The Role Of Mineralocorticoid Receptor Antagonist Regulating Macrophage Phenotype In Experimental Silicosis

Objective:Silicosis is an occupational lung fibrotic disease resulting from inhalation of free crystalline silica, which is one of the most common, progressive and devastating pneumonoconiosis. Despite extensive efforts, no available therapy has been shown to halt or efficiently reverse this disorder. In general, macrophages play an essential role in the pathogenesis of silicosis. Macrophage heterogeneity is associated with the development of pulmonary fibrosis. The activation of mineralocorticoid receptor(MR) induced aorta, cardiac, and kidney perivascular and interstitial fibrosis. It has been demonstrate that MR play an important role in modulating macrophage phenotype. Whether blocking MR attenuated silica-induced fibrosis, and the mechanism is yet unclear.The present study was designed to address the dynamic changes of alveolar and interstitial macrophage in silica-induced pulmonary injury model, aimed to explore the relationship between polarization of macrophage heterogeneity with lung inflammation and fibrosis. Then, we explore spironolactone induce the macrophage phenotype switching normalized via blocking MR, and the role in experimental silicosis inflammatory and pulmonary fibrosis.Method:1 A total 100 mice(6–8weeks old) were lightly anesthesia with isoflurane before oropharyngeal aspiration of silica 40 μL(50mg/ml, n = 50) or sterile saline 40μL(n=50). At 1, 3, 7, 14 or 28 days, animals were sacrificed by exsanguinations with sodium pentobarbital(100 mg/kg). BALF was obtained by cannulating the trachea, injecting and retrieving 1 ml aliquots of sterile saline for 3 times and centrifuged at 300 g for 10 min at 4℃. The BAL cell pellet was resuspended in PBS for total cell counts, differential cell counts and flow cytometry analysis. Cells isolated from BALF were incubated with Propidium Iodide(PI), anti-mouse CD64- Per CP/Cy5.5 and anti- mouse CD206-PE. The interstitial macrophage was obtained by enzymatic method from lavaged lung. Then the cell suspension was stained PI, anti-mouse CD64- Per CP/Cy5.5, anti- mouse CD206-PE and anti-mouse/human CD11b-PE/Cy7. Following incubation, flow cytometer analysis was performed. The non-lavaged lungs were fixed with 4% paraformaldehyde in phosphate-buffered saline(p H 7.2-7.4) for 24 hours and embedded in paraffin. Five micron tissue sections were stained with hematoxylin eosin staining(H&E), Masson’s trichrome staining and alpha smooth muscle actin(α-SMA) immunofluorescence staining. Total RNA was isolated from non-lavaged lung tissue. The relative gene expression level was normalized for expression of the β-actin and calculated using the comparative Ct method formula 2- △ △ Ct method.2 A total 120 mice was randomly divided into four groups: saline group, silica group, silica with 0.9% saline(silica+saline), and silica with 10mg/kg spironolactone(silica+SPI). Animals were sacrificed at 3, 14, 28 days, all detections were as same as above.Results:1 By H&E staining, silica-treated mice were found to have developed alveolar thickening, silicotic lesions and obviously inflammatory infiltrates within the interstitial and alveolar spaces. The inflammation score(IS) revealed a significantly higher degree of affected area in silica group compared to the saline group, which was gradually increased to a peak on day 3. As masson staining show, a lot of blue collagen fiber could be seen in the silica group from day 14. The collagen volume fraction in lungs from silica group was significantly higher compared with the saline group on days 14 and 28(P<0.01).Similarly, the histological analysis showed that α-SMA positive cells(myofibroblasts) were significantly increased after silica challenged.In comparison with saline group, the number of total cells were significantly increased at each time point after silica challenged. The counts of macrophage increased significantly on day 3 and then sustained to day 28(P<0.001).Moreover,a significant increase of neutrophils was observed after silica administration on day 1 and gradually decreased to day 28,which had no difference with saline group.Compared with saline-treated mice, the expression of type I and type III collagen m RNA in the lungs from silica-treated mice were significantly upregulated on days 14 and 28(P<0.05). Moreover, the expression of Arg-1 m RNA in total lung from silica group showed an increasing trend from day 3 to day 7.As shown in figure 5, M1 AMφ(PI-CD64+CD206-) was consistently the predominant cell type of AMφ in saline-treated mice at each time point. However, M2 AMφ(PI-CD64+CD206+) was dramatically increased in the silica--treated mice on day 1 and continued to increase until day 28. The same as AMφ, M2 IMφ(PI-CD64+CD11b+CD206+) was consistently higher than the saline group from day 1 to day 28. Moreover, a moderate positive correlation between M2 AMφ and the FVC(R2=0.6168, P<0.001), and a weak correlation was present between FVC and M2 IMφ(R2=0.1341, P<0.05)2 During the acute inflammatory phase, the IS of silica+SPI group was lower than silica+saline group(P<0.05). Compared with silica+saline group, the CVF of silica+SPI was significantly lower in the fibrosis phase. Similarly, α-SMA-positive areas were also relatively less.The total number of BALF was decreased after spironolactone administered, the neutrophils also showed the same trend. The macrophage of BALF of silica+SPI group was slightly less than silica+saline group, but had no statistically difference. In fibrosis phase(14d, 28d), the number of silica+saline group was significantly increased. However, there was no obviously increased after spironolactone administered. Therefore, the number of macrophages in silica + SPI group was significantly less than silica + saline group(P<0.05).In comparison with silica+saline group, the relative expression of Col Ⅰ,Col Ⅲ m RNA was less in silica+SPI group(P values were <0.05 and <0.01).Compared with the saline group, the proportion of M2 AMφ was significantly increased after silica challenged. While, the proportion of M2 AMφ in silica+SPI group was less than silica+saline group(P<0.01). M2 IMφ mice also showed a corresponding decline in the proportion after spironolactone intervention(P <0.01).Conclusion:(1) The present study demonstrates the detailed dynamic phenotype switching of AMφ and IMφ in experimental silicosis.(2) M2 AMφ / IMφ showed a certain degree of positive correlation with pulmonary fibrosis.(3) MR antagonist(spironolactone) partially normalizes silica-induced AMφ / IMφ M2 polarization, and attenuated silica-induced pulmonary injury and fibrosis.