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.

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.

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.

Peptidylarginine Deiminase Autoimmunity and the Development of Anti-Citrullinated Protein Antibody in Rheumatoid Arthritis: The Hapten-Carrier Model.

OBJECTIVE: The presence of autoantibodies to citrullinated proteins (ACPAs) often precedes the development of rheumatoid arthritis (RA). Citrullines are arginine residues that have been modified by peptidylarginine deiminases (PADs). PAD4 is the target of autoantibodies in RA. ACPAs could arise because PAD4 is recognized by T cells, which facilitate the production of autoantibodies to proteins bound by PAD4. We previously found evidence for this hapten-carrier model in mice. This study was undertaken to investigate whether there is evidence for this model in humans. METHODS: We analyzed antibody response to PAD4 and T cell proliferation in response to PAD4 in 41 RA patients and 36 controls. We tested binding of 65 PAD4 peptides to 5 HLA-DR alleles (DRB1*04:01, *04:02, *04:04, *01:01, and *07:01) and selected 11 PAD4 peptides for proliferation studies using samples from 22 RA patients and 27 controls. Peripheral blood lymphocytes from an additional 10 RA patients and 7 healthy controls were analyzed by flow cytometry for CD3, CD4, CD154, and tumor necrosis factor expression after PAD4 stimulation. RESULTS: Only patients with RA had both antibodies and T cell responses to PAD4. T cell response to peptide 8, a PAD4 peptide, was associated with RA (P = 0.02), anti-PAD4 antibodies (P = 0.057), and the shared epitope (P = 0.05). CONCLUSION: ACPA immunity is associated with antibodies to PAD4 and T cell responses to PAD4 and PAD4 peptides. These findings are consistent with a hapten-carrier model in which PAD4 is the carrier and citrullinated proteins are the haptens.

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.