Microbiota Regulates TLR7Signaling Pathway Against Respiratory Tract Influenza A Virus Infection

ObjectiveAlthough intestinal flora are crucial in maintaining immune homeostasis of the intestine,the role of intestinal flora in immune responses at other mucosal surfaces remains less clear.Inthis study, we show that significant changes in the composition of culturable commensalbacteria reduced the expression levels of components of the TLR7signaling pathway. Ourresults reveal the importance of intestinal flora in regulating immunity in the respiratorymucosa through the upregulation of the TLR7signaling pathway for the proper activation ofinflammasomes.Methods1. FM1virus were increased in chicken embryo allantoic cavity and determinated by LD50.2. To establish the intestinal dysbacteriosis models and probiotics recovery model3. To observation the change of mice mental state and signs in the process of experiment.4. To test the change of lung index; preparation the specimen of Ileum and cecum in micelung tissue and HE staining5. To preparation of the lung tissue homogenate respectively for real-time fluorescencequantitative PCR reaction (RT-qPCR). Aiming at observe the changes of level of mRNAexpressed by the Toll-like receptor7(TLR7), myeloid differentiation factor88(MyD88),interleukin-1receptor associated kinase4(IRAK4), tumor necrosis factor-related kinase6(TRAF6) and nuclear factor-B (NF-B).6. To preparation of the lung tissue homogenate respectively. Using Western-bolt to detectthe TLR7, NF-B protein expression.Results1. Influenza virus was amplified in chick cavity inoculation successfully; hemagglutinationtiter was1:640; theLD50was510-5.56according to Preliminary experiments.2. The three kinds of antibiotics all caused different level of intestinal flora imbalance in mice,successfully established the intestinal dysbacteriosis model and probiotics recovery model3. Mice treated with antibiotics displayed a more serious disrupted structure of the normal tissue than the virus control group. The alveoli, alveolar sacs, alveolar tubes, alveolar septa,and bronchi were twisted, the bronchial epithelium was detached, and the connective tissuedemonstrated hyperplasia. A large amount of lymphocyte infiltration was visible in thepulmonary interstitium, a large number of red blood cells were evident in the pulmonaryvasculature, serious intravascular congestion was observed, and seepage was visible in thebronchial lumen. Compared with the neomycin-only group, the extent of the disease wassignificantly reduced in the probiotic-treated group; the structure of the lung tissue wascomplete, the alveolar interval was thinner, the alveolar walls were diminished, infiltration ofmononuclear cells in the walls of bronchioles decreased, and no seepage was evident in thecavity4. Antibiotics treatment caused an imbalance in the intestinal flora in mice, which was evidentfrom the obvious swelling of the cecum, microscopically evident edema, detachment ofintestinal villi, and mild congestion of the intestinal mucosa. The mice treated with probioticshad reduced swelling of the appendix compared with mice which established dysbacteriosis.These mice still experienced some slight damage to the mucosa compared with those of thenormal group, and intestinal villi were still partly detached.5. ELISA results showed that influenza virus FM1-infected mice displayed reduced secretionofIL-4and IL-10but increased expression of IFN-γand IL-17. Following antibiotics-mediateddestruction of the normal flora, influenza infection increased the levels of IL-4and IL-10butreduced the levels of IFN-γand IL-17. Probiotic treatment reduced the levels of IL-4andIL-10in a pulmonary homogenate, and simultaneously increased the levels of IFN-γ andIL-17. These differences were significant compared with the neomycin group.6. Influenza virus infection significantly increased the pulmonary immunocyte expression ofmRNA in the TLR7signaling pathways; in particular, the levels of TLR7, MyD88, IRAK4,and TRAF6mRNA expression increased. Antibiotic-induced dysbacteriosis reduced mRNAexpression in TLR7signaling pathways, especially the expression of TLR7and NF-κBmRNA. Probiotic treatment significantly restored the initial levels of upregulation of TLR7,MyD88, IRAK4, TRAF6, and NF-κB mRNA expression.7. Western-blot experiments showed that influenza virus infection could significantlyincreased the expression of protein in the TLR7, NF-B; alteration of intestinal flora couldreduce TLR7, NF-B protein expression;and Probiotics treatment can raise the TLR7, NF-Bprotein expression. ConclusionOur results reveal the importance of commensal microbiota in regulating immunity in therespiratory mucosa through the proper activation of TLR7.