TGF-β converts Th1 cells into Th17 cells through stimulation of Runx1 expression

Abstract

Differentiated CD4(+) T cells preserve plasticity under various conditions. However, the stability of Th1 cells is unclear, as is whether Th1 cells can convert into Th17 cells and thereby contribute to the generation of IFN-γ(+) IL-17(+) CD4(+) T cells, the number of which correlates with severity of colitis. We investigated whether IFN-γ(+) Th1 cells can convert into Th17 cells under intestinal inflammation and the mechanisms involved. IFN-γ(Thy1.1+) Th1 cells were generated by culturing naïve CD4(+) T cells from IFN-γ(Thy1.1) CBir1 TCR-Tg reporter mice, whose TCR is specific for an immunodominant microbiota antigen, CBir1 flagellin, under Th1 polarizing conditions. IFN-γ(Thy1.1+) Th1 cells induced colitis in Rag(-/-) mice after adoptive transfer and converted into IL-17(+) Th17, but not Foxp3(+) Treg cells in the inflamed intestines. TGF-β and IL-6, but not IL-1β and IL-23, regulated Th1 conversion into Th17 cells. TGF-β induction of transcriptional factor Runx1 is crucial for the conversion, since silencing Runx1 by siRNA inhibited Th1 conversion into Th17 cells. Furthermore, TGF-β enhanced histone H3K9 acetylation but inhibited H3K9 trimethylation of Runx1- and ROR-γt-binding sites on il-17 or rorc gene in Th1 cells. We conclude that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is possibly mediated by TGF-β induction of Runx1.

Keywords: Colitis; Runx1; TGF-β; Th1; Th17.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1. IL-17+, IFN-γ+, and IL-17+IFN-γ+ CD4+ cells are found in the lamina propria of colitic mice

Naïve splenic CD4+ T cells from CBir1 Tg mice were i.v. injected into Rag−/− mice. Four weeks post T-cell transfer, the severity of colonic inflammation was assessed by histological analysis after hemytoxlin and eosin staining. (A) Histopathology of colon (original magnification, 20×). (B) Pathological scores of accessed colons. Bars show mean +/− SEM of 4 mice from a single experiment representative of 4 experiments. (C) Flow cytometry of CD4+ cells isolated from colon lamina propria of Rag−/− recipient measured for expression of IFN-γ and IL-17. One representative plot out of 4 experiments is shown.

Figure 2. IFN-γ-producing Th1 cells convert into IL-17-producing Th17 cells in inflamed colon

Splenic CD4+ T cells from IFN-γThy1.1 CBir1 TCR Tg reporter mice were cultured under classical Th1 differentiation conditions with mIL-12/anti-IL-4 for 4 days. IFN-γThy1.1+CD4+ double-positive cells were sorted. (A) Gating strategy for sorting of IFN-γThy1.1+CD4+ cells. Representative flow cytometry plot of splenic T cells before sorting, stained for CD4 and Thy1.1. (B-D) After sorting. (B) Representative flow cytometry plot of IFN-γThy1.1+CD4+ cells. (C) Representative flow cytometry plot of IL-17 and IFN-γ. (D) Bar charts of cytokine expression from pool of 2 experiments. (E) Sorted CBir1 Tg IFN-γThy1.1+ CD4+ cells were transferred into Rag−/− mice. 8 weeks post-transfer, lamina propria CD4+ cells of Rag−/− recipient mice were measured for expression of IFN-γ and IL-17 by flow cytometry. One representative plot out of 2 experiments is shown. (F) Bar charts of cytokine expression of lamina propria CD4+ T cells 8 weeks post-adoptive to Rag−/− mice from pool of 2 experiments.

Figure 3. TGF-β and IL-6 stimulate Th1 cells to convert into IL-17-producing Th17 cells

(A) IFN-γThy1.1+ CBir1 Tg CD4+ cells were cultured with splenic APCs and different cytokines in the presence of anti-IL-2 and anti-IFN-γ antibodies for 5 days. IL-17 and IFN-γ expression was determined by flow cytometry. (B) The percentage of IL-17A+ CD4+ cells was quantified and shown as mean +/− SD of three experiments. (C) The total IL-17 production in day 5 culture supernatants was detected by an ELISA and shown as mean +/− SD of three experiments. *p < 0.05, One-way ANOVA with Tukey’s t test.

Figure 4. TGF-β dominates the induction of Th17-associated genes in Th1 cells

IFN-γThy1.1+ CBir1 Tg CD4+ cells were cultured with splenic APCs with TGF-β, IL-6 or TGF-β/IL-6, and anti-IL-2 and anti-IFN-γ antibodies for 4 days. The expression of (A) rorc (B) rorα (C) runx1 and (D) Batf was detected by RT-PCR. Data are shown as mean +/− SEM of 3 experiments, normalized to gapdh expression. *p < 0.05, One-way ANOVA with Tukey’s t test.

Figure 5. TGF-β-induced Runx1 plays a crucial role in Th1 conversion into Th17 cells

IFN-γThy1.1+ CBir1 Tg CD4+ cells were transfected with Runx1 siRNA or control siRNA, allowed to rest for 24 h and stimulated with anti-CD3/anti-CD28 with TGF-β and anti-IL-2/anti-IFN-γ for 2 days. (A) siRNA knockdown efficiency was confirmed by RT-PCR of runx1 at 24 h post transfection. The fold change in runx1 expression is shown as mean +/− SEM of 2 samples from a representative of two experiments. Values are normalized to gapdh expression. (B, D) The expression of (B) il-17, (C) rorc and (D) rorα was detected by RT-PCR. Results are shown as mean +/− SEM of 2 samples from a representative of two experiments, and normalized to gapdh expression. *p < 0.05, Student’s t-test.

Figure 6. Treatment of Th1 cells with TGF-β and IL-6 increases the accessibility of RORγt and Runx1 binding sites

IFN-γThy1.1+ CBir1 Tg CD4+ cells were cultured with splenic APCs with TGF-β/IL-6 and anti-IL-2/anti-IFN-γ for 24 h. Cellular DNA and proteins were fixed, sheared and immunoprecipitated with antibodies against H3K9ac, H3K9me3, or control IgG. The accessibility of multiple Runx1- and RORγt-binding sites on il-17, and rorc genes were analyzed by RT-PCR. Equal volumes of RT-PCR products were subjected to electrophoresis. (A) Runx1-binding site-1 on rorc promoter. (B) Runx1-binding site-2 on rorc promoter. (C) Runx1-binding site on il-17 promoter. (D) RORγt-binding site on il-17 promoter. (E) RORγt-binding site on il-17 CNS-5 enhancer. . Bars indicate fold changes of precipitated DNA after immunoprecipitation with H3K9ac Ab (left) and H3K9me3 Ab (right) of the indicated promoter binding sites and are shown as mean +/− SEM of two experiments. Precipitation from control without TGF-β/IL-6 was set to 1. Normalized on gapdh. Images on the right show representative gel electrophoresis of RT-PCR products, igG, isotype control, C, unstimulated, T, TGF-β/IL-6