IL-10-Producing B Cells Suppress Effector T Cells Activation and Promote Regulatory T Cells in Crystalline Silica-Induced Inflammatory Response In Vitro

Abstract

Long-term exposure to crystalline silica leads to silicosis, which is characterized by persistent lung inflammation and lung fibrosis. Multiple immune cells have been demonstrated to participate in crystalline silica-induced immune responses. Our previous study indicated that B10 could control lung inflammation through modulating the Th balance in experimental silicosis in mice. However, the regulatory mechanism of B10 on CD4⁺ T cells is still unclear. MACS-sorted CD19⁺ B cells from the three different groups were cultured with CD4⁺ T cells either with or without transwell insert plates to evaluate the effects of B10 on CD4⁺ T cells, including Teff and Treg. B10 was eliminated by anti-CD22 application in vivo. Flow cytometry was used to test the frequencies of CD4⁺ T cells, and the expressions of the related cytokines were detected by real-time PCR and CBA. Insufficient B10 elevated the levels of proinflammatory cytokines and promoted Th responses in a way independent upon cell-cell contact in the Teff and B cell coculture system. B10 could both increase Treg activity and enhance conversion of Teff into Treg. Our findings demonstrated that B10 could affect Th responses by the release of IL-10, enhancing Treg functions and converting Teff into Treg.

Figure 1

Insufficient B10 promoted the proinflammatory cytokines in the Teff and B cell coculture system in vitro. (a1) CD19⁺ B cells were purified and checked by flow cytometry. (a2, a3) The percentages of B10 in different groups were tested by flow cytometry. (b1, b2) The CD4⁺CD25⁻ Teff and CD4⁺CD25⁺ Treg were purified and checked by flow cytometry. (c) A schematic of B cell and Teff coculture system with insert plates in vitro. (d, e) The expressions of TNF-α and IL-6 in different groups of the B cell and Teff coculture system with or without insert plates were assayed by real-time PCR. (f, h) The secretions of TNF-α and IL-6 in supernatant of the B cell and Teff coculture system were assayed by CBA. (g, i) The concentrations of TNF-α and IL-6 in supernatant of the B cell and Teff coculture system with insert plates were assayed by CBA. (Data was presented as mean ± SEM (n = 5). ^∗p < 0.05, significantly different compared with the control B + Teff group. ^#p < 0.05, significantly different compared with the silica B + Teff group.)

Figure 2

Insufficient B10 aggravated the Th1 response in the Teff and B cell coculture system in vitro. (a1, a2) The percentages of CD4⁺IFN-γ⁺ Th1 cells in different groups of the B cell and Teff coculture system were studied by flow cytometry. (b, c) The expressions of IFN-γ and T-bet in different groups of the B cell and Teff coculture system with or without insert plates were assayed by real-time PCR. (d) The secretions of IFN-γ in supernatant of the B cell and Teff coculture system were assayed by CBA. (e, f) The concentrations of IFN-γ and IL-2 in supernatant of the B cell and Teff coculture system with insert plates were assayed by CBA. (Data was presented as mean ± SEM (n = 5). ^∗p < 0.05, significantly different compared with the control B + Teff group. ^#p < 0.05, significantly different compared with the silica B + Teff group.)

Figure 3

Insufficient B10 promoted the Th2/Th17 responses in the Teff and B coculture system in vitro. (a, b, c) The expressions of IL-4, IL-13, and GATA3 in different groups of the B cell and Teff coculture system with or without insert plates were assayed by real-time PCR. (d1, d2) The percentages of CD4⁺ IL-17A⁺ Th17 cells in different groups of the B cell and Teff coculture system were studied by flow cytometry. (e, f, g) The expressions of IL-17A, IL-23, and ROR-γt in different groups of the B cell and Teff coculture system with or without insert plates were assayed by real-time PCR. (h, i) The secretions of IL-17A in supernatant of the B cell and Teff coculture system with or without insert plates were assayed by CBA. (Data was presented as mean ± SEM (n = 5). ^∗p < 0.05, significantly different compared with the control B + Teff group. ^#p < 0.05, significantly different compared with the silica B + Teff group.)

Figure 4

Insufficient B10 both suppressed Treg and inhibited the conversion of Teff into Treg. (a1, a2) The percentages of CD4⁺CD25⁺ Treg in different groups of the B cell and Treg coculture system were studied by flow cytometry. (b–e) The expressions of Foxp3, CTLA4, IL-10, and TGF-β in different groups of the B cell and Treg coculture system were assayed by real-time PCR. (f1, f2) The percentages of CD4⁺CD25⁺ Treg in different groups of the B cell and Teff coculture system were studied by flow cytometry. (g–j) The expressions of Foxp3, CTLA4, IL-10, and TGF-β in different groups of the B cell and Teff coculture system with or without insert plates were assayed by real-time PCR. (k, l) The secretions of IL-10 in supernatant of the B cell and Teff coculture system were assayed by CBA. (Data was presented as mean ± SEM (n = 5). ^∗p < 0.05, significantly different compared with the control B + Teff group. ^#p < 0.05, significantly different compared with the silica B + Teff group.)

Figure 5

A schematic representation for the regulatory function of B10 on crystalline silica-induced Teff/Treg in vitro.