Marielle Martin

Transfer of the shared epitope through microchimerism in women with rheumatoid arthritis.

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.

New autoantigens in rheumatoid arthritis (RA): screening 8268 protein arrays with sera from patients with RA

Objective: To identify new IgG autoantibodies in sera from patients with rheumatoid arthritis (RA). Methods: We tested serum samples from 19 patients with RA with given human leukocyte antigen (HLA)-DR genotypes, from 7 patients with spondylarthropathy, 2 patients with lupus, 4 patients with systemic sclerosis and 10 healthy individuals on 8268 human protein arrays. Results: We identified four antigens (peptidyl arginine deiminase 4 (PAD4), protein kinase Cβ1 (PKCβ1), phosphatylinositol 4 phosphate 5 kinase type II γ (PIP4K2C) and v raf murine sarcoma viral oncogene homologue B1 catalytic domain (BRAF)) that were recognised almost uniquely by sera from patients with RA on protein arrays. Using purified proteins, we confirmed that PAD4 and BRAF are recognised almost uniquely by patients with RA. Conclusion: We identified PAD4 and BRAF as RA specific autoantigens.

Male microchimerism and HLA compatibility in French women with sclerodema: a different profile in limited and diffuse subset

OBJECTIVES Male microchimerism (Mc) persisting from pregnancy has been found at greater frequencies and/or higher quantities in women with scleroderma (SSc) compared with controls, suggesting a possible role in disease development. Moreover, women with an HLA-compatible child have a higher risk to develop SSc. We tested the hypothesis, on our French SSc cohort, that women with lcSSc and dcSSc, two distinct clinical subsets, have a different profile in terms of Mc and HLA compatibility in families. METHODS We studied 98 women (52 lcSSc and 46 dcSSc) for male Mc, by real-time PCR, in their whole blood and/or peripheral blood mononuclear cells (PBMC). Similarly, 91 matched healthy women were analysed. Complete HLA-DRB1 typing was obtained for 58 SSc and 68 control families (proband/children). RESULTS Women with lcSSc (N = 50) had male Mc more often in their whole blood than women with dcSSc (N = 40, 20 vs 5%, P = 0.038), but not significantly more than controls. By contrast, women with dcSSc (N = 36) hold Mc more often in PBMC (25 vs 9%), but not significantly and have greater quantities than controls (N = 82, P = 0.048). This contrast is also visible in feto-maternal HLA-DRB1 compatibility, which was increased only among women with lcSSc (N = 33) compared with controls (N = 68, P = 0.003). CONCLUSION For the first time, we showed that women with lcSSc and dcSSc hold male Mc in different blood compartments. Furthermore, a distinct pattern between the two SSc subtypes is observed for feto-maternal HLA-DRB1 compatibility. These results suggest a different mechanism behind each type of disease.

Rheumatoid arthritis–specific autoantibodies to peptidyl arginine deiminase type 4 inhibit citrullination of fibrinogen

OBJECTIVE Autoantibodies to citrullinated proteins are specific for rheumatoid arthritis (RA) and recognize epitopes centered by citrulline, a posttranslationally modified form of arginine. Peptidyl arginine deiminase type 4 (PAD-4), the enzyme that converts arginine into citrulline, is in itself a target for RA-specific autoantibodies. This study was undertaken to assess whether anti-PAD-4 autoantibodies interfere with citrullination in vitro in patients with RA, and to identify peptide targets of anti-PAD-4 antibodies that can activate or inhibit citrullination. METHODS To test whether autoantibodies to PAD-4 influence citrullination, an in-house citrullination assay was developed using purified autoantibodies to PAD-4. To map B cell epitopes on PAD-4, 65 overlapping 20-mer peptides encompassing the entire PAD-4 were analyzed for their reactivity in RA sera. RESULTS Autoantibodies to PAD-4 inhibited PAD-4-mediated citrullination. Three linear peptides on PAD-4 were recognized almost uniquely by PAD-4 autoantibodies in the sera of patients with RA. One peptide was located in the N-terminal, calcium-binding domain of PAD-4, while 2 other peptides were located in the C-terminal, substrate-binding domain of PAD-4. CONCLUSION Autoantibodies to PAD-4 inhibit in vitro citrullination of fibrinogen by PAD-4. Most anti-PAD-4-positive sera recognize peptides located both in the N-terminal domain (211-290) and the C-terminal domain (601-650) of PAD-4.

Autoantibodies to BRAF, a new family of autoantibodies associated with rheumatoid arthritis

IntroductionBRAF (v raf murine sarcoma viral oncogene homologue B1) is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway, known to be implicated in the production of pro-inflammatory cytokines.We have observed that sera from rheumatoid arthritis (RA) patients recognize the BRAFs catalytic domain, which encompasses amino acids 416 to 766. Here, we identify peptide targets of anti-BRAF autoantibodies and test whether anti-BRAF autoantibodies may interfere with BRAF kinase activity.MethodsAnti-BRAF autoantibodies were detected by ELISA (enzyme-linked immunosorbent assay) in the serum of RA patients and controls, using 40 overlapping 20mer peptides encompassing the catalytic domain of BRAF as immunosorbents. To test whether autoantibodies to BRAF influence BRAF kinase activity, we developed an in vitro phosphorylation assay of MEK1 (mitogen extracellular regulated kinase), a major BRAF substrate. MEK1 phosphorylation by BRAF was tested in the presence of purified anti-BRAF autoantibodies from RA patients or control antibody.ResultsWe found that one BRAF peptide, P25 (656 to 675), is specifically recognized by autoantibodies from RA patients. Of interest, anti-P25 autoantibodies are detected in 21% of anti-CCP (cyclic citrullinated peptides) negative RA patients. Anti-BRAF autoantibodies activate the in vitro phosphorylation of MEK1 mediated by BRAF.ConclusionsAnti-BRAF autoantibodies from RA patients preferentially recognize one BRAF peptide: P25. Autoantibody responses to P25 are detected in 21% of anti-CCP negative RA patients. Most anti-BRAF autoantibodies activate BRAF kinase activity.

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, 67FLEDR71, shared by HLA-DRB susceptibility alleles, or 71TRAELDT77, 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 (χ2 = 28.4, p<10−6) and with anti-topoisomerase antibody (ATA) production (χ2 = 43.9, p<10−9) whereas TRAELDT association is weaker in both subgroups (χ2 = 7.2, p = 0.027 and χ2 = 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.

Long term treatment with abatacept or tocilizumab does not increase Epstein-Barr virus load in patients with rheumatoid arthritis - A three years retrospective study

Background Epstein-Barr Virus (EBV) is a widely disseminated lymphotropic herpes virus implicated in benign and malignant disorders. In transplant patients, immunosuppressive drugs (cyclosporine) diminish control of EBV replication, potentially leading to lymphoproliferative disorders (LPD). Rheumatoid arthritis (RA) patients have impaired control of EBV infection and have EBV load ten times higher than controls. As post transplant patients, patients with RA have increased risk of developing lymphomas. Immunosuppressive drugs used to treat RA (conventional disease modifying drugs cDMARDs or biologics bDMARDs) could enhance the risk of developing LPD in RA patients. We have previously shown that long term treatment with Methotrexate and/or TNF alpha antagonists does not increase EBV load in RA. Our objective was to monitor the Epstein-Barr Virus load in RA patients treated with Abatacept (CTLA4 Ig), a T cell coactivation inhibitor, and Tocilizumab, an anti IL6 receptor antibody. Methods EBV load in the peripheral blood mononuclear cells (PBMCs) of 55 patients under Abatacept (in 34% associated with Methotrexate) and 35 patients under Tocilizumab (in 37% associated with Methotrexate) was monitored for durations ranging from 6 months to 3 years by real time PCR. The influences of treatment duration and disease activity score 28 (DAS28) index on EBV load were analyzed. Results Abatacept did not significantly modify EBV load over time. Tocilizumab significantly diminished EBV load over time. No patient (of 90) developed EBV associated lymphoma. Conclusion Long term treatment with Abatacept or Tocilizumab does not increase EBV load in the PBMNCs of patients with RA.

Could microchimerism be a source of disease-associated HLA alleles in patients with scleroderma?

Background and objectives Systemic sclerosis (SSc) is an autoimmune disease divided in two subsets, diffuse cutaneous and limited cutaneous SSc (dcSSc and lcSSc), affecting mostly women. We, and others showed that patients with dcSSc are associated with particular HLA-DRB1*11 and *15 alleles, having in common an amino acid sequence (67FLEDR71). In parallel, cells and/or DNA, originating from bi-directional traffic of cells during pregnancy, referred as maternal or fetal microchimerism (Mc) persist for decades in healthy women and could trigger to SSc under particular circumstances. The aim of our study was to examine whether patients with dcSSc have more often a mother carrying the HLA susceptibility alleles and whether Mc may be a source of FLEDR among patients with dcSSc, as previously described by us and others in patients with RA for RA-associated HLA alleles.1 2 Methods Thirty five patients with dcSSc and 54 healthy women were HLA-DRB1 typed by SSOP methods as well as their mothers (N=89). Among the 35 patients, 13 did not carry the SSc-associated HLA-DRB1*15 alleles and were tested for DRB1*15 Mc by HLA specific quantitative PCR assays in their peripheral blood mononuclear cells and compared to 32 healthy controls who also did not carry HLA-DRB1*15 alleles. Results Initial results, show that women with dcSSc had a tendency to have more often a mother carrying HLA DRB1*15 alleles (non-inherited maternal antigen, NIMA), than healthy controls (22.9% vs 11.1%, p=0.07). However HLA-DRB1*15 Mc levels and frequencies were not different between patients and controls with respectively 4/13 (30.7%) and 9/32 (28.1%) women positive for Mc. HLA-DRB1*11 NIMAs were at similar frequencies for patients and controls and not yet tested for maternal Mc. Conclusions Contrary to our recent publication in RA, patients with SSc do not acquire more often than controls HLA-DRB1*15 susceptibility alleles through Mc. Several mechanisms may explain this negative result as we recently showed that immunosuppressive treatments could decrease Mc levels. Moreover the marginal increase of mothers carrying HLA-DRB1 susceptibility alleles (NIMA) is intriguing and merits further analyses.

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.