Microchimeric cells promote production of rheumatoid arthritis-specific autoantibodies.

BACKGROUND: Women are more likely to develop autoimmune diseases than men. Contribution from microchimerism (Mc) has been proposed, as women naturally acquire Mc from more sources than men because of pregnancy. Women with Rheumatoid Arthritis (RA) who lack RA-associated HLA alleles have been found to harbor Mc with RA-associated HLA alleles in higher amounts than healthy women in prior work. However, an immunological impact of Mc remains to be elucidated. OBJECTIVES: To test the hypothesis that Mc with RA-risk associated HLA alleles can result in the production of RA-associated autoantibodies, when host genetic risk is absent. METHODS: DBA/2 mice are unable to produce RA-specific anti-citrullinated autoantibodies (ACPAs) after immunization with the enzyme peptidyl arginine deiminase (PAD) in a previously developed model. DBA/2 females were mated with C57BL/6 males humanized to express HLA-DR4, which is associated with RA-risk and production of ACPAs, to evaluate DR4+ fetal Mc contribution. Next, DBA/2 females born of heterozygous DR4+/- mothers were evaluated for DR4+ Mc of maternal or littermate origin. Finally, DBA/2 females from DR4+/- mothers were crossed with DR4+ males, to evaluate the contribution of any Mc source to ACPA production. RESULTS: After PAD immunization, between 20 % and 43 % of DBA/2 females (otherwise unable to produce ACPAs) had detectable ACPAs (CCP2 kit) after exposure to sources of Mc with RA-associated HLA alleles, compared to 0 % of unmated/unexposed DBA/2 females. Further the microchimeric origin of the autoantibodies was confirmed by detecting a C57BL/6-specific immunoglobulin isotype in the DBA/2 response. CONCLUSION: Our study demonstrates that Mc cells can produce "autoantibodies" and points to a role of Mc in the biology of autoimmune diseases, including RA.

PAD2 immunization induces ACPA in wild-type and HLA-DR4 humanized mice.

Rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles expressing the "shared epitope." RA is usually preceded by the emergence of anti-citrullinated protein autoantibodies (ACPAs). ACPAs recognize citrulline residues on numerous proteins. Conversion of arginine into citrulline is performed by enzymes called peptidyl arginine deiminases (PADs). We have previously demonstrated that C3H mice immunized with PADs can produce ACPAs by a hapten-carrier mechanism. Here, we address the influence of HLA-DR alleles in this model in mice expressing RA-associated HLA-DRB1*04:01 (KO/KI*04:01), HLA-DRB1*04:04 (KO/KI*04:04), or non-RA-associated HLA-DRB1*04:02 (KO/KI*04:02) after murine PAD2 immunization. Immunization with mPAD2 triggers production of ACPAs in wild-type (WT) and HLA-DR4 C57BL/6 mice. Both I-Ab and HLA-DR are involved in the activation of mPAD2-specific T lymphocytes. Among HLA-DR4 mice, mice expressing RA-associated HLA-DRB1*04:01 are the best responders to mPAD2 and the best anti-citrullinated peptide antibody producers.

Non-Classical HLA Determinants of the Clinical Response after Autologous Stem Cell Transplantation for Systemic Sclerosis.

Systemic Sclerosis (SSc) is a chronic autoimmune disease with high morbidity and mortality. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is the best therapeutic option for rapidly progressive SSc, allowing increased survival with regression of skin and lung fibrosis. The immune determinants of the clinical response after AHSCT have yet to be well characterized. In particular, the pivotal role of the Human Leukocyte Antigen (HLA) system is not well understood, including the role of non-classical immuno-modulatory HLA-E and HLA-G molecules in developing tolerance and the role of Natural Killer cells (NK) in the immunomodulation processes. We retrospectively tested whether the genetic and/or circulating expression of the non-classical HLA-E and HLA-G loci, as well as the imputed classical HLA determinants of HLA-E expression, influence the observed clinical response to AHSCT at 12- and 24-month follow-up. In a phenotypically well-defined sample of 46 SSc patients classified as clinical responders or non-responders, we performed HLA genotyping using next-generation sequencing and circulating levels of HLA-G and quantified HLA-E soluble isoforms by ELISA. The -21HLA-B leader peptide dimorphism and the differential expression level of HLA-A and HLA-C alleles were imputed. We observed a strong trend towards better clinical response in HLA-E*01:03 or HLA-G 14bp Del allele carriers, which are known to be associated with high expression of the corresponding molecules. At 12-month post-AHSCT follow-up, higher circulating levels of soluble HLA-E were associated with higher values of modified Rodnan Skin Score (mRSS) (p = 0.0275), a proxy of disease severity. In the non-responder group, the majority of patients carried a double dose of the HLA-B Threonine leader peptide, suggesting a non-efficient inhibitory effect of the HLA-E molecules. We did not find any correlation between the soluble HLA-G levels and the observed clinical response after AHSCT. High imputed expression levels of HLA-C alleles, reflecting more efficient NK cell inhibition, correlated with low values of the mRSS 3 months after AHSCT (p = 0.0087). This first pilot analysis of HLA-E and HLA-G immuno-modulatory molecules suggests that efficient inhibition of NK cells contributes to clinical response after AHSCT for SSc. Further studies are warranted in larger patient cohorts to confirm our results.

Peptidyl arginine deiminase immunization induces anticitrullinated protein antibodies in mice with particular MHC types.

Autoantibodies to citrullinated proteins (ACPAs) are present in two-thirds of patients with rheumatoid arthritis (RA). ACPAs are produced in the absence of identified T cell responses for each citrullinated protein. Peptidyl arginine deiminase 4 (PAD4), which binds proteins and citrullinates them, is the target of autoantibodies in early RA. This suggests a model for the emergence of ACPAs in the absence of detectable T cells specific for citrullinated antigens: ACPAs could arise because PADs are recognized by T cells, which help the production of autoantibodies to proteins bound by PADs, according to a "hapten/carrier" model. Here, we tested this model in normal mice. C3H are healthy mice whose IEßk chain is highly homologous to the ß1 chain HLA-DRB1*04:01, the allele most strongly associated with RA in humans. C3H mice immunized with PADs developed antibodies and T cells to PAD and IgG antibodies to citrullinated fibrinogen peptides, in the absence of a T cell response to fibrinogen. To analyze the MHC background effect on hapten/carrier immunization, we immunized DBA/2 mice (whose IEßd chain is similar to that of HLA-DRB1*04:02, an HLA-DR4 subtype not associated with RA). DBA/2 mice failed to develop antibodies to citrullinated fibrinogen peptides. Thus, T cell immunization to PAD proteins may trigger ACPAs through a hapten/carrier mechanism. This may constitute the basis for a new mouse model of ACPA-positive RA.

Microchimerism in Ghanaian children recipients of whole blood transfusion for severe anaemia.

BACKGROUND AND OBJECTIVES: Transfusion-acquired microchimerism (TA-Mc) has been reported in major trauma but not in young children despite relative immunodeficiency who, in sub-Saharan Africa, often suffer severe anaemia related to haemoglobinopathies or primary malaria infections. We examined the hypothesis that such massive red cell destructions might provide conditions favourable to TA-Mc, particularly when exposed to massive amounts of parasite antigens. MATERIALS AND METHODS: Twenty-seven female children <5 years transfused with male whole blood for severe anaemia (13 with acute malaria and 14 with other causes) were retrospectively identified, and a blood sample was collected >6 months post-transfusion. Four whole blood samples from paediatric females transfused with blood from female donors and five secondary school female students never pregnant, never transfused were used as negative controls. RESULTS: Nineteen patients (70%) carried male Mc with four (15%) having high levels of Mc (>100 genome equivalent of male cells/million of host cells) compared to three controls (37·5%). There was no difference in frequency or quantity of male Mc between paediatric patients with severe malaria and paediatric patients with other causes of severe anaemia. TA-Mc was not correlated with patient age, duration of whole blood storage or lymphocyte load transfused. After a median of 7 months post-transfusion, acute malaria did not increase the frequency of TA-Mc. One negative control appeared to carry low-level male cells. CONCLUSION: Transfusion-acquired microchimerism appears frequent in young children transfused with whole blood for severe anaemia.

In utero position matters for littermate cell transfer in mice: an additional and confounding source with maternal microchimerism.

Introduction: Feto-maternal cell transfer during pregnancy is called microchimerism (Mc). Its persistence in respective hosts is increasingly studied as to its potential role in immune tolerance, autoimmunity, cancer, and degenerative diseases. Murine models with transgenic reporter genes, heterozygously carried by the mother, allow maternal Mc tracking in wild-type (WT) offspring. However, as gestation in mice is multi-embryonic, an exchange of cells between fetuses carrying the same reporter gene as their mother and negative WT littermate, named littermate Mc (LMc), can occur and be confounded with the maternal source. We propose here to evaluate LMc contribution in mice. Methods: To avoid the maternal confounding source of Mc, transgenic males, heterozygous for a reporter gene, here, the human leukocyte antigen DRB1*04 (DR4+/-), were crossed with WT females (DR4-/-). DR4+/- LMc was specifically quantified by HLA-DR4 quantitative PCR, i) in utero in main organs from 15 DR4-/- fetuses from three litters of 11, nine, and five; and ii) after birth in two litters of eight pups: in two DR4-/- stillborns and four DR4-/- adult mice. Results: At embryonic stages, DR4-/- fetuses having one or two nearby DR4+/- littermates in the same uterine horn were almost seven times more frequently positive for DR4- microchimerism in their organs (p = 0.01) and had quantitatively more LMc (p = 0.009) than those without nearby DR4+/- littermates. Furthermore, LMc persists at birth and into adulthood with interindividual heterogeneity. Conclusions: This study identifies heterogeneity for LMc acquisition according to in utero position and different interpretation of previously published results on maternal Mc in mice.

PAD4 Immunization Triggers Anti-Citrullinated Peptide Antibodies in Normal Mice: Analysis With Peptide Arrays.

The critical immunological event in rheumatoid arthritis (RA) is the production of antibodies to citrullinated proteins (ACPAs), ie proteins on which arginines have been transformed into citrullines by peptidyl arginine deiminases (PAD). In C3H mice, immunization with PAD4 triggers the production of ACPAs. Here, we developed a peptide array to analyze the fine specificity of anti-citrullinated peptide antibodies and used it to characterize the ACPA response after hPAD4 immunization in mice expressing different H-2 haplotypes. Sera from C3H, DBA/2, BALB/c and C57BL/6 mice immunized with human PAD4 (hPAD4) or control-matched mice immunized with phosphate buffered saline (PBS) were used to screen peptide arrays containing 169 peptides from collagen, filaggrin, EBNA, proteoglycan, enolase, alpha and beta fibrinogen, histon and vimentin. Human PAD4 immunization induced antibodies directed against numerous citrullinated peptides from fibrinogen, histon 4 and vimentin. Most peptides were recognized under their arginine and citrullinated forms. DBA/2 and BALB/c mice (H-2d) had the lowest anti-citrullinated peptide IgG responses. C3H (H-2k) and BL6 mice (H-2b) had the highest anti-citrullinated peptide IgG responses. The newly developed peptide array allows us to characterize the ACPA production after hPAD4 immunization in mice on the H-2d, H-2k or H-2b backgrounds. This sensitive tool will be useful for further studies on mice for prevention of ACPA production by PAD tolerization.

Safety and preliminary efficacy of allogeneic bone marrow-derived multipotent mesenchymal stromal cells for systemic sclerosis: a single-centre, open-label, dose-escalation, proof-of-concept, phase 1/2 study.

BACKGROUND: Systemic sclerosis remains an orphan life-threatening autoimmune disease. The unique immunomodulatory, proangiogenic, and antifibrotic properties of mesenchymal stromal cells provide a strong rationale for mesenchymal stromal cell-based therapy for systemic sclerosis, and treatment with mesenchymal stromal cells has shown benefits in preclinical models of this disease. The safety of allogeneic bone marrow-derived mesenchymal stromal cell administration in patients with severe systemic sclerosis has not yet been established. We aimed to test the safety and feasibility of a single intravenous injection of intrafamilial allogeneic bone marrow-derived mesenchymal stromal cells to treat severe diffuse systemic sclerosis. METHODS: We did an open-label, dose-escalation, proof-of-concept, phase 1/2 study at Saint-Louis-Hospital, Paris, France. Eligible patients were aged 18-70 years with severe diffuse systemic sclerosis, who fulfilled the 2013 American College of Rheumatology and European League Against Rheumatism systemic sclerosis criteria, had a minimum modified Rodnan skin score of 15 (range 0-51), had severe lung, heart, or kidney involvement, and had inadequate response or contraindications to conventional immunosuppressive therapy or autologous haematopoietic stem cell transplantation. Patients with severe comorbidities were excluded. The first ten recipients were to receive a single intravenous infusion of 1 × 106 bone marrow-derived mesenchymal stromal cells per kg bodyweight, and the subsequent ten recipients were to be infused with a single dose of 3 × 106 bone marrow-derived mesenchymal stromal cells per kg bodyweight. The primary endpoint was immediate tolerance during infusion and within the first 10 days after infusion, measured as the occurrence of serious adverse events (grade 3 or higher) in all infused patients. Safety was assessed in all participants during the 24-month follow-up period. This study is registered with ClinicalTrials.gov, NCT02213705. FINDINGS: Between March 24, 2014, and Jan 6, 2020, 20 cisgender individuals (13 women and seven men) with severe diffuse systemic sclerosis were enrolled. All 20 patients were included in the primary outcome analysis. No infusion-related severe adverse events and three infusion-related adverse events occurred in the first 10 days after treatment; one patient had grade 1 flushing and another patient had grade 1 nausea and grade 2 asthenia. After ten days and up to a median follow-up of 24·1 months (IQR 20·8-24·5), 36 non-treatment-related severe adverse events in 14 (70%) patients and no treatment-related adverse event were reported. INTERPRETATION: A single infusion of allogeneic bone marrow-derived mesenchymal stromal cells was safe in patients with severe diffuse systemic sclerosis. Future placebo-controlled trials will help to definitively ascertain the efficacy of mesenchymal stromal cell-based cell therapy from various tissue sources in larger number of patients with systemic sclerosis. FUNDING: French Ministry of Health, Capucine Association, Fonds de Dotation de l'AFER pour la Recherche Médicale, and Agence Nationale de la Recherche (Infrastructure Program Ecell), France.

Corrigendum: Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

[This corrects the article DOI: 10.3389/fimmu.2021.651399.].

Grandmaternal cells in cord blood.

BACKGROUND: During pregnancy a feto-maternal exchange of cells through the placenta conducts to maternal microchimerism (Mc) in the child and fetal Mc in the mother. Because of this bidirectional traffic of cells, pregnant women have also acquired maternal cells in utero from their mother and could transfer grandmaternal (GdM) cells to their child through the maternal bloodstream during pregnancy. Thus, cord blood (CB) samples could theoretically carry GdMMc. Nevertheless this has never been demonstrated. METHODS: Using Human Leukocyte Antigen (HLA)-specific quantitative PCR assays on three-generation families, we were able to test 28 CB samples from healthy primigravid women for GdMMc in whole blood (WB) and isolated cells (PBMC, T, B, granulocytes, stem cells). FINDINGS: Five CB samples (18%) had GdMMc which could not be confounded with maternal source, with quantities 100 fold lower than maternal Mc in WB and PBMC. Risk of aneuploidies and/or related invasive prenatal procedures significantly correlated with the presence of GdMMc in CB (p=0.024). Significantly decreased HLA compatibility was observed in three-generation families from CB samples carrying GdMMc (p=0.019). INTERPRETATION: Transgenerational transfer of cells could have implications in immunology and evolution. Further analyses will be necessary to evaluate whether GdMMc in CB is a passive or immunologically active transfer and whether invasive prenatal procedures could trigger GdMMc. FUNDING: Provence-Alpes-Côte d'Azur APEX grant # 2012_06549E, 2012_11786F and 2014_03978) and the Foundation for Medical Research (FRM Grant #ING20140129045).

Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

Background: Cord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition. Patients and Methods: Here we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status. Results: MMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or -DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc. Conclusions: We have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.

Cord blood maternal microchimerism following unrelated cord blood transplantation.

Cord blood transplantation (CBT) is associated with low risk of leukemia relapse. Mechanisms underlying antileukemia benefit of CBT are not well understood, however a previous study strongly but indirectly implicated cells from the mother of the cord blood (CB) donor. A fetus acquires a small number of maternal cells referred to as maternal microchimerism (MMc) and MMc is sometimes detectable in CB. From a series of 95 patients who underwent double or single CBT at our center, we obtained or generated HLA-genotyping of CB mothers in 68. We employed a technique of highly sensitive HLA-specific quantitative-PCR assays targeting polymorphisms unique to the CB mother to assay CB-MMc in patients post-CBT. After additional exclusion criteria, CB-MMc was evaluated at multiple timepoints in 36 patients (529 specimens). CB-MMc was present in seven (19.4%) patients in bone marrow, peripheral blood, innate and adaptive immune cell subsets, and was detected up to 1-year post-CBT. Statistical trends to lower relapse, mortality, and treatment failure were observed for patients with vs. without CB-MMc post-CBT. Our study provides proof-of-concept that maternal cells of the CB graft can be tracked in recipients post-CBT, and underscore the importance of further investigating CB-MMc in sustained remission from leukemia following CBT.

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.

Nonendocrine mechanisms of sex bias in rheumatic diseases.

Rheumatic diseases affect a wide range of individuals of all ages, but the most common diseases occur more frequently in women than in men, at ratios of up to ten women to one man. Despite a growing number of studies on sex bias in rheumatic diseases, sex-specific health care is limited and sex specificity is not systematically integrated into treatment regimens. Women and men differ in three major biological points: the number of X chromosomes per cell, the type and quantities of sex hormones present and the ability to be pregnant, all of which have immunological consequences. Could a greater understanding of these differences lead to a new era of personalized sex-specific medicine? This Review focuses on the main genetic and epigenetic mechanisms that have been put forward to explain sex bias in rheumatic diseases, including X chromosome inactivation, sex chromosome aneuploidy and microchimerism. The influence of sex hormones is not discussed in detail in this Review, as it has been well described elsewhere. Understanding the sex-specific factors that contribute to the initiation and progression of rheumatic diseases will enable progress to be made in the diagnosis, treatment and management of all patients with these conditions.

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.

How twin studies help to understand inflammatory joint disease.

Inflammatory joint disease (IJD) is a group of conditions that target the joints and periarticular structures. The contribution of genetic factors to these conditions is often less than 50%, suggesting a major role for environmental influences. Twin studies are the best means of assessing the role for genetic factors in IJD. Conclusive evidence has been provided by a few studies in vast samples of monozygotic and dizygotic twins, with only one twin in each pair having IJD. These studies have been most successful in ankylosing spondylitis and psoriatic arthritis. The other IJDs have proven more difficult to evaluate. This review demonstrates that genetic and environmental factors are inextricably linked and that ascribing IJDs to one or the other is misguided. Awareness of the limitations and possible sources of bias in twin studies is important when seeking to understand the development of these complex diseases.

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.

Male microchimerism in women without sons: quantitative assessment and correlation with pregnancy history.

PURPOSE: Fetal microchimerism, derived from fetal cells that persist after pregnancy, is usually evaluated by tests for male microchimerism in women who gave birth to sons. We investigated male microchimerism in women without sons and examined correlation with prior pregnancy history. Immunologic consequences of microchimerism are unknown. We studied healthy women and women with rheumatoid arthritis (RA). METHODS: Y-chromosome-specific real-time quantitative polymerase chain reaction was used to test peripheral blood mononuclear cells of 120 women (49 healthy and 71 with RA). Results were expressed as the number of male cells that would be equivalent to the total amount of male DNA detected within a sample containing the equivalent of 100000 female cells. RESULTS: Male microchimerism was found in 21% of women overall. Healthy women and women with RA did not significantly differ (24% vs 18%). Results ranged from the DNA equivalent of 0 to 20.7 male cells per 100000 female cells. Women were categorized into 4 groups according to pregnancy history. Group A had only daughters (n = 26), Group B had spontaneous abortions (n = 23), Group C had induced abortions (n = 23), and Group D were nulligravid (n = 48). Male microchimerism prevalence was significantly greater in Group C than other groups (8%, 22%, 57%, 10%, respectively). Levels were also significantly higher in the induced abortion group. CONCLUSIONS: Male microchimerism was not infrequent in women without sons. Besides known pregnancies, other possible sources of male microchimerism include unrecognized spontaneous abortion, vanished male twin, an older brother transferred by the maternal circulation, or sexual intercourse. Male microchimerism was significantly more frequent and levels were higher in women with induced abortion than in women with other pregnancy histories. Further studies are needed to determine specific origins of male microchimerism in women.