Cbl-b restrains priming of pathogenic Th17 cells via the inhibition of IL-6 production by macrophages

Abstract

E3 ubiquitin ligase Cbl-b is involved in the maintenance of a balance between immunity and tolerance. Mice lacking Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. However, how Cbl-b regulates Th17 cell responses remains unclear. In this study, utilizing adoptive transfer and cell type-specific Cblb knockout strains, we show that Cbl-b expression in macrophages, but not T cells or dendritic cells (DCs), restrains the generation of pathogenic Th17 cells and the development of EAE. Cbl-b inhibits IL-6 production by macrophages that is induced by signaling from CARD9-dependent C-type lectin receptor (CLR) pathways, which directs T cells to generate pathogenic Th17 cells. Therefore, our data unveil a previously unappreciated function for Cbl-b in the regulation of pathogenic Th17 responses.

Keywords: Biological sciences; Immune response; Immunology.

Graphical abstract

Figure 1

Loss or inactivation of Cbl-b in mice leads to the development of severe clinical symptoms of EAE (A) Clinical scores of WT, Cblb^−/−, and Cblb^C373A mice (n = 5/group) immunized with MOG_35-55 in CFA. ∗p < 0.05; Mann-Whitney U test. (B and C) Ex vivo antigen-induced T cell proliferation and cytokine production of WT, Cblb^−/−, and Cblb^C373A mice (n = 3/group for B, n = 5/group for C) immunized with MOG_35-55 in CFA as described in A, determined by CFSE labeling and ELISA. ∗∗p < 0.01, ∗∗∗p < 0.001; Student t test. (D) Flow cytometric analysis of Th1/Th17 responses in dLNs of WT, Cblb^−/−, and Cblb^C373A mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. ∗∗p < 0.01; Student t test. Bracket: the total number of gated CD4⁺ T cells. Data are one representative of three independent experiments.

Figure 2

Cbl-b deficiency in myeloid cells is responsible for heightened EAE and Th17 responses (A) Clinical scores of Rag1^−/− mice receiving CD4⁺ T cells from WT (n = 8) and Cblb^−/− (n = 7) mice, immunized with MOG_35-55 in CFA. (B) Flow cytometric analysis of Th1/Th17 responses in dLNs of Rag1^−/−mice receiving CD4⁺ T cells from WT and Cblb^−/− mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. (C) Clinical scores of Cd4 Cre (n = 10) and Cd4 Cre-Cblb^f/f (n = 9) mice, immunized with MOG_35-55 in CFA. (D) Flow cytometric analysis of Th1/Th17 responses in dLNs of Cd4 Cre and Cd4 Cre-Cblb^f/f mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. (E) Clinical scores of Rag1^−/− (n = 11) and Cblb^−/−Rag1^−/− (n = 10) mice receiving CD4⁺ T cells from WT mice, immunized with MOG_35-55 in CFA. ∗p < 0.05; Mann-Whitney U test. (F) Flow cytometric analysis of Th1/Th17 responses in dLNs of Rag1^−/− and Cblb^−/−Rag1^−/− mice (n = 4/group) receiving CD4⁺ T cells from WT mice on day 8 after immunization with MOG_35-55 in CFA. ∗p < 0.05; Student t test. (G) Clinical scores of LysM Cre and LysM Cre-Cblb^f/f mice (n = 8/group), immunized with MOG_35-55 in CFA. ∗∗p < 0.01; Mann-Whitney U test. (H) Flow cytometric analysis of Th1/Th17 responses in dLNs of LysM Cre and LysM Cre-Cblb^f/f mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. ∗∗p < 0.01; Student t test. (I) Clinical scores of Cd11c Cre and Cd11c Cre-Cblb^f/f mice (n = 6/group), immunized with MOG_35-55 in CFA. (J) Flow cytometric analysis of Th1/Th17 responses in dLNs of Cd11c Cre and Cd11c Cre-Cblb^f/f mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. Bracket: the total number of gated CD4⁺ T cells. Data are representative of two independent experiments.

Figure 3

Hyper-production of IL-6 by Cblb^−/− macrophages promotes a pathogenic Th17 response (A) Th1/Th17 cell differentiation in co-cultures of 2D2 T cells with BMDMs or BMDCs from WT and Cblb^−/− mice (n = 3/group) in the presence of heat-killed M.tb and MOG_35-55. ∗∗∗p < 0.001; Student t test. (B) Th1/Th17 cell differentiation in co-cultures of 2D2 T cells with BMDMs from WT and Cblb^−/− mice (n = 3/group) in the presence of heat-killed M.tb and MOG_35-55 with or without pretreatment with mitomycin C. ∗∗∗p < 0.001; Student t test. (C) Flow cytometric analysis of IL-6-producing cells of dLNs of WT and Cblb^−/− mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA, and stimulated with PMA/ionomycin. The gating strategy and representative plots see Figure S5. ∗∗p < 0.01; Student t test. (D) ELISA of pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-12p40, IL-1β, TGF-β, IL-23, and IL-21) by BMDMs from WT and Cblb^−/− mice (n = 5/group) stimulated with heat-killed M.tb. ∗p < 0.05, ∗∗∗p < 0.001; Student t test. (E) Th1/Th17 cell differentiation in co-cultures of 2D2 CD4⁺ T cells with BMDMs from Cblb^−/− mice that had been treated with Il6 or control siRNA. IL-6-producing macrophages of Cblb^−/− mice treated with Il6 (n = 4) or control siRNA (n = 4) were also determined. The mean fluorescent intensity (MFI) determined by flow cytometry indicates the expression level of IL-6 in CD4⁻ macrophages of Cblb^−/− mice treated with Il6 or control siRNA. ∗∗∗p < 0.001; Student t test. (F) Clinical scores of Cblb^−/− mice immunized with MOG_35-55 in CFA in the treatment with or without Il6 siRNA (n = 5) or control siRNA (n = 5) on day 1, 3, and 7 via tail vein injection. ∗∗p < 0.01; Mann-Whitney U test. (G) Flow cytometric analysis of Th1/Th17 responses and IL-6-producing cells in dLN cells from Cblb^−/− mice treated with Il6 (n = 3) or control siRNA (n = 3) via tail vein injection as in F on day 8 after immunization with MOG_35-55 in CFA. MFI determined by flow cytometry indicates the expression level of IL-6 in F4/80⁺ cells from dLNs of Cblb^−/− mice treated with Il6 (n = 3) or control siRNA (n = 3). ∗p < 0.05; Student t test. (H) Clinical scores of Cblb^−/− mice immunized with MOG_35-55 in CFA in the treatment with or without Il6 siRNA (n = 5) or control siRNA (n = 5) on day 8, 11, and 15 via tail vein injection. Bracket: the total number of gated CD4⁺ T cells. Data are representative of three independent experiments (A, B, E, F, and G) and representative of two independent experiments (C-D).

Figure 4

CNS macrophages, but not neutrophils and microglia, from Cblb^–/– mice with EAE produce more IL-6 Flow cytometric analysis of IL-6-producing macrophages (CD11b⁺Ly6C⁺Ly6G), neutrophils(CD11b⁺Ly6G⁺Ly6C⁺), and microglia (CD11b⁺Ly6G⁻Ly6C⁻TMEM119⁺) cells of CNS of WT and Cblb^−/− mice (n = 4/group) on day 17 after immunization with MOG_35-55 in CFA, and stimulated with PMA/ionomycin. ∗∗p < 0.01; Student t test. Data are representative of two independent experiments.

Figure 5

CARD9-mediated Dectin receptor signaling contributes the clinical symptoms of Cblb^−/− mice with EAE and pathogenic Th17 response (A) Clinical scores of WT, Cblb^−/−, Clec4n^−/−, Clec7a^−/−, Cblb^−/−Clec4n^−/−, and Cblb^−/−Clec7a^−/− mice (n = 5/group) immunized with immunized with MOG_35-55 in CFA. ∗∗∗p < 0.001, Mann-Whitney U test. (B) Flow cytometric analysis of Th1/Th17 responses in dLNs of WT, Cblb^−/−, Clec4n^−/−, Clec7a^−/−, Cblb^−/−Clec7a^−/−, and Cblb^−/−Clec4n^−/− mice (n = 4/group) on day 8 after immunization with MOG_35-55 in CFA. ∗∗p < 0.01, Student t test. (C) Clinical scores of WT and Card9^−/− mice (n = 7/group) immunized with MOG_35-55 in CFA. ∗∗p < 0.01, Mann-Whitney U test. (D) Flow cytometric analysis of Th1/Th17 responses in dLNs WT and Card9^−/− mice (n = 5/group) on day 8 after immunization with MOG_35-55 in CFA. ∗∗p < 0.01, Student’s t test. (E) Flow cytometric analysis of Th1/Th17 responses in dLNs WT and Card9^−/− mice (n = 3/group) on day 8 after immunization with MOG_35-55 in CFA. ∗p < 0.05, Student’s t test. Data are three independent experiments for A and B, and two independent experiments for C, D, and E.