| Background Silicosis is the most prevalent and harmful occupational disease,and it is one of the major public health problems in the world.Pulmonary fibrosis is a major end-stage change of pulmonary diseases characterized by the proliferation of fibroblasts and the accumulation of a large number of extracellular matrix(ECM),accompanied by inflammatory damage and tissue structure destruction.Silicosis is mainly caused by inhalation of a large amount of free silicon dioxide(silica)dust and often occurs in occupational environments.Even if the patient is no longer exposed to silica,the damage to lung function will increase as the disease progresses.With the development of modern industries such as stone countertop manufacturing,sandblasting,jewelry and glass production,most workers in these environments will suffer from acute silicosis or accelerated silicosis due to inhalation of high concentrations of silica in a short period of time.To explore the pathogenesis of silicosis has important theoretical and practical significance for the prevention and treatment of silicosis.Fibroblast-to-myofibroblast transdifferentiation(FMT)and epithelial-mesenchymal transition(EMT)are known to play essential roles in the development of pulmonary fibrosis induced by silica.As far as we know,the molecular mechanism of silica-induced pulmonary fibrosis has not been fully revealed.As a heterogeneous population of immune cell in lung,phenotypic change of alveolar macrophages(AMs)was crucial to the progression of pulmonary fibrosis induced by silica.AMs are the main immune cells in lung tissues,and they are also the main target cells for silica.In different microenvironments,resting macrophages can activate M1 type macrophages that are polarized as classically activated and alternatively activated M2 type macrophages.However,the exact role of phenotypic change of AMs in the progression of silicosis remain unclear.Objective To explore potential pro-inflammatory and pro-fibrofic effects of AMs induced by silica in pulmonary fibrosis progress,we examined the time-and dose-effects of silica exposure on primary mouse AMs polarization,as well as the role of polarized AMs induced by silica on FMT of primary lung fibroblasts and EMT of BEAS-2B cells in vitro.In this study,under the polarization conditions of M1 and M2AMs in vitro,the influence of silica exposure on the polarization of primary mouse AMs was tested.At the same time,the supernatants of AMs exposed to 0,2.5,5 and 10μg/mL silica under M1 polarization conditions for 6 hours and 0,2.5,5 and 10μg/mL silica under M2 polarization conditions for 24 hours were collected,and then made into conditioned medium(CM).The collected CM were used to culture primary lung fibroblasts and BEAS-2B cells for 48 hours respectively.To explore the effects of silica-induced AMs polarization changes on lung FMT and EMT.This study helps to further clarify the pathogenesis of pulmonary fibrosis caused by silica,and provides experimental evidence for finding effective regulatory target molecules.It has essential theoretical and practical significance for the prevention and treatment of silicosis.Research methods Part Ⅰ The study on primary AMs polarization changes induced by silica in miceThe AMs were separated form alveolar lavage of 8-week-old C57BL/6J WT mice,and the purity was identified by flow cytometry(FCM).For M1 polarization experiments,AMs were treated with 100 ng/mLLPS for 24 hours and 20 ng/mL IFN-γfor an additional 24 hours(M1 type conditions).For M2 polarization experiments,AMs were treated with 20 ng/mL IL-4 for 48 hours(M2 type conditions).The expressions of phenotypic markers CD11c or CD206 were measured by immunofluorescence and FCM to evaluate the results of AMs polarization in M1 or M2 type conditions of polarization.Under M1 type polarization conditions,AMs were treated with 5μg/mL silica for 6,12and 24 hours,respectively.FCM was used to analyze the M1 type(F4/80~+CD11c~+)AMs ratio,ELISA and q RT-PCR to detect the activity and mRNA expression levels of iNOS(M1 subtype functional marker).Under M1 type polarization conditions,AMs were treated with 0,2.5,5,and 10μg/mL silica for 6 and 24 hours,respectively.FCM was used to analyze the M1 type AMs ratio;ELISA to detect iNOS activity as well as TNF-αand IL-6 content in cell supernatant;q RT-PCR to analyze the mRNA expression levels of M1 function-related factors(iNOS,TNF-αand IL-6).Under M2 type polarization conditions,AMs were treated with 5μg/mL silica for 6,12 and 24 hours,respectively.FCM was used to analyze the M2 type(F4/80~+CD206~+)AMs ratio,ELISA and q RT-PCR to detect the activity and mRNA expression levels of Arg-1(M2 subtype functional marker).Under M2 type polarization conditions,AMs were treated with 0,2.5,5,and 10μg/mL silica for 6,12 and 24 hours,respectively.FCM was used to analyze the M2 type AMs ratio;ELISA to detect Arg-1 activity as well as TGF-βand IL-10 content in cell supernatant;q RT-PCR to analyze the mRNA expression levels of M1 function-related factors(Arg-1,TGF-βand IL-10).Part Ⅱ In vitro study of the mechanism of silica-induced AMs polarization change mediating pulmonary fibrosisThe AMs were separated form alveolar lavage of 8-week-old C57BL/6J WT mice.At the same time,the supernatants of AMs exposed to 0,2.5,5 and 10μg/mL silica under M1 polarization conditions for 6 hours and 0,2.5,5 and 10μg/mL silica under M2polarization conditions for 24 hours were collected,and then made into conditioned medium(CM).The collected CM were used to culture primary lung fibroblasts and BEAS-2B cells for 48 hours respectively.The control group was cultured with ordinary high-sugar DMEM medium.In addition,primary lung fibroblasts and BEAS-2B cells were cultured for 48 hours by using CM from AMs exposed 10μg/mL silica for 24hours under M2 type polarization condition with 10 ng/mL TGF-βinhibitor(LY364947).Cellular immunohistochemical method was used to detect the protein expression levels of Col I and BEAS-2B cells in lung fibroblasts;Western blotting was used to evaluate the protein expression level of Col I andα-SMA in lung fibroblasts as well as E-cadherin and Vimentin in BEAS-2B cells.Results 1.Under M1 polarization conditions,the proportion of M1 subtype AMs(F4/80~+CD11c~+)and the activity and mRNA expression level of M1 subtype functional marker iNOS were increased after exposure to 5μg/mL silica in 6 hours and 12 hours,with a peak in 6 hours and decreased in 24 hours compared to LPS+IFN-γgroup(P<0.01).After exposure to 0,2.5,5 and 10μg/mL silica for 6 hours under M1 type polarization conditions,the proportion of M1 subtype AMs,activity and mRNA expression level of iNOS were increased in a dose-dependent manner(P<0.01).After exposure to 0,2.5,5 and 10μg/mL silica for 24 hours under M1 type polarization conditions,the proportion of M1 subtype AMs,the activity and mRNA expression level of iNOS were decreased with the increasing of silica dose(P<0.01).After 0,2.5,5 and10μg/mL silica exposure for 6 hours under M1 type polarization conditions,the mRNA expressions of TNF-αand IL-6 were raised in a dose-dependent manner,so did the contents of the supernatant(P<0.01).After 0,2.5,5 and 10μg/mL silica exposure for 24hours under M1 type polarization condition,the mRNA expressions of TNF-αand IL-6were decreased as the dose of silica increased,so did the contents of the supernatant(P<0.01).2.Under M2 polarization conditions,the proportion of M2 subtype AMs(F4/80~+CD206~+)and the activity and the mRNA expression level of M2 subtype functional molecular Arg-1 were increased compared to IL-4 group in a time-dependent manner(P<0.01).After exposure to 0,2.5,5 and 10μg/mL silica for 24 hours under M2type polarization conditions,the proportion of M2 subtype AMs and the activity and mRNA expression level of Arg-1 were increased in a dose-dependent manner(P<0.01);the mRNA expressions of TGF-βand IL-10 increased in a concentration-dependent manner,so did the contents of the supernatant(P<0.01).3.After cultured by CM prepared from AMs exposed 0,2.5,5 and 10μg/mL silica for6 hours under M1 type polarization condition,the protein expressions ofα-SMA and Col I in lung fibroblasts were not statistically different among 5 groups.Meanwhile the protein expressions of E-cadherin and Vimentin in BEAS-2B cells were not observably different among 5 groups.After cultured by CM from AMs exposed 0,2.5,5 and 10μg/mL silica for 24 hours under M2 type polarization condition,results showed that the protein expressions of Col I andα-SMA in lung fibroblasts were raised in a dose-dependent manner(P<0.01).Meanwhile,E-cadherin protein expression levels in BEAS-2B cells were progressively decreased,while Vimentin protein expression levels gradually raised as the dose of silica increased(P<0.01).Lung fibroblasts and BEAS-2B cells were respectively cultured for 48 hours by using the CM from AMs exposed to 10μg/mL silica for 24 hours under M2 type polarization condition with 10ng/mL TGF-βinhibitor(LY364947).Results revealed that Col I andα-SMA protein expressions in lung fibroblasts were observably decreased after inhibition of TGF-βin AMs supernatants exposed to 10μg/mL silica 24 hours,as well as E-cadherin protein expression was increased and Vimentin was decreased in BEAS-2B cells(P<0.01).4.Under M2 type polarization condition,0,2.5,5 and 10μg/mL silica treatment resulted in up-regulation of IRF4 protein expression in AMs in dose-dependent manner(P<0.01),but there was no appreciable differences of IRF5 protein expression among 5groups.Conclusions 1.Silica induces AMs toward M1 subtype polarization and the production of M1 subtype AMs-related cytokines before 12 hours treatment,then inhibits them toward M1 subtype polarization and the production of M1 subtype AMs-related cytokines after 12 hours.2.Silica promotes AMs toward M2 subtype polarization and the production of M2subtype AMs-related cytokines.3.Silica induces alveolar M2 macrophage polarization mediated lung FMT and EMT maybe via the TGF-βsignaling pathway.4.Silica up-regulates IRF4 expression in AMs under M2 type polarization condition. |
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