HIF-2α-dependent induction of miR-29a restrains TH1 activity during T cell dependent colitis

Abstract

Metabolic imbalance leading to inflammatory hypoxia and stabilization of hypoxia-inducible transcription factors (HIFs) is a hallmark of inflammatory bowel diseases. We hypothesize that HIF could be stabilized in CD4⁺ T cells during intestinal inflammation and alter the functional responses of T cells via regulation of microRNAs. Our assays reveal markedly increased T cell-intrinsic hypoxia and stabilization of HIF protein during experimental colitis. microRNA screen in primary CD4⁺ T cells points us towards miR-29a and our subsequent studies identify a selective role for HIF-2α in CD4-cell-intrinsic induction of miR-29a during hypoxia. Mice with T cell-intrinsic HIF-2α deletion display elevated T-bet (target of miR-29a) levels and exacerbated intestinal inflammation. Mice with miR-29a deficiency in T cells show enhanced intestinal inflammation. T cell-intrinsic overexpression of HIF-2α or delivery of miR-29a mimetic dampen T_H1-driven colitis. In this work, we show a previously unrecognized function for hypoxia-dependent induction of miR-29a in attenuating T_H1-mediated inflammation.

Fig. 1. Stabilization of Hypoxia Inducible Factor in colitogenic CD4+ T cells during experimental colitis.

Chronic T cell-mediated colitis was induced in B6.B6.RAG1−/− mice by intraperitoneal injection (0.5 × 106) of naïve CD4+ CD45RBhigh or CD45RBlow T cells. Identification of 2-nitroimidazole EF5 hypoxic adducts in B6.B6.RAG1−/− mice injected with C57BL/6J/6JT cells. 6 weeks post-treatment spleens, lymph nodes and collagenase-digested colon tissues were assessed. a Flow cytometric sub-analysis of cellular profile within Cy5-labeled EF5 (nitroimidazole; injected IP-3h) naïve and effector memory CD4+ T cells from the spleen, mesenteric lymph nodes and the colons, (n = 3–4, one-way ANOVA). b Effector memory CD4+ T cells from the spleen, mesenteric lymph node (MLN) and collagenase digested colons (gated on CD44 + CD62L−) and labeled with EF5-Cy5. Identification of stabilized HIF in dissected colons by IVIS imaging from mice adoptively transferred with 0.5 × 106 B6.ΔODD.luc CD4+ T cells. Prior to euthanizing, mice were injected with Luciferase substrate for 10 min. Freshly dissected colons were flushed and imaged using IVIS. Representative IVIS photograph and corresponding colon micrograph sections were stained with H&E, n = 4. c Healthy colons from CD45RBlow transferred mice, with low luciferase activity in the upper panel. Colon histological sections (lower panel, Scale bar: 184.4 mm). d High luciferase activity in whole colon (upper panel) and histology micrograph with lymphocyte infiltrates in CD45RBhigh transferred mice at 6-week post transfer (lower panel, Scale bar: 184.4 mm). e Image radiance was quantified using IVIS and expressed as photons/sec/cm2/sr. (n = 3, two-tailed T test). f Colon inflammation score (weight/length) in adoptively transferred animals from (c) and (d) (n = 4, two-tailed T test). Male and female mice used in (a) and (b), male recipients and male and female donors used in (c)–(f). Radiance of IVIS image was quantified and expressed as photons/sec/cm2/sr. Data expressed as Mean ±S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. indicated.

Fig. 2. Specific induction of miR-29a in CD4⁺ T cells following experimental hypoxia.

Primary naïve CD4+ T cells were isolated from spleens and MLN of C57BL/6J/6J mice and cultured ex vivo for 8 h with TCR stimulation (anti-CD3/28) in either normoxia (21% O₂) or hypoxia (1% O₂). RNA was prepared and assayed using custom limited-target T cell Q-PCR microRNA array (Qiagen). a Heat-map representing respective increased and decreased T cell-expressed miRNAs following treatment. Fold change over the average of two normoxia samples is depicted as color change (n = 2/group). b Assessment of miR-29a expression by Q-PCR in colon biopsies from patients with inflammatory bowel disease. Patient characteristics included in Supplementary Table 1, (n = 4–10 patients/group, one-way ANOVA). c Normal mouse colon tissue was fractionated into intestinal epithelial cells (IEC) and lamina propria cells (LP) and baseline levels of miR-29a were assessed using Q-PCR, (n = 4, two-tailed T test). d Primary naïve CD4+ T cells were isolated from spleens of C57BL/6J/6J mice and cultured ex vivo under helper T cell polarizing conditions with rmIL-2 and anti-CD3/anti-CD28 stimulation for 3 days, then washed and rested for 48 h, followed by 8 h stimulation (anti-CD3/28) in either normoxia (21% O₂) or hypoxia (1% O₂). miR-29a expression was assessed by Q-PCR (n = 3–6, two-way ANOVA). e Primary human CD4+ T cells were isolated from peripheral blood of healthy volunteers and cultured similarly as mouse CD4+ T cells but with only T_H0 and T_H1 specific stimuli. miR-29a expression was assessed by Q-PCR following TCR-stimulation under normoxia or hypoxia for 8 h, (n = 3–5, two-way ANOVA). Male and female mice used as cell donors in (a, c–e). Data expressed as mean ±S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001 vs. counterparts.

Fig. 3. HIF-2α mediates the induction of miR-29a in CD4⁺ T cells during hypoxia.

Primary mouse naïve CD4⁺ T cells were isolated from spleens and MLN of control (Lck-Cre⁺) or mice with a selective HIF-1α or HIF-2α deficiency in T cells. Isolated CD4⁺ T cells were cultured ex vivo for 8 h stimulation with TCR (anti-CD3/28) in either normoxia (21% O₂) or hypoxia (1% O₂). a miR-29a induction by hypoxia is absent in Hif-2α-deficient CD4⁺ T cells, (n = 2–3, two-way ANOVA). b miR-29a is significantly induced in primary CD4⁺ T cells isolated from Hif-2α-over-expressing mice (LSL-HIF2dPA Lck Cre⁺) cultured ex vivo for 8 h stimulation with TCR stimulation (n = 3, two-tailed T test). Pharmacologic stabilization of HIF-2α with DMOG treatment (1 mM) induced miR-29a in: (c) primary CD4⁺ T cells from WT (B6) mice (n = 3, one-tailed T test) and, (d) CD4⁺ T cells isolated from PBMCs of healthy human volunteers (n = 3–5, two-tailed T test). Hif-2α^loxP/loxP Lck Cre⁺ and Hif-2α^loxP/loxP Lck Cre⁻ mice were treated with two rounds of 3% DSS in drinking water for 5 days with 2 weeks of water in between treatments to induce chronic colitis. e Quantification of miR-29a expression in the total mouse colon tissue, (n = 3, two-way ANOVA). f Schematic representation of murine miR-29b1-a promoter with 3 most proximal putative Hypoxia Responsive Elements (HRE) indicated. More distant putative HREs (4) were omitted. g Chromatin Immuno-Precipitation (ChIP) was performed to assess in vivo DNA-protein interactions at the proximal promoter sequences in primary CD4+ T cells from Hif-2α^loxP/loxP Lck Cre⁺ and Cre^- mice cultured ex vivo for 8 h with TCR stimulation (anti-CD3/28) in either normoxia (21% O₂) or hypoxia (1% O₂) as per Methods (n = 6, two-way ANOVA). Male and female mice used in (a–d, g), male mice used in (e). Data expressed as Mean ±S.E.M from 2 polled independent experiments., *p < 0.05, **p < 0.01.

Fig. 4. T-bet expression in CD4⁺ T_H1 T cells is specifically repressed during hypoxia via miR-29a.

MicroRNA-29a targets T-bet expression in vitro and in vivo. CD4⁺ T cells were isolated from spleens and MLN of WT (B6) mice and cultured ex vivo with anti CD3/CD23 for indicated time under normoxia (21% O₂) or hypoxia (1% O₂). a mRNA expression of miR-29a in CD4⁺ T cells was measured by Q-PCR (n = 3, one-tailed T test), and b mRNA expression of T-bet was measured by Q-PCR following 2 h and 8 h in either normoxia (21% O₂) or hypoxia (1% O₂), (n = 3, pooled 2 experiments, two-way ANOVA). CD4⁺ T cells were isolated from spleens and MLN of WT (B6) mice and cultured ex vivo under T_H1 polarizing conditions. Differentiated T_H1 T cells were stimulated with anti-CD3/anti-CD28 under normoxia (21% O₂) or hypoxia (1% O₂) for 1 day. c T-bet protein was measured by flow cytometry in T_H1 T cells untreated or treated with DMOG (conc. 500 μM). Representative plot shown. d IFNγ secretion from T_H1 T cells treated with DMOG was assessed by ELISA (n = 3–4, one-way ANOVA). e T-bet protein expression was measured by flow cytometry in cells stimulated for 24 h under normoxia or hypoxia (n = 4, one-way ANOVA). f IFNγ secretion in T_H0 or T_H1-skewed cells was assessed by ELISA on day 3 (n = 3, 2-way ANOVA). T-bet protein expression measured by flow cytometry in T_H1 differentiated CD4⁺ T cells transfected with specific mi-29a inhibitor (g, h) or miR-29a mimetic (i) and activated with anti CD3/CD28 in normoxia or hypoxia for 48 h. g Representative flow cytometry profile of T bet expression. h Quantitation of Tbet fluorescence from (g) (n = 3, one-way ANOVA). i Quantitation of Tbet in cells transfected with miR-29a mimic and analyzed by flow cytometry (n = 3, one-tailed T test). T_H1 colitis was induced in LSL-HIF2dPA Lck Cre ⁺ mice and controls by epicutaneous skin sensitization followed by rectal gavage with TNBS as per Materials and Methods. j T-bet protein expression in gated CD4⁺ T cells purified from lamina propria was measured by flow cytometry (n = 3–5, two-tailed T test). k Representative micrographs of TNBS-treated colons from LSL-HIF2dPA Lck Cre⁺ or Cre^- littermate controls stained with H&E, bar = 100 mm. l Histological scores from LSL-HIF2dPA Lck Cre⁺ or Cre^- littermate controls following TNBS trial (n = 4–6, two-tailed T test). Male and female mice used in (a–l). Data expressed as Mean ± S.E.M, *p < 0.05, **p < 0.01 vs. indicated.

Fig. 5. T cell-intrinsic HIF-2α regulates T cell function in Colitis.

Chronic T cell-mediated colitis was induced in B6.B6.RAG1−/− mice by intraperitoneal adoptive transfer of naïve CD4⁺ CD45RB^high or CD45RB^low T cells (0.5 × 10^6/ animal) derived from Hif-2α^loxP/loxP Lck Cre⁺ or Cre⁻ animals. a Weight change during the course of colitis (n = 8–10, 2-way ANOVA, Cre- vs. Cre+). b Colon weight/length ratio at fulminant disease (n = 9–12, one-way ANOVA). c Histological preparations of colons stained with H&E were scored for total inflammation (as per Methods, (n = 5–6, one-way ANOVA). d Representative micrographs of colons at day 56 post-transfer, scale bar: 131.4 mm. Mesenteric lymph node CD4⁺ T cells were purified from adoptively transferred mice and total RNA was assayed by Q-PCR for: e IFNγ mRNA expression (n = 3–7, one-way ANOVA). f T-bet mRNA expression. Gene expression in (e, f) was normalized to 18s rRNA. (n = 3–6, one-way ANOVA). T_H1 colitis was induced in Hif-2α^loxP/loxP Lck Cre⁺ or Lck Cre⁺ control mice by the epicutaneous skin sensitization and subsequent rectal gavage with TNBS. At the time of necropsy mesenteric node lymphocytes were assessed by flow cytometry. g T-bet mean fluorescence intensity (MFI) in CD4⁺ T cells (gated on live CD3+, n = 3–4, one-way ANOVA). h IFNγ expression in mesenteric node lymphocytes was assayed by Q-PCR (n = 4–5, two-tailed T test). i Histological indices including leukocyte infiltration, tissue injury and total inflammation in the course of colitis (n = 6–9, one-tailed T test). j Representative micrographs from Lck Cre⁺ controls or Hif-2α^loxP/loxP Lck Cre⁺ mice 7 days post-challenge, scale bar 100 mm. Male recipients and mixed male/female donors used in (a–f), male and female mice used in (g–j). Data expressed as Mean ± S.E.M, *p < 0.05, **p < 0.01, ***p < 0, .001. vs. indicated.

Fig. 6. T cell-intrinsic miR-29a regulates T cell function in vitro and in vivo.

Naïve CD4⁺ T cell were purified from miR-29a^loxP/loxP Lck Cre ⁺ or Cre^- mice and stimulated with anti-CD3/anti-CD28 for 5 h in normoxia (21% O₂) or hypoxia (1% O₂). Q-PCR assays were employed to measure: (a) microRNA expression of miR-29a, (n = 3, two-way ANOVA). b mRNA expression of T-bet, c mRNA expression of IFNγ. (b, c: n = 3, repeated 2 times, two-way ANOVA). d mRNA level of IFNγ in T_H1-skewed T cells grown in vitro for 3 days and then re-stimulated with anti-CD3/anti-CD28 for 8 h, (n = 3, two-tailed T test). 7-week-old B6.RAG1−/− recipient mice were adoptively transferred with 10⁶ in-vitro differentiated CD4⁺ T_H1 T cells from Lck Cre⁺ and miR-29a^loxP/loxP Lck Cre⁺ mice and weight changes were recorded twice per week. e Weight change chart with transient weight gain followed by continuous weight loss in the course of colitis development, (n = 7–8). f Colon inflammatory score (colon weight/length), (n = 6, n.s). g Representative micrographs of H&E staining in the colon show infiltration of lymphocytes into the intestinal epithelium in Lck Cre⁺ and miR-29a^loxP/loxP Lck Cre⁺ mice. Original magnification ×20, scale bar: 100 mm. Mesenteric lymph node (MLN) cells were isolated and re-stimulated for 5 h in vitro with PMA/Ionomycin in the presence of Brefeldin A, stained for intracellular cytokines and gated on live CD4⁺ lymphocytes. h Representative plots for each mouse group are shown. i Proportion of IFNγ producing CD4⁺ T cells in the MLN (n = 7–8, two-tailed T test). j Proportion of IL-17 producing CD4⁺ T cells (n = 7–8, two-tailed T test. T_H1 colitis was induced in miR-29a^loxP/loxP Lck Cre⁺ mice and controls by the epicutaneous skin sensitization and subsequent rectal gavage with TNBS as per Materials and Methods. k Weight loss curve, (n = 8–12, two-way ANOVA). l Colon length in TNBS-challenged mice (n = 10–18, one-way ANOVA). m Colonic explants were cultured for 24 h and IFNγ release was measured using ELISA, (n = 5, two-tailed T test). n At the time of necropsy, lamina propria lymphocytes (LPL) were purified and CD4⁺ T cells were assayed by flow cytometry for expression of Tbet protein (n = 4, one-tailed T test). o Histological index of colitis. (n = 8–11, One-way ANOVA). p Representative micrographs from TNBS-treated miR-29a^loxP/loxP Lck Cre⁺, miR-29a^loxP/loxP Lck Cre^- or vehicle -treated mice 5 days post-challenge, scale bar is 100 mm. Male and female mice used in (a–d, k–p). Male recipients and mixed male/female donors used in (e–j). *p < 0.05, **p < 0.01, ****p < 0.0001 vs. indicated, Q-PCR microRNA expression normalized to RNU6B and mRNA expression to 18s rRNA.

Fig. 7. Nanoparticle delivery of a miR-29a mimetic attenuates T_H1-mediated TNBS Colitis.

T_H1 colitis was induced in WT mice by TNBS dosing as per Materials & Methods. At 24 h and 72 h post-challenge mice were injected IV with respective mimic microRNA in lipid nanoparticle emulsion (5 µg/mouse). a Schematic of experimental design and treatments in TNBS-colitis model. b Histological indices of colitis (inflammatory, injury, and total) were assessed 5 days post-TNBS challenge, pooled 2 experiments, (n = 6–7, two-tailed T test). c Representative H&E micrographs of colons. Scale bar: 100 mm. d At the time of necropsy, purified lamina propria lymphocytes were assessed by Q-PCR for. the expression of T-bet mRNA, normalized to 18S rRNA, (n = 3–4, one-way ANOVA). e Expression of IFNγ mRNA in purified lamina propria T lymphocytes. Data expressed as Mean ±S.E.M from 2 pooled experiments, (n = 3–4, one-way ANOVA). TNF^∆ARE/+ mice manifest with spontaneous ileitis. f Schematic of miR-29a mimetic treatment schedule of TNF^∆ARE/+ mice. g Flow cytometry staining of IFNγ-positive CD4⁺ T cells and number of CD4⁺ T cells as a percentage of total MLN cells at the conclusion of experiment, 2 pooled experiments, (n = 9–10, one-way ANOVA). h Overall histological inflammatory score, 2 pooled experiments, (n = 13, one-way ANOVA). i Representative H&E micrographs of the ileum in control (untreated), mock-treated and miR-29a mimetic treated mice. Scale bar: 100 mm. Male and female mice used in all experiments. Data expressed as Mean ±S.E.M, *p < 0.05, **p < 0.01 vs. indicated. Schematics in (a, f) were created with BioRender.com.