Function of miR-146a in controlling Treg cell mediated regulation of Th1 responses
I recently had the pleasure of listening an interesting talk on the function of micro RNAs in controlling the function of Tregs. The presenter was Dr. Li-Fan Lu from Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. Presented here is a brief blog on one of his paper.
Regulatory T cells (Tregs) have a suppressive function in body. They limit “collateral damage” resulting from protective immunity to infection and suppress sterile inflammation as well as unwanted immune responses to “self” and allergens. Tregs express FoxP3, an X chromosome encoded transcription factor which plays an important role in differentiation, homeostasis and function of Treg cells. It has been observed that FoxP3 directly or indirectly controls several thousand genes in Tregs. These genes are called as FoxP3 amplified genes many genes that serve as hallmark of Tregs and play important roles in their homeostasis and function. Also included among the FoxP3 amplified genes are those that code for noncoding RNAs called micro RNA (miRNA). These miRNAs serve as important regulators of Treg homeostasis and function both in basal and inflammatory settings. One such miRNA is miRNA-155, the cancer associated miRNA, which is constitutively expressed in high amounts in Treg cells in a FoxP3 dependent manner. It is transiently upregulated in T cells lacking FoxP3, B cells and myeloid cells up on activation. The investigators of this paper have previously shown that miR-155 causes increased response in Tregs to their key survival and growth molecule, IL-2 and is thus responsible for maintaining the numbers of Tregs. Another miRNA constitutively expressed in high amounts in Tregs is miR-146a. Like miR-155, it is induced up on activation of effector T cells and myeloid cells. In the present paper the authors tend to determine the role of miR-146a in Tregs. Their results are as follows:
• Elevated miR-146a expression in Tregs:
miRNA expressions were compared among FoxP3+ Treg cells and FoxP3- non Treg CD4+ cells---miR-146a was more expressed in Tregs in comparison to non-Tregs.
• miR-146a deficiency resulted in increased numbers but impaired function of Tregs:
Firstly, the authors examined FoxP3+ Treg cell subsets in the thymus and in the peripheral lymphoid organs of miR-146a deficient mice. They observed comparable sizes of different thymocyte and peripheral lymphoid and myeloid cell subsets and similar T cell activation status among the wild type and mutant mice. The number of FoxP3+ Tregs in mutant mice was higher in the periphery (but not in thymus) in comparison to wild type mice. These FoxP3+ Tregs also showed heightened proliferative activity as shown by increased expression of Ki67 and increase in other activation markers.
In next set of experiments, the authors transferred mixed bone marrow (1:1ratio of miR 146a deficient or sufficient Tregs mixed with Ly5.1 B6 or Ly5.1 FOXP3 KO) into Rag2-/- (or TCRβδ -/-) mice. They observed that in the mice group Mirn 146a-/-/FOXP3 KO, 40% of the mice died before the day of analysis. In these mice, all Tregs lack mir 146a as FOXP3 KO BM cells fail to generate Tregs; all other BM derived cells originate from both miR 146a deficient and sufficient BMs. The purpose of these experiments was to generate a miR 146a sufficient environment and then study the function of miR146a deficient Tregs in that environment. These mice showed severe clinical conditions and only 40% survived. Histologicaly also, these mice showed massive lymphocyte activation and tissue infiltraion. In contrast, mir 146a-/-/B6 mice had both miR 146a deficient and sufficient Tregs and they showed no clinical or histological signs of immune mediated lesions. So what was the cause of the adverse clinical and histological conditions in the mir 146a-/-/FOXP3 KO mice? The authors observed that the cause of these effects was not due to decrease in number of Tregs as the number of Tregs in KO mice was actually more than in control mice (mir 146a +/+/FOXP3 KO). The mir 146a -/-/ B6 mouse also showed an increase in miR 146a deficient Tregs than in controls (mir 146+/+/B6). These results suggested that miR146a deficiency increases the number of Tregs.
The authors next studied the activation of miR 146a sufficient T effector cells and observed a highly proliferative activity (Ki67), an increased CD62L low cell subset and high levels of activation markers ICOS and CTLA4 in FOXP 3 KO mice. However, both miR 146a deficient or sufficient cells showed control activation in a miR 146 a sufficient environment (B6 Ly5.1+ MICE). These results suggest that miR146 a play an important role in Treg mediated immunological tolerance.
• miR-146a deficiency in Treg cells resulted in dysregulated IFNγ responses: In the next set of experiments the authors isolated miR-146a deficient or sufficient Tregs from mir 146a-/-/B6 or mir146a-/-/FOXP3 KO mice and co-cultured them with wild type responder CD4+ T cells (in the presence of CD3 antibody and irradiated T cell depleted splenocytes). They observed that miR 146a deficient Tregs from mir 146a-/-/B6 mice were as suppressive as miR 146a sufficient Tregs from control animals (mir146a+/+/B6). However, miR 146a deficient Tregs from knock-outs were more suppressive than miR146a sufficient Tregs from controls. These results indicate that deficiency of miR 146a did not diminish the overall suppressive activity of Tregs. They also observed that loss of miR146a in Tregs resulted in increase production of pro-inflammatory cytokine IFNγ by both miR 146a sufficient and deficient Cd4 and CD8 cells. This increase was not observed in the presence of miR146a sufficient Tregs.
• IFNγ blockade prevents the autoimmune disease in mice harboring miR-146a deficient Tregs: The next question was weather this increased production of IFNγ is responsible for clinical condition in Mirn 146a-/-/FOXP3 KO mice. To answer this question, the investigators performed experiments in which IFNγ was blocked by treating these mice with IFNγ blocking antibody. The increase in number of miR 146a deficient Tregs was not affected in the presence of blocking antibody. However, the blocking of IFNγ prevented disease in deficient mice. The blockade of IFNγ with blocking antibody did not cause simultaneous increase in IL4 and IL-17 (Th2 responses). These results suggest that the diseases observed in the miR146a deficient Tregs was IFN-gamma dependent and blockade of IFN-gamma can control Th2 and Th17 responses but not Th1 response.
• miR 146a regulates stat1 in Tregs: Stat 1 is a key transcription factor downstream of IFN-gamma signaling. Thus, the authors transfected CD4+ T cells isolated from miR146a sufficient or deficient with a luciferase reporter construct containing wild type or mutant Stat1 and assessed luciferase activity. miR146a sufficient T but not deficient cells transfected with wild type Stat1 showed suppression of reporter activity. The authors also performed immunoblot analysis of Stat1 protein in Tregs and non-Tregs. There was an increase in production of Stat1 in Mir146a deficient Tregs and non-Tregs.
• miR 146a controls Treg cell mediated regulation of IFNγ responses through targeting Stat1: Based on previous experiments the authors hypothesized that miR146a deficiency causes dysregulated IFN-gamma production, which in turn causes increased expression of Stat1, which can then lead to failure of Tregs to control Th1 response. To test this hypothesis, the authors generated miR146a deficient mice harboring a single function stat1 allele (Mirn 146a-/-/Stat1+/-). The BM cells from these mice were mixed with Ly5.1+ FOXP3 KO mice and transferred into Rag2-/- mice. Thus, the recipients are Mirn 146a-/-/Stat1+/-/FOXP3 KO. Then they measured the amount of Stat1 phosphorylated in Tregs and non-Tregs from these and from control mice (Mirn 146a-/-/Stat1+/+/FOXP3 KO). Both Tregs and non-Tregs from test mice showed nearly 50% reduction in phosphorylated stat1 in comparison to controls. The test mice also developed a much milder and delayed disease. The authors also observed significant reduction in production of IFN-gamma in tests mice
Source: Lu LF, Boldin MP, Chaudhry A, Lin LL, Taganov KD, Hanada T, Yoshimura A, Baltimore D, Rudensky AY. Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses. Cell. 2010 Sep 17;142(6):914-29. PubMed PMID: 20850013; PubMed Central PMCID: PMC3049116.
