The Role Of PM_2.5 In Inhibiting Antiviral Innate Immunity Of A549 Cells And Its Molecular Mechanism

Background:Fine particulate matter(PM_2.5)is one of the hotspots in air pollution and health research in recent years.PM_2.5 refers to particles with an aerodynamic diameter<2.5?m that can be suspended in the atmosphere and can enter the deep lungs through the respiratory tract to exert toxic effects.PM_2.5 has been shown to be a new intermediary for a variety of pathogenic microorganisms(bacterium and viruses)to achieve human-to-human transmission,and long-term exposure to PM_2.5 can cause abnormal immune function in humans.Previous studies have analyzed the components of PM_2.5 and its toxic effects on human health,and paid less attention to the cross-effects of PM_2.5 exposure and viral infection on human immune function.Therefore,this study focused on the effects and mechanisms of PM_2.5 on VSV-activated antiviral innate immune function,providing clues to reveal the effects of PM_2.5 exposure on patients with RNA viral lung infection.Methods:(1)Vitro cell experiment:Human lung adenocarcinoma A549 cells were infected with VSV(MOI=1)carrying GFP fluorescent gene,and/or exposed to 200?g/m L PM_2.5 for 24 h.At same time,a blank control group was established.The viability of A549 cells was observed by CCK-8 method.The fluorescence of GFP was observed by inverted fluorescence microscope.The PM_2.5uptake by A549 cells and GFP fluorescence intensity were detected by flow cytometry.The apoptosis was observed by Hoechst 33342 staining.m RNA expression of VSV-G,IFN-?,ISG15,CCL-5,and CXCL-10 was detected by RT-q PCR.ELISA was used to detect the expression of IFN-?protein in the supernatant of the cell culture medium.Western-Blot assay were used to observe the expression of antiviral pathway-related proteins such as?-Actin,TBK1,IRF-3,p-IRF-3,p-ERK1/2,p-JNK,p-P38 and VSV-G.Pretreatment with MG132 for 2 h observed the fluorescence intensity,VSV-G m RNA and protein expression of PM_2.5+VSV treatment group.Using BGISEQ-500 platform for RNA-seq,observed differential gene expression with different treatment groups,TRIM73 was selected and synthesis si RNA to silence the related gene.TRIM73 m RNA was observed by RT-q PCR.p-IRF-3 and VSV-G were detected by Western-Blot.(2)Vivo experiment:40 6-8 weeks old C57BL/6J mice were purchased from Jinan Pengyue Animal Breeding Company,male and female,and 40 mice were randomly divided into Control group,VSV group,PM_2.5 group and PM_2.5+VSV group by random number table method.The lung VSV infection and/or PM_2.5 exposure model was established.The Control group mice were treated with PBS as a blank control.Body weight changes,food intake and water intake were observed within10 days of the four groups of mice.The wet weight of lung,liver and spleen of mice was also observed.BALF was smeared and stained with Diff-Quick stain to observe the immune cells in BALF.The expression of IFN-?protein in BALF was observed by ELISA.The expression of IFN-?in lung tissue was observed by RT-q PCR.Results:As the concentration of PM_2.5 increased,the SSC-H value,GFP fluorescence and expression of VSV-G protein increased,and all of these have dose-dependent with PM_2.5.PM_2.5with a final concentration of 200 ug/m L inhibited the viability of A549 cells at 3h,6h,12h and 24h,and the difference was statistically significant(P<0.05).The viability of A549 cells was significantly inhibited from 6 h after PM_2.5 and VSV addition,and the difference was statistically significant(P<0.05).PM_2.5 exposure to VSV-infected A549 cells showed nuclear chromatin condensation and cell fragmentation,suggested the occurrence of apoptosis.With the increase of PM_2.5 concentration,Caspase-3 and Caspase-9 were gradually decreased in VSV-infected A549.Compared with the VSV group,the m RNA expressions of IFN-?,ISG15,CCL-5 and CXCL-10 in the VSV+PM_2.5 group were significantly decreased,and the difference was statistically significant(P<0.05).PM_2.5 can synergize with VSV to promote ERK and P38 phosphorylation,and inhibit VSV-induced p-IRF-3 expression.The expression of p-IRF-3 in A549 cells of MG132 pretreatment group was higher than DMSO treatment group.Compare with DMSO treatment group the green fluorescence intensity of GFP and the expression of VSV-G m RNA and protein was decreased in MG132 pretreatment group,the difference was statistically significant(P<0.05).The results of volcano plot showed that compared with VSV group,217 up-regulated genes and 345 down-regulated genes in the PM_2.5+VSV group.The differential gene KEGG Pathway enriched bubble map shows that the pathway with the most differential gene enrichment and the large number of genes is the interaction of cytokines with cytokine receptors.TRIM73 si RNA was transfected into A549 cells for 24h,and then VSV and PM_2.5 were added for 24h.The results showed that TRIM73si RNA had a better silencing effect,and the expression of p-IRF-3 protein increased,the expression of VSV-G protein decreased,the difference was statistically significant(P<0.05).The difference in total food intake between the four groups was statistically significant(P<0.05),and the mice in the PM_2.5+VSV group had the most food intake.Diff-Quick staining of BALF showed an increase in macrophages in BALF in the PM_2.5 and PM_2.5+VSV groups.The expression of IFN-?protein and m RNA in BALF and lung tissue of PM_2.5+VSV group were significantly lower than VSV group(P<0.05).Consistent with the results of vitro studies in this study.Conclusions:1.PM_2.5 can promote the proliferation of VSV and synergistically exert cytotoxicity to induce apoptosis of A549 cells.2.PM_2.5 ubiquitination degradation of p-IRF-3 inhibits IFN-?expression and promotes replication of VSV virus in A549 cells.Accordingly,PM_2.5 inhibits IFN-?downstream antiviral related factors,such as ISG15,CCL-5 and CXCL-10 m RNA expression.3.TRIM73 may be involved in the ubiquitination of PM_2.5 to degrade VSV-induced expression of p-IRF-3 in A549 cells.4.PM_2.5 exposure combined with VSV infection can increase the feed intake of mice for 10 days.Consistently,PM_2.5 can also inhibit the expression of VSV-induced IFN-?m RNA in lung tissue and IFN-?protein in BALD.