Role of IL-10-producing regulatory B cells in modulating T-helper cell immune responses during silica-induced lung inflammation and fibrosis

Abstract

Silicosis is characterized by chronic lung inflammation and fibrosis, which are seriously harmful to human health. Previous research demonstrated that uncontrolled T-helper (Th) cell immune responses were involved in the pathogenesis of silicosis. Lymphocytes also are reported to have important roles. Existing studies on lymphocyte regulation of Th immune responses were limited to T cells, such as the regulatory T (Treg) cell, which could negatively regulate inflammation and promote the process of silicosis. However, other regulatory subsets in silicosis have not been investigated in detail, and the mechanism of immune homeostasis modulation needs further exploration. Another regulatory lymphocyte, the regulatory B cell, has recently drawn increasing attention. In this study, we comprehensively showed the role of IL-10-producing regulatory B cell (B10) in a silicosis model of mice. B10 was inducible by silica instillation. Insufficient B10 amplified inflammation and attenuated lung fibrosis by promoting the Th1 immune response. Insufficient B10 clearly inhibited Treg and decreased the level of IL-10. Our study indicated that B10 could control lung inflammation and exacerbate lung fibrosis by inhibiting Th1 response and modulating the Th balance. The regulatory function of B10 could be associated with Treg induction and IL-10 secretion.

Figure 1. Anti-CD22 attenuates B10 induction in vivo after silica instillation.

(a,c) C57BL/6 mice were treated i.p. with 300 μg anti-CD22 monoclonal antibody or control IgG, and the percentage of CD19⁺ IL-10⁺ regulatory B cells (B10) in the hilar lymph node was assayed by flow cytometry. (b,d) Percentage of B10 in spleen is shown in the graph. (e) CD19 positive gate and IL-10 positive gate were drawn according to their isotype control staining. (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 2. Insufficient B10 enhances accumulation of inflammatory cells in lung after silica instillation.

(a) Total cells, (b) macrophages, (c) lymphocytes, and (d) neutrophils in BALF were counted using Giemsa staining (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 3. Insufficient B10 exacerbates the inflammatory response after silica instillation.

(a) Histopathology changes in mouse lungs after silica instillation observed with H&E staining (×200). a1–d1, day 7; a2–d2, day 28, and a3–d3, day 56. a1–a3, saline group; b1–b3, saline plus anti-CD22 group; c1–c3, silica group; d1–d3, silica plus anti-CD22 group. (b) Secretion of pro-inflammatory cytokine TNF-α in BALF was evaluated by CBA analysis. (c) Secretion of IL-6 in BALF was assayed by CBA analysis (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 4. Insufficient B10 attenuates the fibrotic response during silica-induced lung fibrosis.

(a) Lung sections from C57BL/6 mice were stained with Masson trichrome stain and observed under the light microscope (×200). a1–d1, day 7; a2–d2, day 28, and a3–d3, day 56. a1–a3, saline group; b1–b3, saline plus anti-CD22 group; c1–c3, silica group; d1–d3, silica plus anti-CD22 group. (b) The area of positive collagen deposition in lung sections was visualized by Masson staining and quantified using image-pro plus. (c) Relative IL-13 expression in lung was assayed by real time PCR. (d) Relative expression of TGF-β in lung was assayed by real time PCR (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 5. Insufficient B10 affected the Th1/Th2 balance in the mouse model of silica-induced lung fibrosis.

(a,c) Percentage of CD4⁺ IFN-γ⁺ Th1 cells in the hilar lymph node was assayed by flow cytometry. (b) IFN-γ positive gate were drawn according to their isotype control staining. (d) Secretion of typical Th1 cytokine IFN-γ in BALF was assayed by CBA. (e,f) Relative expression levels of typical Th1 cytokine IFN-γ and Th1 nuclear transcription factor T-bet in lung were assayed by real time PCR. (g) Secretion of typical Th2 cytokine IL-4 in BALF was assayed by CBA. (h,i) Relative expression levels of typical Th2 cytokine IL-4 and Th2 nuclear transcription factor GATA3 in lung were assayed by real time PCR (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 6. Insufficient B10 regulates the Th17 immune response during silica-induced lung inflammation and fibrosis.

(a,c) Percentage of CD4⁺ IL-17⁺ Th17 cells in the hilar lymph node was assayed by flow cytometry. (b) IL-17 positive gate were drawn according to their isotype control staining. (d) Secretion of typical Th17 cytokine IL-17 in BALF was assayed by CBA. (e) Relative expression levels of typical Th17 cytokine IL-17 in lung were assayed by real time PCR. (f,g) Relative expression levels of Th17 related cytokines IL-23 and IL-1β in lung were assayed by real time PCR (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group).

Figure 7. Regulatory T cells are involved in the B10 regulatory mechanism that modulates Th immune responses after silica instillation.

(a,b) Percentage of CD4⁺ Foxp3⁺ Treg in the hilar lymph node was assayed by flow cytometry. (c) Relative expression of Treg nuclear transcription factor (Foxp3) in lung was assayed by real time PCR. (d) To check the efficacy of anti-CD25 in depletion of Treg, the percentage of CD4⁺Foxp3⁺Treg in hilar lymph node and spleen was assayed by flow cytometry. (e) Flow cytometry analysis of B10 percentage differences in hilar lymph node and spleen in Treg-depleted mice and wild-type mice after silica instillation. (f,g) Secretion and relative expression of inhibitory cytokine IL-10 in BALF and lung were assayed by CBA and real time PCR (n = 5; *P < 0.05 compared with the saline control group; ^#P < 0.05 compared with the silica group; and & P < 0.05 for the silica group compared with the silica plus anti-CD25 group).

Figure 8. Schematic representation of the role of B10 in regulating Th immune responses during silica-induced lung inflammation and fibrosis.