Doua F. Azzouz

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.

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.

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.

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.

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.

FRI0114 Allograft inflammatory factor 1 (AIF1) polymorphisms RS4711274 (G/A) and RS2269475 (C/T) may predict etanercept plus methotrexate response in french caucasian patients with rheumatoid arthritis

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

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.

A7.9 Does Telomere Shortening in Women with Rheumatoid Arthritis Predict X Chromosome Inactivation Bias

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).

A7.2 Allograft Inflammatory Factor 1 (AIF1) Polymorphisms in French Caucasians with Rheumatoid Arthritis

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.