Pulmonary Flora Modulates Th17 Response In Silica-induced Pulmonary Fibrosis

BackgroundSilicosis is a disease of interstitial lung fibrosis caused by chronic inhalation of high levels of crystalline-free silica dust.In vivo host cell functional analyses(e.g.transcriptomics and proteomics)combined with microbiome sequencing identified associations between components of the lower respiratory microbiota and macrophage phenotypes,inflammatory cytokine profiles,and Th17 responses.One study using a combination regimen of four antibiotics to reduce the lung microbiota in mice showed reduced lung fibrosis in mice with lung bacterial deficiency.And while silicosis fibrosis and epithelial-mesenchymal transition(EMT)are closely related,the role of lung flora on pulmonary fibrosis in silicosis and the relationship with EMT has not been elucidated.Th17 cells are involved in the pathogenesis of pulmonary fibrosis by promoting fibroblast proliferation and cytokine production.Related studies have shown that lung flora is associated with Th17 responses,but the role in the process of silicosis fibrosis remains to be explored.ObjectiveTo explore the differences in airway flora between silicosis case groups and healthy controls,to investigate the effect of lung flora on EMT,and to elucidate the mechanisms by which lung bacteria regulate fibrosis in silicosis lesions through Th17 cell responses.Methods1.Ten sputum samples were collected from each of the silicosis cases and healthy controls,and the composition distribution,abundance,and diversity of the airway flora were analyzed by 16 S r DNA high-throughput sequencing.2.The lung flora of silicosis mice were cleared by intranasal antibiotic drip and the distribution,richness,and diversity of the lung flora of silica and blank control mice were analyzed.3.The relative expression levels of α-smooth muscle actin(α-SMA),E-calmodulin(E-cad),and vimentin in the lung tissues of mice in each group were measured.4.The relative expression levels of IL-17 A and IL-17 F cytokines secreted by Th17 cells,as well as the relative expression levels of Th17-related cytokines IL-1β,IL-6,TGF-β,IL-21,IL-22 and TNF-α,were measured;the surface markers CD4 and IL-17 were co-localized in mouse lung tissues.Results1.There were significant differences in the abundance,diversity,and community structure of airway microorganisms between silicosis patients and healthy individuals.The common genera were Neisseria,Prevotella,Streptococcus,Fusobacterium,Alloprevotella,Haemophilus,and Veillonella,respectively.2.There were significant differences in the abundance,diversity,and community structure of the lung flora between the silica group and the blank control group.The common genera were Muribaculaceae＿Unclassified,AAP99,TM7 a,Thiobacillus,Acinetobacter,Massilia,Lachnospiraceae＿NK4A136＿group,Tolumonas and Micropruina,respectively.3.The differences in body mass between mice were statistically significant for the main effect of time,the main effect between groups,and the interaction effect between the two,and the differences in lung organ coefficients between groups were statistically significant.The alveolar wall of the mice in the blank control group was thin and structurally intact,with few surrounding infiltrating inflammatory cells and no abnormal distribution of collagen fibers;the alveolar structure of the mice in the silica group was disturbed,with a large number of infiltrating inflammatory cells,thickening of the alveolar wall and proliferation of cellular fibrous nodules,with blue collagen deposits distributed in large numbers in the fibrous nodular masses;the inflammation and fibrosis in the lungs of the mice in the three antibiotic-treated groups were all reduced to varying degrees.4.Compared with the blank control group,the relative expression levels of α-SMA and Vimentin in the lung tissues of mice in the silica group increased,and the expression level of E-cad decreased;compared with the solvent control group,the relative expression levels of α-SMA and Vimentin in the lung tissues of mice in the metronidazole + neomycin model group and the mixed treatment group decreased,and the expression level of E-cad increased.5.Compared with the blank control group,the relative expression levels of IL-17 A and IL-17 F protein in lung tissues of the silica group were both increased;compared with the solvent control group,the relative expression levels of IL-17 A in lung tissues of both the metronidazole + neomycin model group and the mixed treatment group were reduced,and the differences were statistically significant.IL-17 F protein was only reduced in the mixed treatment group,and the difference was not statistically significant.The CD4 and IL-17 co-expression was higher in the silica group,while the CD4 and IL-17 co-expression positive areas were lower in the metronidazole + neomycin model group and the mixed treatment group.6.The results showed that the relative expression levels of IL-1β,IL-6,TGF-β,IL-21,and TNF-α in lung tissues of the silica group were increased and the relative expression level of IL-22 was decreased compared with the blank control group;compared with the solvent control group,the relative expression levels of IL-1β,IL-6,IL-21 and TGF-β in lung tissues of the three groups,the relative expression levels of TNF-α in lung tissues decreased in the metronidazole + neomycin model group and the mixed treatment group,and the relative expression levels of IL-22 in all three antibiotic-treated groups increased.Conclusion1.The structure and relative abundance of airway flora differ between silicosis patients and healthy individuals,and the reduced airway microbial diversity in silicosis patients may be associated with differential genera;the altered diversity,composition,and relative abundance of the lung flora in silicosis mice is accompanied by a dysregulation of the lung flora.2.Decreased alpha diversity,upregulated E-cad protein expression levels and down regulated Vimentin protein expression levels after antibiotic intervention in the lung flora of silicosis mice suggest that the lung flora affects pulmonary fibrosis through EMT.3.Lung flora may attenuate silica-induced pulmonary fibrosis by inhibiting Th17 responses and regulating related cytokines.