Loss of T cell microRNA provides systemic protection against autoimmune pathology in mice
Highlights
► MicroRNA are required to potentiate T cell activation and Th1 effector expansion. ► Excision of Dicer results in profound inhibition of T cell activation. ► The anti-autoimmune function of T cell microRNA is restricted to pathology of the colon. ► A pro-autoimmune function of T cell microRNA is observed systemically. ► Reduction of Dicer within T cells has the potential to mitigate Th1 autoimmunity.
Introduction
The role of T cell microRNA (miR) in autoimmune disease has generated interest both as potential mediators of pathology, and hence putative therapeutic targets, and as biomarkers for disease. Many individual miR expressed within T cells have been linked to a variety of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, primary biliary cirrhosis, ulcerative colitis and psoriasis [1]. As yet, however, most of these linkages remain as in vitro associations, with few systematic studies of the role of miR in autoimmune pathology.
Prediction of the potential roles of individual T cell miR in autoimmune disease largely relies on extrapolation from the biological functions determined through knockout and overexpression studies. These biological functions predict both pro- and anti-autoimmune functions. Anti-autoimmune functions of miR include the role of miR-181 in promoting thymic negative selection [2], through the downregulation of phosphatases and subsequent increased sensitivity of TCR signaling [3]. MiR-101 and miR-184 may also be critical for peripheral T cell tolerance maintenance, due to the suppression of T cell activation [4], [5]. Other miR may have global pro-autoimmune functions within T cells, such as miR-142-3p, which impedes the production of tolerogenic cAMP [6]. Altered expression of each of these miR within T cells is therefore expected to modulate autoimmune susceptibility.
While the miR above may be expected to have relatively predictable effects on autoimmunity, based on the known biological properties, other miR are more likely to have contextual roles in autoimmunity, either due to multiple opposing functions or subset-specific functions. As an example of the first, miR-146a has the immunogenic property of reducing the sensitivity of T cells to tolerance via the Fas pathway [7] and the tolerogenic property of increasing regulatory T cell suppressive capacity [8]. The net effect of enhanced autoimmunity in the miR-146a knockout mouse may indicate that the latter role is dominant over the former, or it may represent the phenotypic dominance of the function of miR-146a in the myeloid lineage [9]. Likewise, miR-155 has seemingly opposing effects, with an enhancement of regulatory T cell homeostasis [10] and also of Th17 differentiation [11]; the latter role is dominant in determining the susceptibility to Experimental Autoimmune Encephalomyelitis (EAE) [11]. Other miR are implicated in particular effector T cell lineages, such as miR-155 suppression of Th2 induction [12], [13], miR-29 suppression of Th1 activity [14], [15], miR-326 amplification of Th17 induction [16] and miR 17–92 suppression of Bcl-6-mediated Tfh activity [17]. The effect of altered expression of these miR are likely to be dependent on the predominant effector lineage in a specific disease, such as the ability of miR-326 overexpression to exacerbate Th17-mediated EAE [16]. With such a diverse set of individual miR, each with opposing functions, it is critical to determine the phenotypic dominance of pro- and anti-autoimmune miR through a whole network approach.
The answer to this simple, yet critical, question of whether the net effect of the T cell miR transcriptome is tolerogenic or immunogenic remains unclear. Deletion of the miR network via Cre-mediated loss of Dicer results primarily in thymic differentiation defects, with no obvious pathological consequence [18], [19]. Careful examination of CD4-Cre Dicer-floxed mice demonstrates a muted immune pathology, with colitis developing at 3–4 months of age, a phenotype associated with decreased regulatory T cell numbers [20]. These results suggest a weak net positive effect of miR on T cell tolerance processes, and are in marked contrast to the rapid and fatal autoimmunity caused by regulatory T cell-specific depletion of the miR network [21], [22], [23]. However, as the wildtype context is that of no autoimmunity, analysis of unchallenged CD4-Cre Dicer-floxed mice constitutes a unidirectional screen, sensitized only to increases, and not decreases, in autoimmune pathology. In order to formally test the net impact of the T cell miR network using a bidirectional screen we eliminated the T cell miR network in both the wildtype (no challenge) context and the TGFβ receptor-deficient context of strong autoimmunity. Unlike the published unidirectional screen, this bidirectional screen found that the primary impact of the Dicer-dependent miR network in T cells is to potentiate autoimmunity, as loss of miR in TGFβ receptor-deficient mice resulted in a profound reduction and delay in autoimmune pathology. These data indicate that the spontaneous colitis observed in CD4-Cre Dicer-floxed mice represents an organ-specific exception where miR has a tolerogenic function, as the phenotypically dominant function of T cell miR at a systemic level is immunogenic.
Section snippets
Mice
CD4-Cre, TGFβRIIfl, and Dicerfl mice were all backcrossed to the C57BL/6 background [24], [25], [26]. Experimental mice were age-matched and housed under specific pathogen–free conditions. Cohorts of mice for the survival test were monitored for ill health and were removed from the study at death or when veterinary advice indicated likely death within 48 h. All mice were used following ethics approval of the University of Leuven mouse facility.
Histological examination
Histological analysis was performed on
The T cell microRNA network is essential for a robust IFNγ response in the absence of TGFβ
T cell-specific excision of Dicer has been demonstrated to result in minor immune pathology, suggesting a weak anti-autoimmune function of the T cell miR network. In order to formally test the function of the T cell miR network during autoimmunity, we created a bidirectional screen where T cell Dicer-deficiency was layered upon either the “no challenge” context (wildtype mice vs CD4-Cre Dicerfl/fl mice) or the “strong autoimmune” context (CD4-Cre TGFβRIIfl/fl mice vs CD4-Cre Dicerfl/fl TGFβRII
Discussion
Understanding the global impact of the miR network in T cells is the critical starting point for investigations into the function of individual miR:mRNA interactions. Previous studies on the effect of the miR network in T cells have either utilized in vitro approaches or have evaluated the effect of Dicer excision in a “no challenge” in vivo context. The use of a unidirectional “no challenge” screen only allows heightened susceptibility to autoimmunity to be detected; employing a bidirectional
Acknowledgments
We thank A. Tarakhovsky for providing Dicer-floxed mice and A. Rudensky for the provision of TGFβRII-floxed mice. The authors would like to thank the NIH Tetramer Core Facility for mouse CD1d tetramer. This work was supported by grants from the VIB and FWO (A.L.). A.L. is a Marie Curie Reintegration Grant Fellow and ERC Start Grant holder. L.T., G.D.H., M.F., K.S., S.S. and S.H-B performed the experiments, L.V.D.B and P.D provided experimental advice and supplied reagents, A.L. and J.D.
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