Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse

Abstract

Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4⁺ and CD8⁺ T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8⁺ T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.

Introduction

Mice transgenic for directed expression of a dominant-negative form of transforming growth factor beta receptor type II (dnTGF-βRII), under the control of the CD4 promoter lacking the CD8 silencer, spontaneously develop an autoimmune biliary ductular disease with similarity to human primary biliary cirrhosis (PBC). In addition, dnTGF-βRII mice spontaneously develop colitis similar to human inflammatory bowel disease (IBD) [1], [2], [3], [4], [5]. The utilization of adoptive transfer experiments has demonstrated that CD8⁺ T cells play a critical role in the autoimmune cholangitis, while CD4⁺ T cells are involved in colitis [2].

MicroRNAs (miRNAs) are small noncoding RNAs that control the translation of mRNAs by promoting the degradation of target mRNAs or preventing their translation [6], [7], [8]. Given the emerging roles of miRNAs in modulating both innate and adoptive immune responses, it is likely that any dysregulation of miRNA expression may contribute to the pathogenesis of autoimmune diseases and chronic inflammation [9]. Dysregulated miRNA expression has been associated with autoimmunity, specifically rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [10], [11], [12]. TGF-β receptor mediated signaling has been shown to directly regulate miRNA biogenesis by at least three mechanisms: 1) direct gene transcription of miRNA genes, 2) direct binding of TGF-β receptor induced SMAD proteins to specific miRNAs, and 3) stabilization of miRNA processing machinery [13], [14], [15]. The net effect of TGF-β signaling is to up-regulate miRNA biogenesis. Our goal was to investigate whether abrogation of TGF-β signaling in mouse CD4⁺ and CD8⁺ T cells affects miRNA expression and involvement in the pathogenesis of cholangitis. We demonstrate herein that, as expected, the overall effect of loss of TGF-β signaling resulted in the down regulation of many species of miRNA in T cells. Surprisingly, however, loss of TGF-β signaling resulted in the up-regulation of several important miRNAs, including miR-21. The level of miR-21 was elevated in both CD4⁺ and CD8⁺ T cells compared with control mice. Furthermore, transfection of miR-21 in B6 T cells resulted in enhanced secretion of IFN-γ and TNF-α, recapitulating the dnTGF-βRII T cell phenotype.