Effect of macrophage-to-myofibroblast transition on silicosis

Abstract

Background: The aim was to explore the effect of macrophage polarization and macrophage-to-myofibroblast transition (MMT) in silicosis.

Methods: Male Wistar rats were divided into a control group and a silicosis group developed using a HOPE MED 8050 dynamic automatic dusting system. Murine macrophage MH-S cells were randomly divided into a control group and an SiO₂ group. The pathological changes in lung tissue were observed using hematoxylin and eosin (HE) and Van Gieson (VG) staining. The distribution and location of macrophage marker (F4/80), M1 macrophage marker (iNOS), M2 macrophage marker (CD206), and myofibroblast marker (α-smooth muscle actin [α-SMA]) were detected using immunohistochemical and immunofluorescent staining. The expression changes in iNOS, Arg, α-SMA, vimentin, and type I collagen (Col I) were measured using Western blot.

Results: The results of HE and VG staining showed obvious silicon nodule formation and the distribution of thick collagen fibers in the lung tissue of the silicosis group. Macrophage marker F4/80 increased gradually from 8 to 32 weeks after exposure to silica. Immunohistochemical and immunofluorescent staining results revealed that there were more iNOS-positive cells and some CD206-positive cells in the lung tissue of the silicosis group at 8 weeks. More CD206-positive cells were found in the silicon nodules of the lung tissues in the silicosis group at 32 weeks. Western blot analysis showed that the expressions of Inducible nitric oxide synthase and Arg protein in the lung tissues of the silicosis group were upregulated compared with those of the control group. The results of immunofluorescence staining showed the co-expression of F4/80, α-SMA, and Col I, and CD206 and α-SMA were co-expressed in the lung tissue of the silicosis group. The extracted rat alveolar lavage fluid revealed F4/80⁺α-SMA⁺, CD206⁺α-SMA⁺, and F4/80⁺α-SMA⁺Col I⁺ cells using immunofluorescence staining. Similar results were also found in MH-S cells induced by SiO₂.

Conclusions: The development of silicosis is accompanied by macrophage polarization and MMT.

Keywords: macrophage; macrophage‐to‐myofibroblast transition; silicosis.

FIGURE 1

Evaluation of the silicosis rat model. (A) HE (hematoxylin and eosin) staining and VG (Van Gieson) staining (bar = 100 μm). (B) Western blot measurement of Col I (type I collagen). **p < 0.01.

FIGURE 2

Macrophage polarization in silicosis rat model. (A) Immunohistochemistry of F4/80, iNOS, and CD206 (bar = 100 μm). (B) Immunofluorescence of iNOS and CD206 (bar = 200 μm). (C) Western blot measurement of iNOS and Arg levels in the lungs. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

Macrophage polarization in MH‐S cell induced by SiO₂. (A,D) Western blot measurement of iNOS and Arg levels in MH‐S cell. (B,E) Immunocytochemical staining of iNOS and CD206 (bar = 50 μm). (C,F) Immunofluorescence of iNOS and CD206 (bar = 100 μm). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

MMT (macrophage‐to‐myofibroblast transition) in silicosis rat model. (A) Western blot measurement of α‐SMA (α‐smooth muscle Actin), vimentin, and Col I (type I collagen). (B) Immunohistochemistry of α‐SMA, vimentin, and Col I (bar = 100 μm). (C) Immunofluorescence of F4/80, CD206, and α‐SMA (bar = 200 μm). (D) Immunofluorescence of F4/80, α‐SMA, and Col I (bar = 100 μm). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

MMT (macrophage‐to‐myofibroblast transition) in the BALF (bronchoalveolar lavage fluid) from silicosis rat. (A) Immunofluorescence of F4/80, CD206, and α‐SMA (α‐smooth muscle Actin) (bar = 100 μm). (B) Immunofluorescence of F4/80, α‐SMA, and Col I (bar = 100 μm).

FIGURE 6

MMT (macrophage‐to‐myofibroblast transition) in MH‐S cells induced by SiO₂. (A) Western blot measurement of α‐SMA (α‐smooth muscle Actin), vimentin, and Col I (type I collagen). (B) Immunocytochemical staining of α‐SMA, vimentin, and Col I (bar = 100 μm). (C) Immunofluorescence of F4/80, CD206, and α‐SMA (bar = 100 μm). (D) Immunofluorescence of F4/80, α‐SMA, and Col I (bar = 50 μm). *p < 0.05.