines were treated with IAA94 or transfected with CLIC1siRNA, regulatory volume decrease

66 (RVD) from both cell lines decreased significantly when compare to control group.

Thus, this study suggests that high CLIC1 expression is associated with enhanced regulatory volume decrease (RVD) rate, which in turn improves metastasis (Wang et al.,

2012). These all suggest that CLIC1 may play a part in regulatory volume decrease

(RVD) and therefore regulate cell volume perhaps through its chloride channel activity.

If this is the case and cell volume seems to be a secondary effect from chloride channel activity, however, to date there is no report of RVD of some other CLICs. Thus, further investigation merit a further work.

67 1.II DENDRITIC CELLS (DCs)

1.II.A INTRODUCTION

In 1973, Ralph M. Steinman and Zanvil A. Cohn reported the identification of a novel cell type in the peripheral lymphoid organ of mice (Steinman and Cohn, 1973). It was distinct from other cells within the lymphoid tissue with features of: 1) irregular large nucleus; 2) cytoplasmic extensions arranged as dendrites of varying length, width, form and number, many of which contain numerous of spherical phase dense mitochondria;

3) large in size. As a result of their distinct morphologies, such cells were named as dendritic cells (DCs), and these cells were soon shown to have important roles in the immune system (Steinman and Cohn, 1974; Steinman et al., 1974). Within 5 years of discovery, spleen DCs were found to be 100 times more effective than lymphocytes and macrophages in stimulating primary allogenic mixed leukocyte reactions (MLR), hence establishing the importance of DC’s in the role of antigen presentation (Nussenzweig et al., 1980; Steinman and Banchereau, 2007; Steinman et al., 1979). The significance of this finding has led to the 2011 Nobel Prize in Physiology and Medicine awarded to

Professor Ralph Steinman for his discovery of DCs and define roles of DCs in adaptive immunity.

In this review, overview features of DCs will be discussed including their subsets, their development and also their functions. Because DC functions are heterogeneous this review will mainly emphasize MHC class II antigen presentation pathway which is the main topic of this thesis.

68 1.II.B DC SUBSETS

1.II.B.i INTRODUCTION

Dendritic cells (DCs) are comprised of heterogeneous subsets with unique cell surface markers and are found in various organs. Currently DC are characterised into three major subsets: conventional dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs) and monocyte derived dendritic cells (moDC). An overview of these DC subsets, featuring commonly nomenclatures, cell surface markers, tissue residency, functions and route of antigen presentation are summaried in Table 8. In this review, only mouse

DC subsets, the main topic of this thesis will be emphasized while human DC subsets are beyond the scope of this thesis.

Conventional dendritic cells (cDCs) can be further subdivided into cDC1 and cDC2 and will be reviewed in details in section 1.II.B.ii. In section 1.II.B.iii, plasmacytoid dendritic cells (pDCs) will also be reviewed. Monocyte derived dendritic cells (moDC), the subset predominantly responsive to inflammatory stimuli, will be reviewed in section 1.II.B.iv.

69 Table 8 Summary of phenotypes of the major mouse DC subsets Mouse DCs DC subset Nomenclature 1 Nomenclature 2 Markers Tissue Main Functions Presentation Lymphoid resident CD8+ MHC II Lymphoid Type 1 T helper Cross-presentation on DCs CD11c organs (Th1) and MHC class I CD8α cytotoxic T DNGR-1 lymphocytes XCR-1 (CTL) immunity CADM1 cDC1 Migratory DCs CD103+ MHC II Nonlymphoid Type 1 T helper Cross-presentation on CD11c organs (Th1) and MHC class I CD103 cytotoxic T DNGR-1 lymphocytes XCR-1 (CTL) immunity CADM1 Lymphoid resident CD4+ MHC II Lymphoid Type 2 T helper MHC class II DCs CD11c organs and Type 17 T CD4 helper immunity Dectin 1&2 CD301b Migratory DCs CD11b+ MHC II Nonlymphoid Type 2 T helper MHC class II CD11c organs and Type 17 T cDC2 CD11b helper immunity Dectin 1&2 CD301b

70 DC subset DC type DC type Markers Tissue Main Functions Presentation Lymphoid resident pDC BST-2 Lymphoid Viral clearance via Poor DCs B220, organs type I Interferon PDCA-1 and circulating pDCs Siglec-H blood MHC II (low) CD11c (low) Induced by Inflammatory DC Ly6C Lymphoid Response to Cross-presentation on inflammation or CD11b organs and inflammation MHC I and MHC TNF-α and iNOS CD11c inflamed class II Monocyte producing DC CD206 nonlymphoid derived DCs (Tip-DC) CD64 sites (moDCs) MHC II CD209α FcεR1

71 1.II.B.ii CONVENTIONAL DCs (cDCs)

Conventional dendritic cells (cDCs) are subdivided into two main subsets; 1) cDC1 are sometime referred to as CD8+ DCs and are found in lymphoid organs or their non- lymphoid equivalent CD103+ DCs (Guilliams et al., 2014); and 2) cDC2 are usually referred to as CD4+ DCs which are found in lymphoid organs, while their equivalent subset in nonlymphoid organs are sometime called CD11b+ DCs (Guilliams et al.,

2014).

cDC1 main function is to cross present intracellular bacteria or virus on MHC class I to

CD8+ T cells and therefore induce Th1 and CTL immunity (Bedoui et al., 2009; Lin et al., 2008a). They are separated from other lineages by their their unique transcription factor usage (see section 1.II.B) and functional characteristics. cDC1 express cell surface markers including X-C motif chemokine receptor 1 (XCR-1) and DC, NK lectin group receptor-1 (DNGR-1). These markers are conserved in both mouse and human cDC1. X-C motif chemokine receptor 1 (XCR-1) is responsible for promoting crosstalk between cDC1 to CD8+ T cells (Crozat et al., 2010), while DC, NK lectin group receptor-1 (DNGR-1) is responsible for capture and routing intracellular antigen into the

MHC class I pathway for stimulation of CD8+ T cells (Ahrens et al., 2012). cDC1 express Toll like receptors (TLR) 3, 4, 11 and 13 (Edelson et al., 2010). TLR3 can recognise viral dsRNA, providing cDC1 with viral sensing capability.

The primary function of cDC2 is to present extracellular antigens such as those from bacteria, fungi and helminth on MHC class II to CD4+ T cells and mount Th2 (Plantinga et al., 2013) and Th17 immunity(Persson et al., 2013) cDC2 express Dectin1&2,

CD301b and MHC class II on their cell surface. Dectin 1&2 recognise fungi (Harman et

72 al., 2013), while CD301 is responsible for expulsion of helminth (Kumamoto et al.,

2013). MHC class II expressed by cDC2 enable them to be the potent antigen presenting cells for extracellular antigen and activate CD4+ T cells (Vander Lugt et al., 2014).

Additionally, cDC2 are equipped with TLR5, 6, 7, 9 and 13, enabling them to sense comprehensive sets of extracellular pathogens.

1.II.B.iii PLASMACYTOID DCs (pDCs) pDCs are a DC subset that is characterised by transient production of type 1 interferon

(IFNs) after viral infection (Cella et al., 1999). However, pDCs also secrete other pro- inflammatory cytokines, such as IL-6 & IL-12, which are necessary for both innate and adaptive immunity (Swiecki and Colonna, 2015). pDCs express CD11clow, bone marrow stromal cell antigen 2 (BST-2), B220, plasmacytoid dendritic cell antigen 1

(PDCA-1) and siliac acid binding immunoglobulin-like lectin H (Siglec-H) on their cell surface (see Table 8). BST-2 is responsible for sorting secreted protein from golgi apparatus to membrane (Blasius et al., 2006), while Siglec-H acts as a receptor for carbohydrate recognition (Zhang et al., 2006). Additionally pDCs are equipped with

TLR7 and 9, enabling them effective detection of double stranded RNA of viruses

(Kawai and Akira, 2011). This makes pDCs key players in viral infection.

As appose to cDCs, pDCs can be found in circulating blood as well as in lymphoid organs, such that they have a role in acute viral clearance from the circulation.

Although, pDCs express MHC class II and co-stimulatory molecules for antigen presentation and the regulation of CD4+ T cells in the same way as cDCs, they are comparatively less effective in this this process (Sapoznikov et al., 2007). Furthermore, unlike cDCs which are all originated from common myeloid progenitors (CMP) (see

73 section 1.II.B), pDCs can also be found in common lymphoid progenitors (CLP) (Onai et al., 2013). However, pDCs of lymphoid lineage are distinct from the myeloid lineage through the expression of the RAG-1 gene.

1.II.B.iv MONOCYTE DERIVED DCs (moDCs)

Monocyte derived DCs (moDCs) are generated within inflamed sites where they express Ly6C, CD11b, CD11c, CD206, CD64, MHC II, CD209α and FcεR1. moDCs differ from cDCs and pDCs due to their monocyte lineage (Plantinga et al., 2013).

There are several other features that separate moDCs from cDCs and pDCs. They express the chemokine CC receptor (CCR7) which enables moDCs to migrate between inflamed tissues and secondary lymphoid organs in response to inflammation (Plantinga et al., 2013). Secondly, high level expression of MHC class II and other co-stimulatory molecules, allow moDCs to present antigens and activate T cells in lymphoid organs

(Hohl et al., 2009). Thirdly, moDCs can produce large amount of TNF-α and iNOS in response to bacterial infection and are thus sometime being described as TNF-α and iNOS-producing DCs (Tip DCs) (Serbina et al., 2003).

Despite moDCs and cDCs share many common features, as mentioned above, it is now accepted that moDCs is a distinct subset from the activated cDCs (Mildner et al., 2013).

74 1.II.C DC DEVELOPMENT

1.II.C.i INTRODUCTION

In the last decade, many research groups have been attempting to understand the development lineage of DC from hematopoietic progenitors. Whilst the precise molecular processes are largely unexplained, it has been widely accepted that the DC development relies on an orchestrated expression of numerous transcription factors and various cytokines for lineage restriction. The transcription factors and cytokines that guide DC development are summarised in Table 9, and their effect on DC development can be universal or subset specific. The developmental pathways from bone marrow stem cells to precursor dendritic cells and to differentiated DC subsets are shown in

Figure 3.

DCs developing from hematopoietic stem cells require several intermediate progenitors

(Geissmann et al., 2010), and precise expression of transcription factors. HSC first differentiate into common myeloid progenitors, then subsequently into macrophages/DC precursor. Under the control of transcription factor and cytokine expression, macrophages/DC precursor can be differentiated to either common DC progenitor or common monocyte precursor. Thus, in this section, the key transcription factors which are important for guiding DC development from bone marrow stem cells to diverse DC subsets will be reviewed. Additionally, down streaming factors such as cytokines that regulate DC differentiation will be also reviewed.

75 Table 9 Transcription factors and cytokines guiding DC development Transcripton factor Family Function Expression Reported by PU.1 ETS family Upregulates Flt3 expression Early stages of Carotta et al., 2010 DC precursors Spi-B ETS family Faciliates PU.1 function Early stages of Sasaki et al., 2012 DC precursors Ikaros Zinc finger Binds to PU.1 gene locus to All DCs Zarnegar and DNA binding activate or repress PU.1 Rothenberg, 2012 protein Gfil Zinc finger Functions downstream of All DCs Spooner et al., 2009 DNA binding Ikaros protein Zbtb46 Zinc finger Binds to MHC class II genes Restricted to all Meredith et al., 2012 DNA binding and modulate cDC cDCs but not protein activation status pDCs Bcl6 Zinc finger Regulates Th2 immune All cDCs but not Ohtsuka et al., 2011 DNA binding responses pDCs protein Id2 Inhibitor of Binds to E2-2 protein and All cDCs but not Moore et al., 2012 DNA binding inhibits pDC development pDCs protein E2-2 E protein Activates pDC specific pDCs but not Ghosh et al., 2010 transcription factors cDCs Batf3 bZIP family Induces cross-presentation cDC1 such as Tussiwand et al., of intracellular pathogens CD8+ DCs and 2012 CD103+ DCs

76 Transcripton factor Family Function Expression Reported by E4BP4 bZIP family Interacts with Batf and cDC1 such as Kashiwada et al., mediate cros- presentation CD8+ DCs and 2011 CD103+ DCs IRF8 Interferon- Interacts with Batf and cDC1 but not Tussiwand et al., regulating mediate cros- presentation cDC2 2012 factor RelB REL- Mediates downstream CD8- DCs Sun, 2012 homology signaling in NFkB complex domain family IRF4 Interferon- Interacts with several genes CD8- DCs and Suzuki et al., 2004 regulating that promote Th2 responses pDCs factor Cytokines Family Function Expression Reported by Flt3 Tyrosine Develops all DCs from MDPs, CDP, pre- Kingston et al., 2009 kinases haematopoietic progenitors DC, and all DCs & Maintains DC homeostasis in tissues M-CSF Tyrosine Produces monocyte lineage MDP, CDP, MacDonald et al., kinases CD103+ DCs and 2005 moDCs GM-CSF Tyrosine Produces cDCs cDCs, Greter et al., 2012 kinases CD11b+DCs

77 Bone marrow Non-lymphoid organs HSC + CMP Batf3 CD103 DC E4BP4 PU.1 SpiB pre-DC MDP M-CSF dependent IRF8 Blood Flt3L+ Flt3L+ GM-CSF dependent Gfil Flt3- + Flt3 dependent Ikaros RelB CD11b DC IRF4 CDP cMoP Flt3L+ Flt3L+ Flt3- MCSFR+ MCSFR- MCSFR+ + Ly6C moDC E2-2 Ly6C+ monocyte ZBt46 monocyte

Id2 Blc6 pDC GM-CSF dependent

pre-DC + Blood Ly6C pDC pDC monocyte

CD8+ DC

Batf3 pre-DC E4BP4 IRF8 moDC pre-DC CD4+ DC

Blood RelB IRF4 Lymphoid organs

Figure 3 Key transcription factors and important cytokines that regulate DC subset differentiation. DC subsets are generated from HSCs through several intermediate progenitors facilitated by several transcription factors and cytokines. MDP gives rise to CDP or cMoP. CDP further differentiate into pre-DC which enter blood to lymphoid and nonlymphoid tissues where they differentiate to cDC1 and cDC2 subsets. CDP also give rise to pDC within the bone marrow and pDC enter the blood to and seed peripheral tissues. Additionally, cMoP gives rise to monocytes circulating in the bloods where they can enter peripheral tissues and differentiated into moDCs. Abbreviations; HSC, hematopoietic stem cells; CMP, common myeloid progenitors; MDP, macrophages/DC precursor; CDP, common DC progenitor; cMoP, common monocyte precursor; pDC, plasmacytoid dendritic cells; moDC, monocyte derived dendritic cells

78 1.II.C.ii TRANSCRIPTION FACTORS

As summarised in Table 9, the patterns of transcription factors determine the routes of

DC lineage development. ETS transcription factors such as PU.1 and Spi-B bind to

DNA and regulate Fms-Related Tyrosine Kinase 3 (Flt3) and macrophage colony stimulating factor receptor (MCSF-R) expression (Carotta et al., 2010; Schonheit et al.,

2013). PU.1 and Spi-B are detected in early precursors such as common myeloid progenitors (CMP), suggesting their necessity for all DC lineages. Additionally, PU.1 and Spi-B are crucial in later stage differentiation of CD11b+ cDC subset from pre-DC through the induction of Irf8 expression, providing multiple functions in DC differentiation (Sasaki et al., 2012; Schonheit et al., 2013).

To segregate DC lineage from macrophage lineage development, macrophages/DC precursors (MDP) rely on two transcription factors Ikaros (Zarnegar and Rothenberg,

2012) and Gfil (Spooner et al., 2009). They are Zinc finger DNA binding proteins, and suppression of these proteins results in surge of monocyte development as part of macrophage lineage.

The divergence of common DC progenitor (CDP) to cDC depends on three different transcription factors: Zbtb46, Bcl6 and Id2. Zbtb46 and Bcl6 are transcriptional repressors that are exclusively expressed in pre DC and all cDCs (Meredith et al., 2012;

Ohtsuka et al., 2011). Zbtb46 modulates MHC II genes. Id2 is required for cDC development, however it is also crucial for the development of other immune cells, such as NK cells (Moore et al., 2012). In addition, E2-2 is another transcription factor that promotes pDC differentiation from common DC progenitor (CDP). Deletion of E2-2 inhibited pDC development, while overexpression of E2-2 enhanced pDC

79 differentiation (Cisse et al., 2008). Interestingly, expression of E2-2 can be inhibited by

Id2 and drive cDC development (Hacker et al., 2003). These suggest that E2-2 works hand in hand with Id2 in the regulation of DC differentiation to their corresponding subsets.

From the bone marrow, pre-DC migrates to both lymphoid and non-lymphoid organs for subset differentiation. Through the expression of the three transcription factors,

Batf3, E4BP4 and Irf8, pre-DC differentiates into cDC1 subset; CD8+ DCs or CD103+

DCs in lymphoid organs or non-lymphoid organs respectively. Because Batf3 has been found to be critical for antigen cross-presentation of intracellular pathogen in cDC1, this suggests that Batf3 is important for cDC1 subset polarization (Tussiwand et al., 2012).

E4BP4 (Kashiwada et al., 2011) and Irf8 (Tussiwand et al., 2012) interact with Batf3 to facilitate antigen cross-presentation, thus suggesting their supporting role in Batf3 signalling. On the other hand, Irf4 and RelB promote cDC2 subset (CD4+ DCs in lymphoid and CD11b+ in non-lymphoid organs) differentiation (Sun, 2012; Suzuki et al., 2004). Irf4 knockout mice displayed cDC2 subset reduction (Suzuki et al., 2004), suggesting its role is cDC2 specific.

1.II.C.iii CYTOKINES

Whilst less is known about the cytokines involving in DC development, there are at least three cytokines including Fms-like tyrosine kinase 3 (Flt3), macrophage colony- stimulating factor (M-CSF) and Granulocyte macrophage colony-stimulating factor

(GM-CSF), that are widely accepted as important cytokines for guiding DC development.

80 Flt3L is highly expressed in macrophages/DC precursors (MDP) and believed to plays a significant role in early DC development. Mice lacking Flt3L gene show a 14 fold decrease in functional DCs in lymphoid organs (McKenna et al., 2000). On the other hand, subcutaneously daily injection of Flt3L for consecutive nine days into the mice increases all DCs in bone marrow and lymphoid organs, with up to 30% of spleen cells expressing CD11c and MHC class II (Karsunky et al., 2003), suggesting Flt3 is crucial cytokine for all cDC and pDC development.

As opposed to Flt3L, the expression of M-CSF receptors (M-CSFR) in macrophages/DC precursors (MDP) responds to M-CSF in bone marrow to differentiating away from the common DC progenitor (CDP) to become common monocyte progenitors (cMoP) (MacDonald et al., 2005). cMoP further differentiates mostly into the bone marrow monocytes, enter into the blood stream and migrate to both lymphoid and nonlymphoid organs where small fraction of these cells can further differentiate into monocyte derived DCs (moDCs).

GM-CSF plays a important role in the late stage of cDC development (Greter et al.,

2012). Circulating pre-DC enters tissues in response to GM-CSF to replenish tissue resident cDCs. Furthermore, in vitro culture of bone marrow stem cells with GM-CSF produced cDCs which are potent in antigen presentation with high expression of CD11c and MHC class II molecules (Rivollier et al., 2012). Recent discoveries from several groups have shown that GM-CSF also controls the development of monocyte derived

DCs (moDCs) which can accumulate in the tissues in response to infection or tissue injury (Reynolds et al., 2016; Ushach and Zlotnik, 2016).

81 1.III FUNCTIONS OF cDCs

1.III.A INTRODUCTION

This part of the review will be describing the most well described function of cDCs, which is antigen presentation. An overview of process involving cDC to achieve a potent antingen presentation are summaried in Figure 4. Whilst antigen presentation is the main focus of this thesis, some other functions of cDCs and other function of other

DC subset will be generally discussed.

As depicted in Figure 4, cDCs continuously survey tissues for self or nonself antigen through their pattern recognition receptors (PPRs). Upon antigens recognition, cDCs uptake the antigens through endocytosis, pinocytosis and phagocytosis. cDCs process internalised antigens through either MHC class II pathway or via antigen cross- presentation through the MHC class I pathway. Fourth, immature cDCs transform into mature cDCs via the cell maturation process, which inhibit their phagocytic capacity and elevates surface expression of MHC class II and other costimulatory molecules.

Finally, mature cDCs migrates from tissues to the secondary lymphoid organs, via lymphatic vessels, where antigen bound cDCs encounter and modulate the activation of

T cells.

82

Figure 4 An overview of cDC function as antigen presenting cells. cDCs patrol peripheral tissues to capture and process antigens. Antigen bound cDCs undergo the maturation processes and migrate to the draining lymph nodes to present antigens to T cells for specific immunity activation. Abbreviations; cDC, conventional dendritic cells; TCR, T cell receptor; IL-12, Interleukin 12; IL-12R, Interleukin receptor, CD40L, CD40 ligand

83 1.III.B TISSUE SURVEILLANCE cDCs are equipped with variety of pattern recognition receptors (PRRs), allowing cDCs to recognise and respond to non-self antigens of infectious organisms, releasing pro- inflammatory cytokines and antimicrobial inflammatory stimulating proteins. An overview of various sets of pattern recognition receptors (PRRs) and how they drive inflammation are summarised in this section and depicted in Figure 5.

As shown in Figure 5, the pattern recognition receptors (PRRs) expressed by cDCs, include Toll like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)- like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and

C-type lectin receptors (CLRs) (Hemmi and Akira, 2005; Kawai and Akira, 2011;

Osorio and Reis e Sousa, 2011). TLRs and CLRs are located on the cell surface and in endosomes while NLRs and RLRs are located in cytosol.

There are at least 12 TLRs that have been identified in mice and they are the major pattern recognition receptors (PRRs) in cDCs. TLRs can be grouped into two main groups, cell surface expressing TLRs (TLR1, 2, 4, 5, 6, 11, 12) and intracellular endosomal TLRs (TLR3, 7, 8, 9,13). TLRs are type I transmembrane proteins that are compose of two domains. An extracellular domain contains leucine rich repeat (LRR) domain that can recognise sets of pathogen associated molecular patterns (PAMPs) which are summarised in Table 10. TLRs also composed of an cytoplasmic domain that can interact with adaptor proteins especially myeloid differentiation factor 88 (MyD88) protein, allowing oligermolerisation and assembly of large multi subunit

84 Carbohydrate LPS /Bacteria/ Flagellin/ Mycoplasma Cell membrane TLRs Virus Bacteria CLRs (1,2,4,5,6,11,12)

MyD88 Endosomes SYK MyD88 IRAK dsRNA TRAF6 TLRs CpG Island dsDNA CARD9 dsRNA Mal-1 IRF-5 (3,7,8,9,13) Bcl-10 I-κB NLRs NF-κB RLRs RIP2 IPS-1 Cytoplasm NF-κB

IRF-3

IRF-5 NF-κB

Proinflammatory Cytokines

IRF-3 Type I Interferon

Nucleus

Figure 5 Pattern recognition receptors (PRRs) used by cDCs to drive inflammation. DCs are equipped with four classes of pattern recognition receptors (PRRs) for pathogen sensing. These pattern recognition receptors (PRRs) can be classified into two main groups; 1) surface membrane toll like receptors (TLRs) and C-type lectin receptors (CLRs) and 2) intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). After pathogen sensing, the activated pattern recognition receptors (PRRs) oligermerises adaptor protein complex and induces downstream signaling cascade for production of pro-inflammatory cytokines as well as antimicrobial peptide type I interferon. Adapted from (Walsh and Mills, 2013).

85

Table 10 Pattern recognition receptors (PRRs) of cDC subsets Cellular PRRs PRRs Ligand/Origin localization cDC1 cDC2

TLR1 Triacyl lipopolysaccharide /Bacteria Cell membrane ++ ++ TLR2 Diacyl lipopeptide /Bacteria Cell membrane ++ ++ TLR3 dsRNA /Viruses Endosome/lysosome +++ +

TLR4 Lipopolysaccharide/ Gram negative Bacteria Cell membrane + + TLR5 Flagellin /Bacteria Cell membrane low + TLRs TLR6 Diacyl lipopeptide /Mycoplasma Cell membrane ++ +++ TLR7 ssRNA /Viruses Endosome/lysosome neg ++ TLR8 ssRNA /Viruses Endosome/lysosome ++ ++ TLR9 CpG DNA /Bacteria Endosome/lysosome +++ + TLR11 Unknown Cell membrane +++ neg TLR12 Unknown Cell membrane ++ + TLR13 Unknown Endosome/lysosome ++ ++

DC-SIGN Carbohydrate/ Virus, Bacteria and Protozoa Cell membrane neg + CLRs DEC-205 Not determined Cell membrane +++ + Dectin-1 Fungal β glucan/Fungi Cell membrane neg ++ NOD1 dsDNA /Bacteria Cytoplasm neg ++ NLRs NOD2 dsDNA /Bacteria Cytoplasm neg ++ Not Not RLRs RIG-1 ssRNA /Viruses Cytoplasm determined determined

86 complexes that initiate intracellular signaling cascade as depict in Figure 5 (Beutler,

2009). Interestingly, whilst there is no obvious difference between cDC subsets in the expression of cell membrane TLRs, there is differential expression of their intracellular

TLRs. cDC1 express higher level of TLR3 and TLR9 than cDC2, whereas, TLR7 is absent in cDC1 but present in cDC2.

Another family of cell membrane PRRs found in DCs are C-type lectin receptors

(CLRs). These CLRs include Dendritic Cell-Specific Intracellular adhesion molecule-3- grabbling Non-integrin (DC-SIGN), Diagnostic utility of thymic epithelial marker

CD205 (DEC205) and DC-associated C-type lectin-1 (Dectin-1) (summarised in Table

10). Whilst ligands for DEC205 are unknown, DC-SIGN and Dectin-1 recognise carbohydrate structures (Koppel et al., 2005) of bacteria, protozoa, viruses and fungal β glucan (Brown, 2006) respectively. Their downstream signaling pathway was postulated to activate NF-κB (Figure 5). However, it is interesting to note that cDC1 express only

DEC-205, whereas DC-SIGN and Dectin1 are absent. cDC2 on the other hand expresses all of these CLRs.

In addition to TLRs and CLRs, there are two additional intracellular PRRs, nucleotide- binding oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid- inducible gene I (RIG-I)-like receptors (RLRs) that reside in the cytoplasm and are responsible for detecting intracellular bacteria and viruses respectively (Vabret and

Blander, 2013). The interaction between RLRs and ssRNA triggers recruitment of adaptor molecule IPS-1 which induces type I interferon production, while interaction of

NLRs with dsDNA activates NF-κB cascade (Figure 5).

87 1.III.C ANTIGEN UPTAKE

1.III.C.i INTRODUCTION

After pathogen detection, cDCs internalize extracellular antigens and process them within the processing compartments. cDCs acquire antigens through three routes; 1) receptor-mediated endocytosis, 2) macropinocytosis and 3) phagocytosis (Liu and

Roche, 2015). An overview of these three routes are shown in Figure 6. Mechanisms underlying these processes will also be discussed below. Generally, cDCs ingest small molecules using receptor-mediated endocytosis that can also been found in many cell types. cDCs also use receptor mediated phagocytosis to engulf and internalise insoluble particulate antigens including necrotic/apoptotic cells and opsonized pathogenic organisms that are larger than 0.5 µm. The receptors display on cDCs include complement (C3a and C5a), scavenger and Fc receptors as well as that for C type lectin.

These receptors all mediate both endocytosis and phagocytosis in cDCs. In case of non specific internalisation of soluble molecules (usually fluid), cDCs acquire this fluid through macropinocytosis sometimes referred to as cell drinking. In this thesis, all of these three routes for antigen uptake will be reviewed individually in more detail in subsequent section.

88

Figure 6 Three possible routes of extracellular antigen uptakes in cDCs cDCs uptake extracellular antigens via three possible endocytic routes such as receptor- mediated endocytosis, macropinocytosis and phagocytosis. Depend on the properties of antigens, endocytosis is responsible for antigen size less than 0.5 µm that internalize in cathrin coated vesicles or cathrin uncoated vesicles, macropinocytosis is responsible for soluble antigen and phagocytosis is responsible for particulate antigens size greater than 0.5 µm, Antigens are processed and form pMHC-II complexes in multivesible bodies (MVBs) before traffic to plasma membrane to initiate antigen-specific adaptive immune responses. Adapted from (Liu and Roche, 2015)

89 1.III.C.ii RECEPTOR-MEDIATED ENDOCYTOSIS

Small molecules are internalised by cDCs through a receptor-mediated endocytosis. As the name suggests, it is mediated by receptors on the cell surface which cluster to form clathrin-coated endocytic vesicles. Some forms of endocytosis are clathrin-independent.

Molecules in clathrin-coated vesicles are then delivered to early endosomes for antigen processing (Liu and Roche, 2015).

cDC1 expresses distinct receptors from cDC2 to mediate the endocytosis. cDC1 expresses DEC-205 and Toll like receptor (TLR11), whereas, these two receptors are absent in cDC2. Interestingly, expression of DEC-205 in cDC1 is associated with their ability to cross presenting extracellular antigens to MHC class I (Carter et al., 2006). On the other hand, cDC2 express Dectin-1, a receptor of which is absent in cDC1, to facilitate antigen presentation via the MHC class II pathway (Carter et al., 2006).

However, most cell surface molecules can also facilitate endocytosis, but their discussion is beyond the scope of this thesis.

It is currently accepted that the receptor-mediated endocytosis can deliver antigens to distinct classes of endosomes in DCs. For example, mannose receptor-endocytosed ovalbumin is preferentially delivered to an early endosomal compartment, and antigen targeting to this compartment generally leads to poor ovalbumin processing (Burgdorf et al., 2007). In contrast, in case of Fc receptor mediated endocytosis, depending on the type of Fc receptor, ovalbumin endocytosed by this route can be transported to late endosomes/lysosomes where it can undergo efficient antigen processing and later presentation to ovalbumin -specific T cells (Platt et al., 2010).

90 1.III.C.iii MACROPINOCYTOSIS

Macropinocytosis represents the main route for antigen uptake in cDCs (Liu and Roche,

2015), allowing cDCs to rapidly and nonspecifically sample surrounding extracellular fluid for antigen detection. As shown in Figure 6, macropinocytosis is actin dependent, and requires small GTP-binding proteins Cdc42 and Rac1 for membrane ruffing and to form intracellular vacuoles called macropinosomes (Lim and Gleeson, 2011). Soluble antigen uptake through macropinocytosis is directed to the late endosome/lysosome and does not transit to early endosomes (Burgdorf et al., 2007).

There are reports showing that the extent of macropinocytosis varies among different

DC subsets. For example, in vitro study of splenic cDCs showed cDCs internalize more soluble fluorescent ovalbumin when compared to pDCs. However, this difference was not observed among cDC1 and cDC2 (Young et al., 2008). While, cDC1 and cDC2 internalize similar quantity of ovalbumin (Jakubzick et al., 2008), cDC2 preferentially internalize soluble ovalbumin protein for presentation on MHC-II to CD4+ T cells, whereas cDC1 preferentially cross- present ovalbumin antigens on MHC-I to induce

CD8+ T cell responses (Jakubzick et al., 2008).

91 1.III.C.iv PHAGOCYTOSIS

1.III.C.iv.a Introduction cDCs as well as other phagocytes can acquire particulate antigens (which are typically

>0.5 µm in diameter) through their specialised and specific process called phagocytosis, which is recognised as a critical component for the innate and adaptive response. In addition to the immune responses, recent studies have also revealed that phagocytosis is crucial for both tissue homeostasis and remodelling.

1.III.C.iv.b Receptor mediated phagocytosis

Unlike macropinocytosis, phagocytosis is a receptor-mediated event. Examples of a handful of the receptors include pattern recognition receptors (such as Dectin 1 for fungal β-1,3-glucans (Carter et al., 2006)), opsonic receptors (such as Fc receptors

(FcR) for antibody opsonised particles (Anderson et al., 1990)), apoptotic corpse receptors (such as αVβ3 for apoptotic cells (Albert et al., 2000)) and complement receptors (such as C3b receptor for opsonised zymosan particle). To date, there are at least 20 receptors have been identified and it is postulated that there are many that remain to be identified (Flannagan et al., 2012). Although many of these receptors can also facilitate the internalisation of ligands by clathrin-mediated endocytosis, their ability to facilitate the internalization of large particles is clathrin independent and does not rely on other components of the clathrin-mediated endocytosis machinery (Tse et al., 2003).

92

Figure 7 Formation of phagocytic cup, phagocytosis and phagosome maturation Clustering of receptors initiate F-actin polymerization and contractility to form phagocytic cup. Once sealed, phagosome under series of fusion and fission to undergo maturation process from early phagosome to late phagosome. Late phagosome fuses with lysosome to form phagolysosome where V-ATPases and proteases are enriched in this low pH environment. Abbreviations; EEA (Early endosome antigen 1), Hrs (Hepatocyte responsive serum phosphoprotein), MON1 (Monensin sensitivity 1), LAMP1 (Lysosome-associated membrane proteins 1).

93 Among these three type of receptors, opsonic receptor mediated phagocytosis is by far the best known and well understood mode of phagocytosis (Flannagan et al., 2012). In this Fc receptor mediated phagocytosis, the process is strictly driven by actin polymerisation which in turn is activated by a signalling cascade involving several elements referred to classical pathway of complement activation (Figure 7).

Additionally, another type of opsonic receptor that is widely studied is complement receptors (CR1, CR3 and CR4) that bind to ligands as mannose binding lectin, C3b and

C4b respectively. It has been demonstrated that complement receptors (CRs) recognize

C3b ligands on serum opsinized zymosan particles and mediate phagocytosis. Unlike Fc receptor mediated phagocytosis that initiates classical pathway complement activation,

CR3 complement receptor activates an alternative pathway.

Fc mediated phagocytosis is the best known in term of its signalling cascade. First, the receptor-ligand interaction activates tyrosine kinases such as spleen tyrosine kinases

(SyK) (Kiefer et al., 1998), followed by recruitment of several adaptor proteins including Grb2, Grb2 associated binder (Gab2) and linker of activated T cells (LAT) and adaptor CrkII protein for the recruitment of downstream signalling components.

Then, this recruitment results in an activation of multiple lipid-modification enzymes including phos-phatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol

4-phosphate 5-kinases (PI4P-5K). Small GTPases including Rac1, Rac2, Rho family and Cdc42 are also activated. When Rac1 and Rac2 are knocked out in neutrophils and macrophages, the cells display significant impaired phagocytosis, (Koh et al., 2005).

Likewise, knockdown Cdc42 greatly inhibits phagocytosis, suggesting it has a direct role in this process (Park and Cox, 2009). Then, nucleation promoting factors such as the Wiskott-Aldrich syndrome protein (WASP)/N-WASP and the Scar/WAVE-family

94 proteins activate the actin nucleation complex Arp2/3, which in turn elicits actin polarisation that drives pseudopod extension and cup formation (Tsuboi and Meerloo,

2007). On the other hand, complement receptor mediated phagocytosis has been reported to activate distinct pathway from classical Fc mediated phagocytosis. Although they share signalling module like kinases, small GTPases and nucleation complexes, complement receptor mediate phagocytosis need an inside-out signal to reach an active conformation for ligand binding before uptake (Dupuy and Caron, 2008).

1.III.C.iv.c Phagosome maturation

Phagosomes, after detachment from plasma membrane, undergo a cascade of programmed changes from an early endosome like organelle – nascent phagosome to late endosome like structure and eventually to lysosome like organelle – phagolysosome

(Figure 7) (Flannagan et al., 2012). The maturation relies on a highly coordinated series of membrane fission and fusion with other organelles such as endosomes and lysosomes, and also depend by several proteins necessary for vesicular traffic such as

Rab small GTPases, SNARE proteins and fission complex (Stenmark, 2009). It was proposed that Rab5 promotes membrane fusion between nascent phagosome to early endosome from an elegant experiment that used Rab5 mutant transfected macrophage cell line and showed that their phagosome maturation was disrupted resulting in the formation of a giant phagosome (Duclos et al., 2000) Thus, the study suggests that Rab5 is a key regulator of fusion and transfer of solute content between nascent phagosome and early endosome without significant increase of phagosome size (Duclos et al.,

2000). Additionally, for late phagosome maturation, Rab7 as well as a proton pump

(vacuolar (V) type ATPases) are recruited to the late phagosome. Rab7 facilitates phagosome-late endsome fusion (Harrison et al., 2003), while vacuolar (V) type

95 ATPases translocates H+ inward across phagosomal membrane at the expense of ATP

(Lennon-Dumenil et al., 2002). To date, it is widely known that acidification of phagosome occurs in two stages. First a poorly understood early acidification step result in a relatively small drop in pH (Hackam et al., 1997). Second, V-type ATPase proton pumps which subsequently trafficked to phagosome at a relatively late stages

(Beyenbach and Wieczorek, 2006). In the final maturation stage, late phagosomes fuse with lysosomes and form phagolysososome, a process that is poorly understood at the molecular level. However, it is currently well accepted that fusion of late phagosomes to lysosomes is coordinated in part by SNARE complex proteins, as blocking of these protein inhibits phagosome-lysosome fusion (Tsai and Discher, 2008). Biochemically, phagolysosomes have two distinct features; 1) they are markedly acidified and 2) abundantly enriched with active enzymes such as cathepsins.

1.III.C.iv.d Phagosome acidification

When phagosomes mature (Figure 7), their phagosome lumen becomes more acidified, largely because of vacuolar (V) type ATPases (Lukacs et al., 1991). Vacuolar (V) type

ATPases at the expense of ATP, pump proton into the lumen of the phagosome, generating acidified environment. Additionally, to maintain this very acidic luminal pH and overcome electrical potential, the influx of anions (Cl-) (Graves et al., 2008) and efflux of cations (K+ and Na+) (Steinberg et al., 2010) are required to compensate for the influx of protons (H+). To date, the chloride chanel (CLC) family proteins especially

ClC-7 is thought to mediate influx of (Cl-) and therefore acts as Cl-/H+ antiporter

(Graves et al., 2008).

96 The degree of phagosome acidification varies between different types of phagocytes. In macrophages, the phagosomal pH can be low as pH 3, whereas DC phagosomes are more alkaline (pH 5) (Savina et al., 2006). In DCs, phagosome pH is regulated in part by NOX2 and NADPH oxidase because the production of ROS by NADPH oxidase 2 or NOX2 consumes large amounts of protons and thereby causes alkalization of the phagosome lumen (Savina et al., 2006). This NOX2 protein complex is composed of cytosolic proteins such as p47phox, p67phox, p40phox, Rac and RhoA and plasma membrane associated proteins such as p22phox and gp91phox. When activated, these cytosolic proteins all move to the membrane to form multiprotein complex on phagosome membrane.

The acidification of phagosome activates more than 50 enzymes including proteases, lipases, nucleases, glycosidases and phosphatases, which all help to degrade large antigens, proteins and dying microbes. Based on the amino acid of the protease active site that catalyzes hydrolysis of substrate peptide bond, proteases found in phagosomes can be classified into three groups including cystein (Cathepsin B, F, H, L, S, Z and

AEP for asparaginylendopeptidase), aspartate (Cathepsin D and E), and serine

(Cathepsin A and G) proteases (Manoury, 2013).

97 1.III.D ANTIGEN PROCESSING

1.III.D.i INTRODUCTION

After cDCs take up antigen it can either be processed along the MHC II or MHC I pathway (cross presentation). Whilst cross presentation, an alternative pathway for captured antigens, will be reviewed, the conventional MHC II pathway will be the major emphasis of this review.

1.III.D.ii MHC CLASS II PATHWAY

1.III.D.ii.a Introduction

MHC class II pathway has two major components. First, extracellular antigens are internalised and then processed to generate smaller peptides of approximately 18-20 amino acids, the size required to bind to MHC class II molecules (Roche and Furuta,

2015). Second, in the endoplasmic reticulum (ER), MHC class II molecules are synthesized and targeted to multivesicular bodies (MVBs) where they meet with processed peptides. Within multivesicular bodies (MVBs), facilitated by accessory molecule HLA-DM, an MHC class II-peptide complex is formed. In this review, section

1.III.D.ii.b will provide an insight on how peptides are processed and generated within endocytic compartments. Then, section 1.III.D.ii.c will give an overview of biosythesis of MHC II molecule and its invariant chain degradation. Section 1.III.D.ii.d will then describe how peptide binds to MHC II molecules and forms MHC II peptide complex.

Section 1.III.D.ii.e will then discuss the possible routes by which MHC II peptide complex can be transported from cytosol to the cell surface.

98

Figure 8 The antigen processing and presentation pathways in cDCs. Extracellular antigens are internalized in endocytic route where they are processed in endocytic compartments before translocate to cell surface through MHC class II pathway or alternatively through MHC class I pathway called cross presentation (Roche and Furuta, 2015). Abbreviations; ER,Endoplasmic Reticulum); MVBs, Multi-Vesicular Bodies; cDC1, conventional dendritic cells 1

99 1.III.D.ii.b Antigen processing

As shown in Figure 8, proteins from exogenous sources that enter endocytic compartments through endocytosis, macropinocytosis and phagocytosis are all destined for a downstream compartments called a multivesicular bodies (MVBs). These are low in pH and high in concentration of proteases to sequentially digest large proteins and generate small peptides suitable for binding to MHC class II molecules (Blum and

Cresswell, 1988). In highly degradative cells such as macrophages, successive cleavages by the proteases generates very short peptides whilst in less degradative cells such as dendritic cells, larger peptides predominate and are used for MHC class II binding.

Members of cathepsin family are the most important multivesicular bodies proteases for antigenic peptide proteolysis (van den Hoorn et al., 2011; Yin et al., 2012). In antigen presenting cells at least 7 cathepsins including cathepsin B, D, E, L, K, S, O are key for protein denaturation and antigen cleavage. However, using immunoblot of dissociated spleen and lymph node cells, it was shown that levels of protease expression of cathepsin S, L, B and D are lower in DC than macrophages (Delamarre et al., 2005), suggesting that proteolytic activity in DCs is less than in macrophages. Further, when lysosomal extracts from DCs and macrophages were incubated with casein, those from macrophages were 20-60 folds more active than DCs (Delamarre et al., 2005).

All cathepsins are synthesized as inactive precursor enzymes whose propeptide covers its catalytic site (Groves et al., 1998). Cathepsin enzymatic activity is controlled by multiple regulatory steps such as acidification and endogenous inhibitors (Groves et al.,

1998). For example, the cathepsin L that has a propeptide that unfolds at acidic pH (pH

100 below 4) to free the active site and allows protease activity (Jerala et al., 1998). Further, endogenous inhibitors such as cystatins can bind to cathepsins and thus also prevent peptide cleavage,. In immature DCs, cystatin F localised to endosomal/lysosomal vesicles and interacted with cathepsin S to prevent its enzymatic activity (Magister et al., 2012). Similarly, in mature DCs, cystatin F in multivesicular bodies binds to cathepsin L to block its enzymatic function (Magister et al., 2012). Thus, these all suggest that acidic environment and endogenous inhibitors are crucial for regulating the acitivity of cathepsin.

Cathepsin S and cathepsin L play a non-redundnant role in antigen processing of DCs.

DC from cathepsin S deficient mice, displayed impaired invariant (Ii) chain degradation and attenuated antigen presentation (Beers et al., 2005; Nakagawa et al., 1999; Shi et al., 1999). However, whilst cathepsin L deficient thymic epithelial cells have impaired invariant (Ii) chain degradation, this was normal in DCs (Honey et al., 2002). Thus, these two studies strongly indicate although cathepsin S plays a non-redundant role in antigen processing, cathepsin L migh not. Similarly, cathepsin B and D play a part in this processing but not as crucial as cathepsin S and L.

1.III.D.ii.c MHC Class II biosynthesis

MHC class II molecules are synthesized in endoplasmic reticulum (ER) (Figure 8) as an inactive form of two heterogeneous dimers alpha and beta sheet glycoprotein

(Cresswell, 1996). Newly synthesized MHC class II are associated with invariant (Ii) chains preventing them from potential peptide binding (Dugast et al., 2005). This invariant chain (Ii), contains targeting motifs that direct the Ii–MHC class II complex to late endosomes or multivesicular bodies where they can encounter antigenic peptide generated as discussed in section 1.III.D (McCormick et al., 2005). Within the

101 multivesicular bodies (MVBs), Ii is proteolytically degraded and dissociates from the

Ii–MHC class II complex, leaving a small fragment (the class II-associated li chain peptide (CLIP) bound to the MHC dimers. This fragment of Ii must be removed from

MHC class II to allow binding of antigenic peptides. The release of CLIP is facilitated by the enzyme HLA‐DM (known as H2‐M in mice), which is an MHC class II‐like protein encoded in the MHC locus. After CLIP removal, the nascent MHC class II molecule can then binds antigenic peptides available in the multivesicular bodies

(MVBs) (Denzin et al., 2005).

1.III.D.ii.d Formation of MHC II peptides complex

Antigenic peptides bind to MHC II molecules and form MHC II peptide complex in the multivesicular bodies. Unlike the MHC class I molecule, the MHC class II molecule has an open peptide binding groove, allowing peptide to extend out of the MHC II structure

(Sercarz and Maverakis, 2003). This means that not only long peptide, but also unfolded or even native proteins with flexible segment can bind to MHC II molecule.

Because of the structure of MHC class II molecule with an opened peptide binding groove, there are two possible models that explain how MHC class II molecules select their antigenic peptide (Trombetta and Mellman, 2005). In the “bind first trim later” model, epitopes of long peptide or whole protein can first be captured by the MHC II binding groove followed by fragmentation by the cathepsins, while the epitopes in

MHC II binding groove are protected from degradation. A study using long polypeptides showed that the polypeptides co-precipitate with MHC class II molecules

(Castellino et al., 1998), suggesting that some polypeptides bind to MHC II binding groove before trimming by cathepsins. Nevertheless, in some scenarios, some proteins

102 must be cleaved first before binding, the so called “cut first bind later” model. In this model, the protein can be cut first by the cathepsins and then be binds to the MHC II binding groove (Blum et al., 2013). Supporting this model, an interesting study employed hen egg lysozyme (HEL) peptide 46-61 which is immunogenic in C3H.SW mice but not in MHC identical C57BL/6 and demonstrated that fine processing to remove Arg61 is required to induce responses, while, failing to remove Arg61 results in unresponsiveness both C3H.SW and C57BL/6 mice (Grewal et al., 1995). Thus, this study suggests that in some polypeptides, cleavage is required before binding to MHC II binding groove. However, to date, it is still unclear which mode of MHC class II antigen processing might employed or it is possible that MHC class II antigen processing might use a bit of both models.

The chaperon protein HLA-DM helps to disassociate class-II-associated invariant chain peptide (CLIP) from the MHC class II binding groove. The removal of CLIP by direct binding of HLA-DM allows repeated binding and disassociation of peptides to MHC class II binding groove until high affinity antigenic peptides are bound to the peptide binding groove (Busch et al., 2005). During this process, another chaperon protein,

HLA-DR facilitates peptide editing, allowing repeated rounds of disassociation and rebinding. Highly stable peptide-MHC II complexes are resistant to HLA-DR editing, while those peptides that bound to MHC class II molecules with low stability are removed by HLA-DR during endosomal processing (Lazarski et al., 2006).

103 1.III.D.ii.e Transportation of MHC II peptide complex to plasma membrane

It has been proposed that there are two possible routes that peptide-MHC II complex are transported from multivesicular bodies (MVBs) to cDC plasma membrane. These are through: 1) direct transport and fusion with membrane (Wubbolts et al., 1996); or 2) through a long tubulation network (Kleijmeer et al., 2001). Both routes require a microtubule network and the vesicles must move along microtubules in a bidirectional manner (Rocha and Neefjes, 2008). The movement of the vesicles is facilitated by two opposing motor proteins, dynein and kinesin which are responsible for directing the vesicle toward nucleus and plasma membrane respectively (Wubbolts et al., 1996). The multivesicular bodies move in the complex and dance until they meet the actin meshwork under the plasma membrane, where they bind to and are retained by actin motor (Rocha and Neefjes, 2008). The actin meshwork must then dissolve to allow multivesicular bodies to contact the plasma membrane for fusion (Rocha and Neefjes,

2008).

At a molecular level, it is currently known that the transportation of MHC II peptide complex within DCs is controlled by various small GTPases namely, Rab, Arf and Arf- like (Arl) family (Paul et al., 2011; Rocha and Neefjes, 2008). For instance, Rab7 mediates the fusion of late endosomes to microtubules (Rocha et al., 2009). Arl14 controls the release of multivesicular bodies (Paul et al., 2011). Arl8b facilitates the expression of cell surface MHC class II peptide complex (Michelet et al., 2015).

Moreover, recent evidence suggests that MHC II peptide complex molecules are constantly recycle back from plasma membrane into endocytic pathways. It has been demonstrated that class II molecules after invariant (li) chain degradation, in the endocytic pathway are ubiquitinated in immature DCs; however, ubiquitination of MHC

104 class II molecule is inhibited during DC maturation (van Niel et al., 2006). Supporting this finding, inhibition of MHC class II ubiquitination in mature DC slows down endocytosis and results in accumulation of MHC class II at the cell surface (Shin et al.,

2006). Thus, these studies strongly indicate that antigen presentation by DCs is essentially controlled by ubiquitination of MHC class II molecule.

1.III.D.iii CROSS PRESENTATION

In addition to the conventional MHC class II pathway, emerging evidence suggests that internalisation of exogenous antigens can also enter the MHC class I pathway and be cross presented to CD8+ T cells by a specialised cDC subset, cDC1 (Heath and

Carbone, 2009; Shortman and Heath, 2010).

Although, the precise mechanisms that contribute to the release of extracellular antigen from the endocytic compartment to the cytosol to gain acccess to the MHC class I processing pathway is largely unknown, some studies suggested that, following uptake, extracellular antigens may escape to the cytosolic pathway by formation of pores in the late endosome or multivesicular bodies (MVBs) (Lin et al., 2008b). Other studies have been proposed that late endosomes fuse to endoplasmic reticulum (ER) and antigens can then be transferred to MHC class I molecules through this route (Burgdorf and Kurts,

2008). Nevertheless, little is known about what contributes to the fate of the antigens and decides the internal route to cross presentation and further discussion of this is beyond the scope of this thesis.

105 1.III.E MATURATION

1.III.E.i INTRODUCTION cDCs exist in in immature or mature states (Mellman and Steinman, 2001). In resting conditions, cDCs are predominantly immature and act as antigen capturing cells which are highly efficient in antigen uptake. In contrast, under some circumstances, immature cDCs undergo a process called “maturation” transforming into mature cDCs. They loose their cacpacity to take up more antigen, becoming antigen presenting cells with increases in antigen processing and presentation activity, ability to migrate to draining lymph nodes and activation of naïve T cells. An overview of functional and phenotypical differences between immature and mature cDCs are summarised in Table

11. This section provides details on how maturation contributes to cDCs antigen presentation to CD4+ T cells.

106 Table 11 Functional and phenotypical differences between immature and mature cDCs Differences Process/Molecules/Chemokines Roles Immature cDCs Mature cDCs Endocytosis Antigen uptake +++ +++ Macropinocytosis Antigen uptake +++ low Phagocytosis Antigen uptake +++ low MHC II biosynthesis Antigen processing +++ low MHC II sort to membrane Antigen processing low +++ Functional MHC II to lysosome Antigen processing +++ low level Antigen processing machinery Antigen processing low +++ T cell tolerance Antigen presentation Yes No T cell immunity Antigen presentation No Yes Secretion of immunostimulatory Antigen presentation ? ? cytokines CD40 Signal 2 low +++ MHC II Signal 2 low +++ CD80 Signal 2 low +++ CD83 Signal 2 low +++ Phenotypic CD86 Signal 2 low +++ level PDL1 Signal 2 low +++ 4-1BBL Signal 2 low +++ OX40L Signal 2 low +++ CD70 Signal 2 low +++ CCR7 Migration low +++

107 1.III.E.ii IMMATURE cDCs

Immature cDCs are generally described as highly endocytic, low expressors of MHC molecules and weak stimulators of naïve T cells (Steinman, 2003). Because of these, immature cDCs are equipped with numbers of pattern recognition receptors (PRRs) (as reviewed in section 1.III.B) and are superior in endocytosis, macropinocytosis and phagocytosis for antigen uptake (as reviewed in section 1.III.C). Although, the rate of

MHC biosynthesis is high in immature cDCs, formation of MHC peptide complexes is inefficient in immature cDCs because of two possible reasons; 1) attenuated antigen processing machinery but 2) high rate in MHC II turn over and sorting to lysosome for degradation. These all contribute to little MHC II peptide complexes on the cell surface.

Additionally, immature cDCs produce very few immunostimulatory cytokines and express low level of costimulatory molecules such as CD40, CD80, CD83 and CD86

(Mommaas et al., 1995). Therefore, it is proposed that that immature cDCs induce T cell anergy and induce immune tolerance (Finkelman et al., 1996; Fu et al., 1996).

1.III.E.iii MATURE cDCs

Upon maturation by in proinflammatory stimuli such as lipopolysaccharide (LPS), CpG motifs and double stranded RNA, mature cDCs transiently change both functional and phenotypical characteristics in order to switch from antigen capturing cells (immature

DCs) to antigen presenting cells (mature DCs). Whilst, mature cDCs downregulate their capacity for antigen uptake, mature cDCs are more potent in antigen processing because of increased production of lysosomal proteases and activated antigen processing machinery (Blankenstein and Schuler, 2002). Moreover, mature DCs are more specialized in antigen presentation as they are capable of forming MHC II peptide complex. The ubiquitination of MHC class II is also decreased, allowing prolonged

108 expression of MHC II peptide complex on the cell surface. Additionally, in contrast to immature cDCs, mature cDCs produce large amount of immunostimulatory cytokines critical for innate and adaptive immune response (Menges et al., 2002) as well as enhanced production of costimulatory molecules such as CD40, CD80, CD83 and

CD86 on their cell surface. Additionally, in order to migrate to the T cell zone in lymphoid organs, mature cDCs downregulate anchor receptors and leave peripheral tissues, while upregulating chemokine receptors such as CC chemokine receptor 7

(CCR7) and home to lymphoid organs (Randolph, 2001). Interestingly, unlike immature cDCs, mature cDCs can trigger both immunity and tolerance depend upon their activation state.

1.III.F MIGRATION

1.III.F.i INTRODUCTION

Mature cDCs constantly migrate from peripheral tissue, via lymph vessels, to draining lymph nodes where migratory cDCs carrying MHC II bound antigens are transformed into lymphoid resident cDCs and subsequently presented to CD4+ T cells (Figure 9)

(Steinman, 2012). Upregulation of CC chemokine receptor 7 (CCR7) on the cDC cell surface directs cDCs to T cell zones in draining lymph nodes (Figure 9) (Forster et al.,

2012). In this review, cellular and molecular events guiding migratory cDC homing to lymph nodes and across lymph nodes to T cell zone will be reviewed.

109

Figure 9 cDCs migration from peripheral tissue to lymph nodes via lymphatic vessel. Migratory cDCs after activated and undergoing maturation by encountering with antigens (blue dot) migrate to lymph nodes through lymph vessel facilitating by upregulation of CC chemokine receptor 7 (CCR7), localise to T cell zone where they present antigens T cells, inducing T cell activation. Adapted from (Forster et al., 2012) & (Teijeira et al., 2014).

110 1.III.F.ii DCs HOMING TO LYMPH NODES

To travel from peripheral tissues to lymphatic vessels, migratory cDCs must upregulate

CC chemokine receptor 7 (CCR7) on their cell surface, which can sense chemoattractant CC chemokine ligand 21 (CCL21) produced by lymphatic endothelial cells (LECs). This initiates cDC movement from peripheral tissues toward lymphatic vessels where CCL21 is constantly produced (Weber et al., 2013). Other chemoattractants such as Sphingosine-1-phosphate (S1P) (Rathinasamy et al., 2010),

CC chemokine ligand 1 (CCL1) (Kabashima et al., 2007) and CXC chemokine ligand

12 (CXCL12) (Qu et al., 2004) are reported to have complementary roles in cDC docking to lymphatic endothelium. Nevertheless, CCR7 is considered to play the central role.

Once docked, cDCs must transmigrate through lymphatic endothelium to enter lymphatic vessels. To achieve this, cDCs employ various molecules including C type lectin like receptor 2 (CLEC2), Sema 3A and CC chemokine receptor 7 (CCR7) which bind to podoplanin, neuropilin and CC chemokine ligand 21 (CCL21) on lymphatic endothelial cells (Fig 9a), facilitating DC transmigration. C type lectin like receptor 2

(CLEC2) binds to podoplanin and enhances DCs actin cytoskeleton rearrangements

(Acton et al., 2012). Sema 3A binds to neuropilin and promote cellular contraction. CC chemokine receptor 7 (CCR7) bind to CC chemokine ligand 21 (CCL21) apart from function as chemoattractant also immobilises cDC and thereby facilitate cDC transmigration across lymphatic vessels.

Transmigrated cDCs first actively crawl inside lymphatic capillaries (Acton et al., 2012) and later are passively transported and mediated by lymph flow (Fig 9b) (Tal et al.,

111 2011). Little is known about molecules involve in intralymphatic crawling and flow in lymphatic vessels (Acton et al., 2012).

1.III.F.iii DCs IN T CELL ZONE LYMPH NODE

cDCs relies on CCL21 and CCL9 expressed by lymphoid stroma cells to migrate to T cell zone in lymph nodes (Cyster, 2005). The CCL21 and CCL9 gradient are tightly regulated within lymph nodes by scavenger receptor chemokine ligand receptor 1 in subcapsular sinus to properly guides cDCs to T cell zone (Ulvmar et al., 2014). In T cell zone, cDCs often adhere closely to one another, creating a tightly packed cluster that incorporates T cells (Lindquist et al., 2004). The movement of mature cDCs slows after arrival in the lymph node (Miller et al., 2003) to allow sufficient time for efficient antigen presentation (Lindquist et al., 2004).

1.III.G ANTIGEN PRESENTATION

1.III.G.i INTRODUCTION

To mount adaptive immune responses, mature cDCs carrying cell surface antigen-MHC

II complex must travel to T cell zone in lymph nodes to present antigen-MHC II complex to naïve CD4+ T cells. The crosstalk between cDCs and naïve CD4+ T cells for adaptive immunity induction requires at least three sequential steps; 1) T cell antigen recognition, T cell activation and T cell polarisation (Figure 10). In this review, section

1.III.G.ii provides information on how T cells recognise antigen carried by cDCs. Then section 1.III.G.iii describes how T cells are activated or repressed through interaction of ligands or costimulatory molecules between cDCs and T cells. Section 1.III.G.iv

112 explains on how activated T cells polarise into T cell subsets under control of cytokines and cDC subsets.

Figure 10 T helper cell polarization. cDCs guide and determine T cell functions through three sequential steps; 1) T cell antigen recognition on MHC class II molecules (signal 1), 2) T cell activation through ligands (signal 2), 3) T cell polarization via instructive cytokines (signal 3). Activated T cells can be polarized into at least six subsets; Type 1 T helper cells (Th1), Type 2 T helper cells (Th2), Type 17 T helper cells (Th17), T follicular helper cells (Tfh), Type 9 T helper cells (Th9) and regulatory T cells (Treg). Each of which functions distinctively. Adapted from (den Haan et al., 2014) and (Carbo et al., 2014).

1.III.G.ii T CELL ANTIGEN RECOGNITION

The first step that initiates antigen presentation is the T cell antigen recognition (signal

1) which has been reported to involve in three possible mechanisms; 1) a direct binding of MHC class II bound antigen and T cell receptor (TCR), 2) antigen cross dressing and

3) exosome secretion. This section provides an overview of these three possible mechanisms.

It has been widely accepted that CD4+ T cells only recognise extracellular antigens on

MHC class II molecules found on cDC cell surface via the direct binding to CD4+ T cell receptor (TCR) (signal 1) (Sporri and Reis e Sousa, 2005). cDCs firstly form an

113 immunolocal synapses with CD4+ T cells where the antigen-MHC II complex on cDC cell surface can interact with CD4+ T cell receptor (TCR) (Alarcon et al., 2011).

Although, T cell receptors (TCR) are structural closely related to immunoglobulin molecules, its antigen recognition is distinct from the immunoglobulin. Whilst, immunoglobulin interact with free floating extracellular antigens, TCR usually only recognise antigens that are displayed on the cell surface, usually by MHC I or II. Most of T cell receptors (TCRs) are consisted of two protein chains; T cell receptor alpha

(TCRα) and T cell receptor beta (TCRβ) chains, whereas a minority of CD4+T cells are made up with T cell receptor gamma (TCRγ) and T cell receptor delta (TCRδ) chains.

Recent discoveries suggest an alternative routes of antigen presentation in which cDCs acquire the preformed peptide–MHCII complexes from neighbouring cDCs termed

“cross dressing” (Li et al., 2012; Smyth et al., 2008; Wakim and Bevan, 2011). It proposed that during interaction between cDCs and CD4+ T cells, MHC II is transferred to neighbouring cDCs. These DCs not only have MHCII peptide complex but also other costimulatory molecules on the cell surface, thus the dressed MHCII and costimulatory molecule cDCs function as expanded cDCs for the amplification of CD4+ T cell proliferation (Zhou et al., 2005). Moreover, several studies have reported that intracellular MHC II transfer occurs not only between cDCs but also between various cells such as DCs and Natural Killer (NK) cells, cDCs and lymph node stromal cells

(LNSCs), and DCs and group 2 innate lymphoid cells (ILC2s) (Markey et al., 2014).

Importantly, cDCs can also present antigen to CD4+T cells through exosome secretion.

Exosomes are small membrane vesicles that are formed in mulivesicular bodies (MVBs)

(Greening et al., 2015). MHCII-bearing exosomes are secreted from cDCs and possess

DC immunological capacity. For instance, DC-derived MHCII-bearing exosomes

114 acquired by MHCII-deficient DCs can stimulate antigen-specific CD4+ T cell proliferation in vitro. In contrast to antigen cross dressing via cell-cell contact which occurs locally and rapidly (within mins), the exosomes can be transferred to distant sites

(Davis, 2007).

1.III.G.iii T CELL ACTIVATION

Apart from T cell receptor (TCR) signalling (Signal 1), cDCs express costimulatory molecules (Signal 2, Figure 10) that can activate or repress CD4+ T cell to either synergise or fine tune T cell receptor (TCR) signalling (Signal 1). In recent years, many of these molecules have been discovered and their mechanisms elucidated. In this section, well characterised costimulatory molecules facilitating in CD4+ T cell activation will be reviewed.

As shown in Figure 10, most of costimulatory molecules belong to two major families;

B7/CD28 family and Tumour necrosis factor (TNF)/ Tumour necrosis factor receptor

(TNFR) family (Chen and Flies, 2013). B7/CD28 family molecules are the best characterised. They are the most important costimulatory molecules on cDCs and have dual roles in both T cell activation and suppression. For example, ligation of B7.1

(CD80) and B7.2 (CD86) molecules binds to CD28 to initiate CD4+ T cell activation

(Rudd et al., 2009). To allow feedback suppression of CD4+ T cell activation and provide immune checkpoint, cytotoxic T lymphocyte-associated protein-4 (CLTA-4) is upregulated on T cells and bind to B7.2 (CD86) molecules on cDCs, inhibiting signal 2

(Rudd et al., 2009). Likewise, cDCs expressed program death-1 ligand (PD-1L) on their surface and this ligand can bind to PD-1 on CD4+ T cell surface and inhibit CD4+ T cell proliferation (Riley, 2009). The other less potent co-stimulatory molecules found on

115 cDCs are belong to TNF/TNFR family such as OX40L and 4-1BBL, CD40 and CD70 which bind to OX40, 4-1BBL, CD40L and CD27 respectively, the binding of these ligands facilitate stimulation of CD4+ T cell activation and proliferation (den Haan et al., 2014). In conclusion, cDCs upregulate distinct sets of costimulatory molecules that guide CD4+ T cells to be either activated or suppressed.

1.III.G.iv T CELL DIFFERENTIATION AND CYTOKINE PRODUCTION

Following CD4+ T cell antigen recognition and activation, naïve CD4+ T cells polarise to specific phenotypes that are best suited to drive an appropriate response against the threat. As shown in Figure 10, Interferon (IFN)-γ-producing Type 1 T helper (Th1) cells are induced in response to intracellular bacteria and viruses, interleukin 4- producing

Type 2 T helper (Th2) cells mediate immunity against helminth parasite infection

(Allen and Maizels, 2011). Interleukin 17-producing Type 17 T helper (Th17) cells are required for immunity to extracellular bacteria and fungi (Mills, 2008), whereas, interleukin (IL)-10-or tumour growth factor (TGF)-β-producing regulatory (Treg) cells are important in all infections to prevent uncontrolled inflammation and immunopathology (Mills and McGuirk, 2004). To achieve this, cDC subsets as well as their secreted cytokines influence CD4+ T cell polarisation. For example, cDC1 are known to guide naïve CD4+ T cells polarisation to Type 1 T helper (Th1) cells through interleukin 12 secretion, whereas, cDC2 influence naïve CD4+ T cells to either Type 2 T helper (Th2) or Type 17 T helper (Th17) differentiation depending on the cytokines millieu (Table 8). Apart from these well described characterised T helper subsets, less well defined Type 22 T helper (Th22), Type 9 T helper (Th9) and T follicular helper

(Tfh) lymphocytes has been identified. This section provides an overview of how naïve

T cells polarise into different T cell subsets.

116 Th1 cells are predominantly induced in responses to intracellular bacteria and viruses infection (Del Prete, 1992). Interleukin 12p70 secreted by DCs and Interferon (IFN)-γ from natural killer (NK) cells promote Th1 cell differentiation. Additionally, Th1 cells themselves secrete Tumour necrosis factor (TNF) α and Interferon (IFN)-γ, which have proinflammatory effects on many innate immune cells. For instance, Interferon (IFN)-γ secreted by Type 1 T helper (Th1) enhances phagocytic activity and nitric oxide production of many phagocytes including macrophages and neutrophils (Gattinoni et al., 2005; Melzer et al., 2008).

Th2 cells are mainly induced in responses to helminth parasite infection as well as in induction of asthma and allergy. Early production of interleukin 4 by Basophils cooperate with DCs drives Type 2 T helper (Th2) polarisation (Oh et al., 2007). Type 2

T helper (Th2) themselves also secrete interleukin 4 and interleukin 5 as their key cytokines. Interleukin 4 drives antibody class switching in B cells as well as drives polarisation of alternatively activated macrophages for tissue repair (Chen et al., 2012).

Additionally, interleukin 5 is found to facilitate in Eosinophil activation (Martinez-

Moczygemba and Huston, 2003).

Th17 cells are predominantly induced in responses to extracellular bacteria and fungi as well as in the immunopathogenesis of various autoimmune diseases. Interleukin 6 and

TGF-β produced by cDCs are the major cytokines driving Type 17 T helper (Th17) differentiation (Mangan et al., 2006). Type 17 T helper (Th17) cells predominantly secrete interleukin 17 and interleukin 22 as their key cytokines which mount pro- inflammatory responses. Interleukin 17 induces other pro-inflammatory cytokines such as interleukin 6, interleukin 1 and Tumour necrosis factor (TNF) α, and also chemokines for chemotaxis of inflammatory cells to the site of inflammation (Ivanov et

117 al., 2006). Interleukin 22 facilitates Type 17 T helper (Th17) differentiation and functions as prominent mediator during proinflammatory response (Zenewicz et al.,

2007).

Treg are crucial in maintenance of immunological tolerance to both self and nonself antigens. In the case of self tolerance, Treg induce tolerance and therefore protect from autoimmunity. For nonself antigens, T regulatory cells (Treg) function to resolve inflammation after the pathogens have been cleared, thus preventing host tissue injury.

Treg cells produce interleukin 10 and tumour growth factor (TGF)-β as their main cytokines which all have anti-inflammatory properties, and therefore function to suppress immune responses (Sonnenberg et al., 2011).

Other T cell subsets that have been discovered recently include Type 22 T helper

(Th22), Type 9 T helper (Th9) and T follicular helper (Tfh). Type 22 T helper (Th22) cells have been reported to produce interleukin 22 cytokine that plays an important role in promoting resistance to extracellular pathogens, particularly to Gram-negative pathogens, such as Klebsiella pneumoniae and Citrobacter rodentium (Sonnenberg et al., 2011). Type 9 T helper (Th9) cells are pro-inflammatory cells that work in a broad spectrum of autoimmune diseases and in allergic inflammation by producing interleukin

9 in response to tumour growth factor (TGF)-β and interleukin 4 (Chang et al., 2010). In recent years, T follicular helper (Tfh) cells have emerged as the key cell type required for the formation of germinal center in secondary lymphoid organs. Tfh cells in human lymphoid organs express the CXC chemokine receptor 5 (CXCR5) and function primarily to provide help to B cells. T follicular helper (Tfh) cells can be distinguished from other T cell lineages by their high expression of interleukin 21 (Fahey et al.,

2011).

118 2 MATERIALS AND METHODS 2.I MATERIALS

2.I.A CHEMICALS AND REAGENTS

Table 12 Chemicals, reagents and materials used in this project Materials Supplier Cat number

0.2-10 ul pipette tips Corning Life 4826

Sciences

1 ml syringes BD Biosciences SYR-500

1-200 ul pipette tips Corning Life 4823

Sciences

10 ml syringes BD Biosciences 309604

100 um cell strainer In vitro 352360

Technologies

100-1000 ul pipette tips Corning Life 4809

Sciences

19 guage needle BD Biosciences 305900

22 gauge needles BD Biosciences 305700

25 gauge needle BD Biosciences NEE-506

3.0 um carboxylate-modified Kisker Products for PSi-3.0COOH silica particles Biotechnologies

6 well plate Corning Life 3335

Sciences

70 um cell strainer In vitro 352350

Technologies

119 8 well chamber slides BD Biosciences 354108

96 U bottom well plate Beckon Dickinson 353077

Pty

Alexa594-SE Molecular Probe A20004

Bafilomycin A1 Sigma-Aldrich B1793

BSA Sigma-Aldrich B8894

Carbonyl cyanide m- Sigma-Aldrich C2759 chlorophenylhydrazone

CD4+ T cells isolation kit II Miltenyi Biotec 30-091-155

CellTrace Violet Life Technologies C34557

CFA Sigma-Aldrich F5881

CFSE Life Technologies C1157

Collagenase D Roche Diagnostics 11088866001

Cyanide Sigma-Aldrich 420-04-2

DMSO Sigma-Aldrich I117

DNase I Roche Diagnostics 1128493200

DQ-bodipy BSA Molecular Probe D-12050

EDTA Sigma-Aldrich 15400-054

Eppendorf tube Eppendorf 0030 125.150

FCS Life-technology 14190-250

FITC succinimidyl ester Molecular Probes F-6185

Fixation and permeable buffer eBioscience 77-5775-40

Flt3L Peprotech 250-31L

Fluordish Coherent Lif Science FD35-100

120 Glutamine Life-technology 25030-081

GM-CSF Peprotech 315-03

Golgi stop Beckon Dickinson 554724

Pty

HBSS Life-technology 14025092

Heat-killed Mycobacterium Bacto Laboratory 231141 tuberculosis

Heparin Sigma-Aldrich H3149

HEPES Life-technology 15630-080

IL-4 Peprotech 214-14

LPS Chemicon LPS25

Mecaptoethanol Sigma-Aldrich M6250

MOG1-125 Anaspec 55150-1000

MOG35-55 Prospec PRO-371

Nigericin Sigma-Aldrich N7143

Paraformaldehyde ProSciTech C004

PBS Life-technology 16000-044

Percoll Sigma-Aldrich P1644-1L

Pertussia toxin Sapphire 180

Biosciences

Red blood cell lysis buffer Sigma-Aldrich R7757

RPMI-1640 Life-technology 11875-093

Streptomycin Life-technology 15140-122

Thioglycollate Sigma-Aldrich 70157

121 Zymosan particle Sigma-Aldrich Z4250

2.I.B ANTIBODIES USED FOR FLOW CYTOMETRY

Table 13 Antibodies used for flow cytometry Labelling Antibody Species/ Supplier Clone Dilution cell Isotype number CD16/32 BD Rat 2.4G2 1:100 1x106 Biosciences IgG2b κ

CD3e-Pacific BD Syrian 500A2 1:100 1x106 Blue Biosciences Hamster IgG2, κ

CD4-Alexa 700 BD Rat (DA) RM4-5 1:100 1x106 Biosciences IgG2a, κ

CD45-PerCP BD Rat 30F11 1:100 1x106 Biosciences (LOU) IgG2b, κ

CD11c-APC BD Armenian HL3 1:100 1x106 Biosciences Hamster IgG1, λ2

CD11b-APC- BD Rat (DA) M1/70 1:100 1x106 Cy7 Biosciences IgG2b, κ

CD45R/B220- BD Rat RA3-6B2 1:100 1x106 PE Biosciences IgG2a, κ

F4/80-FITC eBiosciences IgG2a, BM8 1:50 1x106 kappa Vß11-FITC BD Rat RR3-15 1:100 1x106 Biosciences (F344) IgG2b, κ

6 IFNγ-PE-Cy7 eBiosciences IgG1, XMG1.2 1:50 1x10 kappa

122 CD86-Alexa BD Rat GL1 1:100 1x106 Fluor 700 Pharmingen™ (LOU) IgG2a, κ

CD80-FITC BD Armenian 16-10A1 1:100 1x106 Pharmingen™ Hamster IgG2, κ

CD40-PE BD Rat 3/23 1:100 1x106 Pharmingen™ (LOU) IgG2a, κ

I-A/I-E-FITC Biolegend Rat M5/114.15.2 1:50 1x106 IgG2b, κ

IL17-PE eBiosciences IgG2a, eBio17B7 1:50 1x106 kappa

FoxP3-APC eBiosciences IgG2a, FJK-16s 1:50 1x106 kappa

2.I.C ANTIBODIES USED FOR IMMUNOHISTOCHEMISTRY

Table 14 Antibodies used for immunohistochemistry

Labelling Antibody Species/ Supplier Clone Dilution cell Isotype number CLIC1 In house sheep - 1:1000 5000 RhoA Abcam rabbit - 1:100 5000 IgG-HRP Sigma- donkey - 1:200 5000 Aldrich Strepavidin Sigma- - - 1:100 5000 conjugated with Aldrich cy3 IgG conjugated Sigma- rabbit - 1:100 5000 with cy2 Aldrich

123 2.I.D PARTICLES

2.I.D.i ZYMOSAN PREPARATION

Zymosan (Saccharomyces cervisiae) particles (Sigma-Aldrich Cat #Z4250) were boiled then washed twice in PBS. For opsonisation, 0.5 ml of zymosan particles (14 mg/ml) were mixed with 0.5 ml purified goat IgG (5 ug/ml) (Cat # I9140, Sigma-Aldrich) and incubated at 37°C for 30 min (Jiang et al., 2012). To make FITC conjugated zymosan

(zFITC) Zymosan particles were incubated with FITC succinimidyl ester (1 mg/ml, Cat

#F-6185, Molecular Probes) (Jiang et al., 2012).

2.I.D.ii DQ-BODIPY BSA CONJUGATED BEADS

I covalently coupled 3.0 um carboxylate-modified silica particles (Cat #PSi-3.0COOH,

Kisker Products for Biotechnologies) with Alexa Fluor 594 (R) carboxylic acid, succinimidyl ester (mixed isomers) (Alexa594-SE, Cat #A20004, Molecular Probe) and

DQ green bodipy bovine serum albumin (DQ-bodipy BSA, Cat #D-12050, Molecular

Probe), as described previously (Jiang et al., 2012; Yates and Russell, 2008).

2.I.E CELLS

2.I.E.i BONE MARROW DERIVED DENDRITIC CELLS (BMDCs)

Dendritic cells used in this work were generated from bone marrow derived cells.

Briefly, femur and tibia were removed and bone marrow cells were flushed with PBS containing 0.5% BSA and 2 mM EDTA. After red blood cells were lysed with red blood cell lysis buffer, whole bone marrow cells were counted and cultured with complete medium at 1x106 cells/ml. To generate cDCs, granulocyte macrophage colony-stimulating factor (GM-CSF; 40 ng/ml) and Interleukin 4 (IL-4; 100 ng/ml) were added to the culture every two days for 7 days at 37°C/5% CO2. In some experiments bone marrow cells were cultured with Fms-related tyrosine kinase 3 (Flt3;

124 3 ng) which was added every three days for 11 days to generate a mixed population of cDCs and pDCs.

2.I.E.i.a Isolation of bone marrow cells

Femurs and tibias of mice were removed and isolated from surrounding muscle tissue by rubbing with kimwipe paper. Intact bones were then soaked in 70% ethanol for 2 mins before two times washing with complete medium (RPMI-1640 supplement with

100 U/ml Penicillin, 100 ug/ml Streptomycin, 2 mM L-glutamine, 50 uM 2-

Mecaptoethanol, 10% heat inactivated FCS). Both ends were cut with scissors and the marrow was flushed with complete medium using a 1 ml syringe with 25 gauge needle.

Red blood cells contaminated in marrow were then lysed with red blood cell (RBC) lysis buffer.

2.I.E.i.b Generation of cDCs

Bone marrow was harvested from tibia and femur of 129/SvJ mice or CLIC1 knockout mice. The cells were resuspended at 1x 106 cells/ml in complete medium with 40 ng/ml recombinant murine GM-CSF and 100 ng/ml recombinant murine IL-4. Cells were cultured in T75. Cells were fed on day 3 and 5 of culture, by replacing half the medium in each well with fresh complete medium with GM-CSF and IL-4. On day 3, nonadherent cells were aspirated off following gentle swirling of the flask. Loosely adherent cells, including DC, were harvested by gentle pipetting on day 6 of culture.

2.I.E.i.c Generation of mixed population of DCs

Bone marrow was harvested from tibia and femur of 129/SvJ mice or CLIC1 knockout mice. The cells were resuspended at 1x 106 cells/ml in complete medium with 3 ng/ml recombinant murine Flt3. Cells were culture for 11 days and fed on day 3,6 and 9 by

125 adding 1 ml of complete medium containing 3 ng/ml recombinant Flt3. DCs were then harvested on day 11, by gentle pipetting for nonadherent cells.

2.I.E.ii ISOLATION OF CD4+ T CELLS

CD4+ T cells used in this work were isolated from spleen and lymph nodes from mice and purified using magnetic beads. Briefly, Spleen and lymph nodes were removed freshly form mice and incubated with 0.5 mg/ml collagenase D and 20 µg/ml DNase I

+ for 30 mins at 37°C/5% CO2 to obtain single cell suspension. CD4 T cells were isolated using commercial available CD4+ T cells isolation kit II. Following tissue disassociation, cells were treated with RBC lysis buffer for 5 mins on ice to remove unwanted RBC. PBS was then top up to the cells to about 50 ml and centrifuge again for 5 mins to obtain cell pellet, then resuspended in 40 ul of buffer per 107 total cells. 10 ml of Biotin- antibody cocktail per 107 total cells were added to the buffer, mixed well and incubated for 5 mins on ice. Buffer were then added and followed by addition of 20 ul of Anti-Biotin MicroBeads per 107 per total cells and incubated on ice for another 10 mins. After staining, cell suspension was passed through the magnetic separation. Cell suspension was applied onto column containing magnetic. Flow-through was collected containing unlabeled cells, representing the enriched CD4+ T cells.

2.I.E.iii ISOLATION OF MOUSE IMMUNE CELLS

2.I.E.iii.a Blood

CLIC1+/+ and CLIC1-/- mice were anesthetized using isoflurane then blood was collected in a 1 ml syringe containing 100 U Heparin by cardiac puncture using a 25 gauge needle. Approximately 500 ul of blood was collected from each mouse and placed into a 15 ml Falcon tube.

126 2.I.E.iii.b Spleen

+/+ -/- CLIC1 and CLIC1 mice were sacrificed using CO2. Spleens were removed from the abdominal cavity of these mice. Each spleen was cut into small pieces and incubated in

6 well plates with 5 ml culture medium containing 20 ug/ml DNase I and 0.5 mg/ml collagenase D for 30 mins at 37°C in 5% CO2 incubator. After tissue disassociation, the spleen tissues were transferred to 100 um cell strainer and the tissues were gently grinded using 1 ml syringe plunger and cell suspension collected in a 50 ml Falcon tube.

The cell suspension was centrifuged for 5 mins at 350 x g at room temperature. The pellet was resuspended in 1 ml RBC lysis buffer and incubated for 5 mins at 4 °C. The cells were toped up with 1x PBS to a final volume of 50 ml and centrifuge again for 5 mins 350 x g at 4 °C. The supernatant was discarded and pellet was collected and was resuspended in 5 ml of cell medium RPMI-1640 (Cat #11875-093, Life technology) containing 100 ug/ml Streptomycin (Cat #15140-122, Life technology), 2 mM L- glutamine (Cat #25030-081, Life technology), 50 uM 2-Mecaptoethanol, and 10% heat inactivated fetal calf serum FCS (Cat #14190-250, Life technology). To obtain single cell suspension for immunostaining, the cells were filtered through 70 um cell strainer on top of a 50 ml Falcon tube.

2.I.E.iii.c Cervical lymph nodes and inguinal lymph nodes

+/+ -/- CLIC1 and CLIC1 mice were sacrificed using CO2. Cervical and inguinal lymph nodes were removed freshly from these mice. Cervical lymph nodes were then incubated with 0.5 mg/ml of collagenase D and 20 µg/ml of DNase I for 30 mins at

37°C in 5% CO2 incubator and processed as above for the isolation of splenocytes.

127 2.I.E.iii.d Isolation of peritoneal cavity cells

+/+ -/- CLIC1 and CLIC1 mice were sacrificed using CO2. The abdominal cavity of mice was injected with 10 ml of cell medium using a 25 gauge needle. The mice were then gently swirled and the fluid was withdrawn using 19 gauge needle. Resulting fluid was then transferred to 15 ml Falcon tube and centrifuged for 5 mins at 350 x g at room temperature. Supernatant was discarded and cell pellet then lysed with 1 ml of RBC lysis buffer for 5 mins on ice. After red blood cell lysis, the cell pellet was resuspended in 15 ml of PBS and centrifuged again at 350 x g at room temperature. The cell pellet was collected and passed through 70 um cell strainer, ready for immunostaining.

2.I.E.iii.e Thioglycollate elicited peritoneal inflammation model

Mice were injected intraperitoneally with 1 ml of 3% Thioglycollate medium (Sigma-

Aldrich, Australia). The mice were left for 8 hrs then sacrified and the intraperitoneal cells harvested as described in section 2.I.E.iii.d.

2.I.F MICE

All animal works were approved by the Garvan/St Vincent’s Hospital animal ethics committees. The germ line gene deleted CLIC1-/- mice are on a 129X1/SVJ background and have been previously described(Qiu et al., 2010). In all instances, syngeneic

129X1/SVJ mice or cells derived from them were used as CLIC1+/+ control. 2D2 transgenic mice (C57BL/6 background) were kind gift from Dr Vijay Kuchroo (Harvard

Medical School, Boston, MA)(Korn et al., 2007). Mice were housed in the Garvan

Institute Biological Testing Facility (BTF) and maintained on a 12-hr light/12-hr dark cycle, with standard free chow and water freely available.

128 2.II METHODS

2.II.A IMMUNOSTAINING FOR CELL PROFILING

For whole blood staining, 100 ul of blood was incubated with 1 ul of the following monoclonal antibodies; anti-CD45-PerCP, anti-CD3-Pacific Blue, anti-CD4-Alex Fluor

700, anti-Ly6G-PE-Cy7, anti-CD45R/B220-PE, anti-CD11c-APC and anti-CD11b-

APC-Cy7 for 30 mins at room temperature in the dark. After staining, 2 ml of RBC lysis buffer was added and incubated for further 10 mins at room temperature. PBS was then added to bring the volume to 15 ml and centrifuged for 5 mins at 350 x g. The supernatant was removed and the cell pellet collected and resuspended in 1% paraformaldehyde solution and stored on ice until analysed. Subsets of immune cells can be identified based on this panel: T cells (CD3+), DCs (CD3-CD11c+), B cells

(CD3-CD11c-CD11b-B220+), neutrophils (CD3-CD11c-CD11b+Ly6G+), monocytes

(CD3-CD11c-CD11b+Ly6G-).

For staining of isolated immune cells of lymphoid organs and the peritoneal cavity, the cells were resuspended in 100 ul of staining buffer (PBS containing 2% FBS) and incubated for 15 mins on ice in U bottom 96 well plates in triplicates with 1 ul of purified rat anti-mouse CD16/32 monoclonal antibody that bind to Fcγ III/II Receptor to prevent non-antigen specific binding. Then, cells were incubated for 30 mins on ice with 100 ul of staining buffer containing 1 ul of these monoclonal antibodies anti-

CD45-PerCP, anti-CD3-Pacific Blue, anti-CD4-Alex Fluor 700, anti-Ly6G-PE-Cy7, anti-F4/80-FITC, anti-CD45R/B220-PE, anti-CD11c-APC and anti-CD11b-APC-Cy7.

Stained cells were then centrifuged at 350 x g and washed 4 times with 200 ul of PBS, ready to run on flow cytometer on the same day of experiment. Subsets of immune cells can be identified based on these panels: T cells (CD3+), DCs (CD3-CD11c+), B cells

129 (CD3-CD11c-CD11b-B220+), neutrophils (CD3-CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-CD11b+Ly6G-).

Appropriate isotype control antibodies which have the identical species and fluorescent tags of; CD11c, CD11b, F4/80, Ly6G were used to discriminate nonspecific binding.

BD LSRII flow cytometer (BD Biosciences) was used to acquire data and compensation was performed to correct for potential overlapping fluorescence. FlowJo (Tree Star) software was used to analyse data.

2.II.B DENDRITIC CELL MIGRATION EXPERIMENT

2.II.B.i MATURING BMDCS

1x107 of live BMDCs were incubated in 10 ml of complete medium containing LPS (10 ug/ml) in each well of 6 well plates (Cat #353046, Beckon Dickinson Pty) for 4 hrs at

37°C in a 5% CO2 incubator. After incubation time, BMDCs were transferred into 15 ml falcon tube (Cat # 352096, Beckon Dickinson Pty), then were pelleted at 350 x g at

4 °C for 10 mins, and then extensively washed 4 times with 10 ml PBS, kept on ice, ready for fluorescence labeling.

2.II.B.ii FLUORESCENCE LABELLING

Mature BMDCs were fluorescence labeled with commercially available fluorescent dyes; CellTrace Violet (Cat# C34557, Life Technologies) or carboxyfluorescein succinimidyl ester (CFSE) (Cat# C1157, Life Technologies) for 30 min using a protocol outlined previously (Mitchell et al., 2013; Tang et al., 2006). Briefly, 107 of CLIC1+/+ or

CLIC1-/- BMDCs were incubated in 1 ml PBS containing CellTrace Violet (5mM) or

CFSE (5mM) respectively in a 15 falcon tube (Cat # 352096, Beckon Dickinson Pty) 130 for 30 mins at 37°C in a 5% CO2 incubator. The labelling reaction was stopped by addition of 1 ml complete medium. Labelled BMDCs were then washed for 4 times with PBS. Numbers of live cell were determined using a hemocytometer and trypan blue exclusion. 106 of CellTrace Violet labeled CLIC1+/+ BMDCs were mixed with

1x106 of CFSE labelled CLIC1-/- BMDCs in 100 ul complete medium. The exact proportion of stained cells bearing each flurochrome was then determined by flow cytometer. For a negative control, 50 ul of supernatant from the last wash of these two labelling reaction was mixed and used as a negative control.

2.II.B.iii IN VIVO MIGRATION EXPERIMENT

On the footpad of CLIC1+/+ and CLIC1-/- mice, using a 22 gauge needle, I slowly injected 50 ul of pooled, separately labelled BMDCs, prepared as above. On the left footpad of the same mouse, 50 ul of supernatant from the last wash was injected as a negative control. Mice were kept for 48 hrs and then the mice were sacrificed. Popliteal lymph nodes and inguinal lymph nodes were harvested from both left and right sides.

To extract single cells from these lymph nodes, the lymph nodes were incubated with

0.5 ug/ml collagenase D and 20 ug/ml DNase I for 30 mins at 37°C in a 5% CO2 incubator for tissue dissociation and single cells were obtained from passing through the tissues on 70 um cell strainer.

2.II.B.iv VIABILITY TEST

Separately labelled CLIC1+/+ or CLIC1-/- BMDCs, prepared as above, were resuspended into 1 ml complete medium and plated into 6 well plates and left in at 37°C in a 5%

131 CO2 incubator for 0 min, 24 hrs and 48 hrs. After these incubation times, the cells were stained with 1 ul of 7AAD for 5 mins to differentiate viable cells from dead cells.

2.II.B.v IMMUNOSTAINING

The isolated cells from lymph nodes were stained with the following antibodies to selectively identify DCs: CD45-PerCP (clone 30F11), CD11c-APC (clone N418),

CD11b-APC-Cy7 (clone M1/70). Compensation was performed and isotype controls were included for all antibodies. DCs are characterized by CD45hiCD11c+CD11b+ population from flow cytometer. Samples were run in triplicates.

2.II.B.vi DATA ANALYSIS

Prior to injection of labelled BMDCs, the actual ration of CLIC1+/+ or CLIC1-/- BMDCs had been determined by flow cytometer. The ratio of migrated BMDCs was then corrected for the actual proportion of CLIC1+/+ or CLIC1-/- BMDCs that had been injected. By using calculation equation below, the actual ratio of migrated BMDCs can be obtained.

To achieve corrected ratio of migrated BMDCs, I used the equation below;

Corrected ration of migrated CLIC1+/+ BMDC = A / (A+B)

Corrected ration of migrated CLIC1-/- BMDC = B / (A+B)

When A = corrected number of migrated CLIC1+/+ BMDCs

132 A = (number of migrated CLIC1+/+ from flow dot plot) / (number of stained CLIC1+/+ in pooled BMDCs before injection)

Where B =corrected number of migrated CLIC1-/- BMDCs

B = (number of migrated CLIC1-/- from flow dot plot) / (number of stained CLIC1-/- in pooled BMDCs before injection)

2.II.C CLIC1 CELLULAR LOCALISATION

Resting BMDCs or BMDCs 5 mins after initiation of synchronised phagocytosis were fixed with 4% paraformaldehyde (Cat #C004, ProSciTech) on 8 chamber slides (Cat

#354108, BD Biosciences), then permeabilised with 0.05% saponin (Cat #S4521,

Sigma-Aldrich) (Jiang et al., 2012). After blocking with 2% IgG free BSA (Cat #001-

000-161, Jackson ImmunoResearch Labs) and 1 ug/ml Fc receptor blocking antibody

(Cat #553142, Beckon Dickinson Pty, the cells were stained for; CLIC1 and RhoA.

Briefly, BMDCs were firstly stained with 1:100 rabbit anti-mouse RhoA antibody then

1:100 donkey anti-rabbit Cy2 antibody (Cat#ab6940, Abcam) followed by an in house derived 1:1000 sheep anti-mouse CLIC1 (Jiang et al., 2012), then 1:100 biotinylated donkey anti sheep antibody (Cat #713-065-003, Jackson ImmunoResearch Labs) and finally streptavidin Cy3 (Cat#S6402, Sigma-Aldrich) (Jiang et al., 2012). Confocal images were obtained on a Leica TCS SP confocal microscope (Leica Microsystems,

Germany) and processed using ImageJ64 (NIH, imagej.nih.gov/ij/download/).

2.II.D INTRAPHAGOSOMAL ACIDIFICATION MEASUREMENT

This was undertaken essentially as previously described (Jiang et al., 2012). Briefly, loosely adherent BMDCs on a fluordish (Cat #FD35-100, Coherent Life Science),

133 underwent synchronized phagocytosis (Jiang et al., 2012) with opsonised zFITCs on the heated stage of microscope stage of a Zeiss Axiovert 200M fluorescence microscope and the particle fluorescence was recorded over 60 mins, at a rate of one image per min

(excitation 490 nm, emission 525 nm). In some instances, IAA94 (100 µM) (Cat #I117,

Sigma-Aldrich), a cell permeable CLIC1 ion channel blocker, Chloroquine (Cat

#C6628, Sigma-Aldrich) or DMSO (Cat #D2650, Sigma-Aldrich) were added to the fluordish. To convert the excitation ratio to pH, time lapse recordings over 45 mins were carried out on BMDCs that had phagocytosed opsonised zFITC, incubated in a series of buffers from pH 4 to 8 which also contained bafilomycin A1 (100 nM), nigericin (10 uM), valinomycin (10 uM) and carbonyl cyanide m- chlorophenylhydrazone (10 um) to disrupt membrane channel activity and allow equilibration of intracellular pH with that of the extracellular buffer. There was minimal if any photobleaching. A polynomial equation from this data was then derived and used to convert the real time FITC intensity into pH units.

2.II.E PROTEOLYIS ASSAY

This was performed essentially as previously described (Jiang et al., 2012). In brief, loosely adherent BMDCs on a 42 mm glass coverslip (Cat #CB00400RA1, Menzel- glaser), underwent synchronized phagocytosis with DQ bodipy conjugated silica beads on a heated microscope stage, as above. The fluorescence intensities of calibration

Alexa Fluor 594 dye (excitation 570 nm, emission 620 nm) and green reporter DQ bodipy dye (excitation 490 nm, emission 525 nm) were acquired over 60 mins, at a rate of one image per min, as described above. The ratio of fluorescence intensity of substrate to calibration fluorescence was plotted against time and used for ratiometric data analysis of intraphagosomal proteolysis.

134 2.II.F IN VITRO ANTIGEN PRESENTATION EXPERIMENT

2.II.F.i CELL MIXING EXPERIMENT AND FLOW CYTOMETRY

BMDCs (1x105; 100ul) from 129X1/SVJ or C57BL/6 mice were mixed with 100µl of of purified CD4+ T-cells (2x105) from 2D2 mice in wells of a U bottom 96 well plate

(Cat #353077, Beckon Dickinson Pty) and incubated at 37°C in 5% CO2 for 12 hrs.

Golgi stop (1 ug/ml, Cat #554724, Beckon Dickinson) was added 4 hrs prior to evaluation of intracellular cytokine staining. Cells were then fixed, permeabilised (Cat

#77-5775-40, eBioscience) then stained for with anti-IFNγ-PE-Cy7. In separate experiments, 2D2 CD4+ T-cells were also stained for cell surface activation markers using anti-CD25-APC-Cy7 and anit-CD69-PE-Cy7. Flow cytometry data collection was performed on an LSR II (BD Biosciences, San Jose, CA) and analysed using FlowJo software (Tree Star Inc., USA).

2.II.F.ii IN VITRO T-CELL ACTIVATION

This was undertaken essentially as previously described (Mohammad et al., 2014).

Briefly, 1x105 CLIC1+/+ or CLIC1-/- BMDCs in 100 ul of complete medium were incubated with 1.25 pmoles of 21 amino acids MOG35-55 or 1.25 pmoles of 125 amino acids MOG1-125 peptides or vehicle for up to 4 hrs at 37°C in 5% CO2 in a U bottom 96 well plate, in triplicate. The cells were then washed, and LPS matured after which transgenic 2D2 responder T-cells were added and incubated for a further 16 hrs. The proportion of activated 2D2 T-cells (positive for CD4, Vβ11 and intracellular INFγ) were identified by flow cytometry using the gating strategy described in Fig. S1B and data were analysed using FlowJo software essentially as previously described

(Mohammad et al., 2014).

135 2.II.F.iii COSTIMULATORY MOLECULE EXPRESSION

1x106 BMDCs were resuspended into 200 ul complete medium in wells of a U bottom plate containing LPS at 0.1 ug/ml, 0.0001 ug/ml or 0.00001 ug/ml, for 4 hrs. BMDCs were then washed and stained with 1:100 dilution of anti-CD11c-APC, anti-CD45-

PerCP, anti-CD40-PE, anti-CD80-FITC, anti-CD86-Alex700. Cells were also stained with anti-MHC class II-FITC. All antibodies were from Beckon Dickinson. Appropriate compensation and isotype controls were used. Flow cytometry data collection was performed on an LSR II and analysed using FlowJo software essentially as previously described (Mohammad et al., 2014).

2.II.G IN VIVO ANTIGEN PRESENTATION EXPERIMENT

2.II.G.i EAE INDUCTION BY ANTIGEN PULSED BMDC

Female 8-12 weeks old CLIC1+/+ and CLIC1-/- mice were injected subcutaneously in both flanks 50 ul (1x106 cells) with live GM-CSF and IL-4 generated BMDCs that had been pulsed with 1.25 pmoles of MOG1-125.

cDCs were generated from bone marrow using GM-CSF and IL-4, as described above.

BMDCs were then resuspended in 5 ml of complete medium and plated into wells of a 6

6 well plate (Cat #353046, Beckon Dickinson Pty) at 1 x 10 cell/ml. MOG1-125 (125 ug/ml; 1.25 pmoles) was added to the cells for 4 hrs. Cells were washed three times with PBS to remove any residue peptide. LPS (0.1 ug/ml) was added and cells were incubated for another 4 hrs. These cells were washed and kept on ice at concentration of

1x106 cells in 50 ul. Female 8-12 weeks old CLIC1+/+ and CLIC1-/- mice were then injected subcutaneously in both flanks with 50 ul of these BMDCs. Mice were subsequently injected intraperitoneally with 200 ng of pertussia toxin (Cat #180, 136 Sapphire Biosciences) in 0.2 ml PBS at 24 hrs and 3 days after injection of BMDCs.

The mice were then observed daily in a blinded manner, for clinical neurological signs and scores were assigned based on the following widely used scale (Constantinescu et al., 2011) e.g. scale; 1, flaccid tail; 2, hind limb weakness; 3, complete hind limb paralysis; 4, complete hind limb paralysis and forelimb paralysis; 5, complete paralysis.

137 3 RESULTS 3.I IMMUNE CELL PROFILING

3.I.A INTRODUCTION

In this chapter I will determine immune cell phenotype changes in various tissue compartments associated with germline deletion of the gene for CLIC1.

3.I.B BACKGROUND

Immune cell profiling provides valuable information that could help predict individual susceptibility for particular pathogens and also could guide therapeutic decision making in some immune and inflammatory mediated diseases (Ermann et al., 2015). Under normal physiological conditions, immune cells are found predominantly in lymphoid organs and spleen (Figure 11), and less abundantly in circulating blood. Small numbers are usually present in tissues other than lymphoid organs but this changes significantly if tissue injury occurs. A tissue insult results in the migration of effector immune cells to the site of injury in an effort to restore homeostasis.

Since little is known about the role of CLIC1 in immune cell homeostasis and overall immune responses, I sought to determine the role of CLIC1 in immune cell profiles both under normal physiological conditions as well as under inflammatory challenge.

Using CLIC1+/+ and CLIC1-/- mice, I compared the immune cell profiles of lymphoid organs from CLIC1+/+ and CLIC1-/- mice under normal physiological conditions, from five different organs (Figure 11) including peripheral blood, lymphoid organs (spleens, cervical lymph nodes, inguinal lymph nodes) and the peritoneal cavity. To identify changes associated with immunological challenge, I used the same approach to

138 determine immune cell recruitment under stimulated conditions using thioglycollate elicited peritoneal inflammation.

Figure 11 Schematic drawing of localization of lymphoid organs in mouse. The overview of localization of spleen, cervical lymph nodes and inguinal lymph nodes of the mouse are depicted in the drawing.

139 3.I.C RESULTS

3.I.C.i BLOOD

To determine if CLIC1 gene deletion has any affect on the composition of selected circulating immune cells, fresh peripheral blood from 3 pairs of 8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were collected. The cells were stained using the following antibodies CD45, CD3, CD4, CD11c, CD11b, Ly6G and B220 to identify the following populations: T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3-CD11c-CD11b+Ly6G+) and monocytes (CD3-CD11c-CD11b+Ly6G-). The gating strategy is displayed in Figure

12A. The results are the pooled results of female and male mice.

There was a trend towards an increase in the proportion of T cells in CLIC1-/- mice

(Figure 12B; 33.27 ± 5.16 % vs 19.10 ± 1.89 %, n=6/group, p = 0.06, unpaired T test) as this fell just short of statistical significance. The proportion of monocytes was decreased in CLIC1-/- mice (Figure 12B; 7.74 ± 2.41 % vs 15.64 ± 1.14 %, n=6/group, p

= 0.01, unpaired T test), as was the proportion of neutrophils (Figure 12B; 11.39 ± 1.53

% vs 44.29 ± 1.96 %, n=6/group, p = 0.0004, unpaired T test). The proportion of B cells was unchanged and DCs was unchanged (Figure 12B).

The total average blood WBC count of CLIC1+/+ mice was 1.02 ± 0.12 x 106 cells/100 ul, which was significantly lower than CLIC1-/- mice that was 1.31 ± 0.11 x 106 cells/100 ul (n=6/group, p = 0.001, unpaired t-test). The absolute number of T cell was higher in CLIC1-/- mice (Figure 2C; 0.43 ± 0.06 x 106 cells/100 ul vs 0.19 ± 0.02 x 106 cells/100 ul, n=6/group, p = 0.02, unpaired t-test). There was a trend towards a reduction in the absolute number of monocytes in the CLIC1-/- mice (Figure 12C; 0.09 ± 0.02 x

140 106 cells/100 ul vs 0.16 ± 0.01 x 106 cells/100 ul, unpaired t-test, p = 0.07), as this fell just short of statistical significance, as was for the absolute number of neutrophils

(Figure 2C; 0.22 ± 0.02 x 106 cells/100 ul vs 0.44 ± 0.02 x 106 cells/100 ul, p = 0.001, unpaired t-test). B cells and DC absolute numbers were unchanged (Figure 12C).

Thus, these results showed that under normal physiological conditions, germline deletion of the CLIC1 gene in CLIC1-/- mice directly or indirectly changes the number and proportion of circulating immune cells in the blood i.e. increase the number of T cells whilst reducing in the proportion of circulating monocytes and neutrophils, as for the number of neutrophils.

141

Figure 12 Circulating blood from CLIC1-/- mice has more T cells but less monocytes and neutrophils. (A) Viable cells were first gated based on their size and granularity by FSC-A versus SSC-A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3- CD11c-CD11b+Ly6G+) and monocytes (CD3-CD11c-CD11b+Ly6G-) were identified. Proportion and absolute number of these immune subsets in peripheral blood of CLIC1+/+ and CLIC1-/- mice at steady state were show in B and C respectively (n=6 animals/group). Data representing mean ± SEM were analysed using unpaired t-tests.

142 3.I.C.ii SPLEEN

To investigate if germline deletion of the gene for CLIC1 may have any influence on immune cell phenotype changes in lymphoid organ, spleens from 3 pairs of 8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were collected. The cells were stained with the antibodies as above to identify the following populations: T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3-CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-

CD11b+Ly6G). Gating strategy is displayed in Figure 13A.

The proportion of T cells, B cells, DC, macrophages and neutrophils is indistinguishable between CLIC1-/- and CLIC1+/+ spleens (Figure 13B).

The total average WBC count isolated from CLIC1+/+ spleens was 18.2 ± 0.05 x 107 cells, which was significant higher than CLIC1-/- spleens that was 14.1 ± 0.09 x 107 cells (n=6/group, p = 0.0001, unpaired t-test). The absolute number of T cell was significantly lower in CLIC1-/- spleens (Figure 13C; 4.23 ± 0.17x107 cells vs 5.65 ±

0.45 x107 cells, n=6/group, p = 0.04, unpaired t-test), as was for the absolute number of macrophages (Figure 13C; 0.15 ± 0.02 x107 cells vs 0.29 ± 0.03 x107 cells, n=6/group, p

= 0.03, unpaired t-test). The absolute B cell, DC and neutrophil number was similar between CLIC1-/- and CLIC1+/+ spleens (Figure 13C).

Thus, these results showed consistent results that immune cell composition of CLIC1-/- spleen was altered. Although the proportions of T cells and macrophages were unchanged in CLIC1-/- spleens, the absolute cell numbers of these two cell types were lower in CLIC1-/- spleens because of their lower WBC count.

143

Figure 13 Spleen from CLIC1-/- mice has less T cells and macrophages. (A) Viable cells were first gated based on their size and granularity by FSC-A versus SSC-A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3- CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-CD11b+Ly6G-) were identified. Proportion and absolute number of these immune subsets in peripheral blood of CLIC1+/+ and CLIC1-/- mice at steady state were show in B and C respectively (n=6 animals/group). Data representing mean ± SEM were analysed using unpaired t-tests.

144 3.I.C.iii CERVICAL LYMPH NODES

Data so far suggests that germline deletion gene of CLIC1 is associated with changes in immune cell phenotype in both blood and spleen. To investigate further if CLIC1 gene deletion has any effect on other lymphoid organs, cervical lymph nodes from 3 pairs of

8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were collected. The cells were stained for the same identical antibodies to above study to identify the following populations: T cells (CD3+), DCs (CD3-CD11c+), B cells

(CD3-CD11c-CD11b-B220+), neutrophils (CD3-CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-CD11b+Ly6G). Gating strategy is displayed in Figure 14A.

The results are the pooled results of female and male mice.

The proportion of macrophages was decreased in CLIC1-/- CXLN (Figure 14B; 0.39 ±

0.03 % vs 0.54 ± 0.04 %, n=6/group, p=0.04, unpaired T test). The proportions T/B cells, DCs and neutrophils were similar between CLIC1-/- CXLN and CLIC1+/+ CXLN

(Figure 14B).

The total average of WBC isolated from each lymph node from CLIC1+/+ mice was 4.42

± 0.32 x 107 cells, which was similar to CLIC1-/- mice that was 4.61 ± 0.29 x 107 cells

(n=6/group, p = 0.31, unpaired t-test). There was a trend towards a reduction of the absolute cell number of macrophages in cervical lymph nodes of CLIC1-/- mice (Figure

14C; 0.18 ± 0.02 x106 cells vs 0.24 ± 0.01 x106 cells, n=6/group, p = 0.06, unpaired t- test), as this fell just short of statistical significance. The absolute cell number of T cells,

B cells, DCs and neutrophils in cervical lymph nodes were indistinguishable between these two mouse groups (Figure 14C).

145

Thus, these results showed that CLIC1 gene deletion in mice directly or indirectly reduced the proportion of resident macrophages whilst there was also a trend towards a reduction in the absolute cell number of these but not other immune cells.

146

Figure 14 Cervical lymph nodes from CLIC1-/- mice have less proportion of macrophages. (A) Viable cells were first gated based on their size and granularity by FSC-A versus SSC-A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3- CD11c-CD11b+Ly6G+) and monocytes (CD3-CD11c-CD11b+Ly6G-) were identified. Proportion and absolute number of these immune subsets in peripheral blood of CLIC1+/+ and CLIC1-/- mice at steady state were show in B and C respectively (n=6 animals/group, CXLN=cervical lymph node). Data representing mean ± SEM were analysed using unpaired t-tests.

147 3.I.C.iv INGUINAL LYMPH NODES

Data above indicate that germline deletion gene of CLIC1 is associated with changes in immune cell phenotype especially the proportion and number of resident macrophage in cervical lymph nodes. To investigate another lymphoid organ, we have isolated inguinal lymph nodes from 3 pairs of 8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were collected. The cells were stained for the same identical antibodies to above study to identify the following populations: T cells

(CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3-

CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-CD11b+Ly6G). Gating strategy displayed in Figure 15A. The results are the pooled results of female and male mice.

The proportion of macrophages was reduced in CLIC1-/- IGLN (Figure 15B; 0.17 ± 0.02

% vs 0.34 ± 0.02 %, n=6/group, p=0.04, unpaired T test). The proportion of T cells, B cells, DCs and neutrophils were unchanged in CLIC1-/- IGLN (Figure 15B).

The total WBC isolated from inguinal lymph node of CLIC1+/+ mice was 3.30 ± 0.28 x

107 cells, which is indistinguishable from inguinal lymph node of CLIC1-/- mice that was 3.54 ± 0.31 x 107 cells (n=6/group, p = 0.19, unpaired t-test). In line with the proportion data, the absolute cell number of macrophages was significantly lower in

CLIC1-/-IGLN (Figure 15C; 0.11 ± 0.03 x 106 cells vs 0.15 ± 0.08 x 106 cells, n=6/group, p = 0.01, unpaired t-test).

Thus, these data showed consistent results, as for the cervical lymph nodes that inguinal lymph nodes of CLIC-/- mice had lower number of resident macrophages.

148

Figure 15 Inguinal lymph nodes of CLIC1-/- mice have less macrophages. (A) Viable cells were first gated based on their size and granularity by FSC-A versus SSC-A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3- CD11c-CD11b+Ly6G+) and monocytes (CD3-CD11c-CD11b+Ly6G-) were identified. Proportion and absolute number of these immune subsets in peripheral blood of CLIC1+/+ and CLIC1-/- mice at steady state were show in B and C respectively (n=6 animals/group, IGLN=inguinal lymph node, cell number of DC and macrophages x 10 to display on scale). Data representing mean ± SEM were analysed using unpaired t- tests.

149 3.I.C.v PERITONEAL CAVITY

Data so far suggests that there is an alteration in the number and proportion macrophages in blood, spleen and lymph nodes of CLIC1-/- mice when compare to

CLIC1+/+ mice. To investigate a non-lymphoid organ, cells from peritoneal cavity

(membrane bound and fluid filled abdominal cavity that contain liver, spleen and most of gastro-intestinal tracts) from 3 pairs of 8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were collected. The cells were stained for the same identical antibodies to above study to identify the following populations: T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils

(CD3-CD11c-CD11b+Ly6G+) and macrophages (CD3-CD11c-CD11b+Ly6G). Gating strategy displayed in Figure 16A. The results are the pooled results of female and male mice.

The proportion of T cells was reduced in CLIC1-/- peritoneal cavity (Figure 16B; 7.21 ±

0.71 % vs 10.24 ± 0.10 %, n=6/group, p=0.01, unpaired T test), as for the proportion of

B cells (Figure 16B; 8.92 ± 0.65 % vs 13.38 ± 1.48 %, n=6/group, p=0.05, unpaired T test). In contrast, the proportion of macrophages was increased in CLIC1-/- peritoneal cavity (Figure 16B; 10.24 ± 0.10 % vs 7.21 ± 0.71 %, n=6/group, p=0.03, unpaired T test). However, the proportion of DCs was similar between these mouse groups (Figure

16B). There were no neutrophils in the peritoneal of either mouse groups.

The average total WBC count was 12.2 ± 0.02 x 106 cells for cells isolated from

CLIC1+/+ peritoneal cavity, which is similar to the total count of the cells isolated from

CLIC1-/- peritoneal cavity that was 13.2 ± 0.03 x 106 cells for CLIC1-/- peritoneal cavity

(n=6/group, p = 0.59, unpaired t-test). The absolute cell number of T cells was also

150 reduced in CLIC1-/- peritoneal cavity (Figure 16C; 0.93 ± 0.09 x 106 cells vs 1.23 ± 0.12 x 106 cells, n=6/group, p=0.03, unpaired T test), whilst there was a trend towards a reduction in the absolute cell number of B cells (Figure 16C; 1.16 ± 0.08 x 106 cells vs

1.60 ± 0.18 x 106 cells, n=6/group, p=0.08, unpaired T test) as this fell just short of statistical significance. Additionally, CLIC1-/- peritoneal cavity contains significant more macrophages than CLIC1+/+ peritoneal cavity (Figure 16C; 6.31 ± 0.07 x 106 cells vs 3.63 ± 0.08 x 106 cells, n=6/group, p=0.02, unpaired T test). The absolute number of

DCs was similar between these mouse groups as was the absence of neutrophils (Figure

16C).

Thus, these results further illustrated a consistent finding that CLIC1 gene deletion modulates the proportion and the absolute number of selected immune cell populations in the peritoneal cavity of the mice, as seen in the blood and the spleen.

151

Figure 16 peritoneal cavities of CLIC1-/- mice contain less T and B cells but more macrophages. (A) Viable cells were first gated based on their size and granularity by FSC-A versus SSC-A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), B cells (CD3-CD11c-CD11b-B220+), neutrophils (CD3- CD11c-CD11b+Ly6G+) and monocytes (CD3-CD11c-CD11b+Ly6G-) were identified. Proportion and absolute number of these immune subsets in peripheral blood of CLIC1+/+ and CLIC1-/- mice at steady state were show in B and C respectively (n=6 animals/group, PC=peritoneal cavity, cell number of DC x 10 to display on scale). Data representing mean ± SEM were analysed using unpaired t-tests.

152 3.I.C.vi THIOGLYCOLLATE ELICITED PERITONEAL INFLAMMATION

MODEL

Data so far showed that germline deletion of CLIC1 gene was associated with altered distribution of especially macrophages and T cells in some of tissue compartments. To examine the possible role of CLIC1 in immune cell modulations during inflammatory immune responses, I employed the thioglycollate elicited peritoneal inflammation model. In this model, neutrophils and monocytes start to increase and reach peak level between 4 and 24 hrs post stimulation, while macrophage numbers are increased at 24 hrs and reach peak level at 3 to 4 days (Lam et al., 2013). This model has been used to study neutrophil and monocyte migration in transgenic or gene knockout mice (Muneer et al., 2014). As before, 3 pairs of 8-12 week old female and 3 pairs of 8-12 week old male CLIC1+/+ and CLIC1-/- mice were injected intraperitoneally with 1 ml of 3% aged thioglycollate medium. After 8 hrs post injection, mice were scarified and peritoneal fluid were harvested and peritoneal cells were concentrated by centrifugation and then stained with monoclonal antibodies as listed herein; CD45, CD3, CD4, CD11c, CD11b,

F4/80, Ly6G and Gr1 Gating strategies were performed and demonstrated in Figure 17.

Exudate macrophages can be identified as CD45+CD3-CD11c-F4/80-Ly6G-Gr1+ and tissue resident macrophages are CD45+CD3-CD11c-F4/80+.

Whilst proportion of T cells and DC was similar in CLIC1-/- and CLIC1+/+ thioglycollate elicited peritoneal cavity inflammation (Figure 18A), the proportion of B cells was decreased (Figure 18B; 2.10 ± 0.78 % vs 7.90 ± 0.03 %, n=6/group, p=0.001, unpaired T test), as was the proportion of neutrophils (Figure 18A; 29.42 ± 0.40 % vs

35.61 ± 1.43 %, n=6/group, p=0.01, unpaired T test). In contrast, the proportion of exudate macrophages was increased in CLIC1-/- thioglycollate elicited peritoneal cavity

153 (Figure 18A; 59.10 ± 4.20 % vs 33.30 ± 3.10 %, n=6/group, p=0.01, unpaired T test), was the proportion of tissue resident macrophages (Figure 18A; 9.07 ± 0.33 % vs 5.35 ±

0.34 %, n=6/group, p=0.001, unpaired T test).

The total WBC count isolated from CLIC1+/+ thioglycollate elicited peritoneal cavity was 42.43 ± 0.98 x 106 cells, which is indistinguishable from the number of cells isolated from CLIC1-/- thioglycollate elicited peritoneal cavity, that was 43.38 ± 0.77 x

106 cells (n=6/group, p = 0.09, unpaired t-test). The absolute cell numbers of T cells and

DCs were similar between these mouse groups (Figure 18B). The absolute cell number of B cells was lower in CLIC1-/- thioglycollate elicited peritoneal cavity (Figure 18B;

0.90 ± 0.01 x 106 cells vs 3.32 ± 0.32 x 106 cells, n=6/group, p=0.001, unpaired T test), as was the absolute cell count for neutrophils (Figure 18B; 12.65 ± 0.18 x 106 cells vs

14.95 ± 0.62 x 106 cells, n=6/group, p=0.02, unpaired T test). The absolute number of exudate macrophages was increased in CLIC1-/- thioglycollate elicited peritoneal cavity

(Figure 18B; 25.45 ± 1.54 x 106 cells vs 13.43 ± 2.05 x 106 cells, n=6/group, p=0.01, unpaired T test), as for the number of and tissue resident macrophages (Figure 18B;

3.90 ± 0.14 x 106 cells vs 2.25 ± 0.15 x 106 cells, n=6/group, p=0.001, unpaired T test).

These results demonstrated that during inflammatory immune responses (i.e. thioglycollate stimulation), deletion of gene for CLIC1 in CLIC1-/- mice altered immune cell composition significantly in the peritoneal cavity of CLIC1-/- mice when compare to

CLIC1+/+ mice. This suggests that deletion of CLIC1 gene could result in a change in immune cell recruitment to the site of inflammation.

154 250K 5 5 10 98.8 10

200K CD45+ cells 4 16.7

10 cd3 4 Viable cells cd45 10 150K T cells

3 10 78.5 CD3 SSC-A 90.1 3 100K CD45 10 SSC-A 0 2 :

: 10 50K CD3- cells 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC-A FSC-A SSC-ASSC-A SSC-ASSC-A

250K 5 10 105 5.9 200K DCs 104 Macrophages 4 f4/80 10

cd11c 1.33 150K Neutrophils

3 3 FSC-A 9.77 10 10

100K F4/80 CD11c FSC-A

50K : 98.3 0 : 0 89

0 93.8 3 4 5 0 10 10 10 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K : ly6G SSC-A Ly6G SSC-A SSC-ASSC-A

105 89.5 Monocytes 104

8.56 103 GR-1 B525_B-A: Gr1 102 B cells

0

0 103 104 105 :Ly6G ly6G

Figure 17 Gating strategy used to identify immune cell subsets from peritoneal cavity of mice stimulated with thioglycollate medium. Viable cells were first gated based on their size and granularity by FSC-A versus SSC- A respectively and doublets were excluded. Selected cells were then further separated based on staining for CD45+ population for leukocytes, after which T cells (CD3+), DCs (CD3-CD11c+), macrophages (CD45+CD3-CD11c-F4/80+-), neutrophils (CD3-CD11c- F4/80-Ly6G+) B cells (CD3-CD11c-F4/80-Ly6G-Gr1-), and exudate macrophages (CD3- CD11c-F4/80-Ly6G-Gr1+) were identified.

155

Figure 18 CLIC1-/- mice recruit more monocytes and have more resident macrophages than CLIC1+/+ mice after thioglycollate stimulation. Peritoneal fluid from CLIC1+/+ and CLIC1-/- mice after 8 hrs of thioglycollate stimulation, were harvested and the immune cell phenotypes were determined using flow cytometry. The proportion and the absolute cell number of infiltrating immune cells in peritoneal fluid are showed in (A) and (B) respectively. Data representing mean ± SEM were analysed using unpaired t-tests.

156 3.I.D DISCUSSION

I have demonstrated here that under normal physiological conditions, deletion of CLIC1 gene directly or indirectly altered immune cell composition in blood (Figure 12), spleen

(Figure 13) and peritoneal cavity (Figure 16), while having a minimal effect on immune cell composition in cervical (Figure 14) and inguinal lymph nodes (Figure 15).

Additionally, I have also demonstrated that under inflammatory responses, the proportions and the numbers of most of immune cells at the site of inflammation were altered in CLIC1-/- mice (Figure 18). These all suggest that deletion of CLIC1 gene in the CLIC1-/- mice causes some changes in immune cell composition in which could alter immune cell homeostasis and also modulate overall immune responses under inflammatory challenge.

T cells migrate from the thymus and circulate between secondary lymphoid organs and and peripheral tissues through blood, looking for foreign antigens presented on MHC molecules on antigen presenting cells such as DCs, B cells and macrophages, important for cell mediated immune responses (Friedl and Weigelin, 2008). On the other hand, B cells move within secondary lymphatic tissues to capture free floating antigens in the circulation, receive T cell help and recirculate and become resident in the bone marrow and other lymphoid organs as antibody-secreting plasma cells, crucial for humoral immune responses (Friedl and Weigelin, 2008). Our results showed that the number of

T cells and macrophages (monocytes in blood) changed significantly in most of the tissue compartments examined, whist B cell and DC numbers are mostly unchanged, suggesting that CLIC1 gene deletion in CLIC1-/- mice may alters cell mediated immune responses involving T cells and macrophages rather than the humoral immune responses mediated by B cells.

157 In response to an inflammatory stimulus, circulating immune cells leave the blood and enter tissues in order to fulfill their functions in the immune responses (Yona and Jung,

2010). Several factors may influence the transmigration of immune cells from the blood to the inflamed tissue (Vestweber, 2015). These factors include adhesion molecules, chemokine or other chemoattractants secreted by endothelial cells, cytokines and other pro-inflammatory mediators, and the nature of pathogens (Muller, 2014; Vestweber,

2015). The first two factors are the likely to explain our results.

First, leukocytes must be captured and attached to endothelial cells in the inflamed tissue in order to reach the site of exit. Leukocytes express various integrins such as lymphocyte function-associated antigen 1 (LFA1; also known as αLβ2 integrin), which is expressed by all leukocytes, and macrophage antigen 1 (MAC1; also known as αMβ2 integrin), which is expressed by myeloid cells, as well as the β1 integrin very late antigen 4 (VLA4; also known as α4β1 integrin), which is expressed by effector lymphocytes and monocytes (Herter and Zarbock, 2013). The integrins bind to ICAM1 on the surface of the endothelial cells, slowing down the rolling of leukocytes and arrest the leukocyte to the vascular wall (Herter and Zarbock, 2013). CLICs such as, CLIC3 has been reported to be associated with the recycling back of α5β1 integrin from late endosome to the plasma membrane (Dozynkiewicz et al., 2012). Although there is no direct report on CLIC1’s role in any of integrin recycling as for CLIC3, it is possible that the altered immune cell composition in some of the tissue compartments is due to the difference in the recycling of the integrin to plasma membrane between CLIC1+/+

-/- and CLIC1 mice.

158 Second, chemokines or chemoattractants are also crucial for leukocytes recruitment because chemoattractants produced by endothelial cells and inflammatory cells in the inflamed tissue attract and support the leukocyte recruitment. Several reports have elegantly showed that chemokines produced by platelets enhance leukocyte recruitment to the vascular wall and support immune cell transmigration (Gleissner et al., 2008;

Semple et al., 2011). Our group has found that the platelets of the mice with CLIC1 gene knockout were different from the CLIC1+/+ mice, despite having 15% more platelet count, CLIC1-/- mice have twice prolonged bleeding time when compared to CLIC1+/+ mice (Qiu et al., 2010). Because of this difference, it is plausible that CLIC1-/- platelet may also produce different pattern of chemokines from CLIC1+/+ platelet, which in turn appear in changes of immune cell profile.

This study has some limitations. First, the panel of antibodies used in this study may be insufficient to differentiate the heterogeneity of immune cell subsets especially monocytes and macrophages which are considered so diverse. Second, thioglycollate medium is the only stimulus used in this study. Other stimuli such as TNF, GM-CSF or

LPS could be used to test if there is any difference in immune cells recruitment.

In conclusion, our results have demonstrated that both normal physiological conditions and following inflammatory stimuli, the immune cell composition is altered significantly in CLIC1-/- mice when compared CLIC1+/+ mice. Because of these, it is suggested that CLIC1 gene deletion could alter their basal immune protection and modulate immune responses during inflammatory challenges.

159 3.II DENDRITIC CELL MIGRATION

3.II.A INTRODUCTION

In this chapter I will examine the effect of CLIC1 gene deletion on dendritic cell migration from peripheral footpad to secondary lymph nodes of mice.

3.II.B BACKGROUND

Dendritic cells (DCs) are potent antigen presenting cells that are largely responsible for priming T cell responses. They migrate via afferent lymphatic vessels from inflamed or damaged peripheral sites to the closest draining lymph nodes in order to present foreign antigen to naïve T cells. DCs reside throughout the body and are responsible for providing much of the primary immune surveillance for the detection of infection and malignancy. In the case of infection in peripheral tissue, DCs internalize pathogens and present pathogen derived peptides on MHC I or MHC II molecules. The DCs then migrate to secondary lymphoid organs where they present antigen to T cells with cognate T cell receptors. This induces clonal expansion and activation of T cells capable of responding to the antigen. This is discussed in more detail in literature review

(section 1.III.G).

The migration of DCs from peripheral tissue to lymph nodes is facilitated by the expression of chemokine receptors that are responsive to the chemokine produced in the lymph node microenvironment (Randolph et al., 2008; Sallusto et al., 1999; Sallusto et al., 1998). These receptor-chemokine interactions ensure that DCs migrate to the right place at the right time. Immature DCs constantly patrol peripheral tissues for foreign antigen detection. Upon maturation, they upregulated CC motif chemokine receptor 7

(CCR7) which can sense its ligand CC motif ligand 21 (CCL21) secreted by lymphatic

160 endothelium cells in lymph nodes. As a result, mature DCs migrate from areas like the skin, along lymphatic vessels to the lymph nodes (Weber et al., 2013).

In mice, studies of lymphatic drainage from left footpad injection reveal that lymphatic vessels drain into popliteal or inguinal lymph nodes and then into iliac lymph nodes as shown in Figure 19 (Harrell et al., 2008; Zhang et al., 2013). However, two days after the DC injection onto a footpad, most of the DCs were found in popliteal lymph node and smaller fraction of cells were found in inguinal lymph node (Harrell et al., 2008).

Figure 19 Lymph node drainage in mouse. Diagram of lymph node drainage after dye injection on left footpad of the mouse. Dye injected on footpad drain to popliteal lymph node (PO) and further to iliac (IL) and renal lymph nodes (RE). Alternatively dye drain to inguinal lymph nodes (IN) and occasionally to axillary lymph nodes (AX). Adapted from (Harrell et al., 2008)

CLIC1 has been reported play a part in cell volume regulation which is necessary for migration (Wang et al., 2012). However, there is no data on the role if any of CLIC1 in 161 DC migration. Thus, this study aimed to investigate if germline deletion of the CLIC1 gene had any effect on in vivo DC migration from peripheral tissues to secondary lymph nodes, a key element in their regulation of immune responses.

In order to do this, I used different fluorophores to separately label CLIC1+/+ and

CLIC1-/- BMDCs which were then simultaneously injected into the footpads of

CLIC1+/+ and CLIC1-/- mice. After 48 hrs, I harvested popliteal and inguinal lymph nodes to quantify migrated dendritic cells by flow cytometer.

162 3.II.C RESULTS

3.II.C.i DETERMINATION OF PROPORTION OF CLIC1 AND CLIC1-/-

BMDCs IN POOLED BMDCS PREPARED FOR FOOTPAD INJECTION

I first prepared pooled BMDCs by mixing equal number of CellTrace Violet labelled

CLIC1+/+ BMDCs and CFSE labelled CLIC1-/- BMDCs. To obtain the precise proportion of CLIC1+/+ and CLIC1-/- BMDCs are in the pooled BMDCs, the cells were stained with fluorochrome labelled antibodies to cell surface markers and the cells were then analyzed by flow cytometer. As illustrated in Figure 20, proportion CLIC1+/+

BMDCs were determined as CD11c+CD11b+CellTrace+, whereas, CLIC1-/- BMDCs were determined as and CD11c+CD11b+CFSE+. For example, in the representative experiment in Figure 20, the ratio of CLIC1+/+ and CLIC1-/- BMDCs in the sample prepared for injection was 53.7: 46.1. This data is used for normalization of later experiments.

Figure 20 Proportion of CLIC1+/+ and CLIC1-/- BMDCs in pooled sample prepared for footpad injection After labelling CLIC1+/+ and CLIC1-/- BMDCs with 5mM CellTrace Violet or 5mM CFSE respectively, equal numbers of CLIC1+/+ and CLIC1-/- BMDCs were mixed to prepare as pooled BMDCs. The pooled BMDCs were surface-stained with anti-CD11c and anti-CD11b monoclonal antibodies. The pooled BMDCs were then run on a flow cytometer to determine exact proportion CLIC1+/+ and CLIC1-/- BMDCs as CD11c+CD11b+CellTrace+ CSFE- and CD11c+CD11b+ CellTrace+CFSE+ respectively in the pooled sample. 163 3.II.C.ii GATING STRATEGY USED TO IDENTIFY MIGRATED BMDCs IN

LYMPH NODES

At 48 hrs post injection of BMDCs to footpad the mice, lymph nodes were harvested and a single cell suspension was obtained. The single cells were stained for CD45,

CD11c and CD11b before analyzed by flow cytometer. In order to identify whether the population of migrated BMDCs are CLIC1+/+ or CLIC1-/- BMDCs, the gating strategy shown in Figure 21 was used. First, live cells were gated on forward (FSC-A) and side scatter (SSC-A) plot. Second, immune cells were gated on CD45 positive population.

Third, BMDCs were gated based on the double positive for CD11b and CD11c population. From this, to identify the injected migrated BMDCs, they were gated further for either CellTrace Violet positive cells to identify migrated CLIC1+/+ BMDCs, or

CFSE positive cells to identify migrated CLIC1-/- BMDCs.

164

Figure 21 Gating strategy used to identify migrated BMDCs Cells were isolated from mouse lymph nodes after 2 days post injection with equal numbers of CLIC1+/+ and CLIC1-/- BMDCs. Cells were stained with markers of DC (CD45+CD11b+CD11c+) and analyzed by flow cytometer. Single cells were gated first based on their FSC-A and SSC-A. Immune cells were gated based on their CD45 expression. Then, BMDCs were gated further on CD11c+CD11b+ cells from immune cell population. From this, BMDCs were gated further on either CellTrace Violet positive cells to identify migrated CLIC1+/+ BMDCs, or CFSE positive cells to identify migrated CLIC1-/- BMDCs.

-/- 3.II.C.iii CLIC1 BMDCS MIGRATED TO POPLITEAL LYMPH NODES

MORE EFFICIENTLY THAN CLIC1+/+BMDCS.

Mature DCs are capable of migrating from peripheral tissues to draining lymph nodes.

To unravel CLIC1’s role in DC migration, I compared the homing capacity of CLIC1+/+ and CLIC1-/- BMDCs from peripheral tissues to draining lymph nodes by quantifying

165 the number of migrated BMDCs in draining popliteal lymph nodes of the mice which were previously injected footpad with live BMDCs. Because majority of BMDCs migrate to popliteal lymph nodes after 2 days footpad post injection, popliteal lymph nodes were harvested from the right side of CLIC1+/+ and CLIC1-/- mice 48 hrs post injection of the pooled BMDCs containing a previously quantified ratio of CLIC1+/+ and

CLIC1-/- BMDCs. For a negative control, popliteal lymph nodes were also harvested from the left side of these mice in which supernatant from the last wash of the labelling reaction was injected. Cells from lymph nodes were filtered through a sieve, before stained with anti-CD45, anti-CD11b and anti-CD11c monoclonal antibodies, and analyzed by flow cytometer.

Using the same gating strategy in Figure 21, the proportion of migrated CLIC1+/+ and

CLIC1-/- BMDCs in popliteal lymph node were quantified. The number of cells was corrected to the number of starting cells and expressed as ratio using the formula described in the methods section. The example flow dot plot (Figure 22A), shows that there is a higher proportion of fluorescent labelled CLIC1-/- BMDCs than fluorescent labelled CLIC1+/+ BMDCs when injected cells into the right footpad of CLIC1+/+ mice as demonstrated by their positive labelling for CFSE and CellTrace Violet dyes respective. For the control, the lymph nodes on the left side of the mouse, which had been injected with the supernatant from the last wash of the labelling reaction and no

BMDC, showed no fluorescent-labelled BMDCs found in the popliteal lymph nodes

(Figure 22B). This indicates that the identified labelled BMDCs derive from the injected cells and not non-specific labelling of the cells at the site of injection. When this experiment was performed using a total of 6 mice/group over three separate occasions, the average corrected ratio of migration for CLIC1-/- BMDCs was 0.688 ±

166 0.02 whilst that for CLIC1+/+ BMDCs was 0.312 ± 0.02 (Figure 22C, n=6/group, p<0.0001, unpaired T test). Thus, these results indicate that there are more CLIC1-/-

BMDCs in draining lymph node than CLIC1+/+ BMDCs when injected into CLIC1+/+ recipient mice, suggesting that CLIC1-/- may migrate much faster than CLIC1+/+

BMDCs.

Figure 22 CLIC1-/- BMDCs migrated to popliteal lymph nodes more than CLIC1+/+ BMDCs when injected into CLIC1+/+ recipient mice. Cells from popliteal lymph nodes of CLIC1+/+ mice after 48 hrs injection in the footpad with separately labelled but pooled BMDCs containing CLIC1+/+ or CLIC1-/- BMDCs, were harvested and analyzed by flow cytometer. The ratio of migrated CLIC1+/+ BMDCs or CLIC1-/-BMDCs was determined from flow dot plot by their positive labelling of CellTrace Violet or CFSE dyes respectively (A) when compare to the negative control (B). (C) The average corrected ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1+/+ mice. Data representing mean ± SEM were analyzed using unpaired t-tests.

167 Endogenous CLIC1 expression in most cells CLIC1+/+ recipient mice, this may also influence BMDC migration. To test differences that might occur when the recipient mice are CLIC1-/- mice, I also injected CLIC1+/+ and CLIC1-/- BMDCs into the footpad of CLIC1-/- mice. By using essentially the same protocol as above, I found that, , consistent with the data in CLIC1+/+ recipient mice there was again a higher proportion of fluorescent labelled CLIC1-/- BMDCs than fluorescent labelled CLIC1+/+ BMDCs

(Figure 23C ratio; 0.710 ± 0.01 vs 0.291 ± 0.01, n=6/group with triplicate samples, p<0.0001, unpaired T test) in the draining lymph node.

Figure 23 CLIC1-/- BMDCs also migrated to popliteal lymph nodes more than CLIC1+/+ BMDCs when injected into CLIC1-/- recipient mice. Cells from popliteal lymph nodes of CLIC1-/- mice after 48 hrs injection in the footpad with separately labeled but pooled BMDCs containing CLIC1+/+ or CLIC1-/- BMDCs, were harvested and analyzed by flow cytometer. The ratio of migrated CLIC1+/+ BMDCs or CLIC1-/-BMDCs was determined from flow dot plot by their positive labeling of CellTrace Violet or CFSE dyes respectively (A) when compare to the negative control (B). (C) The average corrected ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1-/- mice. Data representing mean ± SEM were analyzed using unpaired t-tests.

168 To directly assess whether the presence or absence of CLIC1 in the recipient mice has any influence on the ratio of CLIC1+/+ BMDCs in popliteal lymph nodes, I directly compared ratio of CLIC1+/+ BMDCs found in popliteal lymph nodes and found that there is no significant difference in the corrected ratio of migrated CLIC1+/+ BMDCs between CLIC1+/+ and CLIC1-/- recipient mice (Figure 24A, 0.330 ± 0.02 vs 0.307 ±

0.01, n=6/group with triplicate samples, p = 0.310, unpaired T test). Likewise, there is also no significant difference in the corrected ratio of migrated CLIC1-/- BMDCs between CLIC1+/+ popliteal lymph nodes and CLIC1-/- popliteal lymph nodes (Figure

24B, 0.669 ± 0.02 vs 0.693 ± 0.01, n=6/group with triplicate samples, p = 0.393, unpaired T test). Therefore, these results clearly indicate that the presence or absence of

CLIC1 in recipient mice has no influence on the ratio of migrated BMDCs found in popliteal lymph nodes and thus has no influence on the migration of BMDCs.

Figure 24 The presence or absence of CLIC1 in recipient mice has no influence on ratio of BMDCs found in popliteal lymph nodes. Corrected ratio of CLIC1-/- BMDCs found in popliteal lymph nodes of CLIC1+/+ recipient mice were compared to CLIC1-/- recipient mice and displayed in (A). Corrected ratio of CLIC1-/- BMDCs found in popliteal lymph nodes of CLIC1+/+ recipient mice were compared to CLIC1-/- recipient mice and displayed in (B).

.

169 3.II.C.iv FLUORESCENT TOXICITY HAS NO INFLUENCE ON BMDC

MIGRATION BECAUSE OPPOSITE FLUORESCENT CELL LABELING

GIVES CONSISTENT RESULTS

Because fluorescent dye labelling could damage receptors on the cell surface which may has an influence on cell migration, I therefore set up an experiment to identify whether the fluorescence dyes may have any influenced the number of BMDCs in the sampled lymph node. By using essential identical protocol from above, CLIC1+/+

BMDCs and CLIC1-/- BMDCs were fluorescent stained with the “opposite” fluorescent dye i.e. CLIC1+/+ BMDCs were stained with CFSE, while, CLIC1-/- BMDCs were stained with CellTrace Violet.

From 6 experiments and after normalization, the calculated migrated BMDCs showed that more ration of CLIC1-/- BMDCs than CLIC1+/+ BMDCs in popliteal lymph nodes of CLIC1+/+ recipient mice (Figure 25A ratio; 0.705 ± 0.02 vs 0.295 ± 0.02, n=6/group with triplicate samples, p<0.0001, unpaired T test). Likewise, when injected pooled

BMDCs into the right footpad of CLIC1+/+ mice, the result was identical in which there is higher proportion of fluorescent labelled CLIC1-/- BMDCs than fluorescent labelled

CLIC1+/+ BMDCs in popliteal lymph nodes of CLIC1-/- recipient mice (Figure 25B ratio; 0.730 ± 0.03 vs 0.270 ± 0.03, n=6/group with triplicate samples, p<0.0001, unpaired T test). Thus, this study suggests that toxicity from fluorescence dyes has no influence on BMDCs migration at least in our setting. This experiment also showed a consistent result that CLIC1-/- BMDCs can be found in higher ratio than CLIC1+/+

BMDCs irrespective of whether the cells are injected into CLIC1+/+ or CLIC1-/- mice.

170

Figure 25 Toxicity from fluorescent dyes had no influence on BMDC migration as swopping fluorescent dyes on BMDCs gave consistent results. Cells from popliteal lymph nodes of CLIC1+/+ or CLIC1-/- mice after 48 hrs injection into the footpad with pooled BMDCs containing CLIC1+/+ or CLIC1-/- BMDCs previously stained with adverse fluorescent dyes (i.e. CLIC1+/+ BMDCs were stained with CFSE, while, CLIC1-/- BMDCs were stained with CellTrace Violet), were harvested and analyzed by flow cytometer. (A) The average ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1+/+ mice. (B) The ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1-/- mice. Data representing mean ± SEM were analyzed using unpaired t-tests.

Overall, These results clearly demonstrated that there were more CLIC1-/-BMDCs than

CLIC1+/+ BMDCs in popliteal lymph nodes after two days post injection of pooled

BMDCs to both CLIC1+/+ and CLIC1-/- recipient mice. Thus, it is possible that BMDCs bearing no CLIC1 may migrate faster or retained better at least in popliteal lymph nodes.

171 -/- 3.II.C.v CLIC1 BMDCS ALSO MIGRATED MORE EFFICIENTLY TO

MORE DISTAL LYMPH NODES

BMDCs could migrate to several lymph nodes apart from popliteal lymph nodes

(Harrell et al., 2008). While majority of BMDCs migrate to popliteal lymph nodes, a minority of BMDCs could migrate to more distal lymph nodes such as inguinal lymph nodes that are further away from popliteal lymph nodes (Figure 19) (Harrell et al.,

2008). To quantify the number of migrated BMDCs in more distal inguinal lymph nodes, the same protocol was used. Instead of popliteal lymph nodes, inguinal lymph nodes were harvested for analysis. The proportion of migrated BMDCs was analyzed by flow cytometer.

Less number of migrated BMDCs was found in inguinal lymph nodes when compared to popliteal lymph nodes as determined by flow dot plot (Figure 26A). In inguinal lymph nodes after two days post injection of pooled BMDCs, From 6 experiments and after normalization, the calculated migrated BMDCs showed the average corrected ratio of migrated CLIC1-/- BMDCs was higher than CLIC1+/+ BMDCs, when injected the pooled BMDCs into CLIC1+/+ recipient mice (Figure 26C ratio; 0.777 ± 0.01 vs 0.223 ±

0.01, n=6/group with triplicate samples, p<0.0001, unpaired T test). Thus, these provide consistent results that there are more CLIC1-/- BMDCs in distal lymph node compare to

CLIC1+/+ BMDCs when injected into CLIC1+/+ recipient mice.

172

Figure 26 CLIC1-/- BMDCs migrated faster than CLIC1+/+ BMDCs from footpad to more distal inguinal lymph nodes of CLIC1+/+ mice. Cells from inguinal lymph nodes from CLIC1+/+ mice after 48 hrs injection in the footpad with separately labelled but pooled BMDCs containing CLIC1+/+ or CLIC1-/- BMDCs, were harvested and analyzed by flow cytometer. The ratio of migrated CLIC1+/+ BMDCs or CLIC1-/-BMDCs was determined from flow dot plot by their positive labelling of CellTrace Violet or CFSE dyes respectively (A) when compare to the negative control (B). (C) The average corrected ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1+/+ mice. Data representing mean ± SEM were analyzed using unpaired t-tests.

173 To avoid artifacts arisen from the presence of endogenous CLIC1 expression in

CLIC1+/+ recipient mice, I injected CLIC1+/+ and CLIC1-/- BMDCs to footpad of

CLIC1-/- mice and 48 hrs later, inguinal lymph nodes were harvest for analysis. From 6 experiments and after normalization, the calculated migrated BMDCs was higher of

CLIC1-/- BMDCs was higher than CLIC1+/+ BMDCs in CLIC1-/- recipient mice (Figure

27C ratio; 0.708 ± 0.01 vs 0.292 ± 0.01, n=6/group with triplicate samples, p<0.0001, unpaired T test). These results further confirmed that there are more CLIC1-/- BMDCs in distal inguinal lymph nodes irrespective of whether the cells are injected into

CLIC1+/+ or CLIC1-/- mice.

174

Figure 27 CLIC1-/- BMDCs migrated faster than CLIC1+/+ BMDCs from footpad to more distal inguinal lymph nodes of CLIC1-/- mice. Cells from inguinal lymph nodes from CLIC1-/- mice after 48 hrs injection in the footpad with separately labeled but pooled BMDCs containing CLIC1+/+ or CLIC1-/- BMDCs, were harvested and analyzed by flow cytometer. The ratio of migrated CLIC1+/+ BMDCs or CLIC1-/-BMDCs was determined from flow dot plot by their positive labeling of CellTrace Violet or CFSE dyes respectively (A) when compare to the negative control (B). (C) The average corrected ratio of migrated CLIC1+/+ or CLIC1-/- BMDCs when injected into the footpad of CLIC1-/- mice. Data representing mean ± SEM were analyzed using unpaired t-tests.

+/+ 3.II.C.vi ATTENUATED MIGRATION OF CLIC1 BMDCS IS NOT DUE TO

CELL DEATH

Only viable BMDCs migrate to the draining lymph nodes whereas dead cells are phagocytosed and destroyed by tissue resident macrophages (Davies and Taylor, 2015).

Exposure to florescent dyes can result in cell death because of dye toxicity (Ge et al.,

175 2013) and its possible that CLIC1+/+ and CLIC1-/- BMDCs have different sensitivities to this effect. To examine whether exposure to fluorescent dyes has any toxic effect on

BMDC, CLIC1+/+ and CLIC1-/- BMDCs that had previously been labelled with fluorescent dyes were examined for 0 min, 24 hrs and 48 hrs using later using 7AAD staining and flow cytometry to identify dead cells.

The result illustrated that after freshly fluorescence dye labelling, identical percentage of CLIC1+/+ BMDCs and CLIC1-/- BMDCs were stained for 7AAD (dead cells) (Figure

28A) Whilst there was increasing percentage of 7ADD stained cells (dead cells) found after 24 hrs (Figure 28B) and 48 hrs (Figure 28C), there was no difference in cell death between CLIC1+/+ BMDCs and CLIC1-/- BMDCs.

In the experimental settings, I have found that in three replicated experiments, there was no significant difference in number of dead cells between CLIC1+/+ and CLIC1-/-

BMDCs after fluorescent dye labelling (Figure 28D; 1.65 ± 0.04 vs 1.63 ± 0.03 for 0 min, 5.41 ± 0.10 vs 5.39 ± 0.11 for 24 hrs, 6.28 ± 0.11, 6.16 ± 0.08 for 48 hrs, n=3/group, p>0.05, unpaired t test) for all time courses. This indicates that fluorescent dye toxicity has a minimum effect on cell viability and CLIC1 gene deletion has indistinguishing effect on cell viability of both CLIC1+/+ BMDCs and CLIC1-/- BMDCs.

176

177

Figure 28 Attenuated migration of CLIC1+/+ BMDCs is not due to cell death. BMDCs were labelled with either 5mM CellTrace Violet or 5mM CFSE for 30 min and washed extensively with PBS, before being incubated with cell complete medium for 0 min, 24 hrs and 48 hrs. Percentage of dead BMDCs determined as 7AAD positive after 0 min (A), 24 hrs (B) and 48 hrs (C) labelling with fluorescent dyes were analyzed by flow cytometer. (D) Average of percentages of CLIC1+/+ and CLIC1-/- BMDCs death across time course were calculated from 3 replicated experiments.

178 3.II.D DISCUSSION

I have demonstrated that, upon maturation, more CLIC1-/- BMDCs migrate from the site of injection in the footpad and to popliteal (Figure 22) and then to inguinal lymph nodes

(Figure 26) than CLIC1+/+BMDCs. This effect is unchanged irrespective of whether the cells are injected into CLIC1+/+ or CLIC1-/- mice. Additionally, this difference in the cell number homing to the lymph node can’t be explained by cell death (Figure 28) and is probably due to more rapid directed migration of matured CLIC1-/- BMDCs.

Several studies have demonstrated that CLICs involve in immune cell migration through three possible mechanisms. First, many reports have demonstrated that outwardly chloride efflux mediated by chloride channels, enable cell shrinkage and therefore adjust cell shape and volume resulted in an increase in microglia (Ducharme et al., 2007) and monocyte (Kim et al., 2004) migration. The results here however cannot be explained by this mechanism because we would expect to see increased cell volume in CLIC1-/- BMDC which should lead to reduced BMDC migration to secondary lymphoid organs.

CLIC interaction with cytoskeleton or scaffolding proteins may influence cytoskeleton rearrangement, important for cell shape changes and therefore facilitate cell movement or migration. DCs must undergo significant shape changes in order to penetrate from lymphatic endothelium to enter lymphatic vessels (Forster et al., 2012). Several line of evidence indicate that cytoskeleton rearrangement is necessary for immune cell movement in lymphatic vessel and the cytoskeleton proteins are crucial for the rearrangement (Frittoli et al., 2011; Kawai et al., 2005; Lammermann et al., 2009). In line with this, several CLICs such as CLIC4, 5 and 6 interact directly with cytoskeleton

179 or scaffolding proteins (Berryman et al., 2004; Berryman and Goldenring, 2003; Griffon et al., 2003). Although there is no direct evidence to suggest that CLIC1 interacts with the cytoskeletal or scaffolding proteins, by analogy it is quite possible that it does.

CLIC1 thus might modify cell movement and migration through interaction with cytoskeleton elements. However, if this was the case, again, our data still it seems likelier that deletion of CLIC1 should reduce DC migration instead of enhance it.

Another possible mechanism by which CLIC1 might influence DC homing to lymph node is through CLIC1’s effect on surface expression of chemokine receptors on DCs.

As DCs mature, they upregulate CC chemokine receptor 7 (CCR7) which can sense its chemoattractant CC motif ligand 21 (CCL21) secreted by lymphatic endothelium cells in lymph nodes and direct DC to T cell zone in lymph nodes (Sanchez-Sanchez et al.,

2006). Because CCR7 expression was not assessed in this study, it is possible that the difference in expression this chemokine receptors between CLIC1+/+ and CLIC1-/-

BMDCs may result in the difference in migration ability. It would therefore be interesting in the future to measure the expression of these chemokine between

CLIC1+/+ and CLIC1-/- BMDCs.

There are two further potential explanations for our results. First, although this difference in the cell number homing to the lymph node can’t be explained by cell death as displayed in vitro experiment (Figure 28), cell death in vivo may occur differently from in vitro, which our study failed to demonstrate. It might be possible that CLIC1+/+

BMDCs are more susceptible to cell death in vivo after injection into the footpad of the mice.

180 Second, because of the fact that CLIC interact with cytoskeleton elements, it might be possible that CLIC1-/- BMDCs adhere less well to endothelium of lymphatic vessel and therefore migrates faster along the vessel wall.

Although this study was designed such that CLIC1+/+ BMDCs and CLIC1-/- BMDCs must compete with each other for cell migration, this study has some limitations. For example, this study did not allow observation of real time movement of BMDCs in vivo.

Therefore, this study can be improved by utilizing a cutting edge technique such as two- photon microscopy to track a real time movement of BMDCs (Miller et al., 2003). This technique would also overcome the possibility of cell death in vivo, which may cause some artifacts.

In conclusion, the results herein demonstrated that more CLIC1-/- BMDCs migrated from the site of injection in the footpad and homed to popliteal and to inguinal lymph nodes than CLIC1+/+BMDCs in both CLIC1+/+ and CLIC1-/- mice. Our results here are do not fit well with our current limited understanding of the biology of CLIC1.

181 3.III CLIC1 LOCALISATION IN DENDRITIC CELLS

3.III.A INTRODUCTION

Previous study from our group shows that, in macrophages, CLIC1 resides in cytoplasmic vesicle like structure in resting condition, but after phagocytosis, CLIC1 rapidly translocates to phagosome membrane. To identify if this can be also be observed in the other phagocytes such as BMDCs, the topic of this thesis, I will determine the subcellular localization of CLIC1 in BMDCs before and shortly after phagocytosis using confocal microscopy.

3.III.B BACKGROUND

Phagocytosis is one of the several ways of antigen uptake by DCs and can be either receptor or non-receptor mediated processes. Two of the most common receptors mediating phagocytosis are Fc receptor and complement receptors (CR1, CR3 and

CR4). It has been demonstrated that complement receptors 3 (CR3) on DCs recognize

C3b ligands on serum opsinized zymosan particles, the particle used in this study. The receptor-ligand interaction activates F actin polymerization and recruitment of several adaptor proteins, which in turn activate several other molecules including small

GTPases such as Rho, Rac1, Rac2 and Cdc42. More detail can be found in section

1.III.C.iv.

In resting macrophages, CLIC1 resides in uncharacterized cytoplasmic vesicle-like structures. Upon phagocytosis, CLIC1 rapidly translocates to the phagosome membrane, where it is co-located with other membrane proteins like the Rho GTPases,

182 Rac and RhoA, as well as NADPH oxidase components (Jiang et al., 2012). CLIC1 subcellular localisation was discussed in more details in section 1.I.E. As DC are also phagocytic cells, it seem possible that CLIC1 will be expressed in DC and the will undergo similar localization changes associated with phagocytosis.

183 3.III.C RESULTS

To determine the subcellular localization of CLIC1 in BMDCs, we have used immunofluorescence confocal microscopy. BMDCs were fixed and stained with an affinity purified sheep polyclonal antibody to murine CLIC1 and a rabbit anti-murine

RhoA, followed by a cy3 and cy2-labelled anti-sheep and anti-rabbit IgG, respectively.

In resting CLIC1+/+ BMDCs, CLIC1 staining was punctate (Figure 29(b)) in a pattern similar to that we have previously described in macrophages (Jiang et al., 2012). There was a similar staining pattern for the ras homolog family member A (RhoA), which did not co-localise with CLIC1 (Figure 29(a-c)). As expected no CLIC1 staining could be identified in CLIC1-/- control cells (Figure 29(g-i)). To determine whether CLIC1 translocates to phagosome membranes, 5 min after they had undergone synchronised phagocytosis of IgG opsonized zymosan particles, we fixed then stained BMDCs. RhoA can appears on the phagosome membrane at 5 min (Figure 29(d, arrow head)), as it is known to do. At the same time point CLIC1 also appears on the phagosome membrane

(Figure 29(e, arrow)) where is does not colocalises with RhoA (Figure 29(f)). As expected, in CLIC1-/- control cells, whilst RhoA staining was present, no CLIC1 antibody staining was detectable (Figure 29(j-l)).

184

Figure 29 Phagocytosis triggers CLIC1 translocation to BMDC phagosome membrane. Immunofluorescence confocal microscopic images of resting CLIC1+/+ BMDCs (a-c) or BMDCs phagocytosing IgG opsonised zymosan particles (d-f), stained with antibodies to RhoA (green) or CLIC1 (red). Images of resting CLIC1-/- BMDCs (g-i) or BMDCs phagocytosing IgG opsonised zymosan particles (j-l), stained with antibodies to RhoA (green) and CLIC1 (red). Both CLIC1 and RhoA appear on phagosomal membrane after 5 min phagocytosis in CLIC1+/+ BMDCs (f, arrows head (RhoA), arrow (CLIC1)) Only RhoA can be identified on phagosome membrane of CLIC1-/- BMDCs (l, arrows). Scale bar: 10 um.

185 3.III.D DISCUSSION

I have found that upon BMDC phagocytosis of an opsonized particle, cytoplasmic

CLIC1 rapidly translocates to the phagosome membrane where it partially colocalises with RhoA, a known phagosome membrane associated protein. Whilst CLIC1 is on the phagosome membrane this study cannot determine if it is an integral or a peripheral membrane protein. However, it is likely to participate in an event after phagocytosis.

Based on previous studies and because its ion channel like properties, it is possible that it might influence phagosome acidification and as a consequence protein degradation

186 3.IV PHAGOSOMAL ACIDIFICATION AND

PROTEOLYSIS

3.IV.A INTRODUCTION

The previous investigation identifies CLIC1 on the phagosome membrane. Therefore, to investigate if CLIC1 gene deletion in CLIC1-/- BMDCs could influence phagosome acidification and as a consequence protein degradation, in this section, I will investigate the role of CLIC1 in phagosomal acidification and proteolysis of BMDCs.

3.IV.B BACKGROUND

Many studies have demonstrated that proteolysis is an indispensable requirement for effective antigen presentation by DCs (Deussing et al., 1998; Hsing and Rudensky,

2005; Shi et al., 1999). This proteolysis is driven by proteases that reside in the phagosomal or endosomal compartments and function optimally in a defined pH environment. The cathepsins, a class of proteases that are comprised of cysteine and aspartyl proteases, are especially important in processing of peptide for presentation by

MHC class II molecules. Most cysteine proteases are unstable and have weak activity at neutral pH and only function optimally in acidic phagosomal compartments. Thus, efficient antigen processing is a highly pH dependent process.

Unlike macrophages and neutrophils, DCs have developed a more tightly regulated mechanism to sustain their phagosomal pH environment so that peptides are not fully degraded. It is widely accepted that acidification and reactive oxygen species (ROS) production are the two key elements in this regulation. Acidification is mainly, but not

187 exclusively, mediated by the vacuolar ATPase (V-ATPase), which translocate protons from the cytosol into the phagosome lumen (Cross and Segal, 2004). Further, immature

DCs have less efficient phagosomal acidification due to limited recruitment of the V-

ATPase to lysosomes, as compared to macrophages or mature DCs (Trombetta et al.,

2003). Another mechanism that mediates acidification in DCs is the production of ROS from NADPH oxidase 2 (NOX2) leading to an enzymatic multiprotein complex. This multiprotein complex requires the early Rab27a dependent recruitment of gp91phox to the phagosomal membrane (Elsen et al., 2004). Rab27a is believed to regulate DC phagosome pH as Rab27a deficient DCs have a delay in the recruitment of NOX2 to the phagosome, resulting in increased phagosomal acidification and antigen degradation, the consequence of which is a defect in antigen presentation (Jancic et al., 2007).

Further evidence for the involvement of NOX2 and ROS production in antigen presentation came from work in Vav-deficient DCs. Vav, a member of the Guanine

Nucleotide Exchange Factor (GEF) family, catalyzes the exchange of bound GDP to

GTP on Rac, another early component of the NOX2 complex (Crespo et al., 1997).

Vav-deficient DCs also showed a decrease in phagosomal pH, an increase in antigen degradation and consequently failed to present antigen efficiently (Jancic et al., 2007;

Rybicka et al., 2010). It is believed that the NOX2 complex in DCs produce low levels of ROS, resulting in sustained alkalization of the phagosomal lumen and consequent inefficient antigen processing (Savina et al., 2006).

Recently, we have discovered that intracellular chloride channel protein 1 (CLIC1) regulates macrophage phagosomal pH (Jiang et al., 2012) and thus may also play a role in pH regulation of similar structures in DCs. CLIC1, a member of the evolutionarily conserved 6 member CLIC family of chloride ion channel proteins, was first cloned

188 because of its expression in activated macrophages (Valenzuela et al., 1997). Its gene is located in the MHC class III region of chromosome 6 (Littler et al., 2004) near the gene for TNF-alpha, suggesting a potential role in regulation of immune and inflammatory responses. All protein members of the CLIC family are relatively small in size with only a single putative transmembrane region (Jiang et al., 2014). They are unusual, as they exist in both soluble cytoplasmic and integral membrane forms (Valenzuela et al.,

1997). CLIC proteins have to undergo a major structural rearrangement to transform from their glutathione-S transferase (GST) like structure in the soluble form to that of an integral membrane protein (Goodchild et al., 2009; Littler et al., 2004).

189 3.IV.C RESULTS

3.IV.C.i ESTABLISHMENT OF ASSAY FOR MEASUREMENT

PHAGOSOMAL ACIDIFICATION

FITC can be used as an indicator to effectively differentiate pH values between about

5.5 and 7.5 (Chen et al., 2008). I first assessed photobleaching or permanent loss of fluorescence intensity that may occur from long-term image acquisition. BMDCs were fed with FITC conjugated zymosan (zFITC) before the cells were permeabilised and fixed. These cells were incubated in a series of buffers from pH 4 to 8. Fluorescent intensity of zFITC was measured every 60 sec for 45 min for each designated pH. I have found that there was no or negligible photobleaching across 45 min of image acquisition (Figure 30A; n=3/group with > 50 zymosan containing BMDCs analysed).

Thus, this suggests that loss of fluorescence intensity of FITC is negligible and the assay is feasible to measure intracellular pH for at least 45 min. I then established a calibration curve for zFITC intensity versus pH. A calibration curve of average of zFITC fluorescence intensity of at each designated pH was constructed using non-linear sigmoidal regression. The conversion of fluorescence intensity to pH was performed via the built in ‘interpolate’ read out function for zFITC. The optimal pH sensitive region for zFITC is indicated by the box area (Figure 30B; n=3/group with > 50 zymosan containing BMDCs analysed). This data is used for determination of pH within phagosomes of BMDCs in later experiments.

190

Figure 30 pH Calibration curve for zFITC. Fluorescence intensity of zFITC was determined from the known pH buffer from pH 4 to 8. (A) Fluorescent intensity of zFITC was measured every 60 sec for 45 min for each designated pH. (B) A calibration curve of average of zFITC fluorescence intensity of at each designated pH was constructed using non-linear sigmoidal regression.

3.IV.C.ii PHAGOSOMES FROM CLIC1-/- BMDCs DISPLAY IMPAIRED

ACIDIFICATION

The localization of CLIC1 to phagosome membranes suggests that it may regulate phagosomal pH in BMDCs. To investigate this, we monitored the process of phagosomal acidification using live cell imaging of CLIC1+/+ and CLIC1-/- BMDCs that had undergone synchronized phagocytosis of IgG opsonised zymosan particles labelled with the pH sensitive dye FITC (zFITC) (Jiang et al., 2012).

After synchronised phagocytosis, the phagosome of CLIC1+/+ and CLIC1-/- BMDCs slowly acidified (Figure 31A) with consequent decrease in FITC fluorescence of the phagocytosed particle. The rate of decrease in phagosomal pH of CLIC1+/+ and CLIC1-/-

BMDCs started to diverge at about 7 min after phagocytosis. From 7-14 min, the

CLIC1-/- phagosomal pH clearly dropped more slowly than that of CLIC1+/+ BMDC

191 phagosomes. Between 15 and 30 min, the phagosomal pH reached a steady state and over this period, the average phagosomal pH of the CLIC1-/- cells was higher than that of CLIC1+/+ cells (Figure 31B, n=6/group with 10-15 zymosan containing phagosomes analysed per experiment; p=0.02, two-way Repeated-Measures ANOVA). These results show that phagosomes from CLIC1-/- BMDCs have impaired acidification.

Figure 31 CLIC1-/- BMDCs display impaired phagosomal acidification. Live BMDCs that had undergone synchronised phagocytosis of IgG opsonised zymosan particle covalently couple with a pH sensitive fluoresce probe, in the presence or absence of IAA94 (100 µM) were monitored continuously for 30 min using an inverted Zeiss Axiovert 200 M microscope. (A) The phagosomal pH of CLIC1-/- BMDCs was higher than that of CLIC1+/+ BMDCs over the 30 min time course. (B) IAA94 treatment had no effect on the steady state phagosomal pH of CLIC1-/- BMDCs, but impaired the acidification of CLIC1+/+ BMDC phagosomes to the same level as that of CLIC1-/- BMDCs. Data represents mean ± SEM analysed using two-way Repeated-Measures ANOVA or paired t-test respectively.

192 3.IV.C.iii THE CLIC1 ION CHANNEL BLOCKER IAA94 RAISES THE pH OF

CLIC1+/+ BUT NOT CLIC1-/- BMDC PHAGOSOMES

CLIC1 gene deletion in BMDC leads to impaired phagosome acidification. To help further verify that the impaired acidification was directly due to CLIC1 gene deletion, we treated both CLIC1+/+ and CLIC1-/- BMDCs with IAA94, a small molecule blocker of the CLIC family of ion channels, then monitored phagosomal pH as described above.

The average steady state pH of IAA94 treated CLIC1-/- BMDCs, calculated based on the average pH between 15-30 min after synchronized phagocytosis, did not differ significantly from untreated CLIC1-/- BMDCs (Figure 31B; pH 6.02 ± 0.05 vs 5.97 ±

0.01, n=3/group with 10-15 zymosan containing BMDCs analysed per experiment; p=0.564, paired t-test). However, IAA94 treatment of CLIC1+/+ BMDCs significantly raised their average phagosomal pH from 5.63 ± 0.07 to 6.02 ± 0.11 (Figure 31B; n=3/group with 10-15 zymosan containing BMDCs analysed per experiment; p=0.03, paired t-test). Additionally, the pH of these IAA94 treated CLIC1+/+ BMDCs was not different from that of CLIC-/- BMDCs (Figure 31B; pH 5.99 ± 0.03 vs 6.02 ± 0.05, n=3/group with 10-15 zymosan containing BMDCs analysed per experiment p=0.648, paired t-test). These data indicate that the altered phagosomal pH of CLIC1-/- BMDCs is likely to be a direct consequence of gene deletion, and that in our experimental system, the pH effect of IAA94 is due its specific blockade of CLIC1.

3.IV.C.iv CLIC1-/- BMDCs DISPLAY IMPAIRED PHAGOSOMAL

PROTEOLYSIS

Whilst the difference in phagosomal pH between CLIC1+/+ and CLIC1-/- BMDCs is modest, this difference may impact on highly pH dependent processes such as proteolysis. To directly examine this hypothesis, we used live cell imaging to monitor

193 real time proteolysis in BMDC that had engulfed 3 µm silica beads (Jiang et al., 2012;

Yates and Russell, 2008), to which had been coupled Alex Fluor 594 as a reference dye and DQ bodipy BSA as a substrate. The latter becomes more fluorescent as its self- quenching is reduced by proteolysis (Santambrogio et al., 1999). Loosely adhered

CLIC1+/+ and CLIC1-/- BMDCs underwent synchronized phagocytosis with the labelled silica beads, which were then monitored by live cell imagining for 60 min. The graph of the change in the fluorescence signal clearly indicates that CLIC1+/+ BMDCs proteolyse

BSA much faster than CLIC1-/- BMDCs (Figure 32; n=6/group with 15-20 silica bead containing BMDCs analysed per experiment, p=0.005, two-way Repeated-Measures

ANOVA). These rate differences indicate that CLIC1 mediated alteration in phagosomal acidification is also associated with impaired proteolysis in BMDC phagosomes. As proteolysis is a key step in DC antigen presentation, this may have consequences for T-cell activation.

Figure 32 CLIC1-/- BMDCs display impaired phagosomal proteolysis. Live BMDCs that had undergone synchronised phagocytosis of 3 um silica beads, covalently coupled with DQ-bodipy BSA and Alexa Fluor-594, were continuously monitored for 60 min using an inverted Zeiss Axiovert 200 M microscope. The time course of proteolytic activity within the phagosome, measured as gain of fluorescence, showed that CLIC1+/+ BMDCs were more efficient in BSA proteolysis than CLIC1-/- BMDCs. Data represents mean ± SEM analysed using two-way Repeated-Measures ANOVA.

194 3.IV.D DISCUSSION

I have found that phagosomes of CLIC1-/- BMDCs display impaired phagosomal acidification (Figure 31A) and proteolysis (Figure 32). Consistent with this, the CLIC ion channel blocker IAA94, limited phagosomal acidification in CLIC1+/+ BMDCs, but had no effect on those functions in CLIC1-/- BMDCs (Figure 31B), suggesting that

IAA94’s actions are specific for CLIC1. Lack of CLIC1 mediated chloride influx would be expected to lead to higher pH values.

Phagocytosis results in progressive phagosomal acidification, an important component of which is V-ATPase proton pump H+ influx (Feske et al., 2015). As part of this process chloride ions are needed for charge compensation and many lines of evidence from several groups suggests that CLIC proteins can behave as ion channels (Littler et al., 2010b). The first mechanism that CLIC1 might influence phagosomal pH is through its formation of chloride ion channels. Several studies have also demonstrated that oxidation and acidification, which occurs in the phagosome, facilitates CLIC1 transition from the soluble to the integral membrane forms (Fanucchi et al., 2008; Goodchild et al., 2009; Warton et al., 2002) with Cy24-Val46 forming a transmembrane domain

(Goodchild et al., 2011). Further, under oxidizing conditions CLIC1 undergoes a major structural rearrangement (Littler et al., 2004) and in the presence of membranes with cholesterol, monomeric CLIC1 also oligomerized to form a pore comprising 6-8 subunits (Goodchild et al., 2011; Valenzuela et al., 2013). These all suggest that after insertion into a membrane, CLIC1 can form ion channel pores and behave as a chloride ion channel. However, whether CLICs are ion channels is controversial, to a large extent because structural studies of soluble CLICs do not resemble any conventional ion channels but belong to the GST fold superfamily of proteins (Cromer et al., 2007b;

195 Harrop et al., 2001; Littler et al., 2005; Littler et al., 2010a) and display glutaredoxin- like glutathione-dependent oxidoreductase enzymatic activity (Al Khamici et al., 2015).

Thus CLICs must undergo major conformation changes after they insert into the membrane to form ion channels. Whilst there is evidence that CLIC1 can undergo such a structural change under oxidizing conditions (Goodchild et al., 2009; Littler et al.,

2004), to date, there are no high resolution structures of the membrane form of CLICs that provide evidence as to how they are able to conduct chloride ions.

Another possible mechanism by which CLIC1 might influence phagosomal pH is by its interaction with elements of the cytoskeleton, a subject that has previously been discussed in detail (Jiang et al., 2014). CLIC1 binds to actin and other cytoskeleton associated proteins. This binding could either modulate the ion channel activity of the

CLICs or indirectly modify the behaviour of other ion channels. Actin plays a significant role in many cellular functions, one of which is in ion channel regulation

(Ahmed et al., 2000; Gourlay and Ayscough, 2005; Mazzochi et al., 2006). For example, actin polymerisation modulates the neuronal Ca2+ influx through voltage dependent Ca2+ channels (Furukawa et al., 1997; Rosenmund and Westbrook, 1993).

Additionally, the classical chloride ion channel proteins ClC2 and ClC3 directly bind to

F-actin, which modulates their ion channel activity (Ahmed et al., 2000; McCloskey et al., 2007). Actin or actin binding protein has also been reported to interact with CLICs.

For example, CLIC5 interacts directly with the actin-rich component of placenta microvilli (Berryman and Bretscher, 2000) and in rat brain CLIC4 interacts with actin cytoskeleton through dynamin (Suginta et al., 2001). Moreover, the ion channel activities formed by reconstitution of recombinant CLIC5 and CLIC1 in planar bilayer are reversibly inhibited by binding of their C terminal domain to F-actin, an effect that

196 is reversed by F-actin depolymerisation (Singh et al., 2007). This F-actin regulation was specific for CLIC5 and CLIC1 and did not extend to the very similar protein CLIC4.

This implies for the role the C-terminus of at least some of CLICs in ion channel regulation (Singh et al., 2007). It is thus plausible that direct or indirect interaction of

CLIC1 with actin cytoskeleton may exert some regulatory effect on chloride conductance.

CLIC1 might also alter phagosomal pH if it played a role in the phagosome-lysosome fusion, which occurs during phagosome maturation. After phagocytosis, phagosomes progressively acidify in parallel with their maturation and fuse with other acidic organelles, thereby gaining additional membrane and soluble constituents. The fusion process often requires movement of the phagosomes along microtubules where they can fuse with lysosomes, a process requiring actin assembly at the phagosomal membrane

(Blocker et al., 1997). Ezrin, radixin and moesin (ERM) proteins provide a linkage between integral membrane proteins and the actin cytoskeleton (Ivetic and Ridley,

2004) and are downstream effectors of small GTPases (Fehon et al., 2010), which play a part in phagosome-lysosome fusion (Defacque et al., 2000; Erwig et al., 2006; Marion et al., 2011). Whilst there is no direct evidence for CLIC1 in phagosome-lysosome fusion, there are reports that several CLICs interact with ERM. CLIC5 has been purified from placenta microvilli using affinity chromatography with immobilised Ezrin

(Berryman and Bretscher, 2000) and in glomerular podocytes, CLIC5A co-localises and can be co-immunoprecipitated with ERM (Pierchala et al., 2010). CLIC4 is also found alongside Ezrin in apical microvilli of retinal pigment epithelium (Chuang et al., 2010).

Perhaps the most direct evidences supporting CLIC’s role in phagosome-lysosome fusion comes from studies, showing that shortly after macrophage phagocytosis, CLIC3

197 couples to cytoplasmic domain of a C3b transmembrane receptor (CRIg) on phagosomal membranes which increases chloride conductance into the phagosome lumen, and phagosome-lysosome fusion (Kim et al., 2013). Similarly, in cancer cells,

CLIC3 in the late endosome/lysosome compartment works with Rab25 to facilitate recycling of fibronectin binding integrins from late endosome/lysosome to plasma membrane (Dozynkiewicz et al., 2012). Further, in cancer cells CLIC3 plays a role in directing matrix metalloproteinases (MMP) to the late endosome/lysosome compartments independently of Rab25 (Macpherson et al., 2014). Whilst this evidence suggests possible a roles for CLICs in phagosome maturation, using sensitive methods, we have been unable to demonstrate alteration in phagosome-lysosome fusion in

CLIC1-/- macrophages, that as in BMDCs, also display impaired acidification (Jiang et al., 2012).

The study on the effect of drug IAA94 on BMDC phagosomal pH has limitations. The best way of demonstrating that the effect was directly due to CLIC1 would be to transfect the CLIC1-/- BMCDs with CLIC1 to rescue the acidification defect in the

CLIC1-/- BMCDs. This was not done because of time constraints and the well known difficulty of transfecting BMDCs.

Whilst the mechanism of CLIC1 action has not been completely resolved, our studies indicate that phagosomes of CLIC1-/- BMDCs, like macrophages (Jiang et al., 2012), display impaired acidification and as a consequence, impaired proteolysis.

198 3.V ANTIGEN PRESENTATION

3.V.A INTRODUCTION

To identify if an impaired phagosomal acidification and proteolysis in CLIC1-/- BMDCs have any effect on antigen presentation, I will investigate in vitro and in vivo antigen presentation in BMDCs using T cell activation assay and experimental autoimmune encephalomyelitis (EAE) animal model respectively.

3.V.B BACKGROUND

Antigen presentation is a multiple step processes by which antigen presenting cells

(APCs), including macrophages and dendritic cells (DCs) ingest, process and present exogenous antigens, in a complex with MHC class II molecules, to T-cells. APCs first internalize antigen via either endocytosis or phagocytosis, then undertake at least two distinct proteolytic steps. For presentation to CD4 T-cells, there is proteolysis of the antigen and processing of MHC-bound invariant chain (li) to form the class II associated invariant chain peptide (CLIP). If uptake is via phagocytosis, proteolysis of the antigen is initiated by endopeptidases, to fragment the native protein. This is followed by sequential trimming of the peptide ends by amino and carboxypeptidases.

This helps to generate small peptides that have the required lengths of 18-20 amino acids (Blum and Cresswell, 1988; Deussing et al., 1998) to sit in the antigen binding groove on MHC class II molecules. Simultaneously, the invariant chain of MHC II undergoes selective proteolytic cleavage of li, which occupies the antigen binding groove. This cleavage event allows for exogenous peptide loading and formation of the

199 MHC II-peptide complex (Busch et al., 2005; Cresswell, 1996), which is then transported to the plasma membrane of APCs.

The major professional APCs are DCs. For DCs to be able to present antigen effectively, following antigen ingestion, they must next undergo maturation (Chow et al., 2002). This can be triggered by activation stimuli from substances including conserved components of microorganisms or pathogen-associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). PAMPs are recognized by pattern recognition receptors (PRRs) of the innate immune system such as Toll like receptors (TLRs). Activation of PRR signalling in DCs results in inhibition of further endocytosis or phagocytosis of antigens, upregulation of expression of MHCII molecules and their transport to the cell surface and upregulation of expression of cytokines and costimulatory molecules such as CD40, CD80 and CD86 that are necessary for effective T-cell activation (Trombetta and Mellman, 2005).

Activation and clonal expansion of naïve CD4+ T-cells by DCs involves binding of the

T-cell receptors (TCR) to MHC II bound antigen on DCs, coupled with binding of the

DC expressed costimulatory molecule B7 (CD80/86) to CD28 on T-cells (Huppa and

Davis, 2003). In addition, signals provided by distinct cytokines program naïve CD4+ T- cells into different T helper (TH) subsets including Th1 cells. Activated TH1 cells express high levels of intracellular interferon gamma (IFNγ) which is commonly used for flow cytometric detection of T-cell activation, because it is rapidly induced (16 hrs) after encounter with antigen presented by DCs (Mosmann et al., 2005).

200 Experimental autoimmune encephalomyelitis (EAE) is an established model of multiple sclerosis in mice, which has been used widely in laboratory. EAE is triggered by peripheral immunisation of Myelin Oligodendrocyte Glycoprotein (MOG) antigen that actively induce autoreactive T cells. These autoreactive T cells are collected in lymph node and spleen before migrating to central nervous system (CNS) where they recognise their cognate antigens on local antigen presenting cells such as dendritic cells and become activated. Once activated, these autoreactive T cells initiate an inflammatory cascade leading to tissue injury, resulting in inflammation, demyelination, axonal loss and gliosis (Figure 33).

Figure 33 Immunopathogenesis of EAE EAE is mediated by myelin-specific T cells, which are activated in the periphery and translocates into CNS followed by permeabilisation of the blood brain barrier (BBB). These T cells are reactivated by local and infiltrating activated antigen presenting cells (DCs), which present MHC class II associated peptides, resulting in subsequent inflammatory process and eventually in demyelisation and axonal damage.

201 3.V.C RESULTS

3.V.C.i CLIC1-/- BMDC ACTIVATE LESS T-CELLS WHEN A LARGE

PEPTIDE IS USED AS ANTIGEN

CLIC1 gene deletion attenuates both DC phagosomal acidification and proteolysis, one consequence of which may be altered DC mediated T-cell activation. To investigate this, we examined the capacity of BMDCs to present myelin oligodendrocyte glycoprotein (MOG) antigen to CD4+ T-cells from 2D2 mice which express a transgenic MOG35-55 peptide specific TCR (Bettelli et al., 2003). However, 2D2 mice are on a C57BL/6 background and whilst CLIC1-/- and control CLIC1+/+ mice are on a

129X1/SVJ background. Further, as the gene for CLIC1 is in the MHC class III region

(Lehner et al., 2004), these mice could not be backcrossed to alter their genetic background to that of C57BL/6. However, C57BL6 and 129X1/SVJ have the same

MHCII I-Ab/I-Enull haplotype, which suggests that they may be compatible for our in vitro antigen presentation studies. To ensure that, under the conditions of our experiments, no unwanted reactivity was directed by or to 129X1/SVJ cells, we performed a cell mixing experiment. CD4+ T-cells were purified from 2D2 mouse spleens using magnetic beads coated with monoclonal antibody to CD4 and were incubated then with BMDCs from 129X1/SVJ or C57BL/6 mice at a ratio of 1:2

BMDC: T-cell for 16 hrs, with Golgi stop being added for the final 4 hrs. Activated T- cells, were identified by flow cytometry as CD3+CD4+CD45hiVß11+ cells which also stained for intracellular INFγ (Figure 34).

202 250K 250K 105 200K 200K 104 94.5 150K 150K 103 CD45 100K FSC-A 100K SSC-A

2 50K 62.1 50K 10 99.9 0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC-A FSC-W FSC-A

5 10 105 105

4 10 104 104 73.9 96.2 3 0.04 10 103 103 CD4 Vb11

IFNgamma 2 10 102 102 0 0 0

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD3 CD3 CD3

Figure 34 Gating strategy. Coculture of MOG antigen pulsed BMDCs with MOG specific 2D2 CD4+ T cells were stained and then gated to identify activated IFNγ+ MOG specific 2D2 CD4+ T cells.

When 129X1/SVJ BMDCs were mixed with 2D2 T-cells, in the absence of antigen, we could identify no difference in T-cell activation from that of C57BL/6 BMDCs (1.43 ±

0.25 vs 1.47 ± 0.34, n=3/group, unpaired t test, p= 0.69; Figure 35C). To further confirm there are no unwanted responses, T-cells were also labelled with antibody to

CD25/CD69 T- cell activation markers (Figure 35B). Consistent with INFγ responses, in absence of antigen, there was no evidence of T-cell activation in 129X1/SVJ and

C57BL/6 MLR (3.77 ± 0.33 vs 4.27 ± 0.20, n=3/group, unpaired t test, p= 0.27; Figure

35D). These results exclude any artefact from alloreactivity between 129X1/SVJ

BMDC and C57BL/6 2D2 T-cells in our assay format.

203

Figure 35 129X1/SVJ and C57BL/6 cell mixing experiments. BMDCs from 129X1/SVJ or C57BL/6 mice were mixed with purified CD4+ T cells from 2D2 C57BL/6 background mice in 1:2 ratio without antigen for 16 hrs, after which the proportion of activated (A, C) intracellular IFNγ containing and (B, D) surface CD25/CD69 expressing CD4+ T cells were identified by flow cytometry. Data represents mean ± SEM analysed using the unpaired t-test.

To examine the effect of CLIC1 on antigen processing and presentation, CLIC1-/- or

CLIC1+/+ BMDCs were to aliquot into a 96 well plate to which was then added 1.25 pmoles/well of the 21 amino acid MOG peptide MOG35-55 or 1.25 pmoles/well of full length recombinant MOG1-125 peptide. After incubation of the peptides with BMDCs for various time periods, the cells were washed after which BMDCs were matured and antigen processing stopped by the addition of LPS 0.1 ug/ml for 4 hrs. MOG -specific

2D2 T-cells (2 x105/well) were then added for a further 16 hrs during which the last 4

204 hrs of incubation were in the presence of Golgi stop (1 ug/ml). Activated transgenic T- cells, was then determined by staining for intracellular IFNγ+ and analysis using multiparameter flow cytometry (Figure 36).

Figure 36 CLIC1-/- and CLIC1+/+ BMDCs differentially activate CD4+ T cells depending on whether they are pulsed with MOG35-55 or MOG1-125 peptide. Representative dot plots of the time course of the proportion of activated MOG specific 2D2 CD4+ T cells, determined by intracellular expression of IFNγ after 16 hrs coculture with (A) CLIC1+/+ or (B) CLIC1-/- BMDCs that had been previously pulsed with 1.25 +/+ -/- pmoles of the short MOG35-55 peptide, or with (C) CLIC1 or (D) CLIC1 BMDCs that had been previously pulsed with 1.25 pmoles of the full length MOG1-125 peptide.

205 When MOG35-55 peptide, that requires no processing to be presented via MHCII was used as an antigen, CLIC1-/- and CLIC1+/+ BMDCs activated similar proportions of T- cells at all time points (Figure 37A; n=6/group with triplicate samples per time point in each experiment; p=0.21; two-way Repeated-Measures ANOVA). However, when

BMDCs were pulsed with MOG1-125, that does require processing for presentation,

CLIC1-/- BMDCs activated significantly less T- cells at all time points than CLIC1+/+

BMDCs (Figure 37B; n=6/group with triplicate samples per time point in each experiment; p=0.0001, two-way Repeated-Measures ANOVA). This indicated that if antigen processing is required, CLIC1-/- BMDCs present antigen less well than CLIC1+/+

BMDCs and suggested a potential role for CLIC1 in regulating antigen processing and presentation, which may be mediated, at least in part, by modulation of pH and proteolysis.

Figure 37 Antigen pulsed CLIC1-/- BMDCs have a reduced capacity to activate CD4+ T- cells. The proportion of activated (intracellular IFNγ containing) MOG specific 2D2 CD4+ T- cells after 16 hrs coculture with BMDCs pulsed with (A) 1.25 pmoles of the short MOG35-55peptide or (B) with equimolar full length MOG1-125. The fold increase in activated T-cells was calculated relatively to the no antigen control. Data, representing mean ± SEM, were analysed using two-way Repeated-Measures ANOVA.

206 3.V.C.ii THE ANTIGEN PROCESSING INHIBITOR CHLOROQUINE

REDUCES T-CELL ACTIVATION OF A LARGE PEPTIDE ANTIGEN IN

CLIC1+/+ BMDC

Efficient activation of T-cells by large peptide antigens requires both DC antigen processing and presentation to T-cells. To further differentiate these two interdependent processes, we used chloroquine, which inhibits antigen processing by raising the phagosome pH whilst still preserving antigen presentation (Lewinsohn et al., 1998).

Using essentially the same experimental procedure as above, CLIC1-/- or CLIC1+/+

BMDCs were preincubated with 100 ul of culture medium containing 100 mM chloroquine. After 1 hr, the 21 amino acid MOG35-55 peptide or full length recombinant

MOG1-125 were added to the culture for various time periods after which the cells were matured, 2D2 T-cells were added and the cells stained with the same antibody panel as above, to assess activation of MOG reactive 2D2 T-cells.

When BMDCs were incubated with MOG35-55, which requires little or no processing for efficient antigen presentation, a similar fold increase in activated T-cells were found in

CLIC1-/- and CLIC1+/+BMDCs, independent of chloroquine treatment (Figure 38A; n=3/group with triplicate samples per time point in each experiment, p=0.15, two-way

Repeated-Measures ANOVA). However, when BMDCs were incubated with full length

MOG1-125 peptide, which requires antigen processing for effective antigen presentation, the proportion of activated T-cells was very low and similar after incubation with vehicle or chloroquine treated CLIC1-/- BMDC (Figure 38B; n=3/group with triplicate samples per time point in each experiment, p=0.069, two-way Repeated-Measures

ANOVA). Further, antigen presentation by chloroquine treated CLIC1+/+ BMDCs was similar to that of the CLIC1-/- BMDC but was much lower than that of vehicle treated

207 CLIC1+/+ BMDCs (Figure 38B; n=3/group with triplicate samples per time point in each experiment, p=0.001, two-way Repeated-Measures ANOVA). These data further support the hypothesis that CLIC1 is acting to modify antigen processing and therefore reducing the substrate for antigen presentation.

+ Figure 38 Chloroquine reduces CD4 T-cell activation by MOG1-125pulsed CLIC1+/+ but not CLIC1-/- BMDCs. The proportion of activated (intracellular IFNγ containing) MOG specific 2D2 CD4+ T- cells after 16 hrs coculture with chloroquine (100 µM) treated or untreated BMDCs pulsed with (A) 1.25 pmoles of the short MOG35-55 peptide or with (B) equimolar full length MOG1-125. Fold increase in activated T-cells was calculated relative to the no antigen control. Data, representing mean ± SEM, were analysed using two-way Repeated-Measures ANOVA.

3.V.C.iii THE CLIC1 ION CHANNEL BLOCKER IAA94 DIMINISHES T-

CELL ACTIVATION IN CLIC1+/+ BUT NOT CLIC1-/- BMDCs

To further confirm that CLIC1 gene deletion directly caused the defect in antigen processing, we examined the effect of the CLIC1 chloride ion channel blocker IAA94

(Kim et al., 2004; Pope et al., 1991) on antigen process and presentation. Using essentially the same experimental procedure as above, CLIC1-/- or CLIC1+/+ BMDCs were preincubated in culture medium containing vehicle or 100 mM of IAA94. After 1 hr, MOG35-55 or MOG1-125 was added to the culture for 4 hrs after which the cells were 208 matured, 2D2 T-cells were then added and 16 hrs later, and the cells were stained with the same antibody panel as above, for flow cytometric evaluation of 2D2 T-cell activation.

Similar to chloroquine, IAA94 did not modify T-cell activation following MOG35-55 presentation by either CLIC1+/+ or CLIC1-/- BMDCs (Figure 39A; n=3/group with triplicate samples per time point in each experiment; p=0.367, two-way Repeated-

Measures ANOVA). In contrast, when presenting MOG1-125, T-cell activation was reduced when CLIC1+/+ BMDCs were treated with IAA94 (Figure 39B; n=3/group with triplicate samples per time point in each experiment; p=0.003, two-way Repeated-

Measures ANOVA). IAA94 treatment of CLIC1+/+ BMDCs reduced T-cell activation to the same level as vehicle treated CLIC1-/- BMDCs (Figure 39B; n=3/group with triplicate samples per time point in each experiment; p=0.282, two-way Repeated-

-/- Measures ANOVA). As expected, IAA94 treatment of MOG1-125 pulsed CLIC1

BMDCs did not alter their capacity to activate T-cells. These data indicate that, by acting specifically on CLIC1, the ion channel blocker IAA94 acts to reduce DC mediated T-cell activation to MOG1-125 which requires processing but not to MOG35-55 which requires no processing.

209

+ +/+ Figure 39 IAA94 reduces CD4 T-cell activation by MOG1-125 pulsed CLIC1 but not CLIC1-/- BMDCs. The proportion of activated (intracellular IFNγ containing) MOG specific 2D2 CD4+ T- cells after 16 hrs coculture with IAA94 (100 µM) treated or untreated BMDCs pulsed with (A) 1.25 pmoles of the short MOG35-55 peptide or with (B) equimolar full length MOG1-125. Fold increase in activated T-cells was calculated relatively to the no antigen control. Data representing mean ± SEM were analysed using two-way Repeated- Measures ANOVA.

3.V.C.iv REDUCED T-CELL ACTIVATION BY CLIC1-/- BMDCs IS NOT

DUE TO ALTERED EXPRESSION OF COSTIMULATORY MOLECULES

DC maturation is critical for effective antigen presentation to T-cells, in part because it results in an expression of important costimulatory molecules (De Smedt et al., 1996;

Michelsen et al., 2001). To determine if the reduced T-cell activation found on CLIC1-/-

BMDCs was due to decreases in costimulatory molecule expression, we assessed

BMDCs expression of CD40, CD80 and CD86. We also investigated BMDC expression of MHC class II, which is essential for antigen presentation to T-cells. CLIC1-/- and

CLIC1+/+ BMDCs (1x105/well) were dispensed into a 96 well plates and then incubated for 4 hrs with LPS at a concentration of either 0.1, 0.0001 or 0.00001 ug/ml. BMDCs were then washed and stained with antibodies to CD45, CD3, CD11c, CD40, CD80,

210 CD86 and MHC class II. Although expression of these markers was significantly increased with increasing LPS concentration, there was no significant difference in expression of any of these surface markers between CLIC1+/+ and CLIC1-/- BMDCs at any LPS concentration (Figure 40A, B, C, D). This indicates that deletion of CLIC1 gene has no effect on DC expression of CD40, CD80, CD86 and MHC class II and is consistent with the notion that the primary defect is antigen processing, rather than antigen presentation.

Figure 40 CLIC1 has no effect on LPS induced BMDC activation cell surface molecules. The percentage of BMDCs expressing cell surface MHCII or costimulatory molecules after 4 hrs incubation with escalating doses of LPS was measured using flow cytometer. There was no difference between CLIC1+/+ and CLIC1-/- BMDCs in proportion of BMDCs expressing CD40 (A), CD86 (B), CD80 (C) or MHC class II molecules (D) for any LPS dose. Data representing mean ± SEM were analysed using unpaired t-tests.

211 -/- 3.V.C.v CLIC1 BMDC PRESENTING MOG1-125 INDUCE LESS EAE

DISEASE

Our data strongly indicates that CLIC1 deletion has an effect on BMDC processing of peptide, resulting in a reduced in vitro capacity to activate CD4+ T-cells. To determine whether these changes could also be demonstrated in vivo, we studied murine experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (Constantinescu et al., 2011). We induced disease in groups of 6 CLIC1+/+ and

6 CLIC1-/- age and sex matched mice with CLIC1+/+ or CLIC1-/- BMDCs which had been pulsed with MOG1-125 and then matured with LPS. The cells were injected subcutaneously (s.c.) into both flanks of CLIC1+/+ and CLIC1-/- mice. One and 3 days later, the animals were also injected with 200ng of pertussis toxin, which is part of the usual protocol for MOG vaccination induced EAE (Constantinescu et al., 2011). This microbial product is thought to promote EAE development by facilitating the migration of pathogenic T-cells to the CNS (Hofstetter et al., 2002). The mice were observed daily, in a blinded manner, and disease scores were assigned based on a widely used clinical scoring scale (Constantinescu et al., 2011) ranging from 1 for very mild disease

(flaccid tail) to 5 for complete paralysis.

-/- From days 9-17, in the disease development phase of EAE, MOG1-125 pulsed CLIC1

-/- +/+ BMDCs in CLIC1 mice elicited less severe EAE than MOG1-125 pulsed CLIC1

BMDCs in CLIC1+/+ mice (Figure 41A; n=6/group, p=0.003, two-way Repeated-

+/+ -/- Measures ANOVA). Immunisation of CLIC1 mice with MOG1-125 pulsed CLIC1 or

CLIC1+/+ BMDCs lead to essentially identical EAE disease severity (Figure 41B; n=6/group, p=0.222, two-way Repeated-Measures ANOVA). However CLIC1-/- mice

-/- +/+ immunised with MOG1-125 pulsed CLIC1 BMDCs compared to CLIC1 BMDCs, in

212 the disease development phase, there was a trend towards a less severe EAE disease that fell just short of statistical significance (Figure 41C; n=6/group, p=0.064, two-way

Repeated-Measures ANOVA). Overall, these data indicate that CLIC1-/- mice have milder EAE disease, and that there is likely to be a reduced capacity of CLIC1-/-

BMDCs to elicit the initial local response, before secondary amplification of the immune response occurs at more distal sites (see discussion section 3.V.D for further explanation).

213

Figure 41 EAE disease clinical scores of mice immunised with MOG1-125 pulsed BMDCs. EAE disease development, measured as clinical scores, was blindly determined in mice immunised with full length MOG1-125 pulsed BMDCs. EAE disease development was -/- +/+ -/- compared in (A) CLIC1 and CLIC1 mice immunised with MOG1-125 pulsed CLIC1 +/+ +/+ and CLIC1 BMDC respectively or (B) CLIC1 mice immunised with MOG1-125 -/- +/+ -/- pulsed CLIC1 or CLIC1 BMDC or (C) CLIC1 mice immunised with MOG1-125 pulsed CLIC1-/- or CLIC1+/+ BMDC. For analysis purposes, the clinical scores have been separated into disease development stage (days 0 to 17) and recovery phase (days 18 to 25) Data, representing mean ± SEM scores, were analysed using two-way Repeated-Measures ANOVA.

214 3.V.D DISCUSSION

I have found that CLIC1-/- BMDCs has attenuated antigen presentation of a large 125 amino acid MOG peptide (Figure 37B), which requires processing, whilst having little effect on a small 20 amino acid MOG peptide that does not require processing (Figure

37A). When I utilised a modified EAE model in which standard immunisation is replaced by injection of mice with large 125 amino acid MOG antigen pulsed

BMDCs, I have found that CLIC1-/- mice have milder EAE disease (Figure 41). These all suggested that antigen presentation are impaired in CLIC1-/- BMDCs due to its attenuated antigen processing.

To study antigen presentation in vivo, we have modified the EAE model by replacing standard immunisation with injection of mice with antigen pulsed BMDCs. The generation of EAE involves initial antigen presentation in the regional lymph nodes followed by systemic amplification of this immune response in the spleen.

Subsequently, there is antigen presentation in the cervical lymph nodes prior to T-cell entry in the CNS (Mohammad et al., 2014). When EAE is induced with CLIC1-/- antigen pulsed BMDCs in CLIC-/- mice there is complete absence of CLIC1 and disease is reduced as expected, compared with the same situation where CLIC1 is replete (Figure 41A). However, when the same CLIC1-/- BMDCs are used to induce

EAE in CLIC1+/+ mice, the situation is more complex. While the initial immune response is likely to be attenuated by CLIC1-/- BMDCs, antigen presenting cells in the regional lymph nodes, spleen and cervical lymph nodes have intact CLIC1 and would be expected to present antigen competently, diluting the effect of the initial attenuated immune response. In this pathogenic sequence, it might be expected that normal antigen presentation in the spleen and cervical lymph nodes might lead to equivalent

215 responses in CLIC1+/+ mice, as we have demonstrated (Figure 41B). However, when

CLIC1+/+ BMDCs were used to induce disease in CLIC1-/- mice, the initial immune response would not be expected to be amplified. Further, differential initial immune responses elicited by CLIC1-/- and CLIC1+/+ BMDCs would be expected to be maintained through the impaired amplification process resulting in differences in disease. Indeed we found that CLIC1-/- mice injected with CLIC1-/- BMDCs developed less EAE than those injected with CLIC1+/+ BMDCs, but this just failed to reach significance (Figure 41C; p=0.064). This is likely to be due to a number of factors. The magnitude of disease is less in CLIC1-/- mice presumably because of the impaired antigen presentation of key myelomoncytic cell subtypes in these mice, including macrophages and DC, which both contribute significantly to disease (de

Vos et al., 2002; Rawji and Yong, 2013). This attenuated disease is likely to significantly reduce the power of our experimental design to detect small differences in disease. Nevertheless, in our experimental paradigm there was an almost significant reduction in EAE between CLIC1 deficient and replete BMDC induced disease

(Figure 41C), suggesting the likelihood of reduced in vivo antigen presentation by

CLIC1-/- BMDCs.

The reduced antigen presentation by CLIC1-/- BMDCs could be explained by three plausible mechanisms including an impaired in antigen processing, a reduced MHC class II invariant chain (Ii) processing and an altered trafficking of vesicles containing antigen bound MHC class II complexes (MHCII-p).

First, whilst the mechanism of CLIC1 action has not been completely resolved, our studies indicate that phagosomes of CLIC1-/- BMDCs, like macrophages (Jiang et al.,

216 2012), display impaired acidification and as a consequence, impaired proteolysis. In

DCs, important proteolytic enzymes such as cathepsin proteases and γ interferon inducible lysosomal thiolreductase (GILT) have actions that are tightly regulated by local pH (Watts, 2012). Therefore, it is highly likely that the impaired antigen presentation by CLIC1-/- BMDCs could be directly due to the attenuated processing of antigen itself.

Apart from the processing of antigen itself, the impaired phagosomal acidification found in CLIC1-/- BMDCs could influence other steps in antigen presentation.

Phagosomal proteases are also critical for processing the MHC class II invariant chain

(Ii) and phagocytosed antigen (Hsing and Rudensky, 2005). However, because

CLIC1-/- BMDCs can efficiently induce T-cell activation of a short 21 amino acid

MOG peptide, which does not require cleavage into smaller fragments for antigen presentation (Figure 36A), it seems unlikely that Ii proteolysis is impaired. Further supporting this view, MHCII expression was similar on the surface on CLIC1-/-

BMDC cell surface, which requires cleavage of Ii for it to be expressed (Figure 40D)

(Watts, 2012) and participate in T-cell activation.

Another possible way by which deletion of CLIC1 could result in reduced T cell activation is by alteration in trafficking of vesicles containing antigen bound MHC class II complexes (MHCII-p), which must translocate to the cell surface for T cell activation (Roche and Furuta, 2015). Whilst CLIC3 may play a role in endosome trafficking (Dozynkiewicz et al., 2012; Macpherson et al., 2014; Tringali et al., 2012), this is unlikely to explain the actions of CLIC1 because CLIC1+/+ and CLIC1-/-

BMDCs have similar cell surface staining for MHC class II BMDCs (Figure 40).

217 Another plausible explanation for the reduction of EAE in CLIC1-/- mice is that spleens from CLIC1-/- mice have significantly reduced T cells and macrophages, when compared to CLIC1+/+ mice (Figure 13). In the initial stage of EAE, MOG specific autoreactive CD4+ T cells are primed and activated by antigen presented by antigen presenting cells (APCs) in lymph nodes and spleen (Becher et al., 2006). The primed CD4+ T cells escape choroid-plexus blood vessels and cross blood-brain barrier. Reactivation of these primed CD4+ T cells by their cognate antigens presented by local APCs in CNS, leads to amplification of the inflammatory cascade in the CNS

(Gold et al., 2006). Therefore it is possible that the reduced number of T cells and macrophages of CLIC1-/- spleen could lower the magnitude of CD4+ T cells priming and activation in the spleen of CLIC1-/- mice, while this is intact in CLIC1+/+ mice.

In conclusion, our results suggest that in DCs, CLIC1 regulates the antigen processing. As a consequence, deletion of CLIC1 gene in DCs resulted in attenuated antigen presentation both in vitro and in vivo setting. Therefore, in the case of autoimmunity, where these processes are dysregulated resulting in immune mediated tissue destruction, CLIC1 may represent a novel therapeutic target.

218 4 GENERAL DISCUSSION

4.I INTRODUCTION

In this thesis, I described my investigation of the functions of CLIC1 in immune/inflammatory responses. I have showed that several compartments of CLIC1-/- mice have altered immune cell composition both under normal physiological conditions and following inflammatory stimuli. Under normal physiological condition, immune cell composition of blood, spleen and peritoneal cavity altered markedly, whilst more marginal alteration has been observed in cervical and inguinal lymph nodes. Following inflammatory stimuli, the immune cell composition at the site of inflammation was also altered in CLIC1-/- mice. These changes might result in immunomodulation of both basal immune homeostasis and inflammatory state of these mice.

Because DCs play a central role in the immune responses, I then focused on CLIC1 biological function in DCs, and showed that when matured BMDCs were injected subcutaneously into the mice, more CLIC1-/- BMDCs were found in the lymph nodes than CLIC1+/+BMDCs irrespective of whether the recipient mice were CLIC1+/+ or

CLIC1-/-. This difference in the cell number homing to the lymph node was not due to cell death. Whilst the mechanism is uncertain, possible reasons are firstly that CLIC1-/-

BMDCs might be retained better in lymph nodes or secondly CLIC1-/-BMDCs might crawl faster along afferent lymphatic vessels. Whatever the reason, this change in

CLIC1-/- BMDC homing ability to lymph nodes might influence antigen presentation and priming of T cell responses.

219 Then, to investigate subcellular distribution of CLIC1 in BMDCs, I labelled BMDCs with CLIC1 monoclonal antibody, and showed that upon BMDC phagocytosis of the opsonised zymosan particle, cytoplasmic CLIC1 rapidly translocates to the phagosome membrane with a consequence that CLIC1-/- BMDCs displayed impaired phagosome acidification and proteolysis. Consistent with this, the CLIC ion channel blocker

IAA94, limited phagosomal acidification in CLIC1+/+ BMDCs, but had no effect on those functions in CLIC1-/- BMDCs, suggesting IAA94’s actions are specific for

CLIC1. Further, the impairment of phagosomal acidification and proteolysis in CLIC1-/-

BMDCs was associated with impairment of in vitro antigen presentation of a large 125 amino acid MOG peptide, which requires processing, whilst having little effect on a small 20 amino acid MOG peptide that does not require processing. In line with the in vitro data, when experimental autoimmune encephalomyelitis (EAE) is induced with

CLIC1-/- antigen pulsed BMDCs in CLIC-/- mice, there is complete absence of CLIC1 and disease is reduced, suggesting the likelihood of reduced in vivo antigen presentation by CLIC1-/- BMDCs.

4.II IS CLIC1 A PROINFLAMMATORY MOLECULE?

Proinflammatory molecules include cytokines, cytokine receptors, chemokine and their receptors, adhesion molecules, growth factors and transcription factors necessary for triggering and promoting inflammatory responses. There are several reasons why

CLIC1 might be considered to have overall proinflammatory effects.

220 First, in this thesis, when BMDCs bearing no CLIC1 and CLIC1+/+ BMDCs was injected into the mice, CLIC1+/+ BMDCs were found less in the lymph nodes. Whilst lots of other explanations are possible, this suggests that CLIC1 expressing BMDCs could bind to lymphatic vessel more tightly than BMDCs bearing no CLIC1, results in reduced migration of CLIC1+/+ BMDCs to the lymph nodes. The most obvious evidence supporting the interaction of CLIC1 with an adhesion molecule is from the study that showed that an association of CLIC3 with adhesion molecule such as α5β1 integrin and facilitated its recycling back to the plasma membrane (Dozynkiewicz et al., 2012).

Our data also suggests that CLIC1 could involve in generation of chemokine or chemotactic factors necessary for immune cell recruitment. In this thesis, we showed that deletion of CLIC1 in the mice was associated with the altered immune cell composition during inflammatory responses when use thioglycollate elicited as animal model. It is widely known that in response to inflammation, leukocytes recognized chemokine secreted by the cells in the inflamed site. This interaction allows leukocytes to move toward the inflamed site in gradient manner. In our case, when CLIC1 was absent, there were more infiltrating monocytes present in the inflamed site. When

CLIC1 was absent, the pattern of chemokine secreted in the inflamed tissue might also be altered.

The best overall evaluation of the biology of a molecule is how animal models of disease are altered if its deleted or overexpressed. Using a K/BxN arthritis animal model revealed that CLIC1-/- mice are protected from K/BxN arthritis when compared to

CLIC1+/+ mice (Jiang et al., 2012). In line with this existing data, in this thesis, when I used experimental autoimmune encephalomyelitis (EAE) animal model for multiple

221 -/- -/- sclerosis, I found that MOG1-125 pulsed CLIC1 BMDCs in CLIC1 mice elicited less

+/+ +/+ severe EAE than MOG1-125 pulsed CLIC1 BMDCs in CLIC1 mice, whilst, the

-/- -/- same MOG1-125 pulsed CLIC1 BMDCs also elicited less EAE in CLIC1 mice when

-/- +/+ compared to the CLIC1 mice immunized with MOG1-125 pulsed CLIC1 BMDCs.

Because myelomoncytic cell such as DCs contribute significantly to EAE severity, impaired antigen processing by CLIC1-/- BMDCs could lead to the initial attenuated immune response in lymph nodes and therefore reduced severity in EAE. Taken together, these all illustrated CLIC1 is required to elicit inflammation, when CLIC1 absent, reduced and attenuated severity of the autoimmune diseases was observed, indicating that CLIC1 could be a proinflammatory molecule.

4.III IS CLIC1 AN ENZYME?

Studies of crystal structure of soluble CLIC1 have shown that CLIC1 is a structural homolog of the glutathione- S- transferase (GST) superfamily (Dulhunty et al., 2001;

Harrop et al., 2001). Furthermore, in redox state, CLIC1 has glutaredoxin like enzymatic activities (Al Khamici et al., 2015), which catalyze the conjugation of GSH to elective electrophiles, which could detoxify toxins or products of oxidative stress in the cells. During inflammatory responses, innate immune cells produce reactive oxygen species (ROS), which not only harm pathogens but could also damage host tissues.

Because of the ability of binding to GSH, CLIC1 could serve an antioxidant that detoxifies ROS, preventing cellular damage. In this thesis, we found that during inflammatory responses using thioglycollate elicited animal model, immune cell profile in peritoneal cavity changed markedly between CLIC1-/- and CLIC1+/+ mice. Reduced antioxidant in CLIC1-/- mice could result in an accumulation of ROS, which in turn

222 could damage the immune cells to undergo apoptosis or even cell death, which could explain the altered immune cell composition of several compartment of CLIC1-/- mice.

Although there is no direct evidence that link between CLIC1 and matrix metalloproteases (MMPs) enzyme, our results from BMDC migration suggest that

CLIC1 may be associated with matrix metalloproteases (MMPs) at least during BMDC migration. In order for BMDCs to transmigrate from lymphatic vessels to lymph nodes, the cells must disengaged from extracellular matrix (ECM), cross basement membrane and travel to the lymph nodes (Parks et al., 2004). BMDCs disengage from extracellular matrix (ECM) through secretion of matrix metalloproteases metalloproteases (MMPs), which digest extracellular matrix (ECM) and therefore BMDCs can cross the basement membrane (Parks et al., 2004). Although the mechanism by which CLIC1 might be associated with MMPs is unclear, our results indicates that CLIC1 could have inhibitory effect on the activity of MMPs, resulting in decreased transmigrated CLIC1+/+ BMDCs in the lymph nodes when compared to CLIC1-/- BMDCs.

4.IV IS CLIC1 A POTENTIAL TARGET FOR ANTI- INFLAMMATORY THERAPEUTICS?

Although primary function of immune system is to protect host against infection, excessive and unwanted inflammation is a key factor in pathogenesis of many diseases

(Navarro-Gonzalez et al., 2011). Autoimmune diseases are one of these example in which the diseases are the result of the failure of maintenance of tolerance to self and therefore damage host results in autoimmunity (Wang et al., 2015). CLIC1 is associated with pathogenesis of several autoimmune diseases at least in animal model of arthritis

223 (Jiang et al., 2012) and animal model for multiple sclerosis (in this thesis). Therefore, down regulation of CLIC1 gene expression by small interference RNA (siRNA), or inhibition of CLIC1 functional expression by inhibitors, could be a potential therapeutic for these autoimmune diseases. CLIC1 can be a therapeutic target of choice because of many reasons. First, CLIC1 translocates to cell membrane once the protein is oxidized or once the cells are activated, thus this could eliminate the nonspecific action of the therapeutic drugs if CLIC1 is used as a target. Second, CLIC1 as a membrane protein could potentially interact with the protein that is on the external side of the plasma membrane, this would beneficial to the development of the new compound. However,

CLIC1 is expressed in most of the cells as I have discussed in section 1.I.E.i, this could mean that inhibitors may lead to unwanted side effects. Since little is known about membrane-integrated form of CLIC1, a better understanding of structural changes between soluble and membrane CLIC1 is without doubt needed before CLIC1 can be used as a novel target for treatment of inflammatory diseases.

4.V HOW DOES CLIC1 EXERT ITS BIOLOGICAL EFFECTS

CLIC1 is a member of CLIC family proteins, which has been considered as intracellular chloride ion channels, and in sections 1.I.B I have discussed the evidence supporting its ion channel activity. However, it is possible that CLIC1 could exert its biological effects through the chloride ion channel and/or other non-chloride ion channel related activities.

Our data support the chloride ion channel by showing that chloride influx mediated by

CLIC1 could also facilitate cellular event after phagocytosis in BMDCs. Shortly after

224 phagocytosis, I showed that cytoplasmic CLIC1 rapidly translocates to the phagosome membrane of BMDCs. During this step, phagosomes undergo maturation and are acidified by promoting the influx of H+ (proton) by proton pump ATPases. Chloride influx mediated by CLIC1 provides electric shunting for compensating the influx of H+

(proton) inside the phagosomes, and therefore facilitates phagosomal acidification in

BMDCs. The CLIC ion channel blocker IAA94, limited phagosomal acidification in

CLIC1+/+ BMDCs, but had no effect on those functions in CLIC1-/- BMDCs, suggests that phagosomal acidification is driven by chloride ion channel activity mediated by

CLIC1. Furthermore, our results also showed that the CLIC1-/- BMDCs had less efficient in antigen presentation maybe because of the their impairs of the processes in protein degradation/proteolysis or processing of MHC class II invariant chain (Ii).

+/+ Based on our finding that IAA94 treatment of MOG1-125 pulsed CLIC1 BMDCs reduced T-cell activation to the same level as vehicle treated CLIC1-/- BMDCs, whilst,

-/- IAA94 treatment of MOG1-125 pulsed CLIC1 BMDCs did not alter their capacity to activate T-cells, it is likely that the chloride ion channel activity of CLICs is more likely to involve in the fist process. Therefore, our results support that CLIC1 facilitates in chloride ion channel, even though the question still remains whether CLIC1 forms a chloride channel or it acts as a regulator for other chloride channels.

Apart from the chloride ion channel activity, CLIC1 could exert its biological function by interaction with other partners as discussed in section 1.I.A.i. One example might be through its interaction with cytoskeleton elements and small GTPases. Interaction of

CLIC1 with cytoskeleton elements could facilitate cytoskeleton rearrangement, important for cell shape changes. As shown in this thesis, deletion of CLIC1 in leukocytes altered immune cell profiling significantly, and this might be because of the

225 changes in cell shape that in turn could influence the cell movement. For example, interaction of CLIC1 to cytoskeleton element such as such as ERM could direct the trafficking of intracellular organelles including phagosomes along the microtubule, where phagosomes could fuse with lysosomes. Similar to other fusion in the secretory and endocytic pathway, fusion of phagosome to lysosome, to form phagolysosomes, is also dependent on small GTPases such as Rho and Rab family proteins for tethering and docking of phagosomes to lysosomes (Erwig et al., 2006). Interaction of CLIC1 to small

GTPases therefore could modify the tethering and docking steps during the fusion. In our thesis, it would be possible that deletion of CLIC1 could inhibit or limit phagosome movement along microtubule and reduce the tethering and docking of phagosomes to lysosome, result in an impaired phagosome acidification as seen in CLIC1-/- BMDCs.

226 5 CONCLUSION AND FUTURE DIRECTION

5.I CONCLUSION FROM THIS THESIS

I propose here in this thesis that CLIC1 could play multifunctional roles in immune/inflammatory responses. CLIC1 could be a proinflammatory molecule, an enzyme, a chloride ion channel or a regulator for other chloride channels. Whatever

CLIC1 does, these all contribute to the outcome of overall immune/inflammatory responses. Although the mechanisms by which CLIC1 may utilize in immune/inflammatory responses are still unclear, targeting CLIC1 could provide a novel therapeutic drug for treatment inflammatory diseases such as arthritis and multiple sclerosis. However, a better understanding of the mechanism by which CLIC1 may utilize will not only shed light on its role and therapeutic application for immune/inflammatory responses but will also provide a better understanding for other cellular processes.

5.II FUTURE DIRECTION

Limitations of each experiment have already been discussed individually in each of its corresponding section of this thesis. Therefore, the future works and follow up experiments will only be discussed here.

Because CLIC1 is one of the six members of CLIC family proteins, it likely that there is gene redundancy of other CLIC proteins that could interfere with the experiments where

227 CLIC1 gene is deleted but the other CLIC genes are still intact. Therefore, biological functional studies of multiple CLIC knockout mice merit a future work.

Currently, the mechanisms by which CLICs may utilize in phagosome acidification and proteolysis are unclear solely due to the lack of certainty if CLIC1 is forming a chloride ion channel or only acts as a regulator. Mice treated with IAA94, a chloride channel blocker, to completely block and disrupt other intracellular chloride channel proteins should be investigated to identify if the chloride ion channel is the mechanism by which

CLIC1 utilizes in phagosome acidification.

Classical pathway of antigen presentation (MHC class II pathway) of BMDCs has been studied in this thesis. For the future work, investigation on the role of CLIC1 could be expanded to an alternative pathway of antigen presentation (antigen cross presentation),

This will certainly expand the understanding of CLIC1 biology in the overall DC antigen presentation and also T cell priming.

In EAE animal model, the number of mice used in this experiments is 6 pairs per group.

To identify the small difference of disease severity, larger numbers of mice are required.

Other phagocytes such as neutrophils should be investigated in the future. Neutrophils similar to DCs degrade pathogens and also produce reactive oxygen species (ROS), which is crucial in regulating intracellular phagosomal pH.

Thus, the experiments listed above will certainly expand our understanding of CLIC1 both under normal physiological condition and during inflammatory responses.

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