Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1

Abstract

T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.

Figure 1. HIF-1α mRNA is up-regulated in T cells under T_H17 skewing conditions in Stat3 dependent manner

(A) Naïve (CD4+CD25-CD62L^high) T cells were cultured under Th-cell subset inducing conditions, and HIF-1 mRNA was detected by qRT-PCR. (B, C and D) Naïve T cells from wild type (WT, HIF-1α^+/+) or CD4^Cre x HIF-1^flox/flox mice (ko, HIF-1α^−/−) were stimulated in the presence of TGFβ and IL-6. RNA was isolated from these cells and qRT-PCR was performed at different time points during culture to measure HIF-1α (B), IL-17 (C) and RORγt (D) transcript levels. (E) WT or Stat3^−/− (obtained from CD4^Cre x Stat3^flox/flox mice) naïve CD4+ T cells were isolated and cultured under the indicated conditions for 2 days, followed by SDS-PAGE and Western blotting using antibodies against HIF-1α (top), HIF-1β (middle) and tubulin (bottom), respectively. (F) A ChIP assay was used to examine direct Stat3 binding to the HIF-1α promoter. Panels A–D and F depict the mean + s.d. of at least three experiments while Panel E is a representative result. See also Figures S1.

Figure 2. HIF-1α is required for T_H17 development in vitro

(A) Naïve T cells isolated from wild type (WT, HIF-1α^+/+) or CD4^Cre x HIF-1α^flox/flox (HIF-1α^−/−) mice were cultured under T_H17 skewing conditions with anti-CD3/CD28 and TGFβ, IL-6 and anti-IFNγ, IL-12 and IL-4 antibodies for 6 days. Cells were then stained for IL-17 and Foxp3 (see Methods). Numbers represent the percentage of CD4+ cells positive for the indicated marker. (B) FACS-sorted naïve CD4+ T cells from WT or T-HIF-1α−/− mice were activated and cultured under T_H17 skewing for 4 days. Total RNA was isolated and mRNA expression of IL-17, IL-17F and IL-23R genes was assessed by qRT-PCR. For each gene, expression level in HIF-1−/− T cells was set to 1. The mean + s.d. of at least 3 trials is shown. (C) Naïve CD4+ T cells were activated with anti-CD3/CD28 under either neutral (anti-IL-4 and anti-IFNγ Ig) or T_H17-skewing conditions, and transduced with a bicistronic retrovirus expressing HIF-1α-GFP or GFP alone. Intracellular cytokines were stained and analyzed in GFP+ cells. Numbers represent the percentage of gated GFP+ cells. (D) Naïve T cells isolated from WT or T-HIF-1α−/− mice were activated as described for (A) under normoxia or hypoxia (see Methods). Panels C and D represent at least 2 independent experiments. See also Figure S2.

Figure 3. HIF-1α transactivates RORγt transcription and is necessary for RORγt-driven T_H17 differentiation in vitro

(A) Jurkat T cells were co-transfected with a luciferase reporter under the control of a wild type RORγt promoter or one with a mutated HIF-1-binding site. 24hrs post-transfection, cells were either treated with PMA and ionomycin or left untreated prior to analysis of luciferase activity, which was normalized to that of Renilla luciferase. (B) Jurkat T cells were transfected with a RORγt-luciferase reporter plasmid (incorporating the RORγt promoter) along with plasmid encoding either wild type HIF-1 or a HIF-1 mutant (with a DNA binding domain deletion). 24hrs post-transfection, cells were stimulated and luciferase activity was assessed as described in (A). Data are shown for three independent experiments (mean and s.d. of triplicate transfections). (C) A ChIP assay was used to measure direct HIF-1 binding to the RORγt promoter directly. (D and E) Naïve WT or HIF-1^−/− CD4+ T cells were activated with anti-CD3/anti-CD28 under neutral (anti-IL4 and anti-IFNγ) conditions, and transduced with either a bicistronic retrovirus expressing RORγt-GFP or the GFP containing empty vector. Intracellular IL-17 and Foxp3 were stained. The plots shown are gated on GFP+ cells. These experiments were repeated at least twice with consistent results. See also Figures S3.

Figure 4. RORγt, HIF-1α and p300 bind to the IL-17 promoter to regulate its gene expression

(A) A HIF-1α mutant (HIF-1-ΔDBD, with the DNA binding domain deleted) retains the capacity to activate IL-17 promoter-driven luciferase activity in the presence of RORγt. Jurkat T cells were transfected with an IL-17 promoter-driven luciferase reporter along with the indicated plasmids followed by stimulation and assessment as described for Figure 3A. Data are representative of at least 3 experiments (mean and s.d. of triplicate transfections). (B) The interaction between HIF-1 and RORγt was examined with co-immunoprecipitation. FACS-sorted CD4+ T cells were activated and cultured under T_H17-skewing conditions for 5 days. The whole cell lysate were immunoprecipitated with either anti-RORγt (left panel) or anti-HIF-1α antibodies (right panel), resolved by SDS-PAGE and blotted with the indicated antibodies. (C) RORγt interacts with the N-terminus of HIF-1. His-tagged RORγt purified from E. coli was incubated with different fragments of GST-HIF-1 also purified from E. coli as indicated, followed by pull-down with GST beads, resolution by SDS-PAGE and Westen blotting with anti-RORγt (top and bottom) antibodies, or anti-GST (middle). (D and E) FACS-sorted CD4+ T cells from WT or HIF-1−/− mice were activated under T_H17-skewing conditions (as described for B) prior to harvest for ChIP assay utilizing anti-RORγt, anti-HIF-1 or anti-p300 antibodies. (F) Hypoxia enhances the activation of IL-17 promoter-driven luciferase activity by HIF-1, RORγt and p300. Jurkat T cells were transfected with a IL-17 promoter-driven luciferase reporter along with the indicated plasmids under normoxia or hypoxia, followed by stimulation and assessment as described for Figure 3B. Data are representative of at least 3 independent experiments (mean and s.d. of triplicate transfections). (G) Histone hyperacetylation at the IL-17 promoter was detected by ChIP assay in WT and HIF-1α−/− T cells under T_H17 skewing conditions for 5 days. See also Figures S4–S6 and Table S1.

Figure 5. HIF-1α mediated Foxp3 degradation through proteasomal degradation pathways

(A) Naïve T cells from either WT or T-HIF-1−/− mice were cultured and stimulated in the presence of TGFβ and IL-6 and qRT-PCR was performed to measure Foxp3 mRNA at different times. (B) HIF-1α−/− T cells displayed enhanced Foxp3 accumulation during in vitro Treg differentiation. Naïve T cells from HIF-1+/+ and T-HIF-1−/− were isolated by FACS and activated under Treg skewing (5ng/ml TGFβ, 100U IL-2). Cells were stained for Foxp3 after 72 hours. Shown are representative histograms for HIF-1+/+ (red line) and HIF-1−/− (blue line) cells from three experiments. An isotype control is shown in green. Numbers represent the mean percentage of Foxp3+ cells. (C) Foxp3 protein is lost upon culture of WT T cells with IL-6 but remains unchanged in HIF-1α−/− Foxp3+ T cells. Naive T cells were activated under Treg-skewing (TGFβ, 5ng/ml) with the indicated doses of IL-6 for 4 days. Western blotting was used to measure Foxp3 protein level. (D) Hypoxia reduced expression of Foxp3 by naïve T cells during in vitro differentiation. Naïve T cells isolated from Foxp3-GFP reporter mice (CD4+GFP-CD62L^high) were cultured under Treg skewing conditions (see B) in either a hypoxic chamber or under normoxia (blue and red lines, respectively). Numbers represent the mean percentage of CD4+ cells expressing GFP (Foxp3+) from three experiments. (E) HIF-1 interacts with Foxp3 in iTreg cells. FACS-sorted CD4+ T cells were activated and cultured under iTreg-skewing for 4 days. Cell lysates were immunoprecipitated with anti-HIF-1 (left panel) or anti-Foxp3 antibodies (right panel), followed by SDS-PAGE and western blotting. (F, G and H) HIF-1α mediates Foxp3 degradation. 293T cells were cotransfected with Foxp3 +/− ubiquitin and increasing amounts of WT HIF-1 (F) or mutant HIF-1 (p420A and p564A) (G) or a deleted ODD domain (CA5-HIF) (H)). CoIP and Western blots were probed as indicated. (I and J) Naïve T cells were cultured under Treg-skewing conditions in normoxia (N) or hypoxia (H) for 4 days. Cells were harvested and lysed, followed by immunoprecipitation with anti-Foxp3 or control IgG antibodies. The pulled down protein along with an input control were resolved by SDS-PAGE, followed by western blot. Depicted are typical findings from 3 independent experiments. See also Figure S7

Figure 6. Mice lacking HIF-1α in CD4+ T cells are deficient in IL-17 production, have increased numbers of Foxp3 Treg and are more resistant to EAE

(A) EAE was induced in HIF-1+/+ and T-HIF-1−/− mice by injection of MOG_35–55 in CFA and Pertussis Toxin. Disease severity was monitored and scored daily. T-HIF-1−/− mice failed to develop the severe disease seen in HIF-1+/+ mice. Mean scores for HIF-1+/+ and T-HIF-1−/− mice over time are represented (+/− SEM; *p<0.05). Shown is a representative of 4 experiments (N=7–10 per group). (B, C) During peak disease (day12), draining lymph node, splenic, and CNS infiltrating T cells were recovered and stained for IL-17 and IFNγ. The mean percentage of CNS CD4+ cells during peak disease (days 12–14) positive for IL-17 was determined (C). (D and E) Similarly, during the recovery phase (day 21), tissue infiltrating cells were isolated and the percentage of CD4+ that wereFoxp3+ was found (D and E). Panels C and E present the mean (± SEM, *p<0.05) of at least 3 trials and dot plots are representative analyses.

Figure 7. A model for the multi-factorial role of HIF-1α in modulating the T_H17/Treg balance

Stat3 activation by factors such as IL-6 transcriptionally activates HIF-1. HIF-1 levels are further regulated by oxygen tension and other metabolites, representing a key molecular link between metabolic cues and T cell lineage commitment. HIF-1 directly activates RORγt gene transcription and furthermore, recruits p300 to RORγt transcription complexes to the promoters of T_H17 genes (ie IL-17). These activities promote Th17 differentiation. Concomitantly, HIF-1 induces Foxp3 protein degradation via targeting for ubiquitination and proteasomal degradation.