The Role And Mechanism Of Macrophage Polarization In Experimental Silicosis

BackgroundSilicosis is a systemic disease characterized by diffuse fibrosis of lung tissue caused by long-term occupational exposure to high concentration of free silica dust,the pathogenesis of which has not been clarified and there is no effective treatment now.Macrophages are highly plastic and prone to polarization in different microenvironment,different subtypes of macrophages will get its specific functions.Studies have shown that macrophage polarization plays an important role in the regulation of idiopathic pulmonary fibrosis,hepatic fibrosis and renal fibrosis.It has been recognized that pulmonary macrophages are important effector cells involved in the pathogenesis of silicosis.However,the phenomenon of macrophage polarization in silicosis has not got enough attention.Therefore,to construct an experimental silicosis model,to observe the distribution of macrophage subtypes and other biological changes in different stages of silicosis,and to study the correlation,influence and law between macrophage polarization and silicosis,so as to provide reference for further understanding of the pathogenesis of silicosis.Materials and MethodsExperimental silicosis model was established with male C57BL/6 mouse.112 mice(6-8 weeks,18-22g)were randomly divided into experimental and control groups,mice in experimental group were exposed to silica by intratracheal instillation(2.5mg silica particles in 50?l saline per mouse),and mice in control group were instilled with equal volume of sterile saline.Mice were sacrificed at 1,3,7,14,28,42,56 days after silica exposure respectively,8 mice in each group,and lung tissues and bronchoalveolar lavage fluids(BALF)were collected.H&E staining were used to visualize inflammatory infiltrates,and Masson's trichrome staining were used to visualize the fibrosis.Flow cytometry were used to determine the proportion of M1 and M2 macrophages in lung tissues and BALF.Immunohistochemistry were used to detect the distribution of M1 and M2 macrophages in lung tissues.ELISA were used to detect the content of IL-1?,TNF-?,IL-10 and TGF-?1 in BALF.qRT-PCR were used to determine the mRNA expression of fibrosis markers ?-SMA,Collagen I;markers of Ml and M2 macrophages iNOS,CD86,Argl,CD206;cytokines TNF-?,IL-1?,IL-6,IFN-?,IL-10,TGF-?1,VEGF;chemokines CCL2,CCL3,CCL4;matrix metalloproteinase MMP2,MMP9,MMP28 and molecules in STAT/IRF pathway STAT1,STAT3,STAT6,IRF3,IRF5,NF-?B p65,PPAR-y in lung tissues.Western blot was used to detect the protein expression of fibrosis markers ?-SMA,Collagen I;markers of M1 and M2 macrophages iNOS,CD206 and molecules in STAT/IRF pathway STAT1,STAT3,STAT6,IRF3,IRF5,NF-?B p65,PPAR-? in lung tissues.All statistical analysis was performed with SPSS 21.0 software.All data were shown as mean±standard deviation(SD).Comparisons between two subgroups were analyzed by Student's t-test.Comparisons between more than two groups were analyzed by one-way ANOVA.The inspection level ?=0.05.Results1.Results of mouse silicosis model constructionCompared to the saline group,after exposed to silica particles 7 days,there were obvious inflammatory cell infiltration in the lung tissues of mice.On the one hand,lots of inflammatory cells were recruited to alveoli site and lead to the incrassation of alveoli walls.On the other hand,silica particles destroyed the integrity of alveoli by inducing the death of alveoli epithelial cells.On day 28,more inflammatory cells aggregated and cellular nodules formed.On day 56,extensive fibrosis appeared in lung tissue,the structure of alveoli was destroyed serious,and collagen fibers gradually accumulated.Meanwhile,the expression of fibrosis markers ?-SMA and collagen I in lung tissues were elevated significantly at mRNA and protein levels(P<0.05).2.Detection of macrophage subtypes in lung tissues and BALFIn AMs,M1 and M2 macrophages had the same change tendency,both of which increased after silica exposure and reached a maximum at day 14,then decreased to a low level but still higher than saline groups.Besides,the absolute proportion of M1 was always higher than M2 macrophages,the ratio of M1/M2 reflect that M1 macrophages played a leading role in the inflammation stage.In IMs,the proportion of M1 macrophages increased firstly until day 14 and then decreased just in accordance with in AMs.However,M2 macrophages in IMs were inhibited since silica exposure but gradually being induced with the fibrosis happen.In addition,compared to M2 macrophages,M1 macrophages were dominant in IMs before day 42.M1 macrophages were mainly distributed in the inflammatory infiltrating area of lung tissue,and M2 macrophages were abundant in the nodular and fibrotic areas.3.Detection of cytokines,chemokines and matrix metalloproteinase in lung tissues and BALFAfter exposure to silica,in lung tissues,the mRNA expression of inflammatory cytokines TNF-? and IL-6 increased significantly until day 14,then decreased gradually.The mRNA expression of IL-1? increased from day 14 and got a highest level at day 28.The mRNA expression of immunosuppressive cytokine IL-10 was inhibited in the early stage of silica exposure,and then gradually increased until day 56.In BALF,the expression of TNF-? and IL-1? increased significantly until day 28,and then decreased.The expression of IL-10 increased significantly after 1 day of silica exposure,then decreased rapidly,and increased from day 7 to day 56.The expression of chemokines CCL3 and CCL4 in lung tissues only elevated within 7 days after silica exposure.On the contrary,the expression of metalloproteinase MMP2 and MMP28 only elevated after silica exposure 42 days later.4.Detection of the activation of STAT/IRF signaling pathwayAfter silica exposure,the results showed that the expression of IRF5 decreased with the increase of slica exposure time,while the expression levels of IRF3 and PPAR-y increased with the increase of silica exposure time.ConclusionMacrophage polarization exist in the development of silicosis.And M1 macrophage mainly exists in the early stage,which may have the effect of promoting inflammation;M2 macrophage mainly exists in the late stage,which may have the effect of eliminating inflammation,repairing tissue damage and promoting fibrosis.STAT/IRF signaling pathway may be involved in the regulation of macrophage polarization in silicosis.