Macrophages are multifaceted innate immune cells, able to adapt their phenotype to respond to a myriad of conditions, engaging in tissue-specific functions and mediating either inflammatory or anti-inflammatory responses depending on the encountered stimuli. They conduct key roles in the orchestration of immune responses; from pathogen recognition through sterilising inflammation to resolution and repair. The Udalova laboratory has previously demonstrated that IRF5 promotes a pro- inflammatory macrophage phenotype, leading to the secretion of TNF, IL-12, and IL-23, enhancing Th1/Th17-mediated immune responses, and described the cooperation between IRF5 and the transcription factor RelA, which mediate the production of pro-inflammatory genes. The aim of this thesis is to further characterise the activity of IRF5 in macrophage inflammatory responses. I demonstrate that IRF5 not only regulates the transcription of cytokines and chemokines in response to bacterial stimuli, but also anti-microbial peptides, whilst simultaneously down-regulating homeostatic and resolving macrophage functions. My data also suggests that IRF5 plays a role in enforcing monocyte to macrophage differentiation by up-regulating the transcription of key macrophages markers and repressing dendritic cell identity genes. To further characterise the mechanisms of the inflammatory response mounted by macrophages I used an unbiased approach; combining twenty-three transcription factor ChIP-seq data sets with chromatin accessibility information from ATAC-seq, uncovering RUNX1 as a novel partner of IRF5 that binds co-operatively to clusters of enhancers, which control the transcription of pro-inflammatory genes in a signal-dependent manner. This is the first study demonstrating a critical role for RUNX1 in activity of inflammatory macrophages.

The pathogenesis of systemic lupus erythematosus (SLE) and cardiovascular disease (CVD) are tightly linked, and CVD is one of the leading causes of death in lupus patients. There are many risk factors that increase the risk of CVD in SLE patients, including endothelial dysfunction, lipid dysregulation, and abnormal regulation of innate and adaptive immunity. We have previously investigated the role of interferon regulatory factor 5 (Irf5), on atherosclerosis in lupus mouse models. Irf5 has a pro-inflammatory function by activating macrophage and cytokine recruitment and is thus being considered as a potential therapeutic target for the treatment of SLE. We hypothesized that Irf5 deficiency would ameliorate lupus disease as well as improve cardiovascular disease in the Irf5-deficient mouse model. However, while lupus disease did improve in the mouse model, the atherosclerotic plaques were found to be significantly increased in size. This poses a challenge to our current understanding of Irf5, as well as adds complexity to an already difficult clinical problem. Therefore, our aim of this study is to characterize the cells within the atherosclerotic lesions to examine their inflammatory potential. The focus of this study is the infiltration of macrophages into the mouse aortic root as determined by immunohistochemistry staining. In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet. In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus. Together, our results do not support nor refute a role of Irf5 in macrophage infiltration into the aortic root. More samples are needed, as are more methods of identifying macrophages and quantifying them. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring. In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet. In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus. Together, our results do not support a role of Irf5 in macrophage infiltration into the aortic root. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring.

Interferon regulatory factor 5 (IRF5) is a protein with different functions including theactivation of genes that encode different cytokines. Overexpression of IRF5 has been observedto lead to different types of stress in the cells, including an overproduction of cytokines, whichis referred to as a cytokine storm. Clinical states in which dysregulated cytokine release in theform of a cytokine storm can be referred to with an umbrella term: Cytokine storm syndrome.The aim of this study was to test for inhibitors for IRF5 that could be developed and used as apharmaceutical drug to treat Cytokine Storm Syndromes including autoimmune diseases andCOVID-19. The method for this screeing consisted of finding possible inhibitors usingcomputer based drug design which resulted in the selection of 21 possible inhibitors. Thesesubstances were then tested on induced macrophages that are cytokine producing. The abilityfor inhibition is based on the amount of cytokines present in the sample after exposure. Thiswas tested using an ELISA based assay which measures the amount of cytokines in the sample..A handful of substances was found to be effective and substances 11 and 17 stood out asespecially effective. This indicates the possibitily for a drug to be developed that would inhibitIRF5, which could be used for treatment of cytokine storm syndromes.Keywords: IRF5,

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by autoantibody production, inflammation and end organ damage resulting from an overactivation of the immune system through mechanisms that are still not completely understood. One critical component of this pernicious cycle is a loss of tolerance to self-antigen that precedes disease manifestations. Importantly, there has only been one FDA approved treatment for SLE in the last 60 years, though an additional treatment has recently been approved for lupus nephritis. Therefore, there is a need to understand how pathogenic pathways are regulated in the context of SLE. This thesis focused on not only further understanding how interferon regulatory factor 5 (IRF5) drives pathogenicity in a mouse of model of lupus but also discerning the molecular pathways important for blood dendritic cell antigen 2 (BDCA2) inhibition of type I interferon (IFN-I) release from plasmacytoid dendritic cells (pDCs). Gain-of-function polymorphisms in the transcription factor IRF5 are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. This work shows that monoallelic IRF5 deficiency in B cells markedly reduces disease in the FcγRIIB−/−Yaa mouse model of lupus. Mechanistically, B cell receptor and TLR7 signaling synergize to promote IRF5 phosphorylation through IRAK4, and also increase IRF5 protein expression, with these processes being independently regulated. This synergy increases B cell-intrinsic IL-6 and TNF-𝛂 production, both key requirements for germinal center responses, with IL-6 and TNF-𝛂 production in vitro and in vivo being substantially lower with loss of one allele of IRF5. Moreover, this thesis further strengthens the notion of IRF5 as a target for the treatment of SLE by therapeutically targeting IRF5 in the FcγRIIB−/−Yaa mouse lupus model. This work shows that tamoxifen driven deletion of IRF5 after disease associated manifestations have already developed confers protection in the FcγRIIB−/−Yaa mouse model. IFN-I is a family of pleotropic cytokines that are thought to be pathogenic in the context of SLE. PDCs are a major source of IFN-I. As such, they have been shown to play a major role in anti-viral immunity and are thought to be pathogenic in context of SLE. I used the anti-BDCA2 antibody 24F4A to ligate the receptor in a human pDC cell line (Gen 2.2) and applied a variety of proteomics methods, including profiling of post-translational modifications, to evaluate signaling downstream of BDCA2. I found that phosphorylation of phosphoinositide 3-kinase adapter protein 1 (PIK3AP1)/ B cell adaptor protein (BCAP) was increased after BDCA2 engagement by 24F4A. Deletion of BCAP from Gen2.2 cells reversed BDCA2 mediated AKT phosphorylation and IFN inhibition, suggesting that PI3 kinase (PI3K) may be important for BDCA2 mediated IFN inhibition. Treatment of human pDCs with PI3kinase inhibitor followed by TLR9 stimulation confirmed that PI3K activity is critical for BDCA2 mediated IFN inhibition. This work describes a critical threshold of IRF5 expression in B cells necessary for the development of the lupus like disease in the FcγRIIB−/−Yaa mice and that systemic deletion of IRF5 after disease onset reduces the disease manifestations in the same mouse model. Additionally, this work demonstrated that BCAP and PI3K are important pathways to mediate the IFN inhibition observed with engagement of the BDCA2 pathway. This work supports IRF5 as a therapeutic target in SLE and furthermore, describes a molecular pathway important for BDCA2 mediated IFN-I inhibition from pDCs.

The main forms of inflammatory bowel diseases (IBD) are Crohns disease and ulcerative colitis. These are chronic diseases, with periods of progression and remission. They are mostly characterized by digestive symptoms such as diarrhea, abdominal pain and weight loss. They affect young individuals and their frequencies have increased for the last decades. The etiology of these pathologies is not well understood, however genetic and environmental factors are involved. The treatment of IBD aims to control the inflammation and to extend periods of clinical remission. Infliximab is an anti-TNF-α antibody, leading to a clear improvement of the symptomatology. However, about 30 % of the patients do not response to this treatment. Genetic factors are certainly involved in these inter-individual differences. The purpose of our work was to find: 1- genetic factors implicated in the response to Infliximab in Crohns disease and 2- genetic factors predisposing to IBD. First we could show that both genes LTA and TNF, which are closely related, are not associated with the answer to Infliximab in Crohns disease. However, different polymorphisms of the ADAM17 gene were associated with a response to the treatment in our Belgian cohort. Second, we could demonstrate an association between an insertion/deletion in the IRF5 gene and IBD. The insertion allele, predisposing to IBD, is expected to create a new binding site for the SP1 transcription factor.

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disorder characterized by a failure in the mechanism of self-tolerance and production of autoantibodies, potentially affecting any organ in the body. The genetic factors behind the disease have been extensively studied in the past years and to date a list of more than 30 loci have been associated with SLE. However, very little is known about the functional significance of the risk variants. In this thesis, we focused on the analysis of SLE-associated variants in three genes: interferon regulatory factor 5 (IRF5), CD226 and the microRNA 146a. In paper I, we analyzed four polymorphisms in the IRF5 gene in a large set of individuals from different populations. We replicated a strong association of a promoter indel in our meta-analysis, but expression analysis indicated that it is rather another variant, SNP rs10954213 in the poly(A) signal of the gene that is in fact the major contributor to the altered gene expression in leukocytes. In manuscript II, we further characterized the regulation of IRF5 expression, showing that this gene can be up-regulated by estrogen in PBMCs and monocytes, regardless of the genotype, which could to some extent, explain the sex-bias of SLE. In paper III, we investigated the association of CD226 with SLE and the potential functional effect of the associated variants. The genetic analysis showed an association of a three-SNP-haplotype located at the 3’UTR region of the gene. The risk haplotype correlated with lower CD226 protein expression on the surface of cytotoxic and helper T cells, as well as in NK T cells. Reporter assays pointed to rs727088 in the 3’UTR as the main responsible variant for altered gene expression. In paper IV, we described the association of a variant in microRNA miR-146a, involved in the interferon pathway, with SLE in Europeans, which could in addition be correlated with decreased expression of both mature and primary miR-146a in leukocytes. In summary, we have investigated the genetic association of three genes with SLE in a large cohort of individuals and identified variants responsible for functional alterations of these genes, providing further insight into the pathogenesis of SLE.

Η Μυασθένεια είναι μια αυτοάνοση νόσος της νευρομυϊκής σύναψης που χαρακτηρίζεται από την παραγωγή αυτοαντισωμάτων έναντι, συνήθως, του AChR, καθώς και άλλων πρωτεϊνών της σύναψης. Στην παρούσα εργασία, πραγματοποιήθηκε μελέτη γενετικής συσχέτισης για την ταυτοποίηση πολυμορφισμών που πιθανώς εμπλέκονται στην εκδήλωση της Μυασθένειας. Για το σκοπό αυτό, επιλέχθηκαν πολυμορφισμοί οι οποίοι εδράζονται σε γονίδια που αποτελούν σημαντικούς ρυθμιστές της ανοσολογικής απόκρισης και έχουν προηγουμένως συσχετισθεί με άλλες αυτοάνοσες νόσους. Τα υποψήφια γονίδια είναι τα: interferon regulatory factor 5 (IRF-5), TNFα-induced protein 3 (TNFAIP3) και interleukin-10 (IL-10). Στη μελέτη συμμετείχαν 101 μυασθενείς και ισάριθμα υγιή άτομα ως ομάδα αναφοράς, όλοι ελληνικής καταγωγής. Οι μέθοδοι γονοτύπησης που εφαρμόσθηκαν περιλαμβάνουν τον προσδιορισμό αλληλουχίας κατά Sanger, την HRM ανάλυση, την PCR-RFLP και την PCR σε συνδυασμό με ηλεκτροφόρηση σε αγαρόζη, στην περίπτωση ενός in/del 30 bp. Μια στατιστική τάση συσχέτισης (p=0.068) ανιχνεύθηκε για τους πολυμορφισμούς στον υποκινητή της IL-10 μεταξύ των μυασθενών με πρώιμη ηλικία έναρξης της νόσου (early-onset) και αυτών που εμφάνισαν τη νόσο ηλικιακά αργότερα (late-onset). Για τους υπόλοιπους πολυμορφισμούς που μελετήθηκαν, δεν παρατηρήθηκαν στατιστικά σημαντικές διαφορές. Η μελέτη αυτή αποτελεί την πρώτη προσπάθεια συσχέτισης πολυμορφισμών των γονιδίων IRF-5 και TNFAIP3 με τη Μυασθένεια, σε οποιονδήποτε πληθυσμό. Όσον αφορά τους πολυμορφισμούς του υποκινητή της IL-10, περαιτέρω μελέτες σε πολυπληθέστερες ομάδες πιθανώς να αποκαλύψουν μια στατιστικώς ισχυρότερη συσχέτιση. / Myasthenia gravis (MG) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against proteins of the postsynaptic membrane, in the neuromuscular junction. The contribution of genetic factors to MG susceptibility has been evaluated through family and twin studies, however, the precise genetic background of the disease remains elusive. We conducted a case-control association study in 101 unrelated MG patients, of Hellenic origin and 101 healthy volunteers in order to assess the involvement of common genetic variants in susceptibility to MG. We focused on three candidate genes which have been clearly associated with several autoimmune diseases, aiming to investigate their potential implication in MG pathogenesis. These are interferon regulatory factor 5 (IRF-5), TNFα-induced protein 3 (TNFAIP3), also known as A20, and interleukin-10 (IL-10), key molecules in the regulation of immune function. Genotyping was performed by PCR-RFLP, direct automated sequencing, High Resolution Melt curve Analysis (HRM) and PCR-agarose gel electrophoresis analysis in the case of a 30 bp in/del polymorphism. A statistical trend of association (p=0.068) between IL-10 promoter single nucleotide polymorphisms (SNPs) and the subgroups of early and late-onset MG patients was revealed. No statistically significant differences were observed in the rest of the variants examined. As far as we are aware, this is the first worldwide attempt to address the possible association between IRF-5 and TNFAIP3 common genetic variants and the genetic basis of MG.

Le syndrome de Sjögren primitif (SSp) est une maladie auto-immune (MAI) systémique ayant des caractéristiques communes avec le lupus érythémateux. Ces caractéristiques incluent des mécanismes physiopathologiques et des facteurs de predispositions génétiques. Notre équipe et d’autres groupes ont pu mettre en evidence une signature interféron (IFN) dans les glandes salivaires et les PBMCs de patients ayant un SSp. Cette découverte a permis de mettre en évidence de nouvelles voies à explorer dans la pathogénie du SLE et SSp en permettant la focalisation des recherches sur le rôle de l’immunité innée et de la voie IFN.Nous avons confirmé le rôle de 2 gènes importants dans le SSp, impliqués dans les voies des IFN. Le premier est IRF5 sur la voie IFN de type I et STAT4 sur la voie IFN de type II. Nous avons pu mettre en évidence une fonctionnalité de l’allèle à risque d’IRF5 (Polymorphisme Indel situé dans le promoteur). Concernant STAT4, son expression n’était pas altérée par le SNP associé à la maladie. Toutefois, l’ARNm de STAT4 était corrélé à l’expression des gènes IFN de type I. Les dérégulations épigénétique pourraient jouer un rôle important dans la pathogénie de nombreuses MAI, en particulier la méthylation de l’ADN qui est hautement liée à l’extinction de l’expression des gènes. Nous avons étudié la méthylation du promoteur d’IRF5 et nous n’avons pas trouvé de régulation de ce promoteur par le méthylation. Une analyse de la méthylation avec une approche globale du méthylome est en cours dans notre équipe et permettra d’identifier de gènes cibles d’une dérégulation épigénétique pouvant être impliqués dans les MAI.Nous avons essayé de comprendre la relation entre STAT4 et gènes IFN de type I. Ainsi, nous rapportons que l’IL-12 induit spécifiquement l’IFN de type I par intéraction entre deux partenaires cellulaires, les lymphocytes T CD4+ et les cellules dendritiques plasmacytoïdes. Ces résultats pourraient expliquer l’implication des polymorphismes de STAT4 dans les MAI dépendantes de l’IFN de type I. Ces résultats suggèrent également que les MAI dépendantes des IFN de type I et II ne s’opposent pas. Elles seraient seulement le Yin et le Yang d’un facteur d’activation commun, STAT4, capable d’induire les IFNs de type I et II. / Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease (AID) that presents similar characteristics to systemic lupus erythematosus. These characteristics include pathophysiology and genetic factors. Our team and other groups have highlighted an interferon (IFN) signature in salivary glands and PBMCs from patients with Sjögren syndrome. This signature demonstrates new pathways in pSS and lupus, focusing research on innate immunity and in the IFN pathway.We have confirmed the implication of 2 genes in the pSS, and these genes are involved in the IFN pathway. The first gene is IRF5 which is in the type I IFN pathway and the second is STAT4 which is in the type II IFN pathway. We have shown a functional consequence of IRF5 at-risk allele. Regarding STAT4, the associated SNP did not altered STAT4 mRNA expression but was highly correlated with type I IFN genes expression.The epigenetic deregulation could play a triggering role in autoimmune diseases, particularly through DNA methylation which is highly implicated in the suppression of gene expression. We studied the methylation of IRF5 promoter and found no methylation. Our team is currently undertaking a global approach with methylome analysis. This methylome study will assess specific gene methylation patterns and will allow a better understanding of the role of these genes in autoimmune diseases.We further demonstrated that IL-12 specifically induces a type I IFN signature through a CD4+ T cells and pDCs crosstalk. These results could explain the implication of STAT4 polymorphism not only in type II IFN-dependent AIDs but also in type I IFN-dependent AIDs. Our data confirm that type I IFN- and type II IFN-mediated AIDs do not have to be opposed. They are only the yin and the yang of a common STAT4 activation which may induce secretion of both cytokines.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

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