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Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210

Nature Immunology volume 15, pages 393–401 (2014)Cite this article

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Abstract

The microRNA miR-210 is a signature of hypoxia. We found robust increase in the abundance of miR-210 (>100-fold) in activated T cells, especially in the TH17 lineage of helper T cells. Hypoxia acted in synergy with stimulation via the T cell antigen receptor (TCR) and coreceptor CD28 to accelerate and increase Mir210 expression. Mir210 was directly regulated by HIF-1α, a key transcriptional regulator of TH17 polarization. Unexpectedly, we identified Hif1a as a target of miR-210, which suggested negative feedback by miR-210 in inhibiting HIF-1α expression. Deletion of Mir210 promoted TH17 differentiation under conditions of limited oxygen. In experimental colitis, miR-210 reduced the abundance of Hif1a transcripts and the proportion of cells that produced inflammatory cytokines and controlled disease severity. Our study identifies miR-210 as an important regulator of T cell differentiation in hypoxia, which can limit immunopathology.

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Figure 1: Mir210 is induced after T cell activation and regulated during T cell differentiation.
Figure 2: Costimulation via CD28 controls Mir210 expression via the PI(3)K-mTOR pathway.
Figure 3: The induction of Mir210 requires HIF-1α.
Figure 4: miR-210 deficiency has no apparent effect on the development or proliferation of T cells.
Figure 5: miR-210 directly targets Hif1a.
Figure 6: Priming of naive T cells under reoxygenation increases the abundance of miR-210.
Figure 7: miR-210 deficiency, along with reoxygenation, markedly increases TH17 differentiation.
Figure 8: miR-210 in T cells ameliorates IBD in a CD4+ T cell–transfer model of colitis.

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Acknowledgements

We thank A. Roque for animal husbandry; R. Locksley and Z. Wang for access to the cell-sorting facility and assistance with cell sorting; J.J. O'Shea (US National Institutes of Health) for CP550690; K. Shokat (University of California, San Francisco) for GDC-0941 and MLN0128; R. Wang (St. Jude Children's Research Hospital) for organs from Hif1af/fCD4-Cre mice; M. Matloubian (University of California, San Francisco) for RNA from mice infected with lymphocytic choriomeningitis virus; A. Abbas (University of California, San Francisco) for Il2−/− DO11.10 mice; K. Shokat (University of California, San Francisco); J.J. O'Shea (US National Institutes of Health) for kinase inhibitors; and K. Mark Ansel and L. Jeker for critical reading of the manuscript. Supported by the Arthritis Foundation (H.W.), Deutsche Forschungsgemeinschaft (H.F.), the National Basic Research Program of China (2013CB967002 to L.W.) and the Keck Foundation.

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  1. Haopeng Wang and Henrik Flach: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine and Department of Microbiology & Immunology, the Rosalind Russell-Ephraim P. Engleman Medical Research Center for Arthritis, and the Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA

    Haopeng Wang, Henrik Flach & Arthur Weiss

  2. Department of Medicine, University of California, San Francisco, San Francisco, California, USA

    Michio Onizawa

  3. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China

    Lai Wei

  4. Diabetes Center, University of California, San Francisco, San Francisco, California, USA

    Michael T McManus

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Contributions

H.W. and H.F. planned and did experiments, analyzed and interpreted data and wrote the manuscript; M.O. did histology analysis; L.W. contributed critical prepublication data; M.T.M. provided guidance and direction and the Mir210-targeted mice; and A.W. supervised the work, helped conceive of the experiments and edited the manuscript.

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Correspondence to Arthur Weiss.

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Integrated supplementary information

Supplementary Figure 1 Global miRNA expression–profiling studies reveal robust miR-210 induction after T cell activation.

(a) The expression of miR-210 in resting T cells, activated T cells and different helper T cell subsets. The primary data was from ref.57. (b) The expression of miR-210 in CD4+ and CD8+ thymocytes and activated T cells. The small RNA deep sequencing data was from ref.58,59.

Supplementary Figure 2 miR-210 induction in primary T cells in vitro and in vivo.

(a) Adoptively transferred T cells were harvested from various tissues 3 weeks after adoptive transfer. The surface-expression of CD44 and CD62L on adoptively transferred T cells were measured by flow cytometry. (b) Quantitative RT-PCR analysis of miR-210 abundance in resting or anti-CD3–anti-CD28 stimulated mouse CD8+ T cells. Cells were stimulated for a period of 4 d under normoxic (21% O2) or hypoxic (1% O2) conditions and RNA was harvested at the indicated time points. Relative expression was normalized to sno202. (c) Quantitative RTPCR analysis of miR-210 transcripts in P14 transgenic CD8+ T cells from mice infected with LCMV. P14 transgenic CD8+ T cells were injected into BoyJ congenic host mice. The chimeric mice were LCMV-infected 24 hrs after the adoptive transfer and P14 transgenic CD8+ T cells were harvested at the indicated time points, followed by RNA isolation and RT-PCR analysis of miR-210 abundance. Relative expression was normalized to sno202. (d) Quantitative RT-PCR analysis of miR-210 or miR155 abundance in resting or anti-CD3–anti-CD28 stimulated mouse or human primary T cells. Mouse CD4+ (left) and CD8+ (middle) T cells as well as human CD4+ (right) T cells were stimulated for a period of 4 d under normoxic (21% O2) or hypoxic (1% O2) conditions and RNA was harvested at the indicated time points. Relative expression is normalized to sno202 and the ratio of hypoxic vs. normoxic expression of miR-210 or miR-155 at the indicated timepoints is depicted. Data are from one experiment representative of two (a–c) or three (d) independent experiments. (mean and s.d. in b,c).

Supplementary Figure 3 HIF-1α protein is increased by CD28-mediated costimulation and HIF-1α binds the Mir210 promoter region.

(a) Immunoblot analysis with a monoclonal HIF-1α-specific or GAPDH-specific antibody in total protein extracts of resting or anti-CD3–anti-CD28 stimulated primary CD8+ T cells. Cells were stimulated for 24 h under normoxic (21% O2) or hypoxic (1% O2) conditions and protein was harvested at the indicated time points. (b) Naive CD4+ T cells from wild-type (WT) or CD28-deficient (KO) animals were stimulated with anti-CD3 and anti-CD28 for 3 d or kept unstimulated. The cells were harvested and an immunoblot analysis with a monoclonal HIF-1α-specific or GAPDH-specific antibody in whole cell lysates was performed. (c) ChIP-seq analysis of the interaction of HIF-1α with the Mir210 promoter region in polarized TH17 cells. ChIP-seq primary data was from http://th17.bio.nyu.edu/pages/igv.php and ref.60. Naive T cells were polarized towards the TH17 lineage for 48 h. The results of two independent experiments are shown. The blue bar indicates the location of the Mir210 sequence. Data are representative of two (a,b) independent experiments.

Supplementary Figure 4 Analysis of miR-210 abundance during T cell development and identification of miR-210 target genes.

(a) Quantitative RT-PCR analysis of miR-210 abundance in flow cytometry sorted thymocyte populations and in resting or anti-CD3–anti-CD28 stimulated primary CD4+ T cells derived from wild-type or Mir210–/– animals. Relative expression is normalized to sno202. Data are from one experiment representative of three independent experiments. (mean and s.d.; abbreviations: DN, double negative; DP, double positive). (b) miR-210 target gene identification flow chart. miR-210 target genes in mouse T cells were selected by performing a two-step selection process. First, four algorithms, Target scan, PicTar, miRDB and miRanda, were used to predict miR-210 target genes. All targets predicted by miRanda were scored for an empirical probability of target inhibition using mirSVR scores and a stringent mirSVR score cutoff of -1.1. This list was combined with previously reported miR-210 targets, resulting in 69 potential miR-210 target genes (Supplementary Table 1). Next, T cell-expressed target genes were selected according to the Immgen data-base (www.immgen.org), resulting in 21 genes. Their expression was compared by RT-PCR in wild-type or Mir210–/– CD4+ T cells, which were activated for 4 d. Assuming that miR-210–deficiency resulted in a higher expression of direct miR-210 targets, five candidate genes were identified, that exhibited a more than two-fold increased expression in Mir210–/– CD4+ T cells

Supplementary Figure 5 Hif1a 3' UTR harbors a conserved miR-210 target sequence in the arognaute-binding region and hypoxic conditions inhibit T cell proliferation.

(a) AGO binds the miR-210 targeting site in Hif1a 3' UTR in activated T cells. AGO binding site information in primary T cells came from CLIP Base (http://bit.ly/12z7yMe) and ref.61. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITSCLIP) was performed on T cells that were activated by anti-CD3 and anti-CD28 for 4 d at 37°C. Blue shade represents WT replicates, and yellow shade is miR-155-deficient replicates. The black bar indicates the location of the miR-210 seed region in the binding site. Coordinates along the x-axis indicate nucleotide position relative to the start of the Hif1a 3' UTR. The y-axis indicates read counts. (b) Sequence alignment of miR-210 target sequences from multiple species. (c) Naive mouse CD4+ T cells were anti-CD3–anti-CD28 stimulated for up to 4 d under normoxic (21% O2) or hypoxic (1% O2) conditions and cell numbers were determined at the indicated timepoints by using a Vi-Cell® counter. Data are representative of three (c) independent experiments.

Supplementary Figure 6 miR-210 deficiency along with reoxygenation conditions markedly increases TH17 differentiation but not TH1, TH2 or iTreg differentiation

(a) The reoxygenation culture conditions, involving the differentiation of TH17 cells by a priming step under a low O2 concentration (5% O2) followed by transfer to normoxic conditions. (b–c) RT-PCR analysis of miR-210 abundance and immunoblot analysis of HIF8 1α and GAPDH in CD4+ T cells stimulated in nonpolarizing conditions under normoxic or reoxygenation conditions. Relative miR-210 expression is normalized to its expression in naïve T cells. (d) Naive CD4+ T cells from wild-type or Mir210–/– mice were differentiated under TH1, TH2, TH17 (0.2 ng/ml of IL-6) or iTreg skewing conditions under normoxic or reoxygenetion conditions (see scheme in a), followed by intracellular staining of IL-4, IL-17A, IFN-γ or Foxp3. For TH1 conditions, 10 μg/ml anti–IL-4, 100 U/ml IL-2, 3.5 ng/ml IL-12 were added to the cultures; For TH2 conditions, 10 μg/ml anti–IFN-g, 100 U/ml IL-2, 50 ng/ml IL-4 were added to the cultures; For iTreg assays, naive T cells were cultured in the presence of 0.5 ng/ml TGF-β. (e) Naive CD4+ T cells were polarized towards iTregs under normoxic or reoxygenation conditions with varying doses of TGF-β for 3 d, followed by intracellular Foxp3 staining. (f) Wild-type or Mir210–/– CD4+ T cells were differentiated as described in Fig. 7a. Foxp3 expression was assessed by intracellular staining. Data are from one experiment representative of two (b,c,e,f) or three (d) independent experiments. (mean and s.d. in b).

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Wang, H., Flach, H., Onizawa, M. et al. Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210. Nat Immunol 15, 393–401 (2014). https://doi.org/10.1038/ni.2846

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