The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

Abstract

C1q/TNF-related proteins (CTRP) including CTRP3 are a group of secreted proteins which have a complement C1q-like domain in common, and play versatile roles in lipid metabolism, inflammation, tumor metastasis and bone metabolism. Previously, we showed that the expression of C1qtnf3, encoding CTRP3, is highly augmented in joints of autoimmune arthritis models and CTRP3-deficiency exacerbates collagen-induced arthritis in mice. However, the mechanisms how CTRP3-deficiency exacerbates arthritis still remain to be elucidated. In this study, we showed that CTRP3 was highly expressed in Th17 cell, a key player for the development of autoimmune diseases, and Th17 cell differentiation was augmented in C1qtnf3^-/- mice. Th17 cell differentiation, but not Th1 cell differentiation, was suppressed by CTRP3 and this suppression was abolished by the treatment with a receptor antagonist against AdipoR2, but not AdipoR1, associated with suppression of Rorc and Stat3 expression. Furthermore, AdipoR1 and AdipoR2 agonist, AdipoRon suppressed Th17 cell differentiation via AdipoR2, but not AdipoR1. The development of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis was enhanced in C1qtnf3^-/- mice associated with increase of Th17 cell population. CTRP3 inhibited MOG-induced IL-17 production from T cells by affecting both T cells and dendritic cells. These results show that CTRP3 is an endogenous regulator of Th17 differentiation, suggesting that the CTRP3-AdipoR2 axis is a good target for the treatment of Th17 cell-mediated diseases.

Keywords: AdipoR2; CTRP3; EAE; Th17 cells; autoimmune diseases.

Figure 1

CTRP3 inhibits the differentiation of Th17 cells in an autocrine manner. (A) The relative expression of C1qtnf3 mRNA in different Th subsets was determined by real-time PCR, and the expression levels are shown relative to that of Th0 cells. Th0, Th1, Th2 and Th17 cells were prepared as described in the Materials and Methods section. (B) WT or C1qtnf3 ^–/– (KO) naïve CD4⁺ T cells were cultured under Th17-polarizing conditions for 4 days (n = 4 each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The numbers in each panel indicates percentage of IL-17⁺CD4⁺ T cells in total CD4⁺ T cells (2 left panels). The content of IL-17⁺CD4⁺ T cells in total CD4⁺ T cells (%) (center), and IL-17 concentrations (ng/ml) in culture supernatant determined by ELISA (right). Average and SEM are shown. *p < 0.05, ***p < 0.001. Student’s t-test. (C) C1qtnf3 ^–/– naïve CD4⁺ T cells were cultured under Th17-polarizing conditions in the absence or presence of recombinant CTRP3 (50, 100 and 200 ng/ml) for 4 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IL-17⁺CD4⁺ T cells (left), and the proportion of IL-17⁺CD4⁺ T cells is shown in the center panel. IL-17 concentration in the culture supernatant was determined by ELISA (right). Average and SEM are shown. **p < 0.01, ***p < 0.001. Student’s t-test. (D) C1qtnf3 ^–/–naïve CD4⁺ T cells were cultured under Th1-polarizing conditions for 3 days (n = 4 wells each). Intracellular IFN-γ expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IFN-γ ⁺CD4⁺ T cells in total CD4 T cells (left). The population of IFN-γ ⁺CD4⁺ T cells (center). IFN-γ concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. Student’s t-test. (E) The expression of Adipor1 and Adipor2 mRNA in different Th subsets was determined by real-time PCR and relative expression levels to that in Th0 cells are shown. (F) C1qtnf3 ^–/– naïve CD4⁺ T cells were cultured with recombinant CTRP3 (200 ng/ml) under Th17-polarizing conditions in the absence (-) or presence of AdipoR1 blocker (R1 block, 10 μg/ml) or AdipoR2 blocker (R2 block, 10 μg/ml) for 4 days (n = 4 each). Intracellular IL-17 expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17⁺CD4⁺ T cells (left). The IL-17⁺CD4⁺ T cell content (center). IL-17 concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. *p < 0.05, **p < 0.01. Student’s t-test. (G) The effect of AdipoR1 blocker and AdipoR2 blocker on the regulatory effects of CTRP3 on Th17 cell gene expression was examined. In vitro differentiated C1qtnf3 ^–/– Th17 cells (1 x 10⁵ cells in 200 μl/96 well) were treated with CTRP3 (200 ng/ml) in the presence or absence of AdipoR1 blocker (10 μg/ml, R1 block) or AdipoR2 blocker (10 μg/ml, R2 block), and the expression of Ampka, Ppara, Rorc, Stat3 and Mtor mRNA was determined by real-time PCR. The relative expression levels to that of Gapdh are shown. These data are the average from three independent experiments. Average and SEM are shown. *p < 0.05, **p < 0.01. Student’s t-test. All data were reproduced in another independent experiment with similar results.

Figure 2

AdipoRon suppresses Th17 cell differentiation via AdipoR2. (A) C1qtnf3 ^–/– naïve CD4⁺ T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (0, 1, 2 and 3 μg/ml) for 5 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17⁺CD4⁺ T cells (2 left panels). The population of IL-17⁺CD4⁺ T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. **p < 0.01 and ***p < 0.001. Student’s t-test. (B) C1qtnf3 ^–/– naïve CD4⁺ T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (1 μg/ml, Ron) for 4 days (n = 3 wells each), and the effect of AdipoR1 (10 μg/ml, R1) or AdipoR2 blocker (10 μg/ml, R2) on the Th17 cell differentiation was examined. Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The Number in each panel indicates percentage of IL-17⁺CD4⁺ T cells (4 left panels). The population of IL-17⁺CD4⁺ T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. *p < 0.05, **p < 0.01 and ***p < 0.001. Student’s t-test. All data were reproduced in another independent experiment with similar results.

Figure 3

Development of EAE is exacerbated in C1qtnf3 ^–/– mice associated with an augmented Th17 cell differentiation. (A–D) Incidence of affected mice (A), onset time (B), disease score time course (C) and mean maximum disease score (D) are shown after induction of EAE (11 mice for each group). Average and SEM are shown. *p < 0.05 and ***p < 0.001. χ²-test. Mann-Whitney U-test. Student’s t-test. (E, F) Tissue sections of the spinal cord were stained with H&E (E) and LFB (F) at day 28 after induction of EAE. (G, H) CD4⁺ T cells (G), CD4⁺ IL-17⁺ and CD4⁺ IFN-γ⁺ T cells (H) in the whole spinal cord from WT and C1qtnf3 ^–/– (KO) mice at day 21 after immunization were determined by flow cytometry after PMA/ionomycin stimulation (WT, KO: n = 5 each). The numbers in each panel indicate the percentage of IL-17⁺, IFN-γ⁺, and IL-17⁺IFN-γ⁺ T cells (H, 2 left panels). The population of IL-17-single positive, IFN-γ-single positive and IL-17-, IFN-γ-double positive T cells (H, 3 right panels). Average and SEM are shown. ***p < 0.001. Student’s t-test. (I, J) T cell recall response against the MOG peptide. Seven days after MOG/CFA-immunization, LN cells were isolated from WT and C1qtnf3 ^–/– (KO) mice and re-stimulated with MOG_35-55 (0, 25, 50 μg/ml) for 72 h (n = 5 each). Then, the proliferative response was measured by [³H]TdR incorporation (I). IL-17 (J, left) and IFN-γ (J, right) concentrations in the culture supernatant were determined by ELISA. Average and SEM are shown. *p < 0.05. Student’s t-test. (K) C1qtnf3 ^–/– 2D2 T cells and C1qtnf3 ^-/- BMDCs were cultured with 5 μg/ml MOG_35-55 in the presence or absence of CTRP3 (0, 1, 10, 100 ng/ml) for 5 days, and Th17 cell content (left), and IL-17 (center) and IFN-γ (right) concentrations in the culture supernatant were determined (n = 4 wells each). Average and SEM are shown. *p < 0.05 and **p < 0.01. Student’s t-test. (L) WT or C1qtnf3 ^-/- 2D2 T cells were cultured with WT or C1qtnf3 ^-/- BMDCs in the presence of 5 μg/ml MOG_35-55 for 5 days (n = 3 each), and IL-17 (left) and IFN-γ (right) concentrations in the culture supernatant were determined. Average and SEM are shown. *p < 0.05, **p < 0.01 and ***p < 0.001. Student’s t-test. All data were reproduced in another independent experiment with similar results.

Figure 4

DC differentiation is normal in C1qtnf3 ^-/- mice. (A) C1qtnf3 mRNA in immune cells was determined by real-time PCR, and the relative expression levels to that in B cells are shown in left panel. C1qtnf3 mRNA expression in DCs after stimulation with zymosan or LPS was determined by real-time PCR, and the relative expression levels to that in cells without stimulation (-) are shown in right panel. (B, C) Effect of CTRP3 on the differentiation and proliferation of BMDCs. BMDCs were differentiated from WT or C1qtnf3 ^–/– (KO) BMCs (B), or from C1qtnf3 ^–/– BMCs in the presence of indicated concentrations of CTRP3 (C). Then, viable cell number was counted at day 8 by using Cell Counting Kit-8. 4 wells for each. Average and SEM are shown. Student’s t-test. (D) WT or C1qtnf3 ^-/- (KO) BMCs were cultured under BMDC-differentiation conditions, and the expression of CD11c, CD40, CD80 and CD88 was determined by flow cytometry at day 8. (E) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 ^–/– (KO) BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml). Four wells for each. Average and SEM are shown. Student’s t-test. (F) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 ^–/– BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. Student’s t-test. (G) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 ^–/– (KO) BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml). Four wells for each. Average and SEM are shown. *p < 0.05, ***p < 0.001. Student’s t-test. (H) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 ^–/– BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. *p < 0.05. Student’s t-test. All data were reproduced in another independent experiment with similar results.

Figure 5

Regulation of Th17 cell differentiation by CTRP3 through AdipoR2. CTRP3 mainly expressed in dendritic cells and Th17 cells regulates Th17 cell differentiation through activation of AdipoR2, but not via AdipoR1. The CTRP3/AdipoR2 axis enhances PPARα expression and inhibits STAT3 and RORγt expression.