SAT0045 In inflammatory arthritis, chemokine CCL19/21 expression is regulated by nuclear factor of activated T cells (NFATC4) pathways
Authors: V.A. Adarichev, M. Ramez, P.A. Kenny, T.S. Forde
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> 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). <h3>Objectives</h3> To validate the <i>in silico</i> 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. <h3>Methods</h3> In genome-wide association study (GWAS), we correlated susceptibility to antibody-mediated arthritis (serum transfer-induced arthritis <sup>1</sup> and collagen Ab-induced arthritis, CAIA <sup>2</sup>) of inbred murine strains with all available SNPs <sup>3</sup>. CAIA was induced in <i>Nfatc4</i>-ko gene deficient <sup>4</sup> 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 <i>Nfatc4</i>-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. <h3>Results</h3> Using GWAS, <i>Nfatc4</i> gene was discovered to be significantly associated with arthritis severity, p&lt;0.000003. Biological effect was confirmed in <i>Nfatc4</i>-ko gene-deficient mice that exhibited significantly weaker CAIA (55% downregulation, p&lt;0.01) when were compared to wild-type. The most significantly downregulated genes in paws of <i>Nfatc4</i>-ko mice were chemokines <i>Ccl19</i> and <i>Ccl21</i> (11.55-fold, p&lt;0.00006). Using RT-PCR and ELISA, we showed that production of CCL19/21 was basically blocked in LPS-stimulated peritoneal macrophages from <i>Nfatc4</i>-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&lt;0.01). <h3>Conclusions</h3> Transcriptome and proteome analyses confirmed <i>in silico</i> genetic association between severity of inflammatory arthritis and polymorphisms within the <i>Nfatc4</i> gene. We showed that activation of macrophages is under the strong control of NFATc4 regulatory pathways. <h3>References</h3> Ji, H. et al. Genetic influences on the end-stage effector phase of arthritis. Journal of Experimental Medicine 194, 321-330, (2001). Nandakumar, K. S. &amp; Holmdahl, R. Collagen antibody induced arthritis. Methods Mol Med 136, 215-223, (2007). Grubb, S. C., Maddatu, T. P., Bult, C. J. &amp; Bogue, M. A. Mouse phenome database. Nucleic Acids Res 37, D720-730, (2009). Wilkins, B. J. et al. Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic defect in calcineurin-mediated cardiac hypertrophic growth. Mol Cell Biol 22, 7603-7613, (2002). <h3>Disclosure of Interest</h3> None Declared