RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis

Abstract

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-β that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32^-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32^-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4⁺ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.

FIGURE -1.

RGC-32 is preferentially induced in Th17 cells and promotes their differentiation. (A) Quantitative RT-PCR for RGC-32 in naive CD4⁺ T cells from spleens of WT mice stimulated for 48 h with anti-CD3 and anti-CD28 in the presence or absence of indicated cytokines and in (B) Th0, Th1, Th2, Th17, or Treg polarizing conditions (**p, 0.01; mean 6 SEM; n = 3–4 mice per group). (C) Representative histogram of RGC-32 intracellular staining in CD4⁺ T cells cultured under Th17 and Treg conditions. Gray histogram represents background staining in RGC-32^−/− CD4⁺ T cells. (D and E) Naive CD4⁺ T cells were cultured for 72 h in Th17 conditions and intracellular expression of IL-17A was assessed by flow cytometry. A representative profile is shown. Plots were gated on viable singlet CD4⁺ events. (F) ELISA for IL-17A determined in supernatants. (G) Real-time PCR for IL-17A. (H) Quantitative RT-PCR for Th17 signature genes IL-17F, IL-21, IL-22, and IL-23R. (*p, 0.05, **p, 0.01, mean 6 SEM). Data are representative of more than three independent experiments (n = 3–4 mice per group).

FIGURE 2.

Decreased CD4⁺IL-17⁺ T cells in lamina propria lymphocytes of RGC-32^−/− mice. Freshly isolated lamina propria lymphocytes from WT and RGC-32^−/− mice were stimulated in vitro with PMA/ionomycin for 4 h before intracellular cytokine staining for IL-17A. (A and B) Percentage and total number of IL-17A–expressing CD4⁺ T cells. (C) A representative profile is shown. Plots were gated on viable singlet CD4⁺ events (*p, 0.05, **p, 0.01, mean 6 SEM. Data are representative of three independent experiments; n = 2–3 mice per group).

FIGURE 3.

RGC-32^−/− CD4⁺ T cells polarize normally to Th1, Th2, and Treg lineages. Naive CD4⁺ T cells were skewed in vitro under Th1, Th2, and Treg conditions. (A) Intracellular expression of IFN-γ was assessed by flow cytometry. (B) ELISA for IFN-γ determined in supernatants. (C) Intracellular expression of IL-4 was assessed by flow cytometry. (D) ELISA for IL-4 determined in supernatants. (E) Foxp3 expression was determined by intracellular staining. (F) In vitro suppression assay of purified CD4⁺CD25² T responder cells cocultured for 3 d with in vitro–differentiated, purified CD4⁺CD25⁺ iTregs from WT or RGC-32^−/− mice. Proliferation was assessed by [³H]thymidine incorporation added in the last 18 h of culture (left panel). Percentage inhibition at 1:1 and 1:2 Treg/T responder ratio is shown in the right panel. ***p, 0.001. Data represent the mean 6 SD and are representative of more than three independent experiments (n = 3 mice per group). Representative profiles shown for intracellular IL-4, IFN-γ, and Foxp3 are gated on viable singlet CD4⁺ events.

FIGURE 4.

Lack of RGC-32^−/− did not affect expression of IL-2/IL-2R or cell division. (A) Naive CD4⁺ T cells were stimulated under Th17 conditions. After 48 h, IL-2 expression was determined by intracellular staining and (B) ELISA. (C) Percentage of CD25⁺CD4⁺ T cells. (D) Naive CD4⁺ T cells were stained with CFSE prior to culture under Th17 conditions. CFSE dilution due to cell proliferation was determined by flow cytometry on gated CD4⁺IL-17⁺ cells. Results are representative of more than three independent experiments (n = 3 mice per group).

FIGURE 5.

RGC-32 promotes Th17 cell differentiation through TGF-β–dependent and –independent mechanisms. Freshly isolated naive CD4⁺ T cells were stimulated under Th17 conditions. (A and B) RORδ expression was determined by intracellular staining. (C) Quantitative RT-PCR for Rorc. (D) Quantitative RT-PCR for IRF4 and BATF. (E) Western blotting analysis of phospho-STAT3, -SMAD2, and -SMAD3 in unstimulated and Th17 conditions for the indicated times. The graphs in (F)–(H) show cumulative data of densitometry (*p, 0.05, **p, 0.01, mean 6 SEM). Data are representative of three independent experiments (n = 3 mice per group)

FIGURE 6.

IL-21 and IL-23 do not restore defective IL-17 production in RGC-32^−/− CD4⁺ T cells. Naive CD4⁺ T cells from WT and RGC32^−/− mice were cultured under Th17 conditions and IL-21 or IL-23 was added after 24 h of culture. (A and B) Intracellular expression of IL-17A was assessed at 72 h by flow cytometry. A representative profile is shown. Plots were gated on viable singlet CD4⁺ events (*p < 0.05; mean 6 SEM; n = 3 mice per group).

FIGURE 7.

Lack of RGC-32 ameliorates EAE and CNS infiltration. WT (n = 10) and RGC-32^−/− (n = 9) mice were immunized with MOG35–55 peptide. (A) Mean clinical EAE scores 6 SEM are shown and are representative of three independent experiments (*p, 0.05). (B and C) H&E and Luxol fast blue (LFB) staining of cervical spinal cords harvested at the peak of disease. Original magnification 3200. (D) Thirteen days after disease induction, CNSinfiltrating lymphocytes were isolated, stimulated with PMA and ionomycin for 4 h, and stained for IL-17A and IFN-γ. Representative flow cytometric analysis of cytokine profile on gated CD4⁺ T cells is shown. (E and F) Percentage and total number of GM-CSF⁺, IL-17⁺, IFN-γ⁺, and IL-17⁺IFN-γ⁺CD4⁺ T cells in the CNS (*p, 0.05; mean 6 SEM; n = 3 mice per group). (G) Splenocytes from MOG-immunized WT and RGC-32^−/− mice were cultured in the presence or absence of MOG_35–55 (25 µg/ml) for 72 h. Proliferation was determined by [ H]thymidine incorporation. (H) WT CD4⁺ T cells were injected into WT and RGC-32^−/− mice 5 d prior to EAE induction. Mean clinical EAE scores 6 SEM are shown. (I and J) Percentage and total number of IL-17⁺–, IFN-γ⁺–, and IL-17⁺IFN-γ⁺–expressing CD4⁺ T cells in the CNS. *p, 0.05. Data shown are representative of three independent experiments (K). CD4⁺ T cells (7 × 10⁶) from WTor RGC-32^−/− mice were injected into RAG1^−/− mice 1 d prior to EAE induction. Mean clinical EAE scores 6 SEM are shown. *p, 0.05.