Tregs Promote the Differentiation of Th17 Cells in Silica-Induced Lung Fibrosis in Mice

<div><h3>Background</h3><p>Silicosis is an occupational lung disease caused by inhalation of silica dust and characterized by lung inflammation and fibrosis. Previous study showed that Tregs regulate the process of silicosis by modulating the maintenance of immune homeostasis in the lung. Th17 cells share reciprocal developmental pathway with Tregs and play a pivotal role in the immunopathogenesis of many lung diseases by recruiting and activating neutrophils, but the regulatory function of Tregs on Th17 response in silica induced lung fibrosis remains to be explored.</p> <h3>Methodology/Principal Findings</h3><p>To evaluate the role of Th17 and IL-17 in the development of silicosis and their interaction with Tregs, Treg-depleted mice model was generated and exposed to silica to establish experimental model of silica-induced lung fibrosis. Here we showed that silica increased Th17 response in lung fibrosis. Tregs depletion enhanced the neutrophils accumulation and attenuated Th17 response in silica induced lung fibrosis. Both mRNA and protein results showed that Tregs exerted its modulatory function on Th17 cells and IL-17 by regulating TGF-β1 and IL-1β.</p> <h3>Conclusion/Significance</h3><p>Our study suggested that Tregs could promote Th17 cells differentiation by regulating TGF-β1 and IL-1β in silica induced lung fibrosis of mice, which further the understanding of the progress of silicosis and provide a new insight in the regulatory mechanism of Th17 by Tregs in lung inflammation.</p> </div

TGF-β1 and IL-10 decreased in the Tregs depletion group and TGF-β1 may contribute to the Th17 cells differentiation.

<p>The IL-10 (A) and TGF-β1 (B) mRNA (day 3) were assayed by realtime RT-PCR by using −ΔΔCt method. Results (n = 5) are shown as mean±SEM (one-way analysis of variance followed by pair-wise comparison with the Student-Newman-Keuls test. *, as compared with the saline control group, P<0.05; Δ, as compared with the silica group, P<0.05).</p

Th1 type of cytokines took the advantage over Th2 cytokine in the depletion of Tregs.

<p>Typical Th1 IL-2 (A), IFN-γ (B), IL-12 (C) and Th2 IL-4 (D) cytokines (day 3) were assayed by realtime RT-PCR by using −ΔΔCt method. Results (n = 5) are shown as mean±SEM (one-way analysis of variance followed by pair-wise comparison with the Student-Newman-Keuls test. *, as compared with the saline control group, P<0.05; Δ, as compared with the silica group, P<0.05).</p

Depletion of Tregs increased the accumulation of inflammation cells in the lung of experimental silicosis mice.

<p>The total cells (A), macrophages (B) lymphocytes (C) and neutrophils (D), in BALF(day 3) were counted by using Giemsa staining. Results (n = 5) are shown as mean±SEM (one-way analysis of variance followed by pair-wise comparison with the Student-Newman-Keuls test. *, as compare with the saline control group, P<0.05; Δ, as compared with the silica group, P<0.05).</p

The regulatory function of Tregs on Th17 and IL-17A may depend on IL-1β but not IL-6 and IL-23.

<p>The IL-1β (A), IL-6 (B) and IL-23 (C) mRNA were assayed by realtime RT-PCR by using −ΔΔCt method. Results (n = 5) are shown as mean±SEM (one-way analysis of variance followed by pair-wise comparison with the Student-Newman-Keuls test.*, as compared with the saline control group, P<0.05; Δ, as compared with the silica group, P<0.05; +, as compared with 3day of the same group, P<0.05; #, as compared with 7day of the same group, P<0.05).</p

Cell infiltration and alveolar change of the mice lungs in each group at day 3, 7, 28 and 56.

<p>The degree of inflammation was assessed by the histological analysis of six random fields per sample (n = 5).</p

Depletion of Tregs decreased Th17 response in silica induced lung fibrosis.

<p>The RORγ-t (A) and IL-17A (B) mRNA were assayed by realtime RT-PCR by using −ΔΔCt method. Results (n = 5) are shown as mean±SEM (one-way analysis of variance followed by pair-wise comparison with the Student-Newman-Keuls test. *, as compare with the saline control group, P<0.05; Δ, as compared with the silica group, P<0.05; + compared with 3day of the same group, P<0.05; #, as compared with 7day of the same group, P<0.05).</p

Histopathology changes in mouse lungs after instillation with HE staining (×200).

<p>The scale on the graph above was 50 µm; date was day3, day7, day28. Lung sections were stained with H&E. The degree of inflammation was assessed by the histological analysis of six random fields per sample (with n = 5 mice per group).</p

Lipopolysaccharides may aggravate apoptosis through accumulation of autophagosomes in alveolar macrophages of human silicosis

<p>Silica dust mainly attacks alveolar macrophages (AMs) and increases the apoptosis of AMs in silicosis patients. However, it is still unclear whether autophagy is affected. Autophagy mainly has defensive functions in response to stress, contributing to cell survival in adverse conditions, and conversely it has also been implicated in cell death. Lipopolysaccharide (LPS) induces autophagy and apoptosis in macrophages. The role of LPS in autophagy and apoptosis in AMs of silicosis patients is unknown. In this study, we collected AMs from 53 male workers exposed to silica and divided them into an observer (control) group, and stage I, II and III patient groups. We found increased levels of LC3B, SQSTM1/p62 and BECN1，whereas the phosphorylation of MTOR，and levels of LAMP2, TLR4, MYD88, TICAM1, as well as the number of lysosomes decreased with the development of silicosis. LPS stimulation triggered autophagy and increased levels of SQSTM1 in AMs. The autophagy inhibitor, 3-methyladenine (3MA), inhibited LPS-induced apoptosis in the AMs of silicosis patients. Moreover, 3MA reversed the LPS-induced decrease in BCL2 and the increase in BAX and CASP3 levels in AMs. These results suggest that autophagosomes accumulate in AMs during silicosis progression. LPS can induce the formation of autophagosomes through a TLR4-dependent pathway, and LPS may exacerbate the apoptosis in AMs. Blockade of the formation of autophagosomes may inhibit LPS-induced apoptosis via the intrinsic apoptotic pathway in AMs. These findings describe novel mechanisms that may lead to new preventive and therapeutic strategies for pulmonary fibrosis.</p

Mycobacteria-induced expression of MCP-2/CCL8 through TLR2.

<p>(A-B) Real-time PCR of <i>mcp-2/ccl8</i> mRNA in primary peritoneal macrophages from wildtype, TLR2^−/−, TLR3^−/−, or TLR4^−/− mice infected with either <i>M. bovis</i> BCG (A) or <i>M. tuberculosis</i> H37Rv (B) at MOI 5 for 24 h. (B) Western blot of phosphorylation of Akt and p38 in primary peritoneal macrophages from wildtype or TLR2 KO mice infected with <i>M. bovis</i> BCG at MOI 5 for indicated times. Data shown are representative of three independent experiments. (D) Densitometric analysis of three independent experiments as in (C). Data shown are the mean±sem of three independent experiments. **, <i>p</i><0.001, *, <i>p</i><0.05.</p