Effects Of Low-dose LPS Pre-stimulation On Inflammation Induced By Influenza A(H1N1) Virus Infection

Background:Influenza is a contagious respiratory disease caused by influenza A or B virus infection.Symptoms of mild influenza are mainly limited to the upper respiratory tract,manifested as fever,sore throat,runny nose,and cough,headache,muscle pain and fatigue,severe local or systemic excessive inflammatory response,leading to acute lung injury,pneumonia,and even death.At present,it has been found that the mechanism of influenza virus infection causing excessive inflammatory response in the body is mainly the activation of NF-κB（Nuclear factor kappa-light-chain-enhancer of activated B cells）,NLRP3（NLR family pyrindo main containing 3）inflammasome assembly,thereby inducing the release of proinflammatory cytokines,such as interleukin（IL）-6,tumor necrosis factor（TNF）?and IL-1β,etc.Therefore,how to effectively control the excessive inflammatory response caused by influenza virus infection is the key to the prevention and treatment of influenza.The hygiene hypothesis states that chronic exposure to microorganisms or other antigens reduces the incidence of allergic diseases such as asthma.Lipopolysaccharide（LPS）is a component of the cell wall of Gram-negative bacteria and a common endotoxin.Studies based on the hygiene hypothesis have found that long-term exposure to low-dose LPS can prevent the occurrence of allergic excessive inflammatory reactions,but the impact of long-term exposure to low-dose LPS on excessive inflammation induced by infectious diseases such as H1N1 infection is still unclear.Therefore,we hypothesize that low-dose LPS pre-stimulation can prevent the excessive inflammatory response of the body caused by influenza A virus H1N1 infection,and conduct in-depth research on the molecular mechanism,so as to provide new ideas for clinical prevention and treatment of severe influenza and reduce mortality.Objective:This study explored whether low-dose LPS pre-stimulation could inhibit the excessive inflammation induced by influenza A virus H1N1（hereinafter referred to as H1N1）infection,and explained its molecular mechanism,so as to provide theoretical support and experimental basis for the clinical prevention and treatment of influenza.1.Effects of low-dose LPS pre-stimulation on H1N1-infected miceMethods:A low-dose LPS pre-stimulated mouse model was established by intranasally administering 50μL of 2μg/m L LPS to mice every other day for two weeks.At the same time,mice in the control group were set up,and allantoic fluid（AF）was used for intranasal instillation.Two weeks later,10LD₅₀ H1N1 influenza virus was inoculated nasally,and the body weight and survival of the mice were recorded daily.HE（Hematoxylin and eosin）staining was used to observe the pathological injury of mouse lung histopathology.Results:H1N1-infected mice developed clinical symptoms such as piloerection,arched back,decreased activity,and weight loss.Compared with the control group,mice pre-stimulated with low-dose LPS had milder symptoms,slower weight loss,and significantly longer survival time.HE staining showed that the lung tissue structure of mice infected with H1N1 in the control group was disordered,the alveolar septum was significantly thickened,and a large number of inflammatory cells infiltrated around the airway.The pathological damage of the lungs of the mice in the low-dose LPS pre-stimulation group was significantly improved,and the pathological score was significantly reduced.2.Effects of low-dose LPS pre-stimulation on lung inflammation in H1N1-infected miceMethods:A low-dose LPS pre-stimulated mouse model was established by intranasally administering 50μL of 2μg/m L LPS to mice every other day for two weeks.At the same time,mice in the control group were set up,and allantoic fluid was used for intranasal drops.72 hours after nasal inoculation with 10LD₅₀ H1N1 influenza virus,the lung tissue was detected by enzyme-linked immunosorbent assay（ELISA）and real-time polymerase chain reaction（RT-PCR）respectively.The expression levels of cytokines TNF-α,IL-6,IL-1β.Western Blot was used to detect the expression levels of NF-κB signaling pathway and NLRP3 inflammasome-related protein in lung tissue of mice.Results:H1N1 influenza virus infection activated NF-κB signaling pathway and NLRP3 inflammasome,and low-dose LPS pre-stimulation significantly inhibited the expression of NF-κB signaling pathway and NLRP3 inflammasome-related proteins.The expression levels of inflammatory factors TNF-α,IL-6,and IL-1βin the lung tissue of H1N1-infected mice were significantly increased,and low-dose LPS pre-stimulation effectively reduced the expression level of inflammatory factors in lung tissue.3.Effects of low-dose LPS pre-stimulation on A20 expression and inflammatory cytokines in A549 cellsMethods:A low-dose LPS pre-stimulated A549 cell model was constructed by replacing the culture medium with a culture medium containing 1 ng/m L LPS every other day for two weeks,and an A549 cell line knocked out of the A20 gene was constructed using CRISPR（Clustered regularly interspaced short palindromic repeats）.24 hours after inoculation with 102 TCID50 H1N1,the expression levels of A20,NF-κB signaling pathway and NLRP3 inflammasome-related proteins were detected by Western Blot.The expression levels of cytokines TNF-α,IL-6 and IL-1βwere detected by ELISA and RT-PCR respectively.The nuclear translocation of NF-κB p65 was observed by immunofluorescence.Results:Low-dose LPS pre-stimulation significantly up-regulated the expression of A20.H1N1 infection activated the NF-κB signaling pathway and NLRP3inflammasome,and low-dose LPS pre-stimulation significantly inhibited the expression of NF-κB signaling pathway and NLRP3 inflammasome-associated proteins,as well as the nuclear import of NF-κB p65.After knocking out the A20 gene,low-dose LPS pre-stimulation no longer inhibited the NF-κB signaling pathway,the expression of NLRP3 inflammasome-associated protein,and the nuclear import of p65.4.Screening and verification of low-dose LPS pre-stimulation on the interaction proteins related to A20 in H1N1 infection modelMethods:Quantitative proteomics sequencing technology labeled with tandem mass tags（TMT）was used to detect the changes in protein expression profiles during the process of up-regulating A20 expression and inhibiting inflammatory response by low-dose LPS pre-stimulation.The key proteins screened by proteomics sequencing were detected by Western Blot for verification.Results:Through the quantitative proteomics sequencing analysis of TMT markers,it was found that compared with the Control+H1N1 group,the Chronic-LPSlo+H1N1group had a total of 136 differentially expressed proteins,of which 123 proteins were significantly up-regulated and 13 were significantly down-regulated.GO and KEGG enrichment analysis showed that the differentially expressed proteins were mainly involved in multiple signaling pathways related to inflammation,such as PPAR（Peroxisome proliferator-activated receptor）,TNF,NF-κB and NOD（Nucleotide-binding oligomerization domain-like）Related pathways further verified that the up-regulation effect of low-dose LPS pre-stimulation on the expression of PPAR-αand PPAR-γis A20-dependent.Conclusion:1.Low-dose LPS pre-stimulation can inhibit the pulmonary edema of H1N1-infected mice,thereby prolonging the survival time of mice.2.Low-dose LPS pre-stimulation inhibits the expression of NF-κB signaling pathway and NLRP3 inflammasome-related protein,attenuating the inflammatory response induced by H1N1 infection.3.Low-dose LPS pre-stimulation can inhibit the inflammatory response by up-regulating the expression of A20 and promoting the expression of PPAR-αand PPAR-γ.