Gut Microbes Mediates ILC3s To Regulate The Immune Barrier Of Mice Against Influenza Infection

Influenza virus causes respiratory diseases of different degrees in a wide range of hosts,which poses a great threat to human health and the development of breeding industry.Vaccination and treatment with antiviral drugs are used internationally to avert influenza infection.However,due to the rapid mutation of the influenza virus and the lag in the development of vaccines and drugs,it is still hard to prevent and control the influenza pandemic.Therefore,the exploration of the host’s own response has become the latest research focus of influenza prevention and control.In recent years,related studies have suggested that there is a close relationship between intestinal commensal bacteria and lung tissue,which could influence the intestinal environment through diet or drug intervention to regulate the immune response of the respiratory tract and maintain homeostasis.As a non-absorbent broad-spectrum antibiotic,rifaximin induces bacteria to produce some metabolite,such as Indole acid and lipopolysaccharide,which can help maintain normal intestinal flora and regulate immunity,and become a potential adjuvant drug for the prevention and treatment of influenza.The purpose of this experiment is to study the effects of rifaximin treatment on the regulation of intestinal flora function,which can maintain the stability of the intestinal environment of influenza virus-infected mice and regulate the body’s antiviral innate immunity.By using rifaximin to prevent and treat influenza virus infection in simulated clinical practice,the effects of influenza infection and rifaximin on intestinal flora,tryptophan metabolism and immune barrier of lung and colon in mice were evaluated,and the mechanism of antiviral innate immune regulation related to the activation of ILC3 by rifaximin was explored.This study provided a new idea for the treatment of influenza from the perspective of ILC3,and provided a theoretical basis for the follow-up study on the key role of the lung intestinal axis in the regulation of immune response and the resistance to influenza infection.Experiment Ⅰ,rifaximin was pre-administered to the experimental mice,and the A/WSN/1933(WSN,H1N1)influenza virus was infected by intranasal drops.The mouse model of influenza infection was established.Experimental technology to explore the effects of rifaximin pretreatment on body weight,lung and colon damage of mice infected with influenza virus.Flu Mice showed lost weight,sparse coat,reduced food intake,and decreased intestinal contents.Pathological features such as vasodilation and congestion in lung tissue,rupture of alveolar septum,infiltration of red blood cells,and pathological damage such as crypt atrophy and goblet cell reduction in colon tissue.Rifaximin pretreatment could mitigate the barrier damage caused by influenza infection in mice such as weight lost,lung edema,ruptured of alveolar septum and inflammatory cell infiltration in lung tissue,and improved the shortening of intestinal glands,crypt atrophy,and crypt atrophy in the colon.Symptoms such as goblet cell reduction and inflammatory cell increased.Experiment Ⅱ,part of the work in this experiment used 16 S r RNA gene sequencing technology,targeted metabolomics,q RT-PCR and western blotting techniques to explore the funtions of rifaximin pretreatment on the intestinal environment of mice with influenza.The dominant bacteria in the feces of normal mice were Firmicutes,Bacteroidetes,and Proteobacteria.There was no significant change in the early stage of influenza.The relative abundance of Ruminococcaceae＿UCG-013 and Candidatus＿Saccharimonas were significantly reduced,but the pretreatment with rifaximin had no significant effect on the structure of the intestinal flora.Besides,influenza virus infection had a negative impact on the indole acrylic pathway related to the intestinal flora in tryptophan metabolism,and rifaximin drug treatment could improve this metabolic pathway.The results of q RT-PCR and western blot experiments showed that influenza virus infection reduced the expression of tight junction genes and proteins in the intestinal tract,and rifaximin could improve the intestinal mechanical barrier damage caused by influenza virus infection.Experiment Ⅲ,this test analyzed the migration and activation of ILC3 in influenza virus-infected mice,using flow cytometry,ELISA,q RT-PCR,and western blotting to explore the influence of gut microbes on the resistance to lung injury caused by influenza virus infection.The results showed that in the early stage of influenza virus infection,lymphocytes in the lungs of mice were recruited,the content of ILC3 in the intestines increased,there was a tendency to migrate to the lungs,and the expression of tight junction proteins in the lungs was significantly reduced.Rifaximin pretreatment increased the number of ILC3 s in the lungs,increased the content of IL-22,activated the STAT signaling pathway,mitigate the decrease in TJ protein expression caused by influenza virus infection,repaired the damaged barrier structure of the lung,and promoted the body’s resistance viral gene expression,and regulated the anti-viral innate immune response,thereby alleviating the further damage of influenza virus to the body.In summary,influenza virus infection caused lung and intestinal tissue damage in mice,decreased expression of tight junction proteins,changes in the number of lymphocytes and ILC3 in the lung-gut axis,changes in the structure of the intestinal flora and the pathway of indole acrylic acid metabolism.Rifaximin treatment alleviated lung and intestinal tissue damage,promoted the expression of tight junction proteins in the tissue,and improved the indole acrylic acid metabolism of intestinal symbiotic bacteria,thereby increasing the number of ILC3 cells in the lungs and improving the mucosal immune barrier function of lung tissue during influenza infection.