Background/Purpose: Rheumatoid arthritis (RA) is a systematic chronic inflammatory disorder, characterized by severe joint destruction.  Regulatory T cells (Tregs) have been implicated to be important for maintaining peripheral tolerance and controlling disease development. A novel surface protein, Glycoprotein A repetitions predominant (GARP), was recently identified to be specifically expressed on human Tregs and important for the suppressive capacity of Tregs. Several studies suggest that GARP expression in Tregs is post-transcriptionally regulated. In this study, we investigated in detail miRNA regulation of GARP expression, and dissected the functional outcome of miRNA:GARP-mRNA interaction and its resulting effects in patients with RA.

Methods: In silico analysis was performed to predict putative miRNA binding sites (MRE) in the 3’UTR of the GARP mRNA. Luciferase reporter vectors were used to identify specific GARP 3’UTR-recognizing MREs. Ribonucleoprotein immunoprecipitation (RNP-IP) was performed using antibodies against the Ago1- or Ago2-asscociated RISC complex. For cell proliferation and GARP expression, CD25+ and CD25- CD4 T cells were isolated from the peripheral blood. Cells were transfected with miRNA mimics or antagomirs and labeled with CFSE. The proliferating capacity was accessed by FACS. For Treg function assays, CD25+ T cells were transfected with miRNA mimics and co-cultured with CFSE-labeled CD25- T cells. Eight patients with early treatment naive RA (disease duration 3.0±2.4 months, DAS28 5.2±1.2) and 20 age and gender-matched healthy individuals were analyzed for GARP surface expression as accessed by FACS, or GARP mRNA and miRNA expression as measured by RT-PCR.

Results: The distal part of the GARP 3’UTR was capable to down-modulate reporter protein expression. Within this region, one MRE was recognized by its miRNA, miR142-3p. Mutation of this site abrogated the respective miRNA recognition confirming the specificity of the binding to the GARP 3’UTR. GARP mRNAs and miR142-3p were both immunoprecipitated in the Ago2-associated RISC complex by RNP-IP using an antibody against Ago2. Co-transfection with the antagomir of miR142-3p prevented GARP mRNA loading into the Ago2-associated RNP complex. CD25+ CD4 T cells treated with the antagomir showed a higher proliferating capacity upon stimulation. Complementary, treatment of cells with miRNA mimics lead to reduction of proliferation. CD25+ CD4 T cells treated with miRNA mimics showed a reduced suppressive capacity. The up-regulation of miR-142-3p was more prominent in RA patients than in healthy individuals resulting in consequentially diminished up-regulation of GARP.

Conclusion: We identified and characterized miR-142-3p regulation of GARP expression in Tregs. miR142-3p is critical for high GARP expression on Tregs and therefore for Treg function. Delineation of miR-142-3p expression and the linked GARP expression in RA provides an important hint for the reduced Treg function in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mir142-3p-interfers-with-t-cell-proliferation-by-targeting-the-expression-of-garp-in-patients-with-rheumatoid-arthritis/