Fig 1.
Pathology of lung tissues in T-CNT-exposed mice.
Lung tissues at 12 months after T-CNT exposure were collected and tissue sections were stained with H&E. (A, B) Controls, (C, D) Low dose T-CNT, (E-G) High dose T-CNT. Change to the alveolar wall is shown (A-F). Macrophage accumulation in the alveolar space is shown (D-G). Photos are representative of each group. Scale bar: 100 μm (A, C, E), 50 μm (C, D, F, G). Arrow heads indicate T-CNT fibers (G). (H) SEM of the sediment of the dissolved lung tissue of a mouse exposed to T-CNT (high dose). Photos is representative of high dose group. Scale bar: 4 μm.
Fig 2.
Fibrotic lesions in the lung of T-CNT-exposed mice.
(A) Connective tissues in the lung of T-CNT-exposed mice were detected by Azan staining. Photos are representative of each group. Scale bar: 100 μm. (B) The area (mm2) of Azan-stained connective tissue was measured. Data are presented as the average ± standard deviation (SD) of five mice of each group. *p < 0.05, vs controls.
Fig 3.
Alveolar macrophages in T-CNT-expose mice.
(A) CD11b and F4/80 expression of mononuclear cells in BALF from control and T-CNT-exposed mice were detected by flow cytometric analysis. The results are representative of each group. F4/80+CD11blow and F4/80+CD11bhigh populations are shown. (B) The proportion of F4/80+CD11blow and F4/80+CD11bhigh macrophage in BALF from control and T-CNT-exposed mice were detected by flow cytometric analysis. Data are presented as the average ± SD of five mice of each group. *p < 0.05, **p < 0.005, vs controls.
Fig 4.
M1-like and M2-like macrophage phenotype of alveolar macrophages in T-CNT-exposed mice.
(A) CD192 (M1) and CD206 (M2) expression of F4/80+CD11b+ macrophages in BALF, spleen, and LNs was detected by flow cytometric analysis. The results are representative of each group. (B) The proportion of CD192+CD206− M1-like macrophages, CD192−CD206+ M2-like macrophages, and CD192+CD206+ macrophages in BALF, spleen, and LNs was measured flow cytometric analysis. Data are presented as the average ± SD of five mice of each group. *p < 0.05, vs controls.
Fig 5.
Macrophage-related gene expression in lung tissues of T-CNT-exposed mice.
mRNA expression of macrophage-related genes of lung tissues in control and T-CNT-exposed mice was analyzed by real time RT-PCR. (A) Levels of mRNA encoding MCP-1, iNOS, CD192, Arginase-1, Retnla, MGL-1, MGL-2, and CHl3L1. Data are presented as the average of relative expression to control ± SD of five mice of each group. (B) Levels of mRNA encoding IL-1β, IFN-γ, TNF-α, IL-12, IL-10, and IL-13. Data are presented as the average of relative expression to control ± SD of five mice of each group.
Fig 6.
Gene expression showing profibrotic phenotype and collagen type IV expression in the lung tissues of T-CNT-exposed mice.
(A, B) Levels of mRNAs encoding IL-5, TGF-β1, Col1A2, Col3A, ColIV, MMP-2, MMP-9, MMP-12, TIMP-2, and TIMP-3 were detected by real time RT-PCR. Data are presented as the average of relative expression to control ± SD of five mice of each group. *p < 0.05, **p < 0.005, vs controls. (C) Protein expression of collagen type IV was detected by immunohistochemical analysis. Photos are representative of each group. Scale bar: 100 μm.
Fig 7.
Detection of MMP-12-expressing alveolar macrophages in T-CNT-exposed mice.
(A) MMP-12 and F4/80 expression using frozen lung tissues of control and high-dose T-CNT-exposed mice were analyzed by confocal microscopy. Nuclei were stained with DAPI. Photos are representative of five mice of each group. (B) Number (mm2) of F4/80+MMP-12+ alveolar macrophages was counted. Data are presented as the average of relative expression to control ± SD of five mice of each group. *p < 0.05, **p < 0.005, vs controls.