Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses - PubMed (original) (raw)

Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses

Li-Fan Lu et al. Cell. 2010.

Abstract

Foxp3(+) regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR-146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of the IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity.

Copyright © 2010 Elsevier Inc. All rights reserved.

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Figures

Fig 1

Fig 1. miR-146a-deficient Treg cells failed to effectively control immune homeostasis and restrain miR-146a-sufficient effector T cells

(A) Schematic of generation of mixed BM chimeras. (B) Kaplan-Meyer survival plot of mixed BM chimeras generated upon reconstitution of irradiated _Rag2_−/− (or TCRβδ−/−) recipients with 1:1 mixtures of indicated BM cells. 40% of mice in _Mirn146a_−/−/Foxp3KO group died before the day of analysis (arrow). The rest of the mice in this group along with other control groups were euthanized and analyzed. Splenic Treg cell frequencies in _Mirn146a_−/−/Foxp3KO and _Mirn146a_−/−/B6 chimeric mice generated upon transfer of _Mirn146a_−/− BM mixed with Ly5.1+ Foxp3KO BM (C) or Ly5.1+ B6 BM (D) 6-7 weeks after BM transfer. The proportion (E) and absolute numbers (F) of Ly5.1-Foxp3+ Treg cells in indicated chimeric mice. (G) The ratios of Ly5.1−Foxp3+ and Ly5.1+Foxp3+ cells in indicated chimeric mice are shown. Expression of activation markers and Ki67 in miR-146a-sufficient Ly5.1+Foxp3− Teff cell subsets from _mirn146a_−/−/Foxp3KO and mirn146a+//Foxp3KO (H) or _mirn146a_−/−/B6 and mirn146a+/+/B6 BM (I) 6-7 weeks after BM transfer. Data are representative of three independent experiments (n=11-13); values represent the mean +/− s.d., *P<0.05. See also Figure S1 and S2.

Fig 2

Fig 2. Selective dysregulation of IFNγ responses in mice harboring miR-146a-deficient Treg cells

miR-146a-deficient or -sufficient Treg cells isolated from (A) healthy _Mirn146a_−/−/B6 or (B) diseased _Mirn146a_−/−/Foxp3KO chimeric mice were co-cultured with wild-type responder CD4+ T cells at the indicated ratios for 72h in the presence of CD3 antibody and irradiated T cell-depleted splenocytes. (C) Frequencies of IFNγ secreting cells in Foxp3−CD4+ and Foxp3+CD4+ T cells isolated from diseased _Mirn146a_−/−/Foxp3KO are shown. miR-146a-deficient T effector subset appears to make somewhat less IFNγ as compared to Foxp3ko T cell subset because the latter contains an increased frequency of self-reactive T cells due to the presence of Treg “wannabe’s” (Hsieh et al., 2006). The proportions (D) and absolute numbers (E) of IFNγ secreting cells in Foxp3+CD4+ Treg cells isolated from diseased _Mirn146a_−/−/Foxp3KO chimeric mice are numerated. Data are representative of three independent experiments (n=11-13); values represent the mean +/− s.d., *P<0.05. (F) Frequency of IFNγ secreting cells in Foxp3−CD4+ and Foxp3+CD4+ T cells isolated from healthy _Mirn146a_−/−/B6 mice are shown. Data are representative of three independent experiments (n=11-13). See also Figure S3.

Fig 3

Fig 3. IFNγ blockade rescued immune mediated pathology in mice harboring miR-146a-deficient Treg cells

(A) Schematic of IFNγ neutralization experiments using BM chimeras. (B) Splenic Treg cell frequencies in chimeric mice treated with IFNγ neutralizing or control antibodies 6 weeks after BM transfer. (C) Expression of Ki67 and other activation markers in miR-146a-sufficient Ly5.1+Foxp3−CD4+ Teff cells isolated from _Mirn146a_−/−/Foxp3KO mice with IFNγ neutralizing or control antibodies. (D) Frequency of IFNγ, IL-4, and IL-17 secreting cells in miR-146a-sufficient Ly5.1+Foxp3− CD4+ Teff cell subsers from indicated chimeric mice with or without αIFNγ treatment are shown. Ly5.1+Foxp3− CD4+ Teff cells isolated from chimeric mice reconstituted with Foxp3KO BM only served as positive control. Data are representative of two independent experiments (n=9-12).

Fig 4

Fig 4. Loss of miR-146a-mediated Stat1 repression contributes to the IFNγ-mediated Th1 pathology in mice harboring miR-146a-deficient Treg cells

(A) Multiple species sequence alignment of the Stat1 3′ UTR including the putative miR-146a target site sequence (bold). (B) CD4+ T cells isolated from miR-146a-deficient or -sufficient mice were co-transfected with a luciferase reporter construct containing wild-type or mutated Stat1 3′UTR and assessed for luciferase activity 24hrs after transfection. (C) Immunoblot analysis of the Stat1, TRAF6 and IRAK1 protein expression as well as the phosphrylation of Stat1. Densitometric values normalized on the basis of β-actin expression are indicated below the corresponding lanes; fold increase in normalized target protein expression in the absence of miR-146a with or without IFNγ neutralization in the indicated T cell subsets. Data are representative of two independent experiments. (D) Immunoblot analysis of total Stat1 as well as the phosphorylated Stat1 protein in mice with Stat1 hemizygosity. Densitometric values normalized on the basis of β-actin expression as well as fold increase in normalized target protein expression in the indicated T cell subsets are shown below the corresponding lanes. (E) Kaplan-Meyer survival plot of _Rag2_−/− mice reconstituted with 1:1 mixtures of indicated BM cells. Arrows indicate the time of analysis. Frequency of (F) IFNγ or (G) IL-4 secreting cells in Foxp3−CD4+ and Foxp3+CD4+ T cell subsets in diseased mirn146_−/−_Stat1+/+/Foxp3KO and _mirn146_−/−_Stat1_−/+/Foxp3KO chimeric mice. Data are representative of two independent experiments (n=13).

Fig 5

Fig 5. IFNγ production by miR-146a-deficient T cells partially contributed to, but cannot account for Th1 immunopathology in _Mirn146a_−/−/Foxp3KO mice

(A) CD4+CD25−CD62Lhi cells isolated from _Mirn146a_−/− mice or wild-type littermates were cultured in vitro under Th1/Th2 polarizing conditions. Intracellular IFNγ and IL-4 staining was assessed by FACS analysis. Data are representative of three independent experiments (n=4-6). (B) Schematic of generation of mixed BM chimeras. Frequencies of IFNγ secreting cells in (C) CD4+ and (D) CD8+ T cell population isolated from indicated chimeric mice are shown. (E) Flow cytometric analysis of expression of activation markers and Ki67 in miR-146a-sufficient Ly5.1+Foxp3−CD4+ Teff cells from indicated chimeric mice are shown. Data are representative of three independent experiments (n=6-7). Real-time PCR analysis of (F) primary miR-146a, (G) mature miR-146a as well as (H) T-bet and (I) Foxp3 mRNA expression in FACS-purified CXCR3+ and CXCR3− populations from CD4+CD25+ Treg cells (TR) and CD4+CD25−CD62Lhi R naïve T cells (TN). Data are representative of two independent experiments.

Fig 6

Fig 6. Phenotypic analysis of mice subjected Treg cell-specific SOCS1 ablation

Flow-cytometric analysis of (A) thymus and (B) spleen cells of 6-8 week-old Foxp3creSOCS1fl/fl mice and wild-type littermates. Percentages of different thymocyte and splenocyte subsets are shown. (C-F) Cellularity of the thymus and spleen and the protportion and absolute numbers of thymus and splenic Foxp3+CD4+ Treg cells in Foxp3creSOCS1fl/fl mice or wild-type littermates are shown. Data are representative of three independent experiments (n=6-8); values represent the mean +/− s.d.; *P<0.05. (G) H&E-stained sections of the skin, liver, and lung from the indicated chimeric mice. Flow-cytometric analysis of expression of Ki67 and other activation markers in (H) Foxp3−CD4+ Teff cells and (I) Foxp3+CD4+ Treg cells in 6-8 week old Foxp3creSOCS1fl/fl mice (blue) or wild-type littermates (red). Data are representative of two independent experiments (n=6-8). See also Figure S4.

Fig 7

Fig 7. Unrestrained Stat1 activation in SOCS1-deficient Treg cells is associated with dysregulated IFNγ responses

(A) SOCS1-deficient or -sufficient Treg cells were co-cultured with wild-type responder CD4+ T cells at the indicated ratios for 72 h in the presence of CD3 antibody and irradiated (2000 rads) T cell-depleted splenocytes. (B) Immunoblot analysis of total and phospho-Stat1. Densitometric values normalized on the basis of β-actin expression and fold increase in normalized target protein expression in the indicated T cell subsets are indicated below the corresponding lanes. (C) Frequencies of IFNγ secreting cells within Foxp3−CD4+ and Foxp3+CD4+ as well as CD8+ subsets in Foxp3creSOCS1fl/fl mice or wild-type littermates. Data are representative of two independent experiments. See also Figure S5.

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