Search

Whereas genetic susceptibility for Systemic Lupus Erythematosus has been well explored, the precipitants for clinical disease flares remain a mystery. To investigate for dynamic-relationships between gut-microbial communities and Lupus disease activity, we performed taxonomic surveys of fecal 16S rRNA gene amplicon-libraries from Lupus patients and healthy volunteers, obtained at serial timepoints over many months to several years. Individual Lupus patients commonly displayed imbalances in alpha and beta microbiota-diversity, which were uniquely different from healthy individuals as well as from other Lupus patients. Moreover, multivariate analysis of sequential Lupus libraries documented community-wide ecological microbiota instability overtime, most pronounced in patients with Lupus Nephritis LN), a severe form associated with worse prognosis. Lupus gut communities displayed transient spikes of pathogenic bacterial species, with by far the most prevalent being blooms of Ruminococcus blautia gnavus (RG), occurring in nearly half of LN patients often concordant with disease activity flares. RG strains isolated during disease flares, but not those isolated from healthy individuals or patients with inflammatory bowel disease, commonly expressed a novel, highly immunogenic cell wall-associated lipoglycan with conserved structural features that include a diacyl glycerol anchor. Cross-reactive antigenic determinants on these lipoglycans were recognized by murine monoclonal antibodies, and by spontaneously arising Lupus serum IgG antibodies with peak serum antibody responses also concordant with RG blooms. As SLE is frequently characterized by remitting-relapsing disease, despite appropriate treatment, we speculate that gut blooms of pathogenic bacteria, that impair gut barrier function and stoke systemic inflammation, directly contribute to immunopathogenesis.

Imbalances in the gut microbiome are suspected contributors to the pathogenesis of Systemic Lupus Erythematosus, and our studies and others have documented that patients with active Lupus nephritis have expansions of the obligate anaerobe

Throughout our lives we are immersed in, and colonized by, immense and complex microbial communities. These microbiota serve as activators and early sparring partners for the progressive construction of the layers within our immune defenses and are essential to immune homeostasis. Yet, at times imbalances within the microbiota may contribute to metabolic and immune regulatory abnormalities that underlie the development of inflammatory and autoimmune diseases. Here, we review recent progress in investigations of the microbiome, with emphasis on the gut microbiota associated with systemic autoimmunity. In particular, these studies are beginning to illuminate aspects of the pathogenesis of Systemic Lupus Erythematosus, and may suggest that interconnections with specific disease-associated patterns of dysbiosis within gut communities are bidirectional and mutually reinforcing.

Imbalances in the gut microbiome are suspected as contributors to the pathogenesis of Systemic Lupus Erythematosus, and our studies and others have documented that patients with active Lupus nephritis have expansions of the obligate anaerobe, Ruminococcus blautia gnavus (RG). To investigate whether the RG strains in Lupus patients have in vivo pathogenic properties, we colonized C57BL/6 mice with individual RG strains from healthy adults or those from Lupus patients. These strains had a similar capacity for murine intestinal colonization, in antibiotic-preconditioned specific-pathogen-free, as well as germ-free adults, and their neonatally colonized litters. Lupus-derived RG strains induced high levels of intestinal permeability that was significantly greater in female than male mice, whereas the RG species-type strain (ATCC29149/VPI C7-1) from a healthy donor had little or no effects. Lupus RG strain-induced functional alterations were associated dysregulated occluden transcript production in the ileal wall as well as raised serum levels of zonulin, a regulator of tight junction formation between cells that form the gut barrier. Notably, the level of Lupus RG-induced intestinal permeability was significantly correlated with serum IgG anti RG cell-wall lipoglycan antibodies, and to anti-native DNA autoantibodies that are a biomarker for SLE. Strikingly, gut permeability was completely reversed by oral treatment with larazotide acetate, an octapeptide that is a specific molecular antagonist of zonulin. Taken together, these studies document a molecular pathway by which RG strains from Lupus patients induce a leaky gut and autoimmunity that have been implicated in the pathogenesis of flares of clinical Lupus disease.

Maintenance of immune homeostasis involves a synergistic relationship between the host and the microbiome. Canonical interferon (IFN) signaling controls responses to acute microbial infection, through engagement of the STAT1 transcription factor. However, the contribution of tonic levels of IFN to immune homeostasis in absence of acute infection remains largely unexplored. We report that STAT1 KO mice spontaneously developed an inflammatory disease marked by myeloid hyperplasia and splenic accumulation of hematopoietic stem cells. Moreover, these animals developed inflammatory bowel disease. Profiling gut bacteria revealed a profound dysbiosis in absence of tonic IFN signaling, which triggered expansion of TH17 cells and loss of splenic Treg cells. Resolution of dysbiosis by antibiotic treatment averted the TH17 bias, and blocking IL17 signaling prevented myeloid expansion and splenic stem cell accumulation. Thus, tonic IFNs regulate gut microbial ecology, which is crucial for maintaining physiologic immune homeostasis and preventing inflammation.

The X chromosome, hemizygous in males, contains numerous genes important to immunological and hormonal function. Alterations in X-linked gene dosage are suspected to contribute to female predominance in autoimmunity. A powerful example of X-linked dosage involvement comes from the BXSB murine lupus model, where the duplication of the X-linked Toll-Like Receptor 7 (Tlr7) gene aggravates autoimmunity in male mice. Such alterations are possible in men with autoimmune diseases. Here we showed that a quarter to a third of men with rheumatoid arthritis (RA) had significantly increased copy numbers (CN) of TLR7 gene and its paralog TLR8. Patients with high CN had an upregulated pro-inflammatory JNK/p38 signaling pathway. By fluorescence in situ hybridization, we further demonstrated that the increase in X-linked genes CN was due to the presence of an extra X chromosome in some cells. Men with RA had a significant cellular mosaicism of female (46,XX) and/or Klinefelter (47,XXY) cells among male (46,XY) cells, reaching up to 1.4% in peripheral blood. Our results present a new potential trigger for RA in men and opens a new field of investigation particularly relevant for gender-biased autoimmune diseases.

Juvenile idiopathic arthritis (JIA) is one of the most common chronic inflammatory disorders in children, with hallmarks of joint inflammation, synovial hyperplasia, and leukocyte infiltration1. As in other autoimmune diseases, the exact etiopathogenesis, and pathogenic drivers of JIA, remain incompletely understood. In rheumatoid arthritis (RA) and ankylosing spondylitis (AS), several studies have shown that disease susceptibility is strongly associated with sets of immune-response genes, which include specific MHC alleles. In contrast, JIA does not seem to have such a strong association, and there is less occurrence of familial aggregations, and only limited disease concordance in homozygotic twins2,3,4,5. All of these observations suggest a key role in JIA pathogenesis for noninherited factors. Indeed, a number of reports have implicated maternal factors, such as breast-feeding, and whether the mother is a smoker6. Other environmental factors have been cited, such as bacterial or viral infection, antibiotic usage7, and most recently the influence of shifts in the gut microbiome8. The development of state-of-the-art 16S rRNA high-throughput sequencing technology for microbial profiling has opened the door for the characterization of the phylogenetic distribution of taxa in the gut bacterial communities in an individual. With this culture-independent approach, an increasing number of reports have documented decreases in gut microbiome diversity and other forms of dysbiosis in inflammatory and autoimmune diseases that include … Address correspondence to Dr. G.J. Silverman, Laboratory of B cell Immunobiology, Department of Medicine, New York University School of Medicine, Room 804, 450 E. 29th St., New York, New York 10016, USA. E-mail: Gregg.silverman{at}nyumc.org

Background:From a cross-sectional cohort, we have identified a candidate human gut anaerobic pathobiont,Ruminococcus gnavus(RG) of the familyLachnospiraceaethat was linked to active Lupus nephritis (LN)(1). Based on 16S rRNA amplicon analysis, LN patients displayed increased fecal RG abundance, concordant with serum IgG anti-RG antibody responses that appeared intertwined with anti-dsDNA responses implicated in renal pathogenesis. Indeed, monocolonization of germ-free mice is reported to result in generalized inflammation and expansions of Th17 cells. However, RG at low levels are also prevalent in healthy adults, and the temporal dynamics of RG representation within Lupus microbiota ecosystems have not been investigated. Also, genomic sequences of few RG strains have been reported, and these vary greatly in genome structure, gene representation and sequence, which may have broad implications for adaptation to a host with systemic inflammation and/or factors that contribute to immune activation in a susceptible host.Objectives:To investigate the relationships betweenin vivoRG expansions and disease activity that often wax and wane overtime, we initiated longitudinal studies in Lupus patients and controls. As representation of RG strains alone might alter pathogenic potential, we also sought to characterize RG strains from active LN patients.Methods:From our cohort, patients were characterized for demographics, clinical disease activity, and serologies including standard autoantibody and complement levels, and anti-bacterial responses of interest. High throughput 16S rRNA amplicon libraries from fecal samples were analyzed using QIIME 2 and DADA2 (1). Also, individual RG colonies were isolated and subjected to whole genome sequencing. Species and strains were then assigned in part based on multi-locus sequence typing and reference guided genomic assemblies.Results:16S rRNA analysis of 34 samples, at 2-4 timepoints from 14 SLE patients, documented highly conserved patterns of gut community representation overtime in 10/14 patients, based in part on unsupervised hierarchical cluster analysis. Notably, independent of vacillations in clinical disease activity of up to 8 SLEDAI points, conserved microbiome phylogenetic abundance/composition was documented at a family level, and the level of amplicon sequence variants that approximate identification of individual species. In pilot studies, from two active lupus nephritis patients hundreds of fecal bacterial colonies were isolated, with initial assignments by 16S rRNA sequence. From highly redundant whole genome sequence analysis, these Lupus-patient fecal colonies were found to distribute into only four distinct RG strains, which differed from reported strains.Conclusion:Our findings suggest that many Lupus patients have little or no detectable perturbations in representation of theLachnospiraceaefamily or abundance of RG species overtime. Moreover, this seeming microbiota stability was documented even in patients with dramatic changes in disease activity. However, these approaches are inadequate to detect shifts between RG strains. In pilot studies we have isolated and characterized the genomes of four unique RG strains from active LN patients, which include variations in gene content and sequence that may have implications for the host-commensal relationship and immune activation. Broadening of these studies to larger number of SLE patients and healthy subjects, with metagenomic surveys of strain representation in genomic shotgun libraries are currently in progress, in coordination with murine colonization testing for immune modulatory properties of individual strains.References:[1]Azzouzet al.Ann Rheum Dis2019 78(7):947-56Disclosure of Interests: :Gregg Silverman Consultant of: Work with industry is unrelated to the topic in this abstract, Doua Azzouz: None declared, Ze Chen: None declared, Jing Deng: None declared, Zhi Li: None declared, David Fenyo: None declared, Alexander Alekseyenko: None declared

Background A transmissible agent has long been suspected in the pathogenesis of SLE, yet the potential contribution of the human intestinal microbiome has been little examined. We therefore characterized the gut microbiota of patients with SLE, with special interest in those with lupus nephritis (LN). Methods Blood and fecal samples from SLE patients were obtained, with strict inclusion/exclusion of criteria. Fecal 16S rDNA sequencing, as well as cytokine and autoantibody assays were performed. In addition, sera from two independent lupus cohorts were studied for validation. Biomarkers of gut leakiness were assessed. Results Compared to controls, the intestinal microbiome from SLE patients (n=61) showed decreased species richness diversity with reductions in taxonomic complexity most pronounced in those with high disease activity. Notably, SLE patients had an overall 5-fold greater representation of a species in the Lachnospiracaea family of obligate anaerobic Gram-positive cocci, with reciprocal contractions of two other commensal species with putative protective properties. Abundance of the Lachnospiracaea species correlated with serum IgG to a cell wall component, postulated to represent a lipoglycan, from a strain of this same species (p=0.002, n=61, Spearman) but not with 7 other strains. There was also a significant direct correlation between SLEDAI scores and levels of these circulating anti-strain IgG antibodies (p=0.02, n=48). Levels of antibodies to strain-specific bacterial antigen, treated with RNAse/DNAse/proteinase K, were significantly higher in those with active nephritis at time of sampling compared to SLE without renal activity (Cohort 1 p=0.01 n=48; Cohort 2 p=0.006, n=28, Mann-Whitney). Levels of serum IgG anti- strain antibodies also significantly correlated with high-titer serum IgG to native DNA (p Conclusions These findings suggest a novel paradigm for the pathogenesis of LN in which a common intestinal commensal bacteria may contribute to the immune-complex mediated disease process, with features akin to poststreptococcal GN but without outward signs and symptoms of clinical infection. Acknowledgements Judith and Stuart Colton Foundation, NIAMS- P50 AR070591.

Background Several risk loci for Rheumatoid Arthritis (RA) have been identified by Genome Wide Association Studies (GWAS), but they do not include Allograft Inflammatory Factor 1 (AIF1). Nevertheless, a few studies have shown that AIF1 rs2269475 (C/T) is associated with RA1,2. Objectives We propose 1) To examine associations in French Caucasian patients with RA, of the seven most described AIF1 SNPs; 2) To study their linkage disequilibrium with HLA-DRB1 alleles; 3) To evaluate whether AIF1 single nucleotide polymorphisms (SNPs) could predict first line treatment responses in RA. Methods We amplified the AIF1 gene region containing the 7 SNPs and sequenced PCR products on a total of 469 individuals, including 95 Anti-Citrullinated Protein Antibody (ACPA) positive RA patients, 146, patients with scleroderma, 132 healthy controls and 96 additional healthy controls selected from a large database of volunteer bone marrow donors (VBMD) for carrying at least one RA-associated allele. Patients and controls were HLA-DRB1 genotyped. Patients with RA were divided into 2 groups, a first group called “non responders” was defined as patients who did not respond to first-line methotrexate (MTX) combined with Etanercept and a second group called “good responders” was defined as patients who did respond to methotrexate combined with Etanercept. Results Two SNPs were associated with RA: rs4711274 (G/A) and rs2269475 (C/T). The frequency of minor allele carriers was respectively 37% (A) and 36% (T) in patients with RA versus 18% among controls (p=0.0014 and p=0.001). Furthermore, patients with RA-associated HLA-DRB1 alleles carried more often minor alleles for both SNPs (p=0.0005). Preliminary clinical data show that 56% of non-responders (N=16) carried the minor alleles of both rs4711274 and rs2269475 compared to only 21% of good responders (N=24, p=0.02). Conclusions AIF is an inflammation-responsive protein encoded within the HLA class III region on chromosome 6 (6p21.3). As already described in British and Polish Caucasians, we found a significant AIF1 Rs2269475 association with RA. We also found an association with Rs4711274 in linkage disequilibrium with the former. The increased frequency of minor AIF1 alleles in RA was not associated with a particular HLA-DRB1 allele, but to any HLA-DRB1 allele carrying the shared epitope. Finally, patients who failed to respond to Etanercept and MTX carried more often minor alleles of the 2 described AIF1 SNPs. Further analysis on a larger groups of patients is required to confirm whether AIF1 SNPs can predict response to therapy with Etanercept and Methotrexate. References Harney SM, Vilarino-Guell C, Adamopoulos IE, Sims AM, Lawrence RW, et al. (2008) Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis. Rheumatology 47: 1761–1767. Pawlik A, Kurzawski M, Szczepanik T, Dziedziejko V, Safranow K, et al. (2008) Association of allograft inflammatory factor-1 gene polymorphism with rheumatoid arthritis. Tissue antigens 72: 171–175. Disclosure of Interest None declared

International audience; In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10(-4)) and 60% versus 28% (P = 3.10(-8)). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10(-10)) and 82% (P = 5.10(-13)). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P< 0.0001) and correlated with disease activity (p< 0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.

We report the case of a 40-year old man diagnosed with a scleroderma-like disease. Clinical similarities with graft versus host disease prompted initial testing for chimerism employing fluorescence in situ hybridization (FISH). Female cells were observed within peripheral blood mononuclear cells from the patient. Because maternal cells have been detected in healthy immunologically competent adults and patients with autoimmune conditions, we hypothesized that these cells were of maternal origin. Contrary to our expectations, HLA-specific quantitative PCR (QPCR) ruled out maternal microchimerism. However, HLA-specific QPCR testing was positive for the paternal HLA haplotype that the patient did not inherit. We reasoned that the most likely origin of chimerism with non-inherited paternal HLA alleles was from an unrecognized "vanished" twin. The patient had never received a blood transfusion. This report suggests that cells from a vanished twin are a possible source of chimerism. The frequency of chimerism fr...

International audience; BACKGROUND AND OBJECTIVES: Women, having two X chromosomes, are more predisposed than men to autoimmune diseases. The X chromosome contains many genes linked to immunity which may contribute to this gender bias. In a mouse model, a duplication of the innate immunity X-linked toll like receptor 7 (Tlr7) gene has been shown to potentiate autoimmunity in males. We then proposed to investigate whether TLR7 gene and its neighboring paralog TLR8 could have variations in their copy numbers, contributing to the pathogenesis of rheumatoid arthritis (RA) in men. METHODS: A real-time quantitative PCR protocol was developed to assess copy number variation (CNV) of TLR7 and TLR8 gene, using sensitive and optimised ΔΔCt and standard curve methods, in DNA from peripheral blood mononuclear cells of 60 patients with RA (including 49 men) and 64 healthy controls (including 42 men). Among them, 31 men with RA and 18 healthy men were further screened for TLR7/8 CNV in 4 subpopulations: B cells, T cells, granulocytes and the depleted fraction of the former 3. RESULTS: TLR7/8 copy numbers significantly increased with age in PBMCs from all men (P < 0.0001, Spearman's rank correlation test), whether they had RA or not. The increase had mean amplitude of 20%, spanning from the age of 20 until 80, according to the linear-regression-curve's best fit. This age-dependent and disease-independent CNV increase was also observed in all cell subsets. Interestingly, such increase was not observed in women, healthy or with RA, but rather an opposite trend. CONCLUSION AND PERSPECTIVES: For the first time we showed an increased CNV in TLR7 and TLR8 genes which is age and sex-mediated. Several hypotheses could explain such phenomenon. For example, somatically acquired duplications can affect some cells over time and result in an increase with age in men. In parallel, X chromosome monosomy, previously described in aging women could account for the opposite trend in those. Another explanation for men can be due to the presence of feminine microchimerism, arising from feto-maternal or twin sister exchange during in utero life, as previously described. Such cells would carry 2 X chromosomes and contribute to the increased pool of X-linked genes among XY host cells. Investigating these hypotheses would provide better understanding of age-associated X-linked genetic modifications and the role of the X chromosome in gender differences in health and disease. Abstract topicOther.

Rheumatoid arthritis, a chronic inflammatory joint disease, is strongly associated with HLA-DRB1*01 and *04 alleles that have in common similar 5-amino acid motifs in the third hypervariable region of DRB1 (QKRAA, QRRAA, RRRAA), the so called shared epitope (SE). Most patients with RA carry 1 or 2 doses of the SE, with particular genetic combinations at higher risk. In recent work we provided evidence that patients who lack HLA-DRB1*01 and/or *04 alleles can acquire RA susceptibility through fetal, maternal or iatrogenic microchimerism. We also discuss how Mc carrying HLA-DRB1*04 alleles is more likely to be present in the peripheral blood of RA patients compared to Mc carrying HLA-DRB1*01 alleles. We further analyze our results in light of the hierarchy for RA risk with different combinations of the SE. How Mc could contribute to RA susceptibility and whether it also contributes to the hierarchy of risk observed with particular combinations of SE-containing alleles is certainly the beginning of an intrig...

International audience; Embryos during pregnancy and organs during transplantation, express high levels of soluble HLA-G (sHLA-G) molecules for successful implantation and protection against maternal immune cells or recipient's cells. We and others have shown that women with scleroderma (SSc) carry cells/DNA arising from pregnancy, so-called fetal microchimerism (Mc) more often and in higher quantities than healthy women decades after delivery. We hypothesized that high levels of fetal Mc were the consequence of a fetus with a high sHLA-G profile, therefore that children from women with SSc would have this profile more often than children from healthy women. High sHLA-G secretor profile is influenced by at least two variations in the HLA-G 3' untranslated region (UTR): a 14 bp deletion in exon 8 and the presence of cysteine (C) in position +3142 and by one variation in the 5' Upstream Regulatory Region (URR) at position -725. By a previously developed three-step multiplex PCR SNaPshot method, we evaluated 16 HLA-G polymorphisms in DNA samples from the first-born children of 39 women with SSc and 32 healthy women. Contrary to expectations, children from women with SSc did not have a high sHLA-G profile, but rather the opposite. We discuss possible reasons for this result and future orientations for HLA-G studies in SSc.

Many sources of foreign or semi foreign cells, known as microchimerism (Mc), can be found in healthy individuals. We have recently shown in women with end stage renal disease (ESRD) that Mc frequencies and levels are exacerbated prior to kidney transplantation. Is Mc arising from pregnancy a protective factor for renal diseases explaining lower incidence in women? Is Mc helpful in slowing down disease progression? However, natural Mc is not the only actor as post blood transfusion Mc is also found at high levels in women with ESRD. The difficulty is therefore to distinguish the different types of Mc and this is made even more complicated when the recipient receives a potentially chimeric organ. What part does each source of chimerism play in disease and transplant fate, and can one decipher each role knowing that one chimerism may hide another?

Objective Rheumatoid arthritis (RA) is an autoimmune disease that affects mostly women and is associated with HLA–DRB1 genes having in common a shared epitope sequence. In parallel, cells and/or DNA originating from pregnancy (microchimerism) persist for decades and could contribute to autoimmunity. The aim of this study was to examine whether microchimerism may be a source of the shared epitope among women with RA. Methods Women with RA and healthy women who lacked RA-associated genes such as HLA–DRB1*01 (n = 33 and n = 46, respectively) and/or HLA–DRB1*04 (n = 48 and n = 64, respectively), were tested for DRB1*01 or DRB1*04 microchimerism by HLA-specific quantitative polymerase chain reaction assays. As controls, alleles not associated with RA (DQB1*02 and DRB1*15/16) were also analyzed. Results Compared with healthy women, women (42% with RA had a higher frequency and higher levels of DRB1*04 microchimerism versus 8%; P = 0.00002) as well as DRB1*01 microchimerism (30% versus 4%; P = 0.0015). Moreover, no difference in microchimerism was observed for alleles not associated with RA. Conclusion Women with RA had microchimerism with RA-associated HLA alleles, but not with non–RA-associated HLA alleles, more often and at higher levels compared with healthy women. These observations are the first to indicate that microchimerism can contribute to the risk of an autoimmune disease by providing HLA susceptibility alleles.

Background and objectives Men are less often affected by Rheumatoid arthritis (RA) than women. In mice studies, it was shown that a duplication in Toll-like receptor 7 (Tlr7) gene is sufficient to potentiate autoimmunity in males. We therefore propose to investigate whether an increase in copy number (CN) of TLR7, and its neighbouring paralog TLR8 on X chromosome, could contribute to the pathogenesis of RA in men. Materials and methods In a pilot analysis, we had first tested our hypothesis by real-time quantitative PCR assay to assess CN of TLR7 gene and TLR8 compared to Beta-Globin gene (HBB), using the relative standard curve calculation method, on DNA from PBMCs of 49 men with RA and 42 healthy controls. TLR7/8 copy numbers significantly increased in PBMCs from men with RA when compared to healthy men. Nevertheless, because men with RA were significantly older than healthy donors, the increased TLR7/8 CN could be age-dependent. In the current study, we have tested whether TLR7/8 CN varies with age on a large number of controls from birth to 82 years old (160 men and 180 women). We also tested an additional autosomal RPP30 gene to further validate our results. DNA samples from peripheral blood of 49 men with RA versus 160 healthy men and 44 women with RA versus 180 healthy women were tested for TLR7/8 CN compared to mean CN values of HBB and RPP30. Similarly, DNA samples from PBMCs of 53 men with RA versus 42 healthy men and 40 women with RA versus 41 healthy women were analysed. Results TLR7 or TLR8 CN does not vary with age in healthy individuals (women or men). Similar findings were observed in patients with RA. Nevertheless, we confirmed that men with RA had an increased TLR7 and TLR8 CN in peripheral blood compared to healthy men (p < 0.0001 and p = 0.002 respectively). A rather opposite trend was observed in women with RA. Similar results were observed in PBMCs. Conclusions We have presented evidence that copy number of X-linked TLR7 and TLR8 genes increases in men with RA. This increase is not only disease dependent but also sex-dependent. We are currently validating our results by digital droplet polymerase chain reaction to confirm this genetic heterogeneity between men and women with RA.

Background Allograft inflammatory factor 1 (AIF1) is a cytoplasmic inflammatory protein encoded within the HLA class III genomic region on chromosome 6 (6p21.3). Although several risk loci for Rheumatoid Arthritis (RA) have been identified by Genome Wide Association Studies (GWAS), none of them involved AIF1 polymorphisms. However, two studies on small cohorts have shown that AIF1 single nucleotide polymorphism (SNP) Rs2269475 (C/T), causing a non-synonymous change of amino acid, is associated with RA (Harney, SM et al , 2008; Pawlik A et al , 2008). Moreover, AIF1 overexpression in inflammatory synovial tissues and macrophages isolated from synovial fluids of patients with RA, confirms its potential role in RA. Objective We propose to examine the association of the seven most described AIF1 SNPs in our French RA patients. Methods We have tested 99 Anti-Citrullinated Protein Antibody (ACPA) positive Caucasian RA patients who fulfilled ACR/EULAR criteria and 104 healthy Caucasians. We designed AIF1 primers to specifically amplify the AIF1 gene region containing the 7 SNPs: Rs2844475, Rs4711274, Rs2736182, Rs2736181, Rs2259571, Rs2269475 and Rs13195276. PCR products were sequenced (Cogenics Beckman Coulter) and chromatogram results analysed for the 7 SNPs positions in patients and controls. Patients and controls were genotyped for HLA-DRB1. Results Two SNPs out of the 7 were associated with RA: Rs4711274 (G/A) and Rs2269475 (C/T). Regarding Rs4711274, G/A and A/A genotypes were increased when compared with controls (p = 0.0005). The minor A allele was strongly associated with RA (p = 0.0005). Regarding Rs22699475, in linkage disequilibrium with the former, we found a similar pattern with increased T/T and C/T genotypes (p = 0.0009) and increased minor T allele frequency (p = 0.0008) in patients with RA. Interestingly, patients carrying the minor associated AIF1 allele expressed HLA-DRB*04 more often than the patient’s group carrying the C/C or G/G genotype (63.8% versus 44.4%), although the difference was marginal (p = 0.06). Conclusions In this study of French Caucasians with RA, we confirmed Rs2269475 association already described in British and Polish Caucasians. Additionally, we find an association with Rs4711274 in linkage disequilibrium with Rs2269475. Intriguingly, such associations have never been found in GWAS.

Background Rheumatoid Arthritis (RA), like most auto-immune diseases, is a female predominant disease. As a possible explanation for gender bias, we have previously shown that women with RA have non-random X chromosome inactivation (XCI) that could trigger autoimmunity (article in preparation). Intriguingly, this bias in XCI correlates with presence of the shared epitope (SE) and with disease duration. Also associated with presence of the SE, premature immunosenescence, characterised by shorter telomere length, has been described in peripheral blood cells from patients with RA [1]. Moreover, telomeric non coding RNAs have been reported to be enriched near the inactive X chromosome in mammals [2] indicating a potential link between telomere length and XCI. Objectives In this context, we propose to test whether women with RA have shortened telomere length and whether that could influence the epigenetic mechanism of XCI. Methods A total of 73 women with RA and 48 healthy women with no history of autoimmune diseases, who had previously been tested for XCI and HLA-genotyped, were evaluated for telomere length. The relative telomere length was estimated by real-time PCR as originally described by Cawthon [3] with the 2 - ∆∆ Ct method. Results Preliminary results show that women with RA have smaller telomere length than healthy women, although the difference is modest (p = 0.07) and has to be adjusted for age on a larger cohort. Contrary to expectations, shorter telomere length is not correlated with skewed XCI status, disease duration or the presence of shared epitope in our small cohort. Conclusions This preliminary study seems to confirm that women with RA have shorter telomeres than healthy women. Further telomere length measurements have to be done on a larger group of patients with RA and healthy controls, as well as HLA-genotyping them and evaluating their XCI status. This will be a step forward in understanding the relationship between immune senescence, female predisposition and genetic risk (SE) in RA. References SO Schonland et al , Proc Natl Acad Sci U S A 100 , 13471 (Nov 11, 2003). S Schoeftner, M A Blasco, Nat Cell Biol 10 , 228 (Feb, 2008). RM Cawthon, Nucleic Acids Res 30 , e47 (May 15, 2002).

Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc), none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, (67)FLEDR(71), shared by HLA-DRB susceptibility alleles, or (71)TRAELDT(77), shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient's clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ(2) = 28.4, p

Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p

Background Systemic sclerosis or Scleroderma (SSc) is an autoimmune disease affecting mostly women with a higher incidence between 40 and 60 years old. Several HLA class II genes have been associated with SSc depending on the clinical subset of the disease, limited cutaneous SSc (lc-SSc) or diffuse cutaneous SSc (dc-SSc) or on specific autoantibodies, anticentromere antibodies (ACA) or antitopoisomerase (ATA) antibodies, respectively hallmarks of the above clinical subsets. HLA-G is a non classical HLA molecule with tolerance functions that may play a role in inflammatory diseases. A 14 bp sequence insertion/deletion polymorphism (rs16375) in the 3′-untranslated region of the HLA-G gene has been associated to levels of soluble HLA-G (sHLA-G). The insertion is associated with lower levels. Objectives In the current study, the authors propose to analyse this polymorphism in patients with SSc compared to healthy individuals. The authors furthermore stratified patients according to sex, clinical subsets and autoantibody status. Patients and methods The authors genotyped 145 patients with SSc and 95 healthy controls by a previously developed two step multiplex SNaPshot method several HLA-G Single Nucleotide polymorphisms and the -14bp Ins/Del polymorphism. Results Patients with SSc tend to have higher frequency of ins/ins (29/145, 20.0%) genotypes compared with healthy controls (11/95, 11.6%, p=0.008). The frequency of Ins alleles compared to Del alleles is significantly increased among patients with SSc (p=0.024). This association reveals interesting patterns when patients are divided by sex (women, N=110 and men, N=35). Men had similar frequencies of Del/Del, Del/Ins, Ins/Ins genotypes than healthy controls (respectively, 45.7%, 42.9%, 11.4% and 50.5%, 37.9%, 11.6%), whereas women had different proportions of genotypes with Del/Del: 36.4%, Del/Ins: 40.9% and Ins/Ins: 22.7%. The difference in genotype repartition was statistically significant in women with SSc compared with healthy controls (p=0.047) and the difference in allele frequency even stronger (Ins: 43.1% compared with 30.5%, p=0.008). Apart from the gender association an association with dcSSc with the Ins allele (p=0.004), and a marginal association with lcSSc (p=0.08)were also observed. Interestingly both autoantibody subgroups, ATA and ACA, were associated with higher Ins allele frequencies (p=0.018 and p=0.03, respectively). Conclusion Women with SSc but not men have a genotype of low sHLA-G secretor. Further studies need to be conducted to better understand this gender dichotomy and its meaning in tolerance in a disease affecting mostly women in their childbearing years.

Background and objectives It has long been recognised that women have a greater prevalence of autoimmune diseases. Rheumatoid arthritis (RA) does not escape this rule with a women:men ratio of 3:1. X chromosome inactivation (XCI) is a dosage compensation mechanism used by mammals to ensure that XX females and XY males equalise X chromosome gene expression. As a consequence, females are a mosaic of two cell lines, one expressing maternal X-linked and the other expressing paternal X-linked genes with a ratio close to 50:50 when XCI is random. However skewing, defined as a deviation from the 50:50 ratio has been described in females with autoimmune thyroid diseases, scleroderma and juvenile idiopathic arthritis (for review1). The aim of the current study is to test whether women with RA also have a skewed XCI. Methods The highly polymorphic CAG repeat on the first exon of the androgen receptor gene was genotyped, as described elsewhere2 to determine XCI bias in 84 women with RA and 100 healthy women. Results A total of 54 patients and 69 controls were informative for androgen receptor polymorphism. Among them 31.5% of women with RA (17/54) had a skewed XCI (≥80:20) compared to only 17.4% of healthy women (12/69). Only extreme skewing was statistically significant with 18.5% of patients following this pattern and 2.9% of controls (p=0.004). Conclusions Our preliminary data indicate that skewed XCI may be a risk factor for the occurrence of RA in women. Further studies need to be done to analyse whether women who have a skewed pattern have less genetic susceptibilities (shared epitope) or less specific autoantibodies (anti-CCP) as their risk factor is X chromosome linked.