Lipid Uptake by Alveolar Macrophages Drives Fibrotic Responses to Silica Dust

Abstract

Silicosis is a common occupational disease and represents a significant contributor to respiratory morbidity and mortality worldwide. Lipid-laden macrophages, or foam cells, are observed in the lungs of patients with silicosis but the mechanisms mediating their formation remain poorly understood. In this study, we sought to elucidate the mechanisms by which silica promotes foam cell formation in the lung, and to determine whether uptake of lipids alone is sufficient to drive TGF-β production by alveolar macrophages. Consistent with previous reports, we found that foam cells were markedly increased in the lungs of patients with silicosis and that these findings associated with both higher levels of intracellular lipid levels (oxidized LDL, ox-LDL) and elevated transcript levels for the lipid scavenger receptor CD36 and the nuclear receptor PPARγ. Employing a rat alveolar macrophage cell line, we found that exposure to silica dust or ox-LDL alone had a modest effect on the induction of foam cell formation and only silica was capable of inducing the production of TGF-β. In contrast, foam cell formation and TGF-β production were both dramatically increased when cells were exposed to a combination of silica dust and ox-LDL. Moreover, we found that these endpoints were markedly attenuated by either blocking CD36 or inhibiting the activity of PPARγ. Altogether, our findings suggest that foam cell formation and TGF-β production are driven by the simultaneous uptake of silica and lipids in alveolar macrophages and that strategies aimed at blocking lipid uptake by alveolar macrophages might be effective in ameliorating fibrotic responses to silica in the lung.

Figure 1

Alveolar macrophages (AM) from patients with and without silicosis are observed under the microscope. (A,C) AMs of (a) controls, (b) silicosis of stage I, (c) silicosis of stage II, (d) silicosis of stage III were viewed under inverted-microscope at magnification (400×) after Oil red O staining (black arrow) for neutral lipids or HE staining. (B) Percent positive foam cells were quantified. (D) AMs of (a) controls, (b) silicosis of stage I, (c) silicosis of stage II, (d) silicosis of stage III were observed by transmission electron microscopy from patients with and without silicosis. Red arrows highlight vacuolated areas consistent with lipid droplets.

Figure 2

Lipid levels in bronchoalveolar lavage fluid and alveolar macrophages of subjects with and without silicosis. (A–F) Cholesterol, triglyceride and ox-LDL in BALF and AM in control subjects and patients with silicosis. (G,H) Dot plots showing correlation between ox-LDL levels and cholesterol levels in patients with silicosis. Data are presented as the mean ± SD from a duplicate of each sample and from three independent experiments (n = 3); ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 versus the control group, ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001 versus the silicosis I group, *p < 0.05; **p < 0.01; ***p < 0.001 versus silicosis III group.

Figure 3

PPARγ and CD36 transcript levels in human AMs from individuals with and without silicosis. Values were mean ± SD of three independent experiments (n = 3). ^##: p < 0.01; ^###p < 0.001 versus the control group; ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001 versus the silicosis I group; *p < 0.05; **p < 0.01; ***p < 0.001 versus silicosis III group.

Figure 4

Combined exposure to SiO₂ and ox-LDL induces NR8383 foam cell formation. (A) Foam cells readily formed in cells exposed to SiO₂ plus ox-LDL (d) compared with control group (a), SiO₂ group (b) and ox-LDL group (c) as evidenced by Oil Red O staining (400x). (B) Percent positive foam cells were quantified. (C) Total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) levels in NR8383 cells in response to different conditions. (C) CE to TC ratios in NR8383 cells exposed to different conditions. Data are presented as the mean ± SD (n = 3). ^#p < 0.05; ^###p < 0.001 versus the control group; *p < 0.05; **p < 0.01; ***p < 0.001 versus the SiO₂ + ox-LDL group.

Figure 5

Combined treatment with SiO₂ and ox-LDL induced CD36 and PPARγ expression in alveolar macrophages. (A) Western blot for CD36 and PPARγ in NR8383 cells exposed to different conditions. GAPDH was used as loading control. (B) Transcript levels for CD36 and PPARγ in NR8383 cells exposed to different conditions. (C,D) Immunofluorescence staining for CD36 and PPARγ in NR8383 cells exposed to different conditions. DAPI was used to stain nuclei (blue). All images show × 60 original magnifications. Data represent the mean ± SD from three independent experiments(n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 versus the SiO₂ + ox-LDL group. ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001 versus the ox-LDL group.

Figure 6

Inhibition of CD36 blocks foam cell formation in NR8383 cells. (A,B) NR8383 cells (1 × 10⁶/ml) were pretreated with CD36 inhibitor, SSO (10 μM) for 2 h and then treated with SiO₂ + ox-LDL (50 μg/ml) isolated from different individuals for 36 h. Expression of CD36 and PPARγ transcript and protein levels were analyzed. (C,D) Immunofluorescent staining for CD36 and PPARγ (ret) and nuclei (blue) in NR8383 cells exposed to different conditions. (E) Oil Red O staining of NR8383 cells exposed to different conditions (400x). (F) Percent positive foam cells were quantified. (G, H) Intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) levels or the ratio of CE to TC was determined in NR8383 cells exposed to different conditions. All data are expressed as mean ± SD from at least three independent experiments in triplicate. ^#p < 0.05; ^###p < 0.001 versus the DMSO group; ^&&&p < 0.001 versus the SiO₂ + ox-LDL + SSO group; *p < 0.05; **p < 0.01; ***p < 0.001 versus the SiO₂ + ox-LDL + DMSO group.

Figure 7

PPARγ inhibitors reduce foam cell formation in NR8383 cells. (A–C) Protein and transcript levels for CD36 and PPARγ in NR8383 cells exposed to different conditions. (D) Immunofluorescence images of CD36 and PPARγ (red) in NR8383 cells exposed to different conditions. DAPI was used to stain nuclei (blue, 600x). (E) Oil Red O staining in NR8383 cells exposed to different conditions (400x). (F) Percent positive foam cells were quantified. (G,H) Intracellular cholesterol levels and the ratio of CE to TC were determined in NR8383 cells exposed to different conditions. Data are expressed as mean ± SD from three independent experiments. ^###p < 0.001 versus the control group; *p < 0.05; **p < 0.01; ***p < 0.001 versus the SiO₂ + ox-LDL + DMSO group.

Figure 8

Foam cell formation induces the production of TGF-β. (A). TGF-β levels in BALF of patients with silicosis. (B) TGF-β levels in AMs of patients with silicosis. (C–E) The expression of TGF-βin NR8383 cell culture supernatants in different treatment groups. Data are expressed as mean ± SD from three independent experiments. ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 versus the control group; *p < 0.05; **p < 0.01; ***p < 0.001 versus the SiO₂ + ox-LDL + DMSO group; ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001 versus the silicosis I group.

Figure 9

SiO₂ promotes the uptake of ox-LDL from alveolar macrophages, perhaps augmenting CD36-PPARγ signaling.