Longitudinal gut microbiome analyses and blooms of pathogenic strains during lupus disease flares.

OBJECTIVE: Whereas genetic susceptibility for systemic lupus erythematosus (SLE) has been well explored, the triggers for clinical disease flares remain elusive. To investigate relationships between microbiota community resilience and disease activity, we performed the first longitudinal analyses of lupus gut-microbiota communities. METHODS: In an observational study, taxononomic analyses, including multivariate analysis of ß-diversity, assessed time-dependent alterations in faecal communities from patients and healthy controls. From gut blooms, strains were isolated, with genomes and associated glycans analysed. RESULTS: Multivariate analyses documented that, unlike healthy controls, significant temporal community-wide ecological microbiota instability was common in SLE patients, and transient intestinal growth spikes of several pathogenic species were documented. Expansions of only the anaerobic commensal, Ruminococcus (blautia) gnavus (RG) occurred at times of high-disease activity, and were detected in almost half of patients during lupus nephritis (LN) disease flares. Whole genome sequence analysis of RG strains isolated during these flares documented 34 genes postulated to aid adaptation and expansion within a host with an inflammatory condition. Yet, the most specific feature of strains found during lupus flares was the common expression of a novel type of cell membrane-associated lipoglycan. These lipoglycans share conserved structural features documented by mass spectroscopy, and highly immunogenic repetitive antigenic-determinants, recognised by high-level serum IgG2 antibodies, that spontaneously arose, concurrent with RG blooms and lupus flares. CONCLUSIONS: Our findings rationalise how blooms of the RG pathobiont may be common drivers of clinical flares of often remitting-relapsing lupus disease, and highlight the potential pathogenic properties of specific strains isolated from active LN patients.

The gut microbiome in systemic lupus erythematosus: lessons from rheumatic fever.

For more than a century, certain bacterial infections that can breach the skin and mucosal barriers have been implicated as common triggers of autoimmune syndromes, especially post-infection autoimmune diseases that include rheumatic fever and post-streptococcal glomerulonephritis. However, only in the past few years has the importance of imbalances within our own commensal microbiota communities, and within the gut, in the absence of infection, in promoting autoimmune pathogenesis become fully appreciated. A diversity of species and mechanisms have been implicated, including disruption of the gut barrier. Emerging data suggest that expansions (or blooms) of pathobiont species are involved in autoimmune pathogenesis and stimulate clonal expansion of T cells and B cells that recognize microbial antigens. This Review discusses the relationship between the gut microbiome and the immune system, and the potential consequence of disrupting the community balance in terms of autoimmune development, focusing on systemic lupus erythematosus. Notably, inter-relationships between expansions of certain members within gut microbiota communities and concurrent autoimmune responses bear features reminiscent of classical post-infection autoimmune disease. From such insights, new therapeutic opportunities are being considered to restore the balance within microbiota communities or re-establishing the gut-barrier integrity to reinforce immune homeostasis in the host.

Sex-dependent Lupus Blautia (Ruminococcus) gnavus strain induction of zonulin-mediated intestinal permeability and autoimmunity.

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, Blautia (Ruminococcus) gnavus (RG). To investigate whether the RG strains in Lupus patients have in vivo pathogenic properties in a gnotobiotic system, we colonized C57BL/6 mice with individual RG strains from healthy adults or those from Lupus patients. These strains were similar in their capacity for murine intestinal colonization of antibiotic-preconditioned specific-pathogen-free, as well as of germ-free adults and of 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. These Lupus RG strain-induced functional alterations were associated with RG translocation to mesenteric lymph nodes, and 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 with 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 pathway by which RG strains from Lupus patients contribute to a leaky gut and features of autoimmunity implicated in the pathogenesis of flares of clinical Lupus disease.

Systemic Lupus Erythematosus and dysbiosis in the microbiome: cause or effect or both?

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.

Mosaicism of XX and XXY cells accounts for high copy number of Toll like Receptor 7 and 8 genes in peripheral blood of men with Rheumatoid Arthritis.

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.

Soluble HLA-G Expression Inversely Correlates With Fetal Microchimerism Levels in Peripheral Blood From Women With Scleroderma.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5'URR/3'UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p < 0.0001), independently of clinical subtypes, autoantibody profiles, disease duration, or treatments. Moreover, sHLA-G levels were inversely correlated to FMc quantities (Spearman correlation, p < 0.01). Finally, women with SSc had lower sHLA-G independently of the eight HLA-G 5'URR/3'UTR polymorphisms, although they were statistically more often homozygous than heterozygous for HLA-G polymorphism genotypes -716 (G/T), -201 (G/A), 14 bp (ins/del), and +3,142 (G/A) than healthy women. In conclusion, women with SSc display less sHLA-G expression independently of the eight HLA-G polymorphisms tested. This decreased production correlates with higher quantities of persisting FMc commonly observed in blood from SSc women. These results shed some lights on the contribution of the maternal HLA-G protein to long-term persistent fetal Mc and initiate new perspectives in this field.

Evaluation of X Chromosome Inactivation with Respect to HLA Genetic Susceptibility in Rheumatoid Arthritis and Systemic Sclerosis.

BACKGROUND: Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. METHODS: Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. RESULTS: 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the "shared epitope" correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. CONCLUSION: Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity.

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus.

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.

Analyzing HLA-G polymorphisms in children from women with scleroderma.

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.

Male microchimerism at high levels in peripheral blood mononuclear cells from women with end stage renal disease before kidney transplantation.

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<0.00001 and p = 0.0005 respectively). Male Mc was increased in women with ESRD whether they had or not a history of male pregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD.

Chimerism in women with end stage renal diseases: Who's who?

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?

Comparing HLA shared epitopes in French Caucasian patients with scleroderma.

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<10-6) and with anti-topoisomerase antibody (ATA) production (χ(2) = 43.9, p<10-9) whereas TRAELDT association is weaker in both subgroups (χ(2) = 7.2, p = 0.027 and χ(2) = 14.6, p = 0.0007 respectively). Moreover, FLEDR motif- association among patients with diffuse SSc remains significant only in ATA subgroup. The risk to develop ATA positive SSc is higher with double dose FLEDR than single dose with respectively, adjusted standardised residuals of 5.1 and 2.6. The increase in FLEDR motif is mostly due to the higher frequency of HLA-DRB1*11 and DRB1*15 haplotypes. Furthermore, FLEDR is always carried by the most abundantly expressed ß chain: ß1 in HLA DRB1*11 haplotypes and ß5 in HLA-DRB1*15 haplotypes.In French Caucasian patients with SSc, FLEDR is the main presenting motif influencing ATA production in dcSSc. These results open a new field of potential therapeutic applications to interact with the FLEDR peptide binding groove and prevent ATA production, a hallmark of severity in SSc.

How microchimerism can impart HLA susceptibility in patients with rheumatoid arthritis.

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 intriguing story and may offer hope for future therapeutic and/or preventative interventions.

Cells from a vanished twin as a source of microchimerism 40 years later.

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 from this source is not yet known and whether the scleroderma-like disease observed in the patient is anecdotal or implies a potential association with autoimmune disease remains to be elucidated.