Background Nuclear factors of activated T cells (NFAT) is a family of transcription factors critical in regulating genes expression in development and disease. The pathway is an important target for cyclosporine immunosuppression in transplant rejection and in autoimmune diseases such as rheumatoid arthritis (RA).
Objectives To validate the in silico finding of the NFATc4 encoding gene within the major peak controlling antibody (Ab)-mediated arthritis, and to investigate a functional significance and mechanisms implicated in NFATc4-driven regulation of inflammatory arthritis.
Methods In genome-wide association study (GWAS), we correlated susceptibility to antibody-mediated arthritis (serum transfer-induced arthritis 1 and collagen Ab-induced arthritis, CAIA 2) of inbred murine strains with all available SNPs 3. CAIA was induced in Nfatc4-ko gene deficient 4 and in wild-type (WT) C57BL/6J mice with a cocktail of mAbs to collagen type II and LPS injection. Differential gene expression analysis of RNA isolated from arthritic paws of Nfatc4-ko and WT mice was performed using Affymetrix GeneChip® Mouse Gene 1.0 ST Array. Statistical analysis, functional and unsupervised hierarchical clustering was performed using Ingenuity Pathway Analysis and Partek software. Significantly dysregulated genes were replicated using TaqMan RT-PCR with independent RNA samples. Immunohistochemical staining of tissue sections and chemokine production in isolated macrophages were studied.
Results Using GWAS, Nfatc4 gene was discovered to be significantly associated with arthritis severity, p<0.000003. Biological effect was confirmed in Nfatc4-ko gene-deficient mice that exhibited significantly weaker CAIA (55% downregulation, p<0.01) when were compared to wild-type. The most significantly downregulated genes in paws of Nfatc4-ko mice were chemokines Ccl19 and Ccl21 (11.55-fold, p<0.00006). Using RT-PCR and ELISA, we showed that production of CCL19/21 was basically blocked in LPS-stimulated peritoneal macrophages from Nfatc4-ko mice. Cells isolated from articular cartilage of these gene-deficient mice also showed significantly lower production of collagen type I and aggrecan (2-fold, p<0.01).
Conclusions Transcriptome and proteome analyses confirmed in silico genetic association between severity of inflammatory arthritis and polymorphisms within the Nfatc4 gene. We showed that activation of macrophages is under the strong control of NFATc4 regulatory pathways.
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Disclosure of Interest None Declared