The Experimental Study Of Cigarette Smoking Extracts Induce M1/M2 Polarization And Apoptosis In Macrophages

Objective:Chronic obstructive pulmonary disease?COPD?is a type of chronic airway disease characterized by air flow restriction.Cigarette smoke exposure is a risk factor for COPD,in part resulting from damage and dysfunction of macrophages caused by cigarettes.Macrophages are highly heterogeneous cells that can differentiate into classically and alternatively activated macrophages?M1 and M2,respectively?.M1 and M2 macrophages have pro-and anti-inflammatory roles,contributing to the airway inflammation and remodeling in COPD.Besides,cigarette also induce bronchial epithelial cells apoptosis leading to the formation of emphysema.However,the effects and mechanisms through which cigarettes affect macrophage M1/M2 polarization and apoptosis remain elusive.So,this study aims to examine the effects and mechanism of cigarette on the polarization and apoptosis of macrophages.This study consists of three parts.First,we aim to explore the dynamic effects of cigarette smoking extract?CSE?on the polarization of alveolar macrophages?AM?and peritoneal macrophages?PM?.Second,we want to find out the effect of PPAR?on the polarization process of AM caused by CSE.Finally,the effect and mechanisms of apoptosis induced by CSE in Raw264.7,a macrophage cell line,will be clarified in the last part.Methods:Part one:the dynamic effects of CSE on the polarization of AM and PM.AM and PM were isolated from SPF rats which were 6 to 8 weeks old and CSE was freshly prepared according to references each time.AM and PM were cultured with CSE at different concentrations?0.5%,1%and 2%?and CCK-8 assay was used as an indicator of cell viability.The mRNA expression of M1?iNOS,TNF-?,and IL-1??and M2markers?arg1,CD206,and TGF-?1?were measured at 3,6,9,12,and 24 h using qPCR.Expressions of CD86 and CD206 proteins at 12 h were determined using flow cytometry,and the iNOS/arg-1 ratio was used to determine the polarization dominance of M1 and M2.M2 subtypes were detected at 12 h using qPCR and flow cytometry.Part two:the effect of PPAR?on the polarization process of AM caused by CSE.To build the lung emphysema model,male Wistar rats were exposed to cigarette smoke for12 weeks.Mean linear intercept and mean alveolar numbers were used to access the emphysema.The CD86 positive cells and CD206 positive cells in bronchoalveolar lavage fluid?BALF?were determined by flow cytometry assay,and the iNOS positive cells and CD206 positive cells in lung section were accessed by immunohistochemical?IHC?assay.After AM treated by CSE at concentration of 0.5%,1%and 2%for 12h,immunol blot assay was used to detect the protein expression of PPAR?and RXR?,and the mRNA level of TNF-?,IL-1?,iNOS,CD206 and arg1 were determined by qPCR methods.The protein expression of CD80,CD86 and CD206 on AM was detected by flow cytometry.Besides,AM were pretreated with the agonist and antagonist of PPAR?and RXR?to access the role of PPAR?/RXR?heterodimer in the polarization process induce by CSE.Part three:the effect and mechanisms of apoptosis induced by CSE in Raw264.7.Raw264.7 cells were treated with varying concentrations of CSE for 8 h to detect the cell viability and apoptosis.Cells viability was evaluated by CCK 8 assay and the cell apoptosis was access by annexin V/PI staining assay.The reactive oxygen species?ROS?level in cells was detected by DCFH-DA probe assay.The protein expression of caspase3,cleaved caspase3,cleaved PARP,Bcl2,Bax,cytochrome-C,caspase9,CHOP,BiP,P-eif2?,P38,P-P38,JNK,P-JNK,ERK1/2,P-ERK1/2,STAT1,P-STAT1?Ser727?and P-STAT1?Tyr701?were detected by western blot.The intracellular calcium level was evaluated by Fluo-4 AM probe assay.Results:Part one:the dynamic effects of CSE on the polarization of AM and PM.CSE increased the expression of iNOS,TNF?,and IL1?mRNA,and the proportion of CD86-positive cells in AM and PM promoted M1 polarization,and M1 polarization was continuously enhanced as exposure time and concentration increased.CSE reduced the expression of arg1,CD206,and TGF?1 mRNA and the proportion of CD206-positive cells in AM and PM and inhibited M2 polarization.At 9-24 h of CSE exposure,the expression of arg1 in AM and PM gradually increased,showing tendency towards activation of M2 polarization.Besides,CSE might induce M2b and M2d polarization at 12 h.After 12 h of CSE exposure,transformation from M1 to M2 polarization dominance was shown in AM;however,M1 polarization was continuously enhanced in PM within 24 h of CSE exposure.Part two:the effect of PPAR?on the polarization process of AM caused by CSE.Compared with control group,the cigarette exposure group had significant pulmonary emphysema,which was evaluated by the increased mean linear intercept and decreased mean alveolar numbers.The CD86 positive cells in BALF of cigarette exposure group were increased while CD206 positive cells were decreased compared with the control group.The iNOS positive cells in lung tissue section of model group were increased and CD206 positive cells were not changed significantly compared with control group.CSE treatment for 12 h increased decreased the protein level of PPAR?and RXR?.Rosiglitazone,a PPAR?agonist,increased the protein expression of PPAR?and RXR?.Besides,rosiglitazone decreased the expression of the M1 polarization markers?TNF-?,IL-1?,iNOS,CD80 and CD86?induced by CSE while increased the level of M2polarization markers?CD206 and arg1?.The effects of rosiglitazone were inhibited by GW9662 and HX531,the inhibitors of PPAR?and RXR?respectively,which indicated that rosiglitazone regulated the M1/M2 polarization process induced by CSE via PPAR?/RXR?.Part three:the effect and mechanisms of apoptosis induced by CSE in Raw264.7.CSE increased cleaved caspase3 expression and apoptosis,and the effects were attenuated by Z-VAD-FMK,a caspase inhibitor.Levels of cytochrome C and ROS were decreased,while no changes in expressions of Bcl2,Bax and caspase9 were present in response to CSE.CSE increased the expression of CHOP,BiP and P-eif2?.4-PBA,an inhibitor of endoplasmic reticulum?ER?stress,decreased the apoptosis produced by CSE.Phosphorylation levels of P38,JNK and ERK1/2 were increased following incubation with CSE.Only the P38 inhibitor significantly decreased apoptosis,while the ERK1/2 inhibitor promoted apoptosis.Phosphorylation of STAT1 at Ser727 was activated by CSE and attenuated by the P38 inhibitor.Finally,CSE increased intracellular Ca2+levels,and BAPTA,a calcium chelator,partly abrogated the apoptosis and activation of P38 and STAT1 as induced by CSE.Conclusions:CSE promoted M1 polarization in macrophages,exhibiting dynamic regulatory effects on M2 polarization,first as a suppressor and then as a promoter.The polarization change induced by CSE on AM was more sensitive than PM.Cigarette smoke exposure for 12 weeks induced M1 polarization in AM.Rosiglitazone inhibited the M1 polarization induced by CSE while induce M2 polarization via PPAR?/RXR?.CSE induced a caspase3-dependent apoptosis in Raw264.7 cells via ER stress and Ca²⁺/P38/STAT1 pathway.No activation upon the mitochondrial apoptosis pathway and ROS were observed in response to CSE.