Transforming growth factor beta is dispensable for the molecular orchestration of Th17 cell differentiation

Abstract

Interleukin (IL)-17-producing T helper (Th17) cells play a critical role in the pathophysiology of several autoimmune disorders. The differentiation of Th17 cells requires the simultaneous presence of an unusual combination of cytokines: IL-6, a proinflammatory cytokine, and transforming growth factor (TGF) beta, an antiinflammatory cytokine. However, the molecular mechanisms by which TGF-beta exerts its effects on Th17 cell differentiation remain elusive. We report that TGF-beta does not directly promote Th17 cell differentiation but instead acts indirectly by blocking expression of the transcription factors signal transducer and activator of transcription (STAT) 4 and GATA-3, thus preventing Th1 and Th2 cell differentiation. In contrast, TGF-beta had no effect on the expression of retinoic acid receptor-related orphan nuclear receptor gammat, a Th17-specific transcription factor. Interestingly, in Stat-6(-/-)T-bet(-/-) mice, which are unable to generate Th1 and Th2 cells, IL-6 alone was sufficient to induce robust differentiation of Th17 cells, whereas TGF-beta had no effect, suggesting that TGF-beta is dispensable for Th17 cell development. Consequently, BALB/c Stat-6(-/-)T-bet(-/-) mice, but not wild-type BALB/c mice, were highly susceptible to the development of experimental autoimmune encephalomyelitis, which could be blocked by anti-IL-17 antibodies but not by anti-TGF-beta antibodies. Collectively, these data provide evidence that TGF-beta is not directly required for the molecular orchestration of Th17 cell differentiation.

Figure 1.

Differentiation of Th17 cells in the absence of Th1, Th2, or Th1 and Th2 cells. (A) FACS-sorted CD25⁻CD62L^hiCD44^lo naive CD4⁺ T cells isolated from wild-type, Stat-6^−/−, T-bet^−/−, or Stat-6^−/−T-bet^−/− mice were activated by plate-bound anti-CD3 and anti-CD28 in the presence of 5 ng/ml TGF-β, 20 ng/ml IL-6, or both for 72 h. Intracellular cytokines were detected as described in Materials and methods. The percentage of cells positive for IL-17 is shown from one representative out of three independent experiments. (B) Expression of IL-17 and IL-21 in cells treated as in A was determined by quantitative PCR. (C) Naive CD4⁺ T cells from Stat-6^−/−T-bet^−/−/TGF-βRIIDN mice were subjected to Th17 cell differentiation as described in A. Note that these mice and their control littermates are in a mixed genetic background. Results shown are representative of three independent experiments. Four mice per group were used in each experiment.

Figure 2.

Regulation of transcription factors in Th cells by TGF-β and IL-6. FACS-sorted CD25⁻CD62L^hiCD44^lo naive CD4⁺ T cells were activated with plate-bound anti-CD3 and anti-CD28 in the presence of 5 ng/ml TGF-β, 20 ng/ml IL-6, or both for 48 h. Anti–TGF-β was added at a concentration of 10 µg/ml. Expression levels of the transcription factors RORγt, GATA-3, Stat-6, Stat-4, and Tbx21 in the cells were determined by quantitative PCR. Results shown are representative of three independent experiments.

Figure 3.

TGF-β inhibits GATA-3 and STAT-4 expression in T cells during activation. (A) Naive CD4⁺ T cells were activated with plate-bound anti-CD3 and anti-CD28 in the presence of 5 ng/ml TGF-β, 20 ng/ml IL-6, or both for 48 h. Cytokines in the supernatants were determined by multiplexed bead array immunoassay. Data represent means ± SEM. (B) Established Th1 and Th2 cells were activated by plate-bound anti-CD3 for 6 h with or without 5 ng/ml TGF-β, 20 ng/ml IL-6, or both. The expression of transcription factors was determined by quantitative PCR. Data shown are representative of four independent experiments. *, P < 0.03; **, P < 0.002; ***, P < 0.001; #, P < 0.003; ##, P < 0.01; and ###, P < 0.005.

Figure 4.

Stat-6^−/−T-bet^−/− mice develop EAE in an IL-17–dependent manner. (A) Induction of EAE in wild-type and Stat-6^−/−T-bet^−/− mice on the BALB/c background. Stat-6^−/−T-bet^−/− mice were injected with neutralizing antibodies against TGF-β or IL-17 twice weekly, EAE was induced, and clinical disease was scored. Data are from five mice per group and are representative of two independent experiments. (B) Antigen-specific proliferation of T cells from wild-type and Stat-6^−/−T-bet^−/− mice. CD4⁺ T cells were isolated from MBP-immunized mice, mixed with syngeneic spleen cells at a 1:2 ratio, and challenged with MBP. After 2 d, proliferation was measured by [³H]thymidine incorporation. Data represent means ± SEM. (C) Intracellular cytokine staining for IL-10 and IL-17 was performed in lymph node cells isolated from MBP-immunized mice. Cells were stimulated ex vivo with MBP, and their intracellular cytokines were detected as described in Materials and methods. For B and C, results shown are representative of three independent experiments, and three mice were used per group.