Dysbiosis Of Intestinal Microbiota Affects RSV-induced Airway Inflammation

Objective: Respiratory syncytial virus(RSV)is the main pathogen of lower respiratory tract infection in infants and young children in autumn and winter.It is also an important inducer of asthma in children and adolescents.Recent studies found that intestinal microbiota modulates extra-intestinal mucosal immunity through the“gut-lung axis”,affecting the progression of respiratory disease,such as influenza virus infection or asthma.However,the effect of intestinal microbiota dysbiosis on RSV infection and its mechanism are still unclear.Thus,in this study,by using antibiotic-induced intestinal microbiota dysbiosis and acute RSV infection in mice and through experiments in vivo and in vitro,we discuss the effect of intestinal microbiota dysbiosis on RSV infection against immune response and related mechanisms to understand deeply the interaction between intestinal microbiota and respiratory disease,especially respiratory virus infection.Methods: Experimental model of RSV infection in mice with intestinal microbiota dysbiosis was established;Lung pathology was determined by HE staining,the expression of cytokines in lung tissue were detected by real-time PCR,the type and number of immune cells were determined by flow cytometry,to confirm the effect of intestinal microbiota dysbiosis on RSV-induced airway inflammation.Macrophages were sorted out from lung tissue and the expression of Arg1,i NOS,IL-10 and IL-1βm RNA in lung macrophages were determined by real-time PCR.Intestinal microbiota composition was detected by 16 S r DNA high throughput sequencing.Intragastric administration of Clostridium butyricum(C.butyricum)to observe the role of C.butyricum on intestinal microbiota dysbiosis-induced alteration of RSV infection immune response.Co-culture of butyrate sodium and RSV-infected RAW264.7 cells in vitro to prove that butyrate can inhibit pro-inflammatory cytokines secreted by RSV-infected RAW 264.7 cells through inhibiting signal molecular m RNA expression.Results: 1.Streptomycin treatment induces intestinal microbiota dysbiosis: Data from16 S sequencing showed streptomycin treatment significantly reduced the relative abundance of Firmicutes phylum(mainly Lactobacillus and Clostridium at genus level),while increased the relative abundance of Bacteroidetes phylum(mainly Bacteroides genus).2.Intestinal microbiota dysbiosis exacerbates RSV-induced airway inflammation:Streptomycin-treated RSV infected mice showed severe lung histopathology and increased total inflammatory cells in BAL(mainly macrophages and neutrophils),suggesting streptomycin-induced intestinal microbiota dysbiosis exacerbates RSVinduced airway inflammation.3.Intestinal microbiota dysbiosis affects local immune response in lung tissue of RSV-infected mice: the m RNA expression of IFN-γ and IL-17 were significantly increased,while the m RNA expression of IL-5 was significantly reduced in RSVinfected lung tissue in the presence of streptomycin.The number of lung macrophages were significantly increased and the expression of i NOS and IL-1β in macrophages were increased,suggesting intestinal microbiota dysbiosis switch local immune response in RSV-infected mice towards Th1,Th17 and M1 polarization.4.Supplementation of C.butyricum recues streptomycin-induced alteration of RSV immune response: streptomycin treated mice were given gavage with C.butyricum,and then infected with RSV.The lung histopathology was alleviated and total inflammatory cells in BAL were reduced significantly.The expression of IFN-γ and IL-17 were reduced,and the expression of IL-5 in lung tissue was increased.The number of macrophages were reduced,and the expression of i NOS and IL-1β were decreased,while the expression of Arg1 was increased in isolated macrophages,suggesting supplementation of C.butyricum alleviates streptomycin-exacerbated RSV airway inflammation,and switch the immune response towards M2 polarization and away from Th1 and Th17.5.Butyrate promotes the secretion of M2 cytokines in RSV-infected RAW 264.7 cells:At the presence of butyrate,RAW264.7 cells were infected with RSV and the expression of i NOS and IL-1β were significantly decreased,while the expression of Arg1 and IL-10 were increased.At the same time,the expression of NF-κB、P38 and ERK1 were significantly decreased,suggesting butyrate promote M2 polarization while inhibit M1 polarization of RAW264.7 murine macrophages possibly by inhibiting NF-κB,P38 and ERK1 signal pathway.Conclusion: Streptomycin-induced intestinal microbiota dysbiosis aggravate RSV airway inflammation by altering pulmonary immune response.Supplementation with C.butyricum restores such immune dysregulation induced by intestinal microbiota dysbiosis and alleviates RSV airway inflammation.