Sion were measured by real-time PCR. *p,0.05 compared with the Silica group (D30). doi:10.1371/journal.pone.0055827.gstudies using a mouse model have shown the development of fibrosis within the first month after exposure [26,27], while others have detected silicotic nodules, which represent fibrosis, at 15 days after silica administration [28,29]. Doxycycline controlled the expression of hApoA1 protein in the alveolar epithelial cells of our transgenic mice, allowing us to look at chronic treatment with ApoA1 starting 
at different time points after silica exposure. In the present study, lung inflammation and small silicotic nodules were observed on day 7, and 12926553 large silicotic nodules had developed by day 15 after intratracheal silica administration (Fig. 2B). Thus, by initiating doxycycline treatment at 7 days after silica administration (ApoA1_D7 group), we examined the therapeutic effect of AopA1 overexpression on inflammation and early fibrosis, and its effectiveness in established fibrosis was studied by starting doxycycline treatment at 15 days after silica administration (ApoA1_D15 group). The ApoA1_D7 and D15 groups showed decreased levels of lung inflammation and silicotic nodule formation compared with the Silica group. As silicotic nodules were well established by day 15 in the Silica group mice, the Fruquintinib decreases in the silicotic nodule fraction and collagen deposition in the ApoA1_D15 mice indicate that ApoA1 overexpression had a therapeutic effect on established silica-induced lung inflammation and fibrosis in the present study. Given a previous report showing that doxycycline suppressed fibrosis in a beomycininduced lung injury/fibrosis model through the inhibition of MMP production [21], we used UBC-GFP transgenic mice to study theeffect of doxycycline on silica-induced lung fibrosis. The lungs of the silica-exposed doxycycline-treated mice showed similar levels of inflammation, granuloma formation, and collagen deposition compared with the distilled water-treated mice in both the D15 and D30 groups (Figure S3). These data suggest that the doxycycline that was used for the overexpression of hApoA1 did not contribute to the inhibition of lung inflammation and fibrosis in our silica-induced lung fibrosis model. Silica deposits in the lung cause a persistent, toxic, and inflammatory 86168-78-7 response, including the alveolar accumulation of macrophages and neutrophils [26]. We found that in the ApoA1_D7 and D15 groups, lung mRNA levels of the proinflammatory mediators KC, MIP-2 and MCP-1, which are known to recruit neutrophils and macrophages to inflamed sites, were significantly decreased compared to those of the silica group (Fig. 7). We also observed abundant silica particles without inflammatory cell accumulation in the 15755315 airspaces of the ApoA1_D7 and D15 mice, indicating that ApoA1 overexpression in the airspace reduced silica-induced inflammatory cell accumulation and silica nodule formation, despite the presence of silica particles in the lung. We speculate that ApoA1 inhibited the accumulation and migration of alveolar inflammatory cells, particularly macrophages and neutrophils. A decrease in the number or activity of inflammatory cells could account for the observed reduction in the level of TGF-b1, which can induce mesenchymal cell proliferation and extracellular collagen deposition in fibrosis [30]. There areApoA1 Attenuated Silica Induced Lung Fibrosisother possible mechanisms for the protective and therapeutic effect of ApoA1.Sion were measured by real-time PCR. *p,0.05 compared with the Silica group (D30). doi:10.1371/journal.pone.0055827.gstudies using a mouse model have shown the development of fibrosis within the first month after exposure [26,27], while others have detected silicotic nodules, which represent fibrosis, at 15 days after silica administration [28,29]. Doxycycline controlled the expression of hApoA1 protein in the alveolar epithelial cells of our transgenic mice, allowing us to look at chronic treatment with ApoA1 starting at different time points after silica exposure. In the present study, lung inflammation and small silicotic nodules were observed on day 7, and 12926553 large silicotic nodules had developed by day 15 after intratracheal silica administration (Fig. 2B). Thus, by initiating doxycycline treatment at 7 days after silica administration (ApoA1_D7 group), we examined the therapeutic effect of AopA1 overexpression on inflammation and early fibrosis, and its effectiveness in established fibrosis was studied by starting doxycycline treatment at 15 days after silica administration (ApoA1_D15 group). The ApoA1_D7 and D15 groups showed decreased levels of lung inflammation and silicotic nodule formation compared with the Silica group. As silicotic nodules 
were well established by day 15 in the Silica group mice, the decreases in the silicotic nodule fraction and collagen deposition in the ApoA1_D15 mice indicate that ApoA1 overexpression had a therapeutic effect on established silica-induced lung inflammation and fibrosis in the present study. Given a previous report showing that doxycycline suppressed fibrosis in a beomycininduced lung injury/fibrosis model through the inhibition of MMP production [21], we used UBC-GFP transgenic mice to study theeffect of doxycycline on silica-induced lung fibrosis. The lungs of the silica-exposed doxycycline-treated mice showed similar levels of inflammation, granuloma formation, and collagen deposition compared with the distilled water-treated mice in both the D15 and D30 groups (Figure S3). These data suggest that the doxycycline that was used for the overexpression of hApoA1 did not contribute to the inhibition of lung inflammation and fibrosis in our silica-induced lung fibrosis model. Silica deposits in the lung cause a persistent, toxic, and inflammatory response, including the alveolar accumulation of macrophages and neutrophils [26]. We found that in the ApoA1_D7 and D15 groups, lung mRNA levels of the proinflammatory mediators KC, MIP-2 and MCP-1, which are known to recruit neutrophils and macrophages to inflamed sites, were significantly decreased compared to those of the silica group (Fig. 7). We also observed abundant silica particles without inflammatory cell accumulation in the 15755315 airspaces of the ApoA1_D7 and D15 mice, indicating that ApoA1 overexpression in the airspace reduced silica-induced inflammatory cell accumulation and silica nodule formation, despite the presence of silica particles in the lung. We speculate that ApoA1 inhibited the accumulation and migration of alveolar inflammatory cells, particularly macrophages and neutrophils. A decrease in the number or activity of inflammatory cells could account for the observed reduction in the level of TGF-b1, which can induce mesenchymal cell proliferation and extracellular collagen deposition in fibrosis [30]. There areApoA1 Attenuated Silica Induced Lung Fibrosisother possible mechanisms for the protective and therapeutic effect of ApoA1.