A Thesis
entitled
The Role of Antimicrobial Peptide Murine Beta Defensin-3 in Protection against Oropharyngeal Candidiasis
by
Bemnet G. Mengesha
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Master of Science Degree in
Biology
______Dr. Heather Conti, Committee Chair
______Dr. Malathi Krishnamurthy, Committee Member
______Dr. Jianyang Du, Committee Member ______
______Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies
The University of Toledo December, 2017
Copyright 2017, Bemnet G Mengesha
This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of
The Role of Antimicrobial Peptide Murine Beta Defensin-3 in Protection against Oropharyngeal Candidiasis
by
Bemnet G. Mengesha
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology
The University of Toledo
December, 2017
Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused mainly by
Candida albicans. C. albicans is a commensal of oral mucosal surfaces, but can be pathogenic when antifungal immune defense mechanisms are impaired. There is a high prevalence of OPC in patients with human immunodeficiency virus (HIV), implicating
CD4+ T helper (Th) cells in protection against OPC. IL-17 is an important pro- inflammatory cytokine produced by the Th17 subset, and studies in experimental models show increased susceptibility in mice with impaired IL-17 signaling. Deficiencies in IL-17 signaling lead to defects including the production of antimicrobial peptides such as β-
Defensins (BDs). In OPC susceptible IL-17RA knockout mice, expression level of murine
β-defensins-3 (mBD3) is decreased in infected tongue tissue compared to wild type mice suggesting the involvement of mBD3 in protection against OPC. mBD3 has direct antifungal properties against Candida, but can also modulate the immune response through potential interactions with chemokine receptors such as CCR6 necessary for immune cell trafficking and recruitment. Moreover, mDefb3-/- mutant mice expressing low level of
iii murine β-Defensin-3 show high susceptibility to OPC suggesting the involvement of mBD3 in protection against OPC.
Gene expression profile of mDefb3 -/- mice show impaired expression of IL-17 inducing cytokines such as IL1- β and IL-6 and inflammatory cytokine IL-17 following
Candida infection showing mDefb3 -/- mutant mice have defects in IL-17 signaling pathway. In addition, mDefb3 regulates expression of beta defensins mDefb1 (BD1), mDefb2 (BD2), chemokine CXCl2 but not chemokine CXCL1. Loss of expression of Defb3 in mDefb3 -/- mice was compensated by high expression of Defb14, and increased expression level of CCL20 early during infection (day 2 post infection) but was not enough to offset susceptibility to OPC. Using mouse model of OPC, we show that mDefb3 regulate innate immune response against OPC via regulation of AMPs (mDefb1 and mDefb2) and proinflammatory cytokines (IL-17, IL-6 and IL1β).
iv
Acknowledgements
My utmost thanks and sincere gratitude go to my advisor, Dr. Heather Conti for the guidance, encouragement and support during the development and execution of this work.
I would like to express my gratitude to my committee members: Dr. Malathi
Krishnamurthy and Dr. Jianyang Du for serving in my thesis committee and their valuable insight and suggestions.
I would like to thank my family: my wife Mrs. Elizabeth Senbetu and my daughters
Mihret and Saron Gashawbeza for their everlasting love and support, and my parents: my father, Mr. Gashawbeza Mengesha and my mother, Mrs. Bayush Nicola for supporting me spiritually.
Finally, I thank members of Conti lab for the support and congenial work environment.
v
Table of Contents
Abstract ...... iii
Acknowledgements ...... v
Table of Contents ...... vi
List of Figures…………………………………………………………………………...viii
List of Abbreviations ...... x
List of Symbols…………………………………………………………………………...xi
1. Literature Review………………………………………………………………….1
1.1 Introduction ...... ….1
1.2 Pathogenicity mechanisms of C. albicans ...... 2
1.3 Innate recognition of microbial pathogens…………… ...... 3
1.4 Antifungal immunity ...... 5
1.5. Effector mechanisms of antiCandida immunity...... 7
1.5.1 The role of neutrophils and chemokines in antifungal immunity...... 8
1.5.2 The role of antimicrobial peptides in antifungal immunity…………..10
1.6. Hypothesis and Objectives………………………………………………….13
2. Materials and Methods------15
2.1 Preparation of C. albicans culture…………………………………...15
2.2 Induction of OPC…………………………………………………….15
2.3 Candida killing assays ...... 16
2.4 Experimental mice ...... 16
2.5 RNA extraction, cDNA synthesis and gene expression studies ……..17
2.6 Quantification of fungal burden……………………………………...17 vi
2.7 Microscopy ...... 17
2.8 Neutrophil count ...... ….18
2.9 Data analysis------18
3. Results ………...... 19
3.1 rMBD3 and rhBD2 exhibit candidacidal activity in vitro ...... 19
3.2 mDefb3-/- mice are susceptible to OPC ...... 20
3.3 mDefb3 regulates expression of mDefb1 and mDefb2 during oral
Candida infection...... 21
3.4 mDefb3 regulates IL-17 and Th17 inductive cytokines IL1β and IL-6
...... 23
3.5. mDefb3 regulates CCL20 and CXCL2 but not CXCL1 ...... 24
3.6. Immunohistochemical analysis show Defb3-/- mice do not
elicit defects in neutrophil extravasation ------35
4. Discussion……………………………………………………………………………..42
4.1 rMBD3 and rhBD2 show potent candidacidal activity………………42
4.2 mDefb3 is necessary for innate response to OPC………………………44
4.3 mDefb3 contributes to immunity against OPC via regulation of
4.4. AMPs, pro-inflammatory cytokines and chemokines……………….45
References………………………………………………………………………………..52
vii
List of Figures
3-1 Candidacidal activities of recombinant mBD3 and recombinant hBD2 ...... 20
3-2 mDefb3 is important for protection against OPC ...... 22
3-3 Gene expression changes during OPC show the involvement of Defb3 in
Candida clearance ...... 25
3-4 Gene expression changes during OPC show mDefb3 regulates mDefb1
during OPC...... 26
3-5 Altered gene expression during OPC show Defb3 regulates Defb2 during OPC ..27
3-6 Gene expression changes during OPC show Defb14 upregulation in
mDefb3-/- mice during OPC ...... 29
3-7 Altered gene expression during OPC show Defb3 regulate IL-17 expression
during OPC ...... 30
3-8 Gene expression changes during OPC show Defb3 regulate IL1b expression
during OPC ...... 32
3-9 Gene expression changes during OPC show Defb3 regulate IL-6
expression during OPC ...... 33
3.10 Gene expression changes during OPC show CCL20 compensates Defb3
deficiencies during OPC ...... 34
3-11 mDefb3 does not regulate CXCL1 during OPC ...... 36
3-12 Altered gene expression during OPC show mDefb3 regulate CXCL2 during
OPC ...... 37
3-13 Altered gene expression during OPC show mDefb3 regulate Csf3 during OPC ...38
viii
3.14 mDefb3 in oral mucosa does not regulate neutrophil migration during OPC...... 39
3.15 mDefb3 in oral mucosa does not regulate neutrophil migration...... 41
ix
List of Abbreviations
ANOVA ...... Analysis of Variance APC ...... Antigen Presenting Cell ATP ...... Adenosine Triphosphate C. albicans ...... Candida. albicans CFU ...... Colony Forming Unit DNA ...... Deoxyribonucleic Acid FCᵞ…………………..FC-gamma receptor GAPDH ...... Glyceraldehyde 3-phosphate dehydrogenase G-CSF ...... Granulocyte Colony Stimulating Factor H&E ...... Hematoxylin and Eosin HIV ...... Human Immunodeficiency Virus IRAK………………..Interleukin-1 receptor-associated kinase IKK………………….I kappa B kinase KO ...... Knock Out MYD88……………..Myeloid differentiation primary response 88 NF-kb……………….Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells nTh………………….Natural T Helper OD...... Optical Density OEC...... Oral Epithelial Cells OPC ...... Oropharyngeal Candidiasis PAMP ...... Pathogen Associated Molecular Patterns PBS ...... Phosphate Buffered Saline PRR ...... Pattern Recognition Receptor qPCR ...... Quantitative Real Time PCR STAT...... Signal Transducers and Activators of Transcription TGF ...... Transforming Growth Factor Th ...... T Helper TLR ...... Toll-like Receptor TNF ...... Tumor Necrosis Factor TRAF6………………TNF receptor-associated factor 6 WT ...... Wild Type YPD...... Yeast Peptone Dextrose
x
List of Symbols
°C ...... Degrees Celsius α ...... Anti β ...... Beta δ ...... Delta γ ...... Gamma μM ...... micro molar μL ...... microliter kg...... kilogram ml ...... Milliliter ng...... Nano gram
xi
Chapter 1
LITERATURE REVIEW
1.1. Introduction
Candida spp. are commensal microorganism of most healthy individuals. They colonize the gastrointestinal, genital mucosa as well as human skin without causing diseases (Sparber and LeibundGut-Landmann 2015). Candida albicans (C. albicans), the most common Candida spp. in human mucosa, can be a major pathogenic fungi of humans when host immunity is compromised (Brown et al. 2012). Diseases such as HIV/AIDS
(Brown et al. 2012; Garber, 2001;), patients receiving chemotherapy and radiotherapy
(Pfaller and Diekema, 2007), use of antibiotics (Pfaller and Diekema, 2007) and congenial immune defects (Bishu et al., 2014) compromise antifungal immunity. Infections caused by C. albicans could be either superficial or disseminated. Superficial infections occur on mucosal surfaces including oral and vagina whereas systemic infections include infections in the kidneys and could be life threatening (Mayer, Wilson, and Hube, 2013).
Oropharyngeal candidiasis (OPC) is superficial candidiasis, and about 85% of its incidence involves C. albicans either alone or in combination with other species of
1
Candida (Sangeorzan et al., 199; Redding et al., 1999). Common non-albicans Candida species implicated in OPC include C. glabrata, C. tropicalis, C. parapsilosis, C. krusei and
C. dubliniensis although their extent is much lower than C. albicans. Due to high incidence of OPC in HIV patients during the progression of HIV to AIDS, and the association of both
HIV/AIDS and OPC with defects in CD4+ lymphocytes, CD4+ count and clinical variables such as OPC have been used as predictors of progression of HIV to AIDS (Rabeneck et al., 1993). Thus, OPC is considered as one of the first HIV/AIDS defining clinical signs suggesting the vital importance of CD4+ T cells in the human host response against
Candida infection in the oral cavity (Rabeneck et al., 1993).
Both innate and adaptive immunity play a role in protection against fungal infections. However, the contribution of innate immunity to fungal infection on mucosal surfaces is more pronounced. In this regard, IL-17 mediated antifungal innate immunity plays a critical role in protection against OPC (Dongari-Bagtzoglou, Villar, and Kashleva,
2005). Therefore, in this study, the role of antimicrobial peptide β defensin-3, effector molecule downstream of IL-17 signaling pathway mediated antifungal immunity, is discussed.
1.2. Pathogenicity mechanisms of Candida albicans
C. albicans is a dimorphic fungus which exists between a unicellular yeast and a filamentous multicellular (hyphal) form. C. albicans first grow as commensal with manageable number on host epithelial surfaces but become pathogenic and could be life threatening under invasive conditions. There are several steps in C. albicans tissue invasion
2
including adhesion to epithelial surfaces, epithelial penetration and invasion, vascular dissemination and endothelial colonization under invasive conditions (Gow et al., 2011).
C. albicans is equipped with different arsenal of tissue penetration and destruction which depends on the morphological stage of the pathogen. Thus, morphological switch, yeast-to hyphae transition, is considered as one of the most important virulence factors mediating pathogenesis (Cheng et al., 2012). Defects in important transcription factors involved in this morphological switch including Enhanced Filamentous Growth 1 (EFG1) and Candida
PseudoHyphal regulator 1 (CPH1), and efg1 mutants are unable to form the infective stage
(true hyphae) and are avirulent in mouse infection model (Whiteway and Bachewich,
2007). Candida also produces cytolytic peptide toxin candidalysin and is important virulence factor involved in disrupting epithelial barriers by damaging epithelial membranes during OPC (Moyes et al., 2011).
1.3. Innate recognition of microbial pathogens
Host cell surface receptors recognize pathogens in order to activate intracellular signaling pathways leading to stimulation of inflammatory mediators (Janeway and
Medzhitov, 2002) through expression of various genes involved in inflammatory and immune response (Akira, Uematsu, and Takeuchi, 2006). There are four classes of pattern recognition receptors (PRRs): Toll-like receptors (TLRs), C-type lectin receptors (CLRs),
NOD-like receptors (NLRs) and RIG-I like receptors (RLRs) (Netea et al. 2015). PRRs recognize pathogen associated molecular patterns (PAMPs) through conserved microbial components (Akira et al., 2006) such as lipopolysaccharide (LPS) and peptidoglycans
(PGNs) (Scott et al. 2007). CLRs and TLRs are known mediators of C. albicans recognition 3
(Gauglitz et al. 2012) although CLRs play a major role in innate recognition of C. albicans
(Netea et al., 2015).
Epithelial cells (ECs) are one of the first line of defense involved in protection against pathogen attack via different mechanisms including formation of physical barriers
(Naglik et al., 2011), expression of a range of PRRs including TLRs (1-6, 8-10) and CLRs
(dectin 1) (Naglik and Moyes 2011) and production of inflammatory mediators and antimicrobial peptides (Altmeier et al., 2016). On the pathogen side, cell wall is the outer layer structure which mediates interaction with the host (Shibata et al. 2007). Thus, the host defense system targets primarily the cell wall of C. albicans (Zheng et al., 2015) which is composed of chitin, glucans and manno-proteins (Cheng et al. 2012). Two classes of
PRRS mainly mediate recognition of cell wall of C. albicans: Toll-like receptors (TLRs) and the C-type lectin receptors (Cheng et al. 2012). TLRs are expressed by a variety of immune and non-immune cells including dendritic cells, B cells, specific types of T-cells as well as fibroblasts and epithelial cells (Akira, Uematsu, and Takeuchi, 2006). For example defects in TLR2 (Villamón et al., 2004) and TLR4 increases susceptibility to
Candida infection (Netea et al.,. 2004). Both TLR2 and TLR4 use Toll/β receptor (TIR) domain of TLRs for signal transduction and myeloid differentiation factor 8 (MyD88) as adaptor (Fitzgerald, 2001). One well-studied mechanism is ligand binding to TLRs through
PAMP-TLR interaction which induces receptor oligomerization and intracellular signal transduction (Mogensen, 2009).
4
CLRs are expressed mainly by epithelial cells and myeloid cells (Netea et al., 2015;
Drummond and Lionakis, 2016). They detect C. albicans via recognition of β (1, 3 ) and β
(1, 6) glucans (Netea et al., 2015). For example, a CLR receptor dectin 1 is a β-glucan receptor which is expressed by various cell types including macrophages and monocytes
(Brown et al., 2002), neutrophils, dendritic cells and ECs (Naglik and Moyes, 2011).
Recognition of β-glucan initiates innate immune response via induction of cytokines and internalization of fungus (reviewed in (Netea et al., 2015) via signals through spleen tyrosine kinase (SYK) and, caspase activation and recruitment domain containing 9
(CARD9).
Despite the fact that the host innate immune system has various mechanisms to deter fungal colonization, fungi also developed different ways of evading the host recognition by PRRs. C. albicans and Aspergillus fumigatus, for example mask their inner cell wall layer (beta glucan) by mannose (Netea et al., 2015; Slesiona et al., 2012). This help
C. albicans evade recognition by CLRs especially by dectin 1 (Netea et al., 2015)
1.4. Antifungal immunity
Antifungal immunity against OPC caused by C. albicans is mainly mediated by a proinflammatory cytokine interleukin-17 (IL-17) both in humans and in mice (Trautwein-
Weidner et al. 2015; Bar et al. 2014; Conti and Gaffen, 2015). IL-17 is secreted by T helper
(Th17) cells (Huppler et al. 2014; Veerdonk et al., 2009). Th17 cells are subset of CD4+ cells that produce IL-17 cytokine involved in protection against extracellular microbes such as fungi (Annunziato et al. 2007). Differentiation of Th17 cells from naïve CD4+ T-cells 5
is mediated by inductive cytokines transforming growth factor beta (TGF-β), IL-6 and IL-
1β in presence of IL-23 to maintain and expand Th17 cells (Annunziato et al. 2007;
Robinson et al., 2013). Moreover, Th17 specific transcription factor retinoic acid receptor- related orphan receptor gamma t (RORγt) is required for their commitment (Acosta-
Rodriguez et al. 2007; Weaver et al. 2007). However, the role of inductive cytokines in differentiation of Th17 depends on signal transducer and activator of transcription 3
(STAT3) to induce a critical transcription factor RORγt. Owing to the importance of IL-17 for Candida clearance and the time required for differentiation of IL-17 producing Th17 cells, the possibilities of Th17 as major source of IL-17 for innate response to OPC required at the time of encounter was not clear (Gladiator and LeibundGut-Landmann 2013). Thus, early sources of IL-17 involved in Candida clearance before CD4+ cells differentiate into
IL-17 producing Th17 cells (Cua and Tato, 2010) has been point of a research interest.
Results from acute model of oral candidiasis has also revealed protective role of early response from γδ T cells and nTh17 against oral Candida infections (Conti and
Gaffen, 2015). However, the role of ILCs remains controversial as recombinase activating gene (Rag1) deficient mice with intact ILCs are susceptible to OPC (Hernandez-Santos et al., 2011). As a result, in addition to CD4+ Th17 cells, CD8+ Th17 cells and Type 17 innate cell types including γδ T cells and innate lymphoid cells (ILC) that express ROR γt produce IL-17 (Sutton, Mielke, and Mills 2012). More importantly, gamma-delta (γδ) T cells have been identified as major innate source of IL-17 during pathogen infection and environmental stimuli before the adaptive Th17 cells response is activated (Martin et al.
6
2009). Further studies also showed the important role played by γδ cells in fungal clearance during OPC (reviewed in (Conti and Gaffen, 2015)
IL-17 mediated antifungal immunity is initiated upon binding of IL-17 to its receptors indicating the critical role of regulation of IL-17 receptors in IL-17 mediated inflammation/ and immunity including production of neutrophils and defensins (Kao et al., 2004). As a result, defects in IL-17 receptor lead to susceptibility to OPC (Huppler et al. 2014).
There are six members of IL-17 family of cytokines designated as IL-17A-IL17F of which the functions of only two members has been studied in detail in the context of C. albicans infection (reviewed in (Conti and Gaffen, 2010) . Both IL-17A and IL-17F bind to the same receptor and play a role in fungal clearance during OPC although the involvement of IL-17A in gene regulation is much higher than that of IL-17F (Wasilewska et al. 2016). Signaling of IL-17A and IL-17F cytokines is mediated by either homodimer or hetero dimerization of IL-17RA and IL-17RC. IL-17RA -/- mice are highly susceptible to OPC and their defect in controlling C. albicans is evident starting from the first day of infection (Ye, 2001a). Moreover, IL-17 regulates production of neutrophils and antimicrobial peptides (Conti et al., 2016), and IL-17 modulates neutrophil recruitment by inducing chemokine CXC (Ye 2001b).
1.5. Effector mechanisms of antiCandida immunity
Specific interaction of PRRs on the surface of the responding cell with C. albicans initiates signaling pathway (Wells et al., 2008). For example, signaling initiated upon 7
recognition of PAMPs via Toll-like receptors (TLRs) involve different adaptor proteins such as MyD88, TRAF6, IRAK and IKK activate NF-kB. Translocation of NF-kB to nucleus, which in turn triggers expression of AMPs, proinflammatory cytokines and neutrophils (Katzenback, 2015). Thus, host immune cells such as neutrophils, monocytes, macrophages and molecules such as complement, cytokines, chemokines and host defense peptides with anti-microbial properties are involved in antiCandida innate immune responses (Scott et al., 2007). Thus, increased susceptibility of IL-17RA-/- mice to OPC correlates with defects in genes involved in neutrophil recruitment and trafficking, and reduction in the levels of AM Ps (Kao et al., 2004)). Therefore, it is generally agreed that
IL-17 exerts its candidacidal activity via induction of proinflammatory cytokines, antimicrobial peptides and chemokines (Jin and Dong, 2013) via signaling pathway regulating production of neutrophils, chemokines and antimicrobial peptides, and recruitment of neutrophils by chemotactic and granulpoitic chemokines (Conti et al., 2009;
Huppler et al. 2014).
1.5.1. The role of neutrophils and chemokines in antifungal immunity
Neutrophils are the most abundant (Kruger et al., 2015) and the first immune cells rapidly trafficked into infection site to perform their effector functions (Kruger et al., 2015;
Drescher and Bai, 2012). Neutrophils respond rapidly to potentially pathogenic microorganism (Miramo´n et al., 2012) including C. albicans infection (Miramo´n et al.,
2012; Gazendam et al., 2014). They are critical leucocytes in battle against invading pathogens through various antimicrobial defense mechanisms (Drescher and Bai, 2012;
Cheng et al., 2012). Neutrophils kill microbes intracellularly upon phagocytosis or 8
extracellular by degranulation of antimicrobial proteins and the release of Neutrophil
Extracellular Traps (NETs) (Urban et al., 2009). They kill both opsonized and un- opsonized C. albicans via two independent mechanisms. Killing of opsonized Candida depends on FCγ receptor and NADPH oxidase activity while killing of un-opsonized
Candida depends on complement receptor-3 (CR3) and CARD9 but not dectin-1
(Gazendam et al., 2014). Due to their vital role in protection against microbial attack, their depletion implicated in increases host susceptibility to microbial infections in both humans and experimental mice models (Trautwein-Weidner et al., 2015). For example, neutrophil depleted mice fail to control C. albicans hyphal growth starting from the onset of the infection (Trautwein-Weidner et al. 2015). As a result, C. albicans has been reported to be the most frequent fungal infections in neutropenic patients (Mohammadi and Foroughifar,
2015). IL-17 increases granulopoiesis by regulating granulocyte colony-stimulating factor
(G-CSF) (Forlow, 2001).
Chemokines, on the other hand, are mainly involved in modulation of cell activation (Russo et al., 2014) and recruitment (Kruger et al., 2015). Their chemotactic activity on target immune cells to infection sites depend on concentration gradient which ultimately help in mounting immune response (reviewed in (Prasad and McCullough,
2013). Moreover, chemokines have been implicated in modulating the balance between neutrophil release and retention (Kruger et al., 2015; Russo et al., 2014) and neutrophil functions (Swamydas et al., 2016). Recent studies on CXC chemokine receptor CXCR1 and its ligand CXCL5 have revealed that mice defective in CXCR1 show increased susceptibility to systemic candidiasis due to defect in killing of the fungus by neutrophils 9
rather than defects in neutrophil trafficking to infection site (Swamydas et al., 2016). For example, IL-17RA-/- mice show reduced levels of CXC chemokines and/or lack of their recruitment to infection site (Huppler et al. 2014) including reduced CXCL1, CXCL5 and
Csf3 expression (Conti et al. 2009a).
1.5.2. The role of antimicrobial peptides in antifungal immunity
Antimicrobial peptides (AMPs) are broad-spectrum antimicrobial effector proteins involved in host protect against various pathogenic organisms including bacteria, fungi and viruses (Diamond et al., 2009). AMPs are implicated mainly in the host innate immune responses in various tissues including oral cavity (reviewed in Dale et al., 2006). AMPs are diverse types and their structure varies accordingly (Diamond et al., 2009). However, they share some structural features such as small polypeptides (fewer than 50 amino acids), broad-spectrum antimicrobial activity at physiological concentration and positive net charge (reviewed in (Zasloff, 2009; Ganz, 2003; Shai, 2002). Mature host defense peptides are released through proteolytic cleavage of precursor proteins (reviewed in Wang, 2014) and their cationic properties is critical for targeting the negatively charged surface of microorganisms (Reviewed in (Wang, 2015).
Three major families of AMPs exist in the oral cavity: α-helical peptides without cysteine (cathelicidin, LL37); peptides with three disulfide bonds (the α- and β- defensins) and histatins (reviewed in (Dale et al., 2006) and calprotectin (Hans and Hans, 2014).
Depending on species, different type and number of antimicrobial peptides are produced
(Lehrer, 2004). Defensins are important AMPs of the innate immune system. There are 10
three different type of defensins: alpha, beta and theta-defensins. Cluster of genes encoding human α-defensin and the β-defensin are mapped to chromosome 8p23 (Linzmeier et al.,
1999) while the defensin gene cluster region is mapped to mouse chromosome 8 A2 (Amid et al., 2009). α-defensins are mainly packaged in neutrophil granules (HNP1, HNP2, and
HNP3) or secreted by intestinal Paneth cells (HD5, HD6), while β-defensins are expressed in mucosal and epithelial cells (Droin et al., 2009). Alpha and beta defensin families of
AMPs have six disulfide-linked cysteine framework and triple-stranded β sheets but variations exist in the length of peptide segments between the six cysteine and the pairing of cysteins that are connected by disulfide bonds (Ganz, 2003). Anti-microbial properties of both alpha and beta defensin have been documented (Ganz et al., 1985). For example,
Paneth cells of the mice small intestine produce alpha defensins (cryptdins 1-5) and antibacterial properties of cryptidin1 was shown (Selsted et al., 1992; Huttner and
Quellette, 1994). ϴ-defensins were discovered in non-human primate Rhesus macaque
PMNs (Lehrer, 2004), and are circular in form due to ligation of two truncated α-defensins between the N-and C-terminal peptide (reviewed in (Wang, 2014). Mature theta-defensin peptide is formed from cyclization of alpha defensin like precursor peptides (reviewed in
(Ganz, 2003). ϴ-defensins have been shown to have antibacterial and fungicidal activities
(Tang et al., 1999).
Different cell types including epithelial cells produce antimicrobial peptides in response to Candida infection (Diamond et al., 2009). For example, epithelial cells (ECs) and neutrophils (Wiesner and Vilcinskas, 2010) produce defensins and the cathelicidins.
In addition, epithelial AMPs also include calprotectin and adrenomedullin (Hans and 11
Hans, 2014). Expression pattern of antimicrobial peptides vary depending on pathogen, host as well as tissue type. Generally, AMPs are induced by pathogens such as fungi and cytokines (Diamond et al 2009). Some AMPs are constitutively expressed while others are induced in response to infection or inflammation (Ganz, 2003). For example, expression of different murine β-defensins (mBD-1-4) significantly increases in airway mucosa of influenza virus infected mice (Chong, Thangavel, and Tang, 2008). Some AMPs such as human β-defensin-1 (hBD1) in intestinal mucosa (hDefb1) are constitutively expressed while hBD 2-4 encoded by Defb4, Defb3, and Defb4, respectively are induced upon microbial encounter (Kelly et al., 2013).
As effector molecules, AMPs are downstream component of innate immune system. They counteract pathogenic activities through direct antimicrobial activities and/or indirectly via immune modulation (Tomalka et al., 2015). The antimicrobial mechanism of
AMPs is mainly through disruption microbial cell membranes (Cederlund, Gudmundsson, and Agerberth 2011). Moreover, their multifunctional nature makes them important components of immune system, as a single peptide could be capable of elimination of more than one kind of pathogenic microorganisms (Diamond et al., 2009). AMPs are shown to act early during innate immune response during infection in oral epithelium (Shai, 2002).
Recently, β-defensin-3 has been shown to be involved in protection against OPC (Conti et al., 2016).
AMPs in the oral cavity are involved in first line of defense against various pathogens in host innate immune system (reviewed in (Dale et al., 2006; Wiesner and 12
Vilcinskas, 2010). Another important antimicrobial peptide present in the oral cavity include histatins (Wiesner and Vilcinskas, 2010). They are present in salivary glands, epithelial cells and in neutrophils (Dale et al., 2006). Unlike most cationic peptides which utilize cytolysis as fungicidal mechanism (Sun et al., 2008), an important histatin (Hst 5) induces selective leakage of intracellular ions and ATP from yeast cell resulting in gradual cell death ( Koshlukova et al., 2000; Vylkova et al., 2007). To exert its function, Hst5 binds to C. albicans cell wall protein (Ssa1 and Ssa2, members of Heat Shock Protein, (Hsp) 70)
(Li et al. 2003). Direct antimicrobial activities of AMPs function mostly via disruption of the microbial cell membrane (Shai, 2002). However, some AMPs such as beta defensins have been shown to function beyond antimicrobial properties: modulating innate immune system via chemotaxis and modulation of expression of proinflammatory cytokine and other AMPs. The findings of Niyonsaba et al. (2004) demonstrated specific chemotactic activity of human β-defensin-2 (hBD2) but not hBD1 to tumor necrosis factor-(αTNFα) treated human neutrophils.
1.6. Hypothesis and Objectives
OPC is superficial fungal infection mainly caused by C. albicans on mucosal surfaces.
The disease poses significant challenges to patients with defects in IL-17 RA signaling,
HIV/AIDS patients, immunosuppressed patients under high exposure to antibiotics and corticosteroids, patients receiving chemotherapy and radiotherapy. Candia albicans is not the natural commensal of mice and rodents, and thus mouse models of innate immunity to
Candida infection have been the preferred model to study antiCandida immunity (Conti and Gaffen, 2015). Therefore, it has been possible to induce Candida infection on mucosal 13
surfaces by inducing immunosuppression using corticosteroids or creating specific gene knockout mice responsible for Candida clearance.
Therefore, this project was set out to study the contribution of beta defensin-3 in antiCandida immunity in OPC mouse model. The overall hypothesis is that Defb3-/- mice are more susceptible to OPC than WT. The hypothesis was based on our previous findings that showed the mRNA as well as murine beta defensin-3 (mBD3) protein expression levels were significantly down regulated in susceptible IL-17RAKO mice. In general, WT mice infected with C. albicans (CAF2-1) in the oral mucosa clears infection at 3-4 days post infection (p.i). In contrast, Defb3-/- exhibits high fungal burden until day 5 p.i. which provides the basis for the molecular and genetic dissection of innate immunity to OPC.
Based on this hypothesis, our overall objective was to determine the mechanisms underlying susceptibility to OPC in Defb3 mutant mice by characterizing host innate immune responses against OPC.
14
Chapter 2
Materials and Methods
2.1. Preparation of C. albicans culture
For each OPC experiment, a single colony of C. albicans (CAF 2-1) was cultured overnight in YPD broth. C. albicans broth was adjusted to OD600=1.2 and the adjusted culture was centrifuged at 3200 RPM for 5 minutes. The culture was poured off and the pellet (cells) was washed with 1x PBS and was reconstituted with equal amount of PBS.
In Candida killing experiment, candida cells were counted at exponential phase of the overnight culture (see below for details).
2.2. Induction of OPC
OPC infection was performed by sublingual inoculation with 2x 106 Candida cells in a pre-weighed cotton balls (0.0025g) soaked in C. albicans for 75 minutes under anesthetic conditions as previously described (Conti et al. 2009b). Clinical isolate of
C. albicans (CAF2-1) was used for all OPC and candidacidal assays. Oral pre-swabs were obtained before every experiment to verify the absence of commensal fungi. Whenever immunosuppressed wild type controls were used, immunosuppression was induced by cortisone acetate (225 mg/kg i.p.) on days -1, 1, and 3. Fungal loads in tongue were
15
determined by dissociation of tongue tissue on a gentleMACS (Miltenyi Biotec), followed by plating serial dilutions on YPD with antibiotics.
2.3. Candida killing assay
Candidacidal activity of human beta defensin 2 (hBD2) and murine orthologue of hBD2 (murine beta defensin-3, mBD3) were examined using recombinant human beta defensin2 (rhBD2)and recombinant murine beta defensin 3 (rMBD-3) peptides purchased from Bio Basic Inc. as previously described (Vylkova et al. 2007). Briefly, C. albicans cells were grown in Yeast Extract-Peptone-Dextrose (YPD) broth medium overnight, washed twice with 10mM sodium phosphate buffer (NaPB,NA2HPO4-, NaH2P)4, PH 7.4) and re-suspended in 10mMNaPB buffer at a concentration of 104 cells/mL. 75uL of the respective molar concentrations of the peptides (hBD2, 0, 0.5, 1, 2, 4, and 8 µM) and
(mBD3, 0, 10, 20, 200, 500nM) were incubated at 370C for 1h. Reaction was stopped by adding 400uL of buffer and 25uL of the suspensions were plated on YPD agar plates in triplicate (500 cells/plate). Control (0) was equal amount of 10mM NaPB buffer. After incubation for 48h at 300C, colonies were counted and data were expressed as percent
Candida killing using the formula as [1-(number of colonies after peptide treatment/colonies after inoculation with buffer only] X 100.
2.4. Experimental mice
Defb3-/- mice were acquired from MMMRC (UC Davis) through a material transfer agreement (MTA). Animal protocols were performed in accordance with Institutional
16
Animal Care Unit Committee (IACUC) protocol approved by the University of Toledo.
Genotyping of mice was carried out by GenoTyping Center of America.
2.5. RNA extraction, cDNA synthesis and gene expression studies
Liquid nitrogen flash-frozen tongue tissue were placed in Miltenyi gentleMACS™
M tubes with extraction buffer from illustra TM RNAspin Mini Kit (GE Healthcare). Tissues were homogenized using a Miltenyi gentleMACS™ Octo Dissociator. All subsequent extraction protocols were followed as described in GE Healthcare kit. After first strand cDNA synthesis was performed using 100ng RNA using Superscript III Kit, real-time quantitative PCR was performed using PerfeCTa SYBR® Green FastMix. Gene expression data was normalized to glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH).
2.6. Quantification of fungal burden
Fresh tissue samples collected from different mice cohorts were suspended in
500uL 1X PBS and homogenized using gentleMACS™ Octo Dissociator. 100uL of homogenate was plated on YPD plates and incubated at 30°C for 48h. Colonies were counted and data were expressed as colony forming units per gram of tongue tissue
(CFU/g).
2.7. Microscopy
Evos® FL microscope was used for tissue imaging and Candida cell count.
17
2.8. Neutrophil count
Tissue sections stained with hematoxylin and eosin (H&E) were imaged at 20x magnification. Based on relative position with respect to basal cells, tongue tissue was divided into Supra-basal and Sub-basal and neutrophils were counted with their respective locations. Neutrophils were identified based on their purple color and multi-lobed nucleus.
Data was expressed as neutrophil count at Supra-basal and Sub-basal for each mouse cohort.
2.9. Data analysis
Data were analyzed on Prism (GraphPad 7 Software), using ANOVA with Tukey’s multiple comparison test. Statistical significance was declared at P<0.05.
18
Chapter 3
Results
3.1. rMBD3 and rhBD2 exhibit candidacidal activity in vitro
To study candidacidal potency of recombinant human β-defensine-2 (rhBD2) and murine β-defensine-3 (rMBD3) peptides and determine their lethal dose 50 (LD50), we evaluated the Candida killing activities of the two peptides at different concentrations. We used nano-molar concentrations (10, 20,200 and 500nM) for mBD3 while µM concentrations (0.5, 1, 2, 4 and 8µM) for hBD2 (Fig. 3.1). Measured amount of C. albicans
(CAF2-1) cells in suspensions were incubated at 30°C with different concentrations of hBD2 or mBD3 in 10 mM sodium phosphate buffer with PH 7.4. After 1 h, the candidacidal activity was analyzed by plating 25uL of the suspension in triplicate and determined the colonies after 48h. Results were expressed as LD50 (Fig.3.1A&B). Results of treatment of C. albicans with the recombinant peptides showed marked difference between mBD3 and hBD2. The concentration of mBD3 necessary to kill 50% of the microorganisms (50% lethal dose, LD50) was 200nM while that of hBD2 was 6 micro Molar (Fig. 3.1 A&B).
19
A
1 0 0 8 0
g
g
n i
n 8 0
l
i
l l
l 6 0 i
i
k
k
6 0
a
a
d
d i
i 4 0 d LD50 d
n 4 0 n
a
a
C
C 2 0 2 0
% %
0 0 10 20 2 0 0 5 0 0 0 2 4 6 8 1 0 h B D 2 ( M ) m B D 3 [n M ] Fig. 3.1A, B. Candidacidal activities of recombinant mBD3 and recombinant hBD2. (A) mBD3 has more potent candidacidal activity than (B) hBD2 with LD50 of 200nM and 6µM concentrations, respectively. C. albicans cells were incubated with the indicated concentration of recombinant mBD3 (A) or hBD2 (B) at 370C for 1h and plated on YPD agar plate. Colonies were counted after incubation for 48h at 300C and % Candida killing was expressed as [1-(number of colonies after peptide treatment/colonies after inoculation with buffer only] X 100.
3.2. mDefb3 mice are susceptible to OPC
To assess the contribution of Defb3 in protection against OPC on mucosal surfaces in vivo, we challenged Defb3-/- mice and littermate controls (WT) with C. albicans. Cotton balls immersed in Candida suspension were placed sublingually for 75 minutes under anesthetic condition. Mice were administered 1mL of 0.9% saline at the beginning of infection when Candida containing cotton ball was placed sublingually and during removal of inoculation cotton balls. Mice were monitored until the end of each experiment and tongue tissue were harvested for quantification of fungal burden. WT mice treated with cortisone acetate (225 mg/kg i.p.) were included as susceptible control during the establishment of OPC mouse model at UT.
OPC infected cortisone acetate treated wild type mice (WT-OPC Cort) exhibited the highest fungal burden and it was evident early during infection (Fig.3.2). This was 20
followed by OPC infected mDefb3 mutant mice (Defb3-/- OPC) (Fig.3.2). WT-OPC Cort has previously been shown to be the highly susceptible to OPC (Conti et al., 2016). The kinetics of disease progress also showed that all Candida infected cohorts had similar level of colony forming units per gram of tongue tissue (CFU/g) at day 1 p.i. (Fig.3.2). For example, Defb3-/-OPC displayed similar fungal load as that of WT-OPC at day 1 p.i. but significantly increased at days 4 &5 p.i. (Fig.3.2).
3.3. mDefb3 regulates expression of mDefb1 and mDefb2 during oral Candida infection
In order to understand the mechanisms by which Defb3 contributes to immunity to
OPC, we studied gene expression pattern of selected genes previously reported to have potential involvement in antiCandida immunity in the oral mucosa (Conti et al., 2009). In this study, expression of murine beta defensin genes Defb3, Defb1, Defb2 and Defb14 were induced in WT-OPC upon Candida infection compared with WT-Sham (Figs. 3.3-3.6).
However, expression of all beta-defensin genes studied were impaired in mDefb3-/-OPC, except mDefb14, compared with WT-OPC (Figs. 3.3-3.6).
21
1 0 6 ** D e fb 3 -/-S h a m 1 0 5
e W T -S h a m u
s 1 0 4 s W T -O P C i
t **
U e 3 D e fb 3 -/-O P C
F 1 0
u
C g
n W T -O P C C o rt 2
o 1 0
t
g / 1 0 1
1 0 0 1 2 3 4 5 D a y
Fig.3.2. mDefb3 is important for protection against OPC. Indicated mice were subjected to OPC. Mice were euthanized on various days p.i. and tongue tissue was harvested, weighed and re-suspended in 500uL 1X PBS. Tongues were homogenized and 100uL of homogenate was plated on YPD agar plates. Statistical analysis was performed ** P<0.01(WT-Sham, n=4-5, Defb3-/- OPC, n=12-15, Defb3-Sham, n= 4-5, WT-OPC Cort, n= 8-10).The experiment was conducted twice.
The highest level of expression of Defb1 was at day 2 p.i both in WT-OPC as well
as Defb3-/- OPC but expression was significantly impaired (P<0.0001) in Defb3-/- OPC
suggesting regulation of mDefb1 by mDefb3 (Fig.3.4).
On the other hand, the highest mDefb2 expression was observed in WT-OPC at
day1 p.i. but was markedly suppressed in mDefb3-/-OPC (Fig. 3.5). Defb2 expression of
was attenuated in Defb3-/- OPC throughout the duration of the experiment (day1-5)
suggesting the correlation of Defb2 expression with susceptibility of Defb3-/- to OPC.
Similarly, Tomalak et al. (2015) reported importance of mDefb1 and mDefb2 for Candida
clearance during OPC.
mDefb14 expression was highly induced upon Candida infection in both WT as
well as Defb3-/- mice (Fig. 3.6). However, the expression pattern of Defb14 differed
22
between the infected cohorts. For example, Defb14 expression in WT-OPC was highest at days 1&2 but in Defb3-/-OPC it was suppressed at day 1 p.i (Fig.3.6). Moreover, Defb14 expression in mDefb3-/- OPC was significantly (P<0.0001) higher than that of WT-OPC which could suggest defects in mDefb3 expression was compensated by strong induction of mDefb14 (Fig. 3.6).
3.4.Defb3 regulates IL-17 and Th17 inductive cytokines IL1-β and IL-6
The important role IL-17 plays in Candida clearance during OPC has been well documented (Bishu et al., 2014; Conti et al., 2016; Conti et al., 2009). Moreover, proinflammatory cytokines IL1-β and IL-6 play a role as Th17 inductive cytokines (Bishu et al., 2014). Therefore, we studied gene expression pattern of IL.17, IL1-β and IL-6 in mDefb3-/- mice during OPC (Fig. 3.7-3.9). IL-17 expression was undetectable in WT-Sham as well as mDefb3-/- Sham in agreement with previous findings, which showed significant upregulation of IL-17 during OPC (Trautwein-Weidner et al., 2015). IL-17 was strongly induced during OPC both in WT-OPC (day1&2 p.i) as well as Defb3-/-OPC (day1 &4) compared with their respective non-infected (sham) cohorts. However, IL17 expression was significantly attenuated (P<0.0001) in Defb3-/-OPC (Fig. 3.7). The highest expression
IL-17 in WT-OPC as well as its suppression in Defb3-/-OPC correlated with the pattern of infection (Fig.3.7).
IL1-β was highly induced in WT-OPC at day1 p.i. but markedly declined after day1p.i (Fig. 3.8). Similarly, IL-1 β expression in Defb3-/- was induced at day1 but declined after day1 p.i. (Fig. 3.8). However, IL1-β expression in response to Candida infection was 23
significantly compromised (P<0.0001) in mDefb3-/-OPC compared with WT-OPC
(Fig.3.8).
The gene expression pattern of IL-6 showed that it was strongly induced at day1 p.i
(Fig.3.9) in both WT-OPC as well as Defb3-/- OPC but declined after day1 p.i (Fig.3.9).
However, IL-6 was significantly attenuated in Defb3-/- OPC suggesting its regulation by
Defb3 during oral candidiasis.
3.5. mDefb3 regulates CCL20 and CXCL2 but not CXCL1
Previous studies revealed that during Candida infection, immunocompetent mice express
Th17 signature genes (Conti et al., 2009; Saus et al., 2010) including chemokines such as
CCL20, CXCL1, CXCL2 and CSF3 that are involved in regulation of neutrophil recruitment to infection sites (Trautwein-Weidner et al., 2015). Further studies also corroborated strong induction of neutrophil-recruiting chemokines CXCL1 and CXCL2 during oral Candida infection. Therefore, due to the vital role of neutrophils during infection, we wanted to study expression pattern of neutrophil chemokines involved in neutrophil trafficking in
Defb3 deficient mice (Figs. 3.10-3.13). Moreover, we studied the number of neutrophils trafficked into infection site in H&E stained tongue tissue in C. albicans infected mice cohorts (Fig.3.14 - 3.15).
24
Fig.3.3. Gene expression changes during OPC show the involvement of Defb3 in Candida clearance. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n=4-6). D1-D5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test.
25
Fig.3.4. Gene expression changes during OPC show Defb3 regulates Defb1 during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test.
26
Fig.3.5. Altered gene expression during OPC show Defb3 regulates Defb2 during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test.
CCL20 was highly induced during OPC both in WT as well as in Defb3 deficient mice (Fig. 3.10). In WT-OPC, CCL20 was highly induced at day1&2p.i. but declines significantly after day2p.i. (Fig.3.10). On the contrary, CCL20 expression was impaired in
Defb3-/-OPC at day 1 but significantly (P<0.0001) upregulated at day2 compared with WT-
27
OPC. Enhanced CCL20 transcript accumulation in mDefb3-/- OPC at day 2 p.i. could be a compensatory mechanism for defects in mDefb3 (Fig.3.10). On the other hand, CXCL1 expression in WT-OPC was significantly upregulated at day1 &2 but declines sharply after day2 p.i. (Fig.3.11).
Similar, in Defb3-/--OPC, there was a sharp increase in expression of CXCL1 at day1 but declines significantly after day2 p.i.(Fig. 3.11). However, there was no statistically significant (P<.0.05) difference between expression CXCL1 in WT-OPC
(day1, 2) and Defb3-/-OPC (Fig. 3.11). This could suggest that mDefb3 does not regulate
CXCL1.
It has been shown that neutrophil recruiting chemokine CXCL2 is highly induced during OPC (Altmeier et al., 2017). Therefore we studied gene expression profile of
CXCL2 in order to understand whether or not Defb3 mediated immunity against OPC involves CXCL2 (Fig. 3.12).
28
Fig.3.6. Gene expression changes during OPC show Defb14 upregulation in Defb3-/- mice during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test
29
Fig.3.7. Altered gene expression during OPC show Defb3 regulates IL-17 expression during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test
30
Our result showed that CXCL2 was significantly upregulated (P<.0001) in WT-OPC at day1 p.i but there was a sharp decline after day1 p.i. (Fig.3.12). However, CXCL2 was markedly impaired in mDefb3-/-OPC (Fig.3.12) suggesting its regulation by mDefb3 during
OPC.
Csf3, the gene coding for G-CSF, has been shown to be induced during OPC
(Altmeier et al., 2016 and Conti et al., 2009). In our study, WT-OPC showed statistically significant (P<0.0001) induction upon Candida infection at day1 p.i but declined after day1 p.i. (Fig. 3.13). On the other hand, Csf3 expression was attenuated in Defb3-/-OPC compared with WT-OPC although there was significant upregulation of the gene in Defb3-
/- OPC at day2,4 p.i. compared with the sham cohorts. Moreover, Csf3 induction started at day2 p.i in Defb3 -/-OPC (Fig.3.13). The results could indicate that mDefb3 regulate Csf3 during OPC (Fig. 3.13).
31
Fig.3.8. Gene expression changes during OPC show Defb3 regulate IL1b expression during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test
32
Fig.3.9. Gene expression changes during OPC show Defb3 regulate IL-6 expression during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by ANOVA and Tukey's multiple comparisons test
33
Fig.3.10. Gene expression changes during OPC show CCL20 compensates Defb3 deficiencies during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by an ANOVA and Tukey's multiple comparisons test
34
3.5. Immunohistochemical analysis show Defb3-/- mice do not elicit defects in neutrophil
extravasation
Owing to the importance of neutrophils during infection, we studied potential neutrophil migration defects in tongue tissues of Defb3 deficient mice and WT controls.
Hematoxylin and eosin (H&E) stained tongue tissue sections were evaluated for number of neutrophils migrated to supra and sub-basal tongue sections (Fig. 3.14 A-D). Supra- basal neutrophils indicate those located above the basal cells towards the dorsal surface of the tongue. The results of tissue imaging as well as neutrophil quantification data show that mDefb3 deficient mice were not defective in neutrophil migration to site of infection (Fig.
3.14A,B-3.15A,B).
35
Fig.3.11. mDefb3 does not regulate CXCL1 in Defb3-/- mice during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ns=statistically not significant at p < 0.05 by an ANOVA and Tukey's multiple comparisons test.
36
Fig.3.12 Altered gene expression during OPC show Defb3 regulate CXCL2 during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by an ANOVA and Tukey's multiple comparisons test.
37
Fig.3.13 Altered gene expression during OPC show Defb3 regulate Csf3 during OPC. RNA extracted from tongue tissue harvested from C. albicans infected mice or control (Sham) was used for gene expression studies (qPCR, n= 4-6). Day1-Day5= Days p.i. Means of scatter plots indicate mean + SEM. ****p < 0.0001 by an ANOVA and Tukey's multiple comparisons test.
38
-/- Defb3 OPC WT-OPC
A B
Supra
Sub Day1
Day2
Figure 3-14 A, B. mDefb3 in oral mucosa does not regulate neutrophil migration during OPC. Representative sections of each cohort with the supra and sub basal layers in indicated. Tissue section were stained with H&E, imaged at 20x magnification and neutrophils were separately counted for each Sub and Supra-Basal locations. Blue arrows indicate neutrophils.
39
-/- Defb3 OPC WT-OPC
C D
Supra Sub Sub Supra
Figure 3-14 C, D. Defb3 in oral mucosa does not regulate neutrophil migration during OPC. Representative sections of each cohort with the supra and sub basal layers in indicated. Tissue section were stained with H&E, imaged at 20x magnification and neutrophils were separately counted for each Sub and Supra-Basal locations. Neutrophils have cleared by day3 p.i.
40
A
1 0 6
5
t 1 0
W T -O P C S u p ra -B a s a l n
u 4
o 1 0 D e fb 3 -/-O P C S u p ra -B a s a l
c
l i 3
h 1 0
p
o r
t 1 0 2
u e
N 1 0 1
1 0 0 1 2 3 D a y B
1 0 6
5
t 1 0
W T -O P C S u b -B a s a l
n u 4
o 1 0 D e fb 3 -/-O P C S u b -B a s a l
c
l i 3
h 1 0
p
o r
t 1 0 2
u e N 1 0 1
1 0 0 1 2 3 D a y
Figure 3-15 A, B. mDefb3 in oral mucosa does not regulate neutrophil migration.
Quantifications of images as seen in Figure 3-14A, B.
41
Chapter 4
Discussion
4.1. rmBD3 and rhBD2 show potent candidacidal activity
Production of AMPs is one of the earliest innate immune response during mucosal fungal infections suggesting their involvement as a first line of defense against pathogenic microorganisms (Tomalak et al., 2015). AMPs including human β- defensins 1-3 are shown to have fungicidal activities (Tomalak et al., 2015). Defensins have been of research interest due to their antimicrobial properties and immunomodulatory role (Vylkova et al.,
2007). Furthermore, correlation between high susceptibility to OPC in IL-17 receptor knockout and decreased Defb3 expression has been established (Conti et al., 2016). Thus, we conducted in vitro and in vivo experiments to study the role of Defb3 in protection against oral candidiasis.
Mouse β-defensin-3 (mBD3) is the murine homolog of human β-defensin-2 (hBD-
2), and is encoded by the mDefb3 gene. It is mapped to the proximal portion of chromosome 8 and consists of two exons separated by a 1.7-kb intron. (Bals et al., 1999). mBD3 is expressed at a low basal level but highly induced in the epithelia of various organs upon pathogen infection (Bals et al., 1999). In our study, both recombinant mBD3 and hBD2 showed antimicrobial activities to C. albicans (Fig. 3.1 A&B). Previous studies also 42
showed antifungal activities of recombinant mBD3 (Jiang et al., 2009) as well as recombinant hBD2 (Joly et al., 2004). Studies on Candida killing capacity of hBD 1-3 also showed that candidacidal activity of recombinant hBD2 to be the most potent followed by hBD3 andhBD1 in their order (Feng et al., 2005)..
Candidacidal potency of recombinant mBD3 in this study was about 30 fold higher than that of hBD2 (0.2µM vs 6 µM) of mBD3 and hBD2, respectively. Similar activities were reported for hBD2 (Vylkova et al., 2007). While the observed candida killing activities between hBD2 and its murine homolog mBD3 in this experiment could confirm their functional similarity as reported earlier (Jiang et al., 2010), such pronounced potency differences could be attributed to low sequence similarities (only 40% amino acid similarity) between them. Earlier studies on characterization of mBD3 and hBD2 also revealed their structural and functional conservation but low sequence identity (Bals et al.,
1999).
Beta defensins use different mechanisms to limit fungal growth. They inhibit of fungal adherence to epithelial cells (Feng et al., 2005; Niyonsaba et al., 2004) as well as disrupt fungal cell membrane (Feng et al., 2005).
Lack of effective vaccines and increasing problems of drug resistant fungal isolates is challenging the fight against the increasing incidence of fungal infections. Moreover, targeting the IL-17 pathway as therapeutics for the treatment of autoimmune diseases such as Rheumatoid Arthritis (RA) could exacerbate the problems of fungal infections. 43
Therefore, AMPs could be potential antifungal therapeutic target either as broad-spectrum antimicrobial treatment or as antifungal treatment without excessive proinflammatory side effects when α-IL-17 treatments are used.
4.2. mDefb3 is necessary for innate response to OPC
Defects in the expression of AMPs in mouse is associated with high susceptibility to Candida infection on mucosal surfaces (Tomalak et al., 2015 and Conti et al., 2009).
This has been attributed mainly to direct antifungal properties of salivary AMPs in the oral mucosa such as mBD1 and mBD3 (Joly et al. 2009; Tomalka et al. 2015; Conti et al.,
2009). However, studies on antiCandida activity of mBD3 has mainly been in vitro
(Edgerton et al., 2000). Therefore, we studied the role of mDefb3 in protection against OPC in vivo. Here, we show that mDefb3 is necessary for innate immune response to OPC. In our study, mDefb3-/- OPC mice sustained high fungal burden compared with WT-OPC (Fig.
3.2) suggesting the involvement of Defb3 in protection against OPC. However, unlike WT mice with C57BL/6 background which clears Candida at day3 p.i. (Tomalka et al., 2015 and Conti et al. 2016), WT-OPC (littermate controls) did not clear infection at day3 p.i. which could be due to their mixed genetic background (129sv/C57BL/6) (Fig.3.2). While increased susceptibility of Defb3-/- at day4 &5 p.i. show the contribution of Defb3-/- in protection against OPC, the observed similarity of fungal loads (CFU/g) between Defb3-/-
OPC and WT-OPC during the first three days could either be due to protection conferred by innate immune response compensating for deficiency of Defb3 during the first days until it was breached after day3 (Fig.3.2). Similar pattern was reported in OPC susceptible
IL-17 conditional deletion mutant (IL-17RA ΔK13) mice (Conti et al., 2016). 44
4.3. mDefb3 contributes to immunity against OPC via regulation of AMPs, pro-
inflammatory cytokines and chemokines
Due to the high mortality rate of immunocompromised patients mainly due to secondary infections involving fungal infections such as C. albicans, development of antifungal therapies has been a point of research interest. BD3 has been one of the effector molecules whose increased expression level correlate with Candida clearance (Conti et al.
2016; Conti et al., 2009). Thus, to understand the role of beta defensins (BDs) in Candida clearance in Defb3 deficient mice during OPC, we studied kinetics of expression of oral mucosa related IL-17 target genes with possible antiCandida effector functions during mucosal candidiasis. These include AMPs such as mBD1, mBD2, mBD14 and mBD3 that are important regulators of the innate immune system (Rohrl et al. 2010).
Cytokines and pathogens are potent inducers of AMPs. Moreover, commensals induce AMPs. For example, commensal bacteria are shown to be potent inducers of hBD2
(Dale et al., 2006) which could be one mechanism by which human immune system exerts antiCandida immunity in immunocompetent humans. mDefb3 is induced by various stimuli including proinflammatory cytokines and microbial infections (reviewed in (Navid et al. 2012). mDefb3 is highly induced in lung, liver, renal tissue, esophageal tissue and tracheal tissue after stimulation in addition to its expression in unstimulated tissues such as tongue, skin and footpads (Jiang et al., 2009 and Conti et al. 2009b).
45
Besides direct candidacidal activity, beta defensins elicit protection against C. albicans infection via recruitment of neutrophils, dendritic cells and T-cells (Feng et al
2005; Niyonsaba et al 2004; Yang et al., 1999). For example, hBD2 has been shown to be chemotactic to immature dendritic cells and memory T cells via binding to CCR6 (Wu et al., 2003). Moreover, β-defensins regulate other AMPs (Tomalka et al., 2015) .
Previous studies in IL-17RAKO mice as well as conditional deletion of IL-17RA in superficial oral and esophageal epithelial cells (Il17raΔK13) also showed correlation between reduced expression of Defb3 and susceptibility to OPC (Conti et al. 2009b; Conti et al., 2016). Moreover, it is interesting to see that following high induction of mDefb3 in
WT-OPC at day2&4 p.i, the fungal burden was reduced dramatically which could be due to the involvement of mBD3 in fungal clearance during OPC (Figs3.2&3.3). Moreover, mDefb1 was also impaired in mDefb3-/-OPC at day1 p.i (Fig. 3.4) in accordance with its established role in Candida clearance (Tomalka et al. 2015). On the other hand, Defb2 expression was attenuated in Defb3-/- OPC throughout the duration of the experiment
(day1-5) suggesting the correlation of Defb2 expression with susceptibility of Defb3-/- to
OPC. Similarly, Tomalka et al. (2015) reported the importance of mDefb1 and mDefb2 for
Candida clearance during OPC. Based on our findings and previous reports, it is evident that both mDefb1 (Tomalak et al., 2015) and mDefb3 (Fig.5) regulate mDefb2 in protection against OPC. While regulation of mDefb2 both by mDefb3 and by mDefb1 show complex interaction of beta defensins in providing protection against OPC, it could also show the role of mBD2 as important effector protein in candida clearance. Although mDefb3
46
regulates mDefb1 (Fig.3.4), mDefb1 does not regulate mDefb3 (Tomalak et al., 2015) during OPC. Further studies on synergistic of mDefb2 and mDefb3 is recommended.
mDefb14 is the functional homolog of hDefb3 and is expressed in the tongue, thymus, tonsil, and kidney (Hinrichsen et al., 2008). mBD14 has broad antimicrobial activity against different species of bacteria and the fungus C. albicans (Hinrichsen et al., 2008).
Although increased expression levels of beta defensins such as mDefb14 in OPC susceptible mice was not expected, there is no indication of its contribution to fungal clearance. Thus, it could simply show active immune response due to continued high fungal load in Defb3 deficient mice as the mice are susceptible to OPC (Simpson-Abelson et al.,
2015.
Research findings have identified IL-17A as major proinflammatory cytokine regulating protection against OPC (Conti et al., 2016) via regulation of different cytokines, chemokines and various antimicrobial peptides such as beta defensins at mucosal surfaces
(Aujla, Dubin, and Kolls 2007; Altmeier et al., 2016) including hBD2 (Liang et al. 2006 and Kao et al. 2004) and mDefb3 (Conti et al., 2009b). IL-17 exerts its activity via its receptor signaling as binding of IL-17 to the IL-17RA promote expression of downstream target genes by activation nuclear factor kappa-B(NF-κB ) via the adaptor protein Act1, tumor necrosis factor receptor-associated factor 6 and the CCAAT/enhancer-binding proteins C/EBP-β (Trautwein-Weidner et al., 2015).
47
IL-17 expression was attenuated at day1p.i in mDefb3-/-OPC (Fig. 3.7) consistent with its role in protection against OPC. This could suggest the role of Defb3 in regulating
IL-17 during OPC. Increased IL-17 expression in Defb3-/-OPC at day 4, after low expression at day 2&3 p.i, compared with WT-OPC could show the activity of antifungal immune response but it was not enough offset susceptibility to OPC (Fig.3.7). In addition to IL-17 mediated antifungal immunity via regulation of AMPs (Conti et al., 2009), our finding show that mDefb3 also regulate IL-17 (Fig. 3.7). This gives an additional insight into the regulation of IL-17 mediated immunity to OPC via mDefb3-regulated expression of IL-17. Moreover, it is known that chemokine receptor 6 (CCR6) is highly expressed in
Th17 cells (Yamazaki et al., 2008) and earlier studies suggested the vital role of beta defensins interaction with CCR6 as a possible means of their chemotactic activities (Yang et al., 1999). Therefore, mBD3 could play a chemo-attractant role to IL17 producing Th17 cells to regulate IL-17 mediated immunity against OPC.
IL1b, IL-6 and transforming factor beta (TGF-β) initiate expression of IL-17 (Liang et al. 2006). Thus defect in any one of IL1b, IL-6 or TGF-β could affect expression of IL-
17 there by suppresses antifungal immunity. Given IL1-β is necessary for differentiation of CD4+ Th cells into IL-17 producing Th17 cells and IL-17 as critical for antifungal host defense, it is not surprising to see reduced expression levels of IL1-β and IL-17 (Figs.
3.7&3.8). Moreover, low expression level of IL-6 cytokine (Fig.3.9 ) involved in differentiation of CD4+ Th17 cells into IL-17 producing Th17 cells could show mDefb3 mediated regulation of IL-17 could also be via its effect on differentiation Th17 cells.
48
Chemokine CCL20 is a cysteine-cysteine (CC) inflammatory chemokine produced by various cell type including Th17 cells, neutrophils, natural killer cells, B cells, dendritic cells and macrophages (Zhao et al. 2014; Guesdon et al. 2015). CCL20 is known to bind with high specificity to CC-chemokine receptor CCR6 (Zhao et al. 2014; Guesdon et al.
2015) which in turn activates the receptor (Hoover et al. 2002). CCL20 is highly inducible under inflammatory conditions (Zhao et al. 2014). CCL20 functions both as chemotactic and antimicrobial (Guesdon et al. 2015). As chemokine, CCl20 upregulation modulates trafficking of leucocytes such as immature DCs, effector or memory CD4+ T lymphocytes and B-lymphocytes to infection sites. Thus, CCL20/CCR6 axis is involved in recruitment of various immune cells to infection site (Yamazaki et al., 2008).
IL-17RA signaling regulate CCL20 expression during OPC (Conti et al. 2009b;
Zhao et al. 2014) and its expression is attenuated in IL-17RAKO (Conti et al. 2009b).
However, our results show strong induction of CCL20 in mDefb3-/- OPC compared with
WT-OPC (Fig.3.10). Moreover, previous studies have also shown chemotactic activity of mBD2 and mBD3 for murine immature DCs expressing CCR6 (Yang et al., 1999). As a result, hBD2 bind to CCR6 and compete with CCL20 for binding sites (Rohrl et al. 2010).
Therefore, the observed higher expression of CCL20 in Defb3 deficient mice could be
CCL20 compensating for defects in mDefb3 binding sites. CCL20 has additional function besides its chemotactic activities. Antimicrobial properties of CCL20 against various bacteria and fungi (Yang 2003), protozoan and candida (Guesdon et al. 2015) has been documented. However, in this study, there was no strong correlation between CCL20 expression levels and immune response to OPC in mDefb3-/- mice (Fig.3.10). 49
mDefb3-/- mice showed no defect in neutrophil chemokine CXCL1 expression (Fig.
3.11) but expression of CXCL2 in mDefb3-/-OPC was significantly (P<0.001) impaired compared with WT-OPC (Fig.3.12). Moreover, mDefb3 regulate Csf3 expression during
OPC (Fig. 3.13) but no correlation with neutrophil migration (Fig. 14-15). Csf3 has been suggested to contribute to innate immunity against OPC via involvement in increased granulopoiesis and neutrophil mobilization from the bone marrow (Altmeier et al., 2016).
However, there was no defect in neutrophil migration in Defb3-/- OPC in our study
(Fig.3.13-15). In similar studies, in IL-17RA or IL-17RC or mice depleted of cytokines
IL-17A and IL-17F with high susceptibility to OPC, there was no defect in granulocyte colony-stimulating factor (G-CSF), CXC-chemokine response and neutrophil recruitment or function (Trautwein-Weidner et al. 2015). Thus, mDefb3 mice show high susceptibility to OPC without defect in neutrophil migration suggesting Defb3 could clear C .albicans via neutrophil independent mechanisms as reported in IL-17 mediated immunity to OPC in oral epithelia (Altmeier et al., 2015). Moreover, IL-1 receptor (IL-1R) has been shown to modulate fungal clearance in oral mucosa via regulation of chemokine production at the sites of infection (Altmeier et al., 2015). Therefore, Defb3 as downstream effector in IL-
17R signaling could exerts its antiCandida effector mechanisms via different mechanisms including antimicrobial peptides without defects in neutrophil migration to infection sites.
Similarly, OPC-susceptible Defb1-/- mice also showed no neutrophil migration defects
(Tomalka et al., 2015).
In conclusion, although mDefb3 regulate expression of mDefb1, mDefb2, IL-17, IL-6, 50
IL-1β, CXCl2 and Csf3, its candidacidal activities seem to be correlated with expression of
AMPs (Defb1 and mDefb2) and proinflammatory cytokines (IL-17, IL-6 and IL1β) as impaired expression of CXCL2 and Csf3 did not deter neutrophil migration. Further studies on gene expression profile and characterization of networks of genes in mDefb3 deficient mice using next generation sequencing; and T-cell trafficking during OPC is recommended.
51
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