TGF-β-mediated enhancement of TH17 cell generation is inhibited by bone morphogenetic protein receptor 1α signaling

Abstract

The cytokines of the transforming growth factor-β (TGF-β) family promote the growth and differentiation of multiple tissues, but the role of only the founding member, TGF-β, in regulating the immune responses has been extensively studied. TGF-β is critical to prevent the spontaneous activation of self-reactive T cells and sustain immune homeostasis. In contrast, in the presence of proinflammatory cytokines, TGF-β promotes the differentiation of effector T helper 17 (T_H17) cells. Abrogating TGF-β receptor signaling prevents the development of interleukin-17 (IL-17)-secreting cells and protects mice from T_H17 cell-mediated autoimmunity. We found that the receptor of another member of TGF-β family, bone morphogenetic protein receptor 1α (BMPR1α), regulates T helper cell activation. We found that the differentiation of T_H17 cells from naive CD4⁺ T cells was inhibited in the presence of BMPs. Abrogation of BMPR1α signaling during CD4⁺ T cell activation induced a developmental program that led to the generation of inflammatory effector cells expressing large amounts of IL-17, IFN-γ, and TNF family cytokines and transcription factors defining the T_H17 cell lineage. We found that TGF-β and BMPs cooperated to establish effector cell functions and the cytokine profile of activated CD4⁺ T cells. Together, our data provide insight into the immunoregulatory function of BMPs.

Fig. 1.. BMP signaling regulates T cell activation and lineage commitment.

(A) qRT-PCR analysis of BMPR1α mRNA expression in sorted CD4⁺ naive, activated and T_reg cells. Data are means ± SD pooled from 3 independent sorts. (B) Western blot analysis of BMPR1α abundance in lysates from sorted CD4⁺ naive, activated and T_reg cells. Blots (left) are representative of three independent experiments. Normalized band intensity data (right) are means ± SD pooled from all experiments. (C) qRT-PCR analysis of BMPR1α in sorted, naive CD4⁺ T cells stimulated with antibodies against CD3 and CD28 for the indicated times. Results are means ± SD pooled from three independent experiments. (D) Flow cytometry analysis of IFN-γ and IL-17 in CD4⁺ T cells stimulated with antibodies against CD3 and CD28 for 4 days and BMP2/4/7 or TGF-β and IL-6, as indicated. Dot plots (left) representative of four independent stimulations. The frequency of cytokine producing cells (right) are means ± SD pooled from all experiments. (E) Western blot analysis of p-STAT3 and STAT3 in CD4⁺ T cells stimulated for the indicated times with IL-6 or IL-6 and TGF-β in the absence or presence of BMP2/4/7. Blots (top) are representative of four independent experiments. Normalized band intensity data (bottom) are means ± SD pooled from all experiments. *P<0.05, **P< 0.01, ***P< 0.001, ****p< 0.0001 as determined by Student’s t- test.

Fig. 2.. BMPR1α restricts CD4⁺ T cells IL-17 production.

(A) Western blot analysis of total and phosphorylated Smad1/5/8 and Smad 2/3 in activated wild-type (WT) and BMPR1α^T− CD4⁺ T cells. Blots (left) are representative of three independent experiments. Normalized band intensity data (right) are means ± SD pooled from all experiments. (B) Flow cytometry analysis of the indicated cell surface markers on activated WT and BMPR1α^T− CD4⁺ T cells. Histograms (left) are representative of four independent analyses. Mean florescence intensity (MFI) data (right) are means ± SD from all experiments. (C and D) Flow cytometry analysis of cytokine production by CD4⁺ T cells from WT and BMPR1α^T− mice stimulated with antibodies against CD3 and CD28 in medium alone (C), or in the presence of TGFβ and IL-6 (D), as indicated. Contour plots are representative of four independent stimulations. The frequency of cytokine producing cells are means ± SD pooled from all experiments. *P<0.05, **P< 0.01, as determined by Student’s t-test.

Fig. 3.. BMPR1α reduces the generation of pro-inflammatory CD4⁺ T cells in vivo.

(A) Flow cytometry analysis of the indicated activation markers on CD4⁺ T cells isolated from the popliteal lymph nodes of WT and BMPR1α^T− mice injected with CFA in the footpad and analyzed after 5 days. Contour plots are representative of three independent experiments. The frequency of naive, activated, and CD25+ cells and activation marker MFI are means ± SD from all experiments. (B) ELISPOT analysis of cytokine production by T cells isolated from WT and BMPR1α^T− mice 7 days after booster immunization with class II MHC restricted LCMV gp61-80 peptide in CFA. Images (left) were quantified spot forming units (SFU). Quantified data (right) are means ± SD pooled from three independent experiments. (C) Analysis of footpad thickness in WT and BMPR1α^T− mice injected with CFA in the footpad and analyzed after 5 days. Images (top) are representative of three independent experiments. Quantified data (bottom) are means ± SD pooled from all experiments. *P< 0.05, **P< 0.01, ***P< 0.001, as determined by Student’s t-test.

Fig. 4.. Cell-intrinsic BMPR1α expression suppresses the generation of Th17 CD4⁺ T cells.

(A and B) Flow cytometry analysis of IFN-γ and IL-17 production (A) and surface marker expression (B) on co-cultured TCR transgenic WT and BMPR1α^T− CD4⁺ T cells stimulated with antigenic peptide and the indicated cytokines for 6 days. Contour plots and histograms (left) are representative of three independent experiments. The frequency of cytokine producing cells and marker mean fluorescence intensity (MFI) (right) are means ± SD from all experiments. (C and D) Flow cytometry analysis of surface marker expression (C), IFN-γ and IL-17 production (D) and Rorc abundance (D) in lymph node T cells isolated 5 days after TCR transgenic WT and BMPR1α^T− mice were immunized with antigenic peptide and CFA. Histograms and contour plots (left) are representative of three independent experiments. The frequency of cytokine producing cells and marker MFI (right) are means ± SD from all experiments. *P< 0.05, **P< 0.01, ***P< 0.001, as determined by Student’s t-test.

Fig. 5.. BMPR1α alters Th1/Th17 gene expression.

(A) Principal component analysis of genes expressed by activated BMPR1α-sufficient and -deficient CD4⁺ T cells. Analysis included gene expression data from three samples of BMPR1α-sufficient and -deficient cells. (B) Genes differentially expressed in activated CD4⁺ T cells from WT and BMPR1α^T− mice. Analysis included genes differentially expressed between three samples of BMPR1α-sufficient and - deficient cells. Colors show log₂ fold change of the expression difference. (C) Metascape GO analysis of genes differentially expressed by activated WT and activated BMPR1α-deficient CD4⁺ T cells. (D) Transcript levels, fold change expression and p-values of genes differentially expressed in activated CD4⁺ T cells from WT and BMPR1α^T− mice, related to inflammatory, immune and metabolic responses. Vertical bars represent average values of gene expression levels (fpkm) ± SD. All data are representative of three biological replicates.

Fig. 6.. BMPR1α mediates TCR and cytokine-dependent signaling.

(A) Western blot analysis of Smad4 in naive and activated CD4⁺ T cells. Blots (top) are representative of four independent experiments. Normalized band intensity data (bottom) are means ± SD from all experiments. (B) Western blot analysis of total and phosphorylated Smad 2/3 in CD4⁺ T cells from WT and BMPR1α^T− mice stimulated for 1 hour with plate-bound antibodies against CD3 and CD28 in the presence of indicated concentrations of TGF-β. Blots (top) are representative of four independent stimulations. Normalized band intensity data (bottom) are means ± SD from all experiments. (C) Western blot analysis of STAT1 and STAT3 phosphorylation in lysates from WT or BMPR1α^T− CD4⁺ T cells directly isolated and lysed (−IFN-γ and −IL-6) or activated for 5 min with plate-bound antibodies against CD3 and CD28 in the presence of IFN-γ or IL-6. Blots (left) are representative of four independent experiments. Normalized band intensity data (right) are means ± SD from all experiments. (D) Western blot analysis of Jnk, Erk and p38 phosphorylation in lysates from WT or BMPR1α^T− CD4⁺ T cells activated with Con A for 2 days. Blots (left) are representative of four independent experiments. Normalized band intensity data (right) are means ± SD from all experiments. (E) Western blot analysis of Erk phosphorylation in WT CD4⁺ T cells activated with plate-bound antibodies against CD3 and CD28 in the presence of inhibitors DMH1 or dorsomorphin for 24 hours. Blots (left) are representative of three independent experiments. Normalized band intensity data (right) are means ± SD from all experiments. *P< 0.05, as determined by Student’s t-test.

Fig. 7.. Adoptive transfer of BMPR1α-deficient CD4⁺CD45RB^high cells induces more severe colitis.

(A to F) TCRα⁻ recipient mice received a transfer of either WT or BMPR1α^T− CD4⁺CD45RB^high cells. (A to C) Weight loss, bleeding and diarrhea were scored over time. (D) Total colon length was assessed on day 21. (E) Histological analysis of colonic cross-sections by hematoxylin and eosin staining was performed on the indicated mice on day 21. Scale bars equal 100 μM. (F) Flow cytometry analysis of cytokines and Rorc in CD4⁺ T cells isolated from the colons of recipient mice. (G) qRT-PCR analysis of IL-6, IL-1β, IL-17, Rorc and IFN-γ transcripts in CD4⁺ T cells isolated by flow cytometry sorting from colons of recipient mice. All results are representative of four mice per group analyzed in three independent experiments. Quantified data are means ± SD from all experiments. *P< 0.05, **P< 0.01, ***P< 0.001, ****p< 0.0001, as determined by Student’s t-test.