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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

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.

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.

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...