Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis

Abstract

STAT3, an essential transcription factor with pleiotropic functions, plays critical roles in the pathogenesis of autoimmunity. Despite recent data linking STAT3 with inflammatory bowel disease, exactly how it contributes to chronic intestinal inflammation is not known. Using a T cell transfer model of colitis, we found that STAT3 expression in T cells was essential for the induction of both colitis and systemic inflammation. STAT3 was critical in modulating the balance of T helper 17 (Th17) and regulatory T (Treg) cells, as well as in promoting CD4(+) T cell proliferation. We used chromatin immunoprecipitation and massive parallel sequencing (ChIP-Seq) to define the genome-wide targets of STAT3 in CD4(+) T cells. We found that STAT3 bound to multiple genes involved in Th17 cell differentiation, cell activation, proliferation, and survival, regulating both expression and epigenetic modifications. Thus, STAT3 orchestrates multiple critical aspects of T cell function in inflammation and homeostasis.

Figure 1. Intrinsic Requirement for STAT3 in T Cell-Dependent Colitis

(A–E) CD4⁺CD45RB^hiCD25⁻ naive T cells from control (Stat3^fl/fl) or Stat3^−/− (Cd4 Cre; Stat3^fl/fl) mice were transferred into Rag2^−/− mice, which were monitored for evidence of colitis. (A) Weight loss (percentage of initial weight at day 0) was calculated for each mouse over 9 weeks. Data show mean (±SEM) weight changes for each group (n = 4–5 mice) and are representative of four independent experiments. (B and C) Colitis was assessed histologically at 9 weeks and severity was scored by markers of inflammation (Izcue et al., 2008). Each point represents an individual mouse and the data are pooled from three independent experiments. Unreconstituted Rag2^−/− mice and mice that received naive T cells with CD4⁺CD25⁺ Treg cells were included as controls. (D and E) Systemic inflammation was assessed by measuring (D) spleen weights and (E) mean (±SEM) concentrations of inflammatory cytokines and chemokines in the serum. Data are representative of two independent experiments. ^*p < 0.05, ^**p < 0.005, ^***p < 0.0005.

Figure 2. Loss of STAT3 Abrogates Th17 but not Th1 Cell Responses

(A–C) Rag2^−/− mice were injected with naive T cells from control (Stat3^fl/fl) or Stat3^−/− (Cd4 Cre; Stat3^fl/fl) mice. (A) IL-17A production by CD4⁺ T cells in the colon LP, MLNs, and spleen was measured. (B) The overall proportion of IL-17A⁺ T cells represents data pooled from two independent experiments. (C) The proportions of IFN-γ⁺ CD4⁺ T cells in the tissues of Rag2^−/− mice were enumerated and pooled from two independent experiments. See also Figure S1. ^*p < 0.05, ^***p < 0.0001.

Figure 3. STAT3 Inhibits Regulatory T Cell Conversion in Colitis

(A) The proportions and (B) absolute numbers (mean ± SEM) of Foxp3-expressing CD4⁺ T cells in the tissues of control (Stat3^fl/fl) or Stat3^−/− (Cd4 Cre; Stat3^fl/fl) mice are shown. Data are representative of three independent experiments. (C and D) Colitis was induced in Rag2^−/− mice as described in Figure 1. Intracellular Foxp3 expression in the tissues is shown in (C), and in (D), proportions of Foxp3⁺ cells among CD4⁺ T cells represent data pooled from four independent experiments. See also Figure S2. ^*p < 0.05, ^**p < 0.01.

Figure 4. STAT3 Is Directly Involved in Multiple Aspects of Th17 Cell Differentiation

(A) ChIP-Seq was performed on Th17 cells with antibodies against phospho-STAT3 (second panel) and histone 3 lysine 4 trimethylation (H3K4me3) (lower panels). The locations of conserved noncoding sequences (CNSs) are shown in the top panel. The arrows indicate transcription directionality. Scales are constant for all genes and islands. See also Figure S3 and Table S1. (B and C) The effect of STAT3 on gene expression in control or Stat3^−/− Th17 cells was determined by (B) microarray analysis and (C) quantitative PCR.

Figure 5. STAT3 Is Required in Lymphopenia-Induced Expansion of CD4⁺ T Cells

(A) Rag2^−/− mice were reconstituted with naive (RB^hi) T cells from control (Stat3^fl/fl) or Stat3^−/− (Cd4 Cre; Stat3^fl/fl) mice. The absolute numbers of CD4⁺ T cells in the tissues were quantified. Data are pooled from two independent experiments. (B) The absolute numbers of CD4⁺ T cells in the tissues of Rag2^−/− mice cotransferred with a 1:1 ratio of wild-type (CD45.1) and Stat3^−/− naive T cells. See also Figure S4. (C) The absolute numbers of CD4⁺ T cells in the spleen and LN of Rag2^−/− mice 2 weeks after reconstitution with wild-type (CD45.1) or Stat3^−/− T cells are shown. Data are representative of two independent experiments. (D and E) Bone marrow (BM) cells from wild-type (CD45.1) or Stat3^−/− mice were transferred into irradiated Rag2^−/− mice. The proportions of CD4⁺ and CD8⁺ T cells in the thymus and spleen are shown in (D), and as shown in (E), absolute numbers (mean ± SEM) of CD4⁺ cells in the spleen and LN were determined. (F) BM cells from wild-type (CD45.1) and Stat3^−/− mice were cotransferred into irradiated Rag2^−/− mice in a 1:1 ratio and the absolute numbers of CD4⁺ T cells in the tissues were calculated. ^*p < 0.05, ^**p < 0.01, ^***p < 0.0005.

Figure 6. STAT3 Has a Nonredundant Role in T Cell Survival and Proliferation

(A and B) Control (Stat3^fl/fl) or Stat3^−/− (Cd4 Cre; Stat3^fl/fl) naive T cells were transferred into Rag2^−/− mice and cell division was assessed by CFSE dilution. (B) The absolute numbers of CD4⁺ TCR-β⁺ cells in the spleen at day 5 and 7 are shown; data are representative of two independent experiments. (C) Control and Stat3^−/− CD4⁺ T cells were cultured for 3 days in medium or with the indicated cytokines and the proportion of apoptotic (Annexin-V/PI positive) cells was determined by flow cytometry. Means (±SEM) of apoptotic cells from three independent experiments are shown. See also Figure S5. ^*p < 0.05; ^**p < 0.005.

Figure 7. STAT3 Directly Regulates Genes Involved in CD4⁺ T Cell Survival and Proliferation

(A) STAT3 binding (second panel) and H3K4me3 modifications (lower panels) were determined by ChIP-Seq. Conserved noncoding sequences are shown in the top panel. Arrows indicate directionality of transcription. See also Table S1. (B and C) Control or Stat3^−/− T cells were cultured for 3 days with the indicated cytokines and the effect of STAT3 on gene expression was assessed by (B) microarray and (C) quantitative PCR (mean ± SEM; n = 2 samples).