Activation and Inactivation of the Complement Anaphylatoxins
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Activation and Inactivation of the Complement Anaphylatoxins During Chronic Neutrophilic Inflammation of the Cystic Fibrosis Airway Matthew Stott BSc Thesis submitted for the Degree of Doctor of Philosophy September 2018 Institute of Infection and Immunity Cardiff University School of Medicine Heath Park Cardiff CF14 4XN DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ……………………………………………………… (candidate) Date ………………….…………….……… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of ………(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………….…………… (candidate) Date …………………………….…………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ……………………………………….……….…… (candidate) Date …………………….………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ……………………………………………..…..….. (candidate) Date ………………………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ……………………………………………..……… (candidate) Date ………………………………….……… I Dedication I would like to dedicate this thesis to my wife Frankie, who gave me unwavering support and encouragement from beginning to end. Her calming influence has kept me positive and focused throughout, thank you. II Acknowledgements I wish to thank Dr. Eamon McGreal for his supervision throughout my project. He has been and will continue to be an excellent role model and mentor who has continually supported and challenged me. I would like to thank Dr. Carmen van den Berg who initially introduced me to the laboratory 10 years ago and encouraged me to apply for this project. Her encouragement and guidance throughout my PhD will always be greatly appreciated. I would like to thank Prof. Paul B. Morgan who gave me crucial feedback throughout my study. He invited me to join his laboratory and the Complement Biology Group and gave me the opportunities to progress my development. Thank you to Dr. Julian Forton for collecting the patient samples and providing expertise on cystic fibrosis. Thank you to Dr. Peter Monk for the RBL-C5aR1 and RBL-C3aR cell lines that were crucial for this project. I would like to thank Complement UK for the opportunity to study for a PhD in complement biology. I also wish to thank Alexion for my funding via their educational grant. I would like to thank Dr. Samuel Touchard for his expertise in statistics. I wish to thank James Wheeldon for his critical feedback and encouragement during my time in the Henry Wellcome Building. Thank you to the members of the Complement Biology Group, particularly Dina Fathalla, David Walters and Wiola Zelek, for their help and feedback over the past 4 years. III Summary Cystic fibrosis (CF) is a fatal genetic disease that affects 1/2500 people in the UK. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause dehydration of mucosal membranes, leading to mucus obstruction of the small airways. The CF lungs are susceptible to recurrent infection promoting chronic neutrophilic inflammation. Neutrophils recruited to the CF lungs become dysfunctional and are ineffective at clearing pathogens, perpetuating inflammation. Neutrophil serine proteases (NSPs) released by neutrophils collaterally remodel the airway, reducing lung function and causing mortality. The complement anaphylatoxins (C5a and C3a) are important mediators of inflammation. C5a and C3a are chemotactic for monocytes and granulocytes, they also stimulate degranulation and the generation of reactive oxygen species (ROS). C5a is particularly potent towards neutrophils and is critical for orchestrating their response towards pathogens. C5a and C3a are elevated in the CF airway. Furthermore, in addition to complement activation these anaphylatoxins can be generated by non-complement proteases including NSPs. The mechanisms by which C5a and C3a promote chronic neutrophilic inflammation in the CF airways are not fully understood. In my study I show that C5a and C3a correlate with markers of neutrophilic inflammation (neutrophil count and CXCL8) in bronchoalveolar lavage (BAL) fluid from paediatric CF patients. I further characterise the generation of functionally active C5a-like and C3a-like forms by NSPs. Moreover, I demonstrate that atypical C5a production by NSPs cannot be prevented by therapeutic complement inhibitors. I show for the first time that NSP-generated C5a-like fragments are resistant to inactivation by carboxypeptidase B, an important regulator of C5a activity. I also further characterise the interaction between C5a and soluble glycosaminoglycans (GAGs), these are abundant during chronic neutrophilic inflammation of the CF airway. Additionally, I show that GAG interaction influences C5a activity. In conclusion, the CF airway environment modifies C5a function; these mechanisms could promote chronic neutrophilic inflammation. IV Abbreviations A. fumigatus - Aspergillus fumigatus AAT – alpha-1-anti-trypsin ABPA – allergic bronchopulmonary aspergillosis ACP - acylation-stimulating protein aHUS - atypical hemolytic uremic syndrome AMD - age-related macular degeneration ARDS – acute respiratory distress syndrome BAL – bronchoalveolar lavage BCA - bicinchoninic acid BLTR – leukotriene receptor BMI – body mass index BN-PAGE – blue native PAGE BSA – bovine serum albumin C – complement component (e.g. C5a) CaCC – calcium activated chloride channel C. albicans - Candida albicans CAR - coxsackie and adenovirus receptors CD – cluster of differentiation (e.g. CD44) CDR – complementarity determining region CF – cystic fibrosis CFTR - cystic fibrosis transmembrane conductance regulator CG – cathepsin G CHIPS - chemotaxis inhibitory protein expressed by Staphylococcus aureus COPD – chronic obstructive pulmonary disease CPB – carboxypeptidase B CPN – carboxypeptidase N CR – complement receptor CS – chondroitin sulphate CT - computerised tomography C3aR – C3a receptor C5aR – C5a receptor C/EBPa - (CCAAT/ enhancer binding protein a) Da - dalton DAMP – damage associated molecular pattern DNA – deoxyribonucleic acid V ECL – enhanced chemiluminescence substrate ECM – extracellular matrix EGTA - ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid ELISA – enzyme linked immunosorbent assay ENaC – epithelial sodium channel ER – endoplasmic reticulum ERS – European Respiratory Society FasL – first apoptosis signal ligand FcR – fragment crystallisable region receptor FEV1 – forced expiratory volume in 1 second fMLP – formyl-Met-Leu-Pro FPR – formyl peptide receptor FRET - Forster resonance energy transfer ESL-1 - E-selectin-ligand g - gram GAG - glycosaminoglycan GI – gastrointestinal GPI - Glycosylphosphatidylinositol HA – hyaluronic acid HMGB - high mobility group box 1 protein HRP – horse radish peroxidase HS – heparan sulphate HTA – Human Tissue Act ICAM - intercellular adhesion molecules Ig - immunoglobulin IL – interleukin ITAMs - immunoreceptor tyrosine-based activation motifs JAM - junctional adhesion molecule k – kilo (103) (prefix) L – litre LFA - lymphocyte function-associated antigen-1 LPS – lipopolysaccharide LTB4 – leukotriene B4 m – metre (unit) m – milli (10-3) (prefix) M - Molar M – mega (106) (prefix) VI MAC – membrane attack complex MAPK - mitogen-activated protein kinase MASP - mannose-binding lectin associated protease MBL – mannose binding lectin MCP-1 – macrophage chemoattractant protein-1 MES - 2-(N-morpholino)ethanesulfonic acid MG – alpha-2-macroglobulin MHC – major histocompatibility complex MMP – matrix metalloprotease MMR – macrophage mannose receptor MPO – myeloperoxidase n – nano (10-9) (prefix) NADPH - Nicotinamide adenine dinucleotide phosphate nC5a – native human C5a (generated by C5-convertase) NE – human neutrophil elastase NET – neutrophil extracellular trap NLR – NOD-like receptor NMR - nuclear magnetic resonance spectroscopy NOD - nucleotide-binding oligomerisation domain NRA - orphan nuclear receptor NSP – neutrophil serine protease ORAI - calcium release-activated calcium channel protein p – pico (10-12) (prefix) P. aeruginosa – Pseudomonas aeruginosa Pa - pascal PAF – platelet activating factor PAFR – protease activated receptor PAGE – polyacrylamide gel electrophoresis PAMP – pathogen associated molecular pattern PAR - protease activated receptor PBS – phosphate buffered saline PCR – polymerase chain reaction PGP - Pro-Gly-Pro PI3K - phosphatidylinositol 3-kinase PK3 – Protein kinase 3 PLC - phospholipase C PMA - phorbol 12-myristate 13-acetate VII PMN – polymorphonuclear leukocyte PNH - paroxysmal nocturnal hemoglobinuria PRR – pathogen recognition receptor PR3 – proteinase 3 PSGL-1 - P-selectin glycoprotein ligand-1 RBL – rat basophil leukaemia rhC5a –