T helper 17 cell activation in response toCandida albicans in autoimmune polyendocrinesyndrome type 1

Iulia Karlsson

Degree project in biology, Master of science (2 years), 2010Examensarbete i biologi 45 hp till masterexamen, 2010Biology Education Centre and Biology Education Centre and Department of Medical Sciences,Uppsala University, Uppsala UniversitySupervisor: As. Prof. Anna Lobell

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Table of Contents

Abbreviations .........................................................................................................................2

Abstract..................................................................................................................................3

Introduction

Autoimmune polyendocrine syndrome type 1 .....................................................................4

Chronic mucocutaneous candidiasis....................................................................................4

Th17 cells...........................................................................................................................5

Previous findings ................................................................................................................5

Hypothesis and goals ..........................................................................................................6

Methods

Cell purification..................................................................................................................7

Cell culture .........................................................................................................................7

Intracellular cytokine staining.............................................................................................7

ELISpot ..............................................................................................................................7

Real-time RT-PCR .............................................................................................................8

Statistical analyses..............................................................................................................8

Results

Population of rare Th17 cells abtained by flow cytometry without ionomycin pre-stimulation ...................................................................................................................9

C. albicans increased the amount of IL-17A+ cells in controls and APS-1 patients..............9

Blocking of IL-1β and IL-6 cytokines did not modify Th17 reactivity.................................9

IL-17A contribution to pathogenesis.................................................................................10

The exacerbated Th17 cell reactivity in APS-1 is C. albicans-specific ..............................10

Treg cells number and Foxp3 mRNA expression were not affected by C. albicans stimulation, both in APS-1 patients and in controls......................................................10

Upregulated IFN-γ mRNA after stimulation with C. albicans ..........................................10

IL-1β and IL-6 mRNA levels vere not elevated by C. albicans stimulation ......................11

Discussion............................................................................................................................12

References............................................................................................................................14

Figures 1-7 ...........................................................................................................................16

Acknowledgements ..............................................................................................................23

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Abbreviations

AIRE autoimmune regulator

APECED autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

APS-1 autoimmune polyendocrine syndrome type 1

CMC chronic mucocutaneous candidiasis

FACS fluorescence-activated cell sorting

HCA human Candida albicans

PBMCs peripheral blood mononuclear cells

PGN peptidoglycan

TGF tumor growth factor

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Abstract

Rare autosomal recessive disorder - autoimmune polyendocrine syndrome type 1 (APS-1), also known as autoimmune polyendocrine candidiasis ectodermal dystrophy (APECED) - is caused by mutation in the autoimmune regulator gene and characterised by chronic mucocutaneous candidiasis (CMC), hypoparathyroidism and adrenal failure. CMC is C. albicans-caused infection that occurs in 100% of APS-1 patients, most often coming as initial symtpom and setting on very early in patient’s life. Patients in a constant need of antifungal drugs are most often on a life-long drug treatment, which creates a major risk to become colonized with drug-resistant strains. The main risk factors that predispose to such severe opportunistic infection are defects of cell-mediated immunity, including dysregulated Th-cell reactivity. Th17 cells are thought to be involved in several autoimmune and inflammatory conditions and could probably contribute to the pathogenesis of CMC in APS-1 as well. We studied Th17 reactivity ex vivo in response to C. albicans and other antigens in four APS-1 patients and sex- and age-matched healthy controls. After 3 days of stimulation we could detect C. albicans-specific enlargement of Th17-cell fraction, increase in IL-17A mRNA expression and a dramatic increase of IL-17A cytokine production (p < 0.05) in peripheral blood mononuclear cells (PBMCs) purified from APS-1 patients. In healthy controls the changes in corresponding cell population, mRNA and protein expression were significantly smaller compared to APS-1, suggesting an important role for Th17 cells and IL-17A in chronic mucocutaneous candidiasis accompanying APS-1.

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Introduction

Autoimmune polyendocrine syndrome type 1 (APS-1)

Autoimmunity is a state of an organism when its immune system attacks self matter. It is the result of a complex interaction between genetic and environmental factors, most of which have not been identified (Burek and Talor 2009). In human autoimmune diseases (as a group), a large number of antibodies and T cells directed against functional cell structures (e.g. nucleic acids, nuclear molecules, receptors) can be found. Autoantibodies and autoimmune T cells can also be present in other clinical conditions, such as cancer or acute tissue damage, as well as in individuals with no clinical manifestations of a disease. In order to avoid autoimmune conditions, B and T cells must bypass multiple checkpoint mechanisms during their maturation, and the failure of both central and peripheral tolerance mechanisms is favouring the development of autoimmunity.

In APS-1, one mechanism of central tolerance – negative selection – is impaired, which allows self-reactive thymocytes to escape clonal deletion and cause substantial damage to tissues and organs of the body. Several genes expressed by medullary thymic epithelial cells are important for maintenance of self-tolerance, and autoimmune regulator (AIRE) is shown to be one of them (Peterson, Org et al. 2008). Aire-deficient (Aire0/0) mice demonstrate a role for Aire in negative selection of T cells in the thymus (Anderson, Venanzi et al. 2005) through activating the expression of peripheral tissue-specific antigens. Additional roles for Aire in negative selection have also been reported (Mathis and Benoist 2007).

Mutations in AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED; also known as APS-1), which is a rare monogenic recessive disorder characterized by autoimmune processes localized at endocrine glands and caused by the presence of autoantibodies and T helper (Th)-cells specific for multiple self antigens (Peterson, Org et al. 2008). Several endocrine glands and other organs may be destroyed or damaged by the patient’s immune system (Rautemaa, Hietanen et al. 2007). Most commonly, patients with APS-1 have chronic mucocutaneous candidiasis (CMC), hypoparathyroidism and Addison’s disease, although the range of clinical features varies among the patients (Perheentupa 2006).

Chronic mucocutaneous candidiasis (CMC)

CMC in APS-1 patients is caused by Candida albicans (C. albicans) infection, including localised invasion of the mucosa that can lead to oral and oesophageal squamous cell carcinoma (Rautemaa, Hietanen et al. 2007). The molecular basis of the increased susceptibility to CMC in APS-1 patients is still poorly understood.

C. albicans is a dimorphic organism capable of colonizing, infecting and persisting on mucosal surfaces. It is a human commensal fungus that under certain circumstances can invade mucosa through a phenotype switch from yeast to hyphal form, and may seek to induce a protective host immune response to permit its own survival (Romani 1999). Among

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the fungal pathogens, Candida spp. are the most predominant causes of invasive infections and C. albicans is the most common causative species of candidaemia (Shao, Huang et al. 2007).

In adult immune-competent individuals, immune defence against fungal infections is achieved through a cooperative effort of the innate and adaptive immune systems. When infectious fungi can breach early lines of defence, generation of a dominant Th1-cell response mediated by interleukin-12 (IL-12) will ensue. Through production of the signature cytokine interferon-gamma (IFN-γ) and providing help for opsonizing antibodies, Th1 cells can optimally activate phagocytes at cites of infection. Dendritic cells producing IL-10 and T regulatoty (Treg) cells activated by this cytokine are needed to provide a proper coordination and regulation of overall immune response against fungi (Romani 2008). Unopposed Th1 responses may generate unnecessary tissue damage, elaborate Th2 cytokines (IL-4; IL-6; IL-13) and lead to chronic infections or allergic responses (Hohl, Rivera et al. 2006). Therefore, regulatory mechanisms are important to establish host protection against fungus.

The main risk factors that predispose to severe C. albicans infections are defects of cell-mediated immunity, including dysregulated Th-cell reactivity (Romani 2008). These defects occur in several disorders and immune compromised states in humans, such as HIV and immuno- and myelo-suppressive therapies for autoimmune and neoplastic diseases (Patterson 2005). Patients in a constant need for antifungal drugs are also at risk to become colonized with drug-resistant strains.

Th17 cells

For the last decades, effector CD4+ T cells have been thought to branch into nothing but two cell lineages - Th1 and Th2 cells, with lineage characteristics based on the cytokine expression profile, and lineage commitment determined by cytokine milieu. However, recent data demonstrate that a new Th-cell subset, IL-17-producing CD4+ T cells, have emerged to have crucial role in immunity towards pathogens (Dong 2006). These effector cells are commonly called Th17 cells (Harrington, Hatton et al. 2005; Isaksson, Ardesjö et al. 2009; Sharma, Hou et al. 2009; Yun, Binyan et al. 2009). Their development is dependent on IL-23, IL-21, IL-1, IL-1β, IL-6, transforming growth factor β (TGF-β), transcription factors RORC (in humans) and ROR-γt (in mice). They are claimed to produce IL-17 (also called IL-17A), IL-17 F, IL-21 (Miossec, Korn et al. 2009) and IL-22 (Yun, Binyan et al. 2009) both in mice and humans.

IL-17 is associated with the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and asthma, and is mainly involved in host defence against bacterial and fungal infections (Dong 2006). Autoimmunity and inflammation are thought to be induced by overexpression of IL-17. Therefore the cell population that preferentially produce this cytokine is often called pathogenic (Langrish, Chen et al. 2005).

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Previous findings

Peripheral blood mononuclear cells (PBMCs) isolated from APS-1 patients and sex- and age-matched healthy controls and cultured with C. albicans revealed increased expression of the cytokine IL-17A and mRNA expression of IL-17A, IL-1β, IL-6 and RORC. Data obtained from Aire0/0 mice, the animal model for APS-1, shows an increased Th17 cell activation together with elevated susceptibility to C. albicans infection in an induced model of candidiasis.

The elevated expression of cytokines IL-6 and IL-1β is thought to somehow contribute to IL-17A enhanced production and increased patient susceptibility to candidiasis (Ahlgren, K. et al. unpublished data).

Hypothesis and goals

The major goals of this study are:

1. Development of a method to directly measure human Th17 cells

a. Intracellular staining of IL-17A in Th cells by flow cytometry. b. ELIspot assay using anti-IL-17A coated plates. c. Cell sorting by flow cytometry followed by RNA isolation, cDNA synthesis and real-

time PCR.

2. Investigate the mechanism behind the enhanced Th17 reactivity in APS- 1 patients

a. Block IL-6 and IL-1β in cultures with specific antibodies. Is the IL-17A expression altered after the ablation?

b. Compare the responces to C. albicans and other antigens. Are there significant differences that could suggest the specificity of Th17 response?

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Methods

Cell purification

Blood samples were obtained from healthy donors buffy coats (Blodcentralen, Academic hospital,Uppsala), from venal bleeding of four APS-1 patients, one CMC patient negative for APS-1 and sex- and age-matched healthy volunteers. APS-1 patients were on the life-long anti-fungal therapy and most of them became therefore infected with resistant strains. AIRE-intact CMC patient was HIV-negative. Blood from patients and healthy volunteers was collected into sodium heparin –coated tubes (456051; Vacuette) by authorised personnel.

PBMCs were isolated from fresh blood (within 12 h after bleeding) by Ficoll-Paque™ Plus (17-1440-02; GE Healthcare) according to manufacturer’s instructions. To determine the concentration of PBMCs, purified cells were dissolved in PBS + 2 mM EDTA and counted through Burker chamber or/ and NucleoCounter™ (900-0001; ChemMetec A/S).

Cell culture

Cells were cultured in TB-1 Clonal Express Lymphocyte Medium (C-78610; PromoCell, Heidelberg, Germany) supplemented with 1 % L-glutamine and 1 % heat-inactivated human serum in the presence or absence of the highly virulent C. albicans strain CA 6 (1 CFU per cell; isolated from a clinical specimen, grown to obtain pure germinated yeasts, heat inactivated), Mycobacterium tuberculosis H37RA (5 µg/ml; Difco, BD Diagnostic systems, Sparks, MD), PGN from Staphylococcus aureus (10 µg/ml; tlrl-pgnsa; InvivoGen) or zymosan - cell wall from Saccharomyces cerevisiae (10 µg/ml; tlrl-zyn; InvivoGen). Where indicated, recombinant human interleukin-1 β (IL-1β; 10 ng/ml; PHC0815; Invitrogen BioSource), anti-human IL-1β neutralizing antibody (10 µg/ml; AB-201-NA; R&D Systems) or anti-human interleukin-6 neutralizing antibody (10 µg/ml; AB-206-NA; R&D Systems) were added to cultures. Alternatively, cells were cultured in medium without additional stimuli. After three days of culture in 24-well plates, at 37 °C (98 % rh, 5 CO2), in 2 x 106

cells/ ml concentration and in 1 ml volume, cells were harvested and washed in PBS.

Intracellular cytokine staining

Intracellular staining for IL-17A, Foxp3 and IFN-γ was performed on PBMCs stimulated for three days with various stimuli in the presence of GolgiPlug (BD Biosciences) for the last five hours of culture. CD3-FITC- and CD4-PerCP-stained cells were fixed and made permeable with BD Cytofix/Cytoperm™ Solution (554722; BD Biosciences) according to manufacturer’s instructions. Cells were incubated with PE-labeled anti-IL-17A (12-7178; eBioscience) and Alexa Fluor® 647 anti-Foxp3 (560045; BD Pharmingen) or with PE-labeled anti-IFN-γ (554552; BD Pharmingen), then cells were washed and data were acquired on a FACS Canto II (BD Biosciences) and analyzed with FACSDiva Version 6.1.3 software. For compensations, fluorochrome positive controls and unstained control, BD CompBead Plus (560497; BD Biosciences) was used according to manufacturer’s instructions. A fraction of experimental cells was also applied as unstained control, isotype controls for PE-labeled

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mouse γ1 PE (345816; BD Biosciences) and Alexa Fluor® 647-labeled Rat IgG1, k (557731; BD Biosciences).

ELIspot

ELISpotPLUS assay kit (Mabtech) for human IL-17A was used on PBMCs (4 x 105 cells/ well at 2 x 106 cells/ ml) cultured with or without various stimuli (see Cell culture) for three days to enumerate cytokine-producing cells per well.

Real-time RT-PCR

Total RNA were extracted from cell lysates using RNeasy mini kit (Qiagen) according to manufacturer’s instructions. Random hexamers and Superscript III Reverse Transcriptase kit (Invitrogen) were used for cDNA synthesis. Transcripts were quantified using Quanti-Tect™ SYBR® Green chemistry according to the manufacturer’s instructions (Qiagen) and amplification was performed using real-time quantitative PCR on MyiQ Cycler (Bio-Rad laboratories). The PCR primers were as follows (designed with Beacon Designer and produced by CyberGene AB): forward primer 5’-CGGCTACCACATCCAAGGAA-3’ and reverse primer 5’-GCTGGAATTACCGCGGCT-3’ for 18s rRNA; forward primer 5’-TTCTCTGCATCACCACATGC-3’ and reverse primer 5’-TGTGAGCGATCACCATCTTC-3’ for GusB; forward primer 5’-CAGATGTAGCGGATAATGGAACTC-3’ and reverse primer 5’-AAAGGAGACAATTTGGCTCTGC-3’ for IFN-γ; forward primer 5’-ATTGGAAGAAACAACGATGACTCC-3’ and reverse primer 5’-TGTCCTCAGAATTTGGGCATCC-3’ for IL-17A; forward primer 5’-ACTACTTCAAGTTCCACAACATGC-3’ and reverse primer 5’-GAGTGTCCGCTGCTTCTCTG-3’ for Foxp3; forward primer 5’-TATAGCCTGGACTTTCCTGTTGTC-3’ and reverse primer 5’-GCTGACTGTCCTGGCTGATG-3’ for IL-1β; forward primer 5’-CCTCTTCAGAACGAATTGACAAAC-3’ and reverse primer 5’-TTTCAGCCATCTTTGGAAGGTTC-3’ for IL-6. The PCR protocol for SYBR Green real-time PCR was at 95 °C for 13 min, followed by 50 or 55 cycles of denaturation for 15 s at 95 °C, annealing step of 30 s at 60 °C and extension step of 30 s at 72 °C. For each sample, mRNA concentration is expressed as arbitrary units and normalized by deviding the mRNA starting quantity (SQ)-value of the gene of interest over SQ-value of 18s rRNA..

Statistical analysis

Unpaired two-tailed t-test was used for statistical analysis; p values of 0.05 or less were considered significant. GraphPad Prism™ software was used for the statistical analysis. The pilot data and recall data reported were obtained from two to seven experiments.

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Results

Population of rare Th17 cells obtained by flow cytometry without ionomycin pre-stimulation

The difficulties in detecting IL-17A+CD3+CD4+ T Th17 cells by flow cytometry, encountered in previous experiments conducted in our group (Ahlgren, K. et al. unpublished data), inspired us to improve the method. Instead of a solution containing GolgiPlug, Ionomycin, Phorboldibutyrat and PBS and inducing cytokine expression, protein transport was inhibited by adding GolgiPlug alone in concentration recommended by manufacturer. In addition, distinct flourochromes were applied to stain for cell markers of Th17 cells (see Materials and methods). We obtained distinguishable populations of CD3+CD4+ T cells (Fig. 1 A, B) and avoided auto-fluorescence without substantial loss of relevant cells (Fig. 1 B). However, the frequency of Th17 cells was less than one percent and thus very low (Fig. 1 C, D). The frequency of CD4+ T cells stained with isotype control was in the same range as the IL-17A-stained CD4+ T cells.

C. albicans increased the amount of IL-17A+ Th cells in controls and APS-1 patients

To study whether there is an unbalanced Th17 cell reactivity that facilitates susceptibility to CMC in humans caused by C. albicans, we collected PBMCs from APS-1 and AIRE+/+ CMC patients (CMC patients that have no mutations of AIRE), healthy controls and blood donors. We then cultured these cells in the presence or absence of C. albicans.

First, IL-17A mRNA expression and the number of Th17 cells were measured after 3 days of stimulation. In blood donors, we could detect high mRNA levels of IL-17A in C. albicans-stimulated compared to unstimulated samples (p = 0.01; Fig. 2 B), whereas the number of Th17 cells, as measured by flow cytometry, did not differ significantly (Fig. 2 A). Importantly, we could detect higher numbers of Th17A mRNA in both APS-1 patients and sex- and age-matched healthy controls after C. albicans stimulation compared to unstimulated samples (Fig. 2 C). This comes in agreement with our previous data suggesting an important role for IL-17A in antifungal immune response. Unexpectedly, AIRE+/+ CMC patient had no IL-17A mRNA expression, both in C. albicans-stimulated and unstimulated samples (Fig. 2 D).

Blocking of IL-1β and IL-6 cytokines did not modify Th17 reactivity

Our preliminary data showed higher IL-1β and much higher IL-6 mRNA expression levels in PBMCs isolated from APS-1 patients in response to C. albicans. To examine whether IL-1β and/or IL-6 cytokines are responsible for the observed enhanced C. albicans-induced Th17 reactivity, monoclonal antibodies against these cytokines were added to PBMCs cultures in the presence of C. albicans. In addition, IL-1β recombinant protein was cultured with donor PBMCs and C. albicans in attempt to mimic some APS-1 characteristics in vitro. After three days, mRNA expression of IL-17A, IFN-γ, Foxp3, IL-1β and IL-6 was measured in these samples and the changes in Th17, Th1 and Treg cell frequency in response to blocking and stimulation were examined. Neither Th17 cell expansion (Fig. 2 A) nor IL-17A mRNA

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elevated expression (Fig. 2 B) towards C. albicans were altered in the presence of anti-IL-6, anti-IL-1β or recombinant IL-1β protein. The same result was obtained for Th1 proliferation and IFN-γ mRNA expression (data not shown).

Blocking effect was additionally evaluated through IL-17A ELISpot, which revealed persistent, C. albicans-elevated IL-17A protein amounts, insensitive to lack of IL-1β and/or IL-6 (Fig. 3 A).

IL-17A contribution to pathogenesis

In order to confirm our result on the increased amount of IL-17A in PBMCs in response to candidal infection, we measured production of IL-17A protein by PBMCs isolated from APS-1, AIRE+/+ CMC and healthy control individuals after 3 days of culture with or without C. albicans. Much higher amount of IL-17A was produced in APS-1 patients compared to controls after stimulation with fungus (p < 0.05; Fig. 3 A), which is in agreement with mRNA expression of IL-17A detected in the same samples. However, cells from AIRE+/+ CMC patient produced 17 times lower amount of this cytokine compared to sex- and age-matched healthy control (Fig. 3 B).

The exacerbated Th17 cell reactivity in APS-1 is C. albicans-specific

In order to understand whether the C. albicans-induced Th17 cell reactivity was specific, we cultured PBMCs from APS-1, AIRE+/+ CMC and healthy individuals in the presence or absence of three alternative antigens: M. tuberculosis, zymosan and PGN. After three days, we detected that, in all studied cell-samples, the amount of IL-17A protein (Fig. 3 A) and mRNA (Fig. 3 B), as well as the amount of Th17 cells (data not shown) did not differ between patients and controls in response to any of alternative antigens.

Treg cell number and Foxp3 mRNA expression were not affected by C. albicans-stimulation in APS-1 patients or controls

To try to understand the mechanism behind the exuberated C. albicans-induced Th17 cell response in APS-1 patients, we decided to take a closer look at Treg cells. After 3 days of culture in the presence of C. albicans, the number of Foxp3-producing CD3+CD4+ Treg cells did not differ significantly between blood donors, APS-1 patients and healthy controls (Fig. 4 A, B, C). Neither the blocking of IL-1β and IL-6 affected the Treg activity pattern. However, AIRE-intact CMC patient, which had around 40 % less Treg cells in unstimulated sample compared to control, demonstrated a two-fold increase in the amount of Treg after three days of C. albicans stimulation (Fig. 4 D).

Upregulated IFN-γ mRNA after stimulation with C. albicans

The Th1-cell pathway is a physiological immune response towards candidial infections in healthy individuals (Romani 2008). We measured induced IFN-γ mRNA expression in all groups of samples stimulated by C. albicans (Fig. 5 A, B). In healthy controls, the mean C. albicans-induced IFN-γ mRNA expression was significantly higher compared to unstimulated

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samples (p = 0.046). In APS-1 patients, the upregulation of Th1 reactivity was also notable but not significant. Interestingly, although the frequency of Th1 cells was elevated by C. albicans stimulation in samples from all individuals except the CMC patient (Fig. 5 C, D), samples from APS-1 patients demonstrated a larger Th1 proliferation compared to controls (not significant). The AIRE+/+ CMC-patient demonstrated no significant Th1 cell expansion in response to any of the antigens tested. In addition, PGN stimulation facilitated a mild increase of IFN-γ-producing Th1 cells in blood donors, APS-1 and controls.

IL-1β and IL-6 mRNA levels were not elevated by C. albicans stimulation

When we measured the IL-1β mRNA expression in blood donor samples stimulated with C. albicans, we observed a down-regulation in the presence of the blocking anti- IL-1β antibody (Fig. 6 A). This result, however, is statistically insignificant, and auto-feedback regulation is not known for IL-1β. We could not detect differences in IL-1β mRNA expression (Fig. 6 B) between APS-1 and controls in response to C. albicans, as we could detect in our previous studies. The reason for this may be a lower sample size. Interestingly, in response to M. tuberculosis, IL-1β mRNA level was elevated in both APS-1 patients (p < 0.05; Fig. 6 B) and healthy controls (not significant) when compared to unstimulated samples. Notably, the AIRE+/+ CMC patient demonstrated a largely compromised IL-1β response towards M. tuberculosis (Fig. 6 C).

The presence of anti-IL-6 antibody did not, to any detectable extent, affect IL-6 mRNA production in blood-donor PCMCs stimulated by C. albicans (Fig. 7 A). IL-6 (Fig. 7 B).

In response to M. tuberculosis, IL-6 mRNA expression was measured in all groups and stimulations, and appeared to be significantly upregulated only in blood donors (p < 0.05). However, cells obtained from AIRE+/+ CMC patient failed to maintain IL-6 response towards any of the antigens tested (Fig. 7 C).

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Discussion

Th17 cells play an important role in host defense (Huang, Na et al. 2004) and are probably involved in autoimmune conditions such as rheumatoid arthritis, psoriasis, multiple scleroses, inflammatory bowel disease and may also participate in development of corticosteroid-resistant asthma (Miossec, Korn et al. 2009). C. albicans is demonstrated to be among those pathogens that induce a physiological Th17 response, and humans that are unable to mount Th17 responses (e.g. hyper IgE syndrome patients with mutation in STAT3) often end up with recurrent candidiasis in the skin (Milner, Brenchley et al. 2008). We observed an increased C. albicans-induced Th17 cell reactivity and elevated IL-17A production in APS-1 patients, which shows that not all CMC patient groups are characterised by a lack of Th17 reactivity towards C. albicans, but also that enhancement of the Th17 reactivity can accompany the disease.

IL-17A is a cytokine that is produced by several immune cells but mainly by Th17, and induces chemokine and antimicrobial peptide production by tissue cells, which results in the recruitment of neutrophils and inflammation (Acosta-Rodriguez, Napolitani et al. 2007). Unregulated Th17 responses or overwhelming IL-17 production from T cells and other sources is associated with chronic inflammation and severe inflammatory conditions. In APS-1 patients, it is thought that the CD4+CD25+ regulatory T cell subset is impaired (Kekalainen, Tuovinen et al. 2007), which can be one reason accounting for the uncontrolled Th17 cell response and chronic candidiasis in these patients observed in our study. However, our data shows that the frequency of CD3+CD4+Foxp3+ T cells (Treg) is similar in healthy individuals and APS-1 patients in response to C. albicans, and it comes in agreement with our previous studies (data not shown). Therefore, the role of Treg cells in fungal opportunistic infections in APS-1 patients must be further investigated.

The polarization of human naïve CD4+ T cells towards the Th17 cell lineage is promoted by the cytokines IL-1β, IL-6 in combination with tumor growth factor-β (TGF-β), IL-21 or IL-23. IL-17A subsequently induces the production of IL-6 and IL-1β, creating a positive feedback loop that commits naïve T cells to the Th17 lineage (Miossec, Korn et al. 2009). In addition, it has been shown that in presence of IL-6, which strongly reduces retinoic acid receptor α (RARα) expression levels, the generation of Th17 cells can not be inhibited (Nolting, Daniel et al. 2009). However, we could not demonstrate that the blockade of IL-6 or IL-1β by monoclonal antibodies can affect C. albicans-elevated IL-17A production or Th17 cell expansion. It may be possible to get a clearer picture whether or not these cytokines can modulate the Th17 response through more effective blocking, for example targeting IL-6 or IL-1β receptors instead of proteins, but this approach may be unspecific.

Aiming to find a robust method to directly measure human Th17 activity, we came to a conclusion that ELISpot is the most suitable method to use when aiming to evaluate the activity of such rare cells. Comparatively short procedure with small amount of steps gives less chance to damage scarce material, and clear output provides with the end data directly.

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The major finding of this study is the important role for Th17 cells in life-long chronic mucocutaneous candidiasis in APS-1 patients. AIRE-intact person suffering from a CMC entirely lacks IL-17A mRNA in peripheral blood mononuclear cells, whereas those in APS-1 express a dramatically increased IL-17 mRNA upon stimulation with C. albicans. It remains unclear, therefore, whether IL-17A cytokine produced by Th17 and other white blood cells contributes to pathogenesis of the infection or to its clearance. It can not be denied, however, that an enhanced Th17 recruitment in APS-1 patients was not evident for other antigens tested and is strongly C. albicans-specific.

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Yun, L., Y. Binyan, et al. (2009). "Memory IL-22-producing CD4+ T cells specific for Candida albicans are present in humans." European Journal of Immunology 39(6): 1472-1479.

16

FIGURE 1

Figure 1. Flow cytometry plots of human PBMC cultured for 3 days in the presence (HCA) or absence (medium) of C. albicans, stained for surface - CD3-FITC, CD4-PerCP - and intracellular - IL-17A-PE markers. A – lymphocyte gate; B – T helper cell gate; C – Th17 cells of unstimulated PBMCs from blood donor; D – Th17 cells of C.albicans-stimulated PBMCs.

C

A B

D

0,21 %

99,79

0,02 %

99,98 %

17

FIGURE 2

IL-17A

med

ium HCA β

HCA + IL

-1β

HCA + a

nti IL

-1

HCA + a

nti IL

-6M

.tb β

M.tb

+ IL

-1

0.00

0.05

0.10

0.15

0.20

0.25 **

**

IL-1

7A

/18

s rR

NA

CD3+CD4+IL-17A+ T cells

med

ium HCA

HCA + IL

-1b

HCA + an

ti IL-

1b

HCA + a

nti IL

-6M

.tb

M.tb

+ IL

-1b

0.0

0.2

0.4

0.6

0.8

% o

f al

l lym

phoc

ytes

C

IL-17A

0

2

4

6

8

mediummedium HCA M. tb Zymo PGN

ControlAPS-1

IL-1

7A

/18s

rR

NA

D

IL-17A

Med

ium HCAM

tbZym

oPGN

0.0

0.5

1.0

1.5

2.0

2.5 Aire+/+ CMCControl

IL-1

7A

/18

s rR

NA

Figure 2. IL-17A production and Th17 reactivity. A - Flow cytometry data, representing frequency of CD3+CD4+IL-17A+ Th17 cells in PBMCs taken from blood donors and cultured in the presence or absence of different agents. B – Relative IL-17A mRNA expression by PBMCs from blood donors; * p < 0.05. C – Relative IL-17A mRNA expression by PBMCs from APS-1 patients and healthy controls. D – Relative IL-17A mRNA expression by PBMCs taken from a CMC patient and its sex- and age-matched control.

A B

18

FIGURE 3

A

IL-17A

0

100

200

300

400

500

APS-1 Control

medium HCA M.tb Zymo PGN HCA+

αIL-1β

HCA+

αIL-1βαIL-6

HCA+

αIL-6

* *

num

ber

of s

pots

B

IL-17A

med

ium HCAM

.tb

Zymos

anPGN

0

10

20

30

40

AIRE +/+ CMC Control

num

ber

of s

pots

Figure 3. ELISpot analyses of IL-17A protein produced in human PBMCs in response to 3-day culture with different agents. A – number of IL-17A producing cells in PBMCs from APS-1 patients and sex- and age-matched controls. B - number of IL-17A producing cells in PBMCs taken from a CMC patient and its sex- and age-matched control.

19

FIGURE 4

CD3+CD4+FoxP3+ T cells

med

ium HCA

HCA + IL

-1b

HCA + a

nti IL

-1b

HCA + a

nti IL

-6M

.tb

M.tb

+ IL

-1b

0

5

10

15

% o

f al

l lym

ph

ocyt

es

FoxP3

med

ium HCA β

HCA + IL

-1β

HCA + a

nti IL

-1

HCA + a

nti IL

-6M

.tb β

M.tb

+ IL

-1

0.0

0.1

0.2

0.3

0.4

0.52.3

2.4

Fo

xP3

/18

s rR

NA

Figure 4. Foxp3 production and Treg cell reactivity. A – Flow cytometry data representing frequency of CD3+CD4+Foxp3+ T cells in PBMCs taken from blood donors and cultured in the presence or absence of different agents. B - qPCR data on Foxp3 mRNA expression in PBMCs from blood donors. C - Flow cytometry data representing frequency of Treg cells in PBMCs from APS-1 patients and healthy controls. D - Flow cytometry data representing frequency of Treg cells in PBMCs taken from a CMC patient and its sex- and age-matched control.

A B

C D

20

FIGURE 5

IFN-γγγγ

0.0

0.5

1.0

1.5

2.0

2.5

medium HCA M.tb PGNZymosan

APS-1Control

*

IFN

-γ/1

8s r

RN

A

IFN-γγγγ

Med

ium HCAM

tbZym

oPGN

0.0

0.1

0.2

0.3

0.4Aire+/+ CMCCtrl

IFN

-γ/1

8s r

RN

A

Figure 5. IFN-γ production and Th1 cell reactivity. A – Relative IFN-γ mRNA expression by PBMCs from APS-1 patients and healthy controls. B – Relative IFN-γ mRNA expression by PBMCs taken from a CMC patient and its sex- and age-matched control. C - Flow cytometry data representing frequency of Th1 cells in PBMCs from APS-1 patients and healthy controls. D - Flow cytometry data representing frequency of Th1 cells in PBMCs taken from a CMC patient and its sex- and age-matched control.

A B

C D

21

FIGURE 6

IL-1ββββ

med

ium HCA β

HCA + IL

-1β

HCA + a

nti IL

-1

HCA + a

nti IL

-6M

.tb β

M.tb

+ IL

-1

0.00

0.05

0.10

0.15

0.20

0.25

IL-1

β /18

s rR

NA

IL-1ββββ

0.0

0.5

1.0

1.5

2.0

2.5

medium HCA M.tb Zymosan PGN

*

APS-1 Control

IL-1

β /18

s rR

NA

IL-1ββββ

Med

ium

C. albi

cans

M tb

Zymos

anPGN

0.0

0.5

1.0

1.5

2.0

2.5

Aire+/+ CMC Control

IL-1

β /18

s rR

NA

Figure 6. IL-1β mRNA expression. A - Relative IL-1β mRNA expression by PBMCs from blood donors. B - Relative IL-1β mRNA expression by PBMCs from APS-1 patients and healthy controls; * p < 0.05. C - Relative IL-1β mRNA expression by PBMCs taken from a CMC patient and its sex- and age-matched control.

A

C B

22

FIGURE 7

IL-6

med

ium HCA β

HCA + IL

-1β

HCA + a

nti IL

-1

HCA + a

nti IL

-6M

.tb β

M.tb

+ IL

-1

0.0

0.2

0.4

0.6 *

IL-6

/18s

rR

NA

IL-6

0

10

20

30

40

medium HCA M.tb Zymosan PGN

APS-1 Control

IL-6

/18s

rR

NA

IL-6

Med

ium

C. albi

cans

M tb

Zymos

anPGN

0102030405060708090

Aire+/+ CMC Control

IL-6

/18

s rR

NA

Figure 7. IL-6 mRNA expression. A - Relative IL-6 mRNA expression by PBMCs from blood donors; * p < 0.05. B - Relative IL-6 mRNA expression by PBMCs from APS-1 patients and healthy controls. C - Relative IL-6 mRNA expression by PBMCs taken from a CMC patient and its sex- and age-matched control.

A

C B

23

Acknowledgements

The project was performed in the Endocrine Autoimmunity group, Department of Medical Sciences, Uppsala University. My special gratitude to:

� Anna Lobell (supervisor), for governing my wild scientific enthusiasm and keeping my both feet on the logical ground; for reading my reports a dosen times and still noticing every single clause;

� Kerstin Ahlgren (technical supervisor), for teaching me everything one could teach in the lab, and sharing some Friday midnights by the FACS machine;

� Åsa Hallgren, for being an absolute best example of how one can be polite all the time and still keep everything and everyone running on the schedule;

� Brita Ardesjö Lundgren, for helping in our most important experiments and treating my problems as her own;

� Magnus Isaksson, Mohammad Alimohammadi and Fredrik Rorsman, for providing us with the crucial material - human blood.

And to all, for always treating me with trust and respect. Thank You!