Mechanism Of TLR4-mediated ATP Release In The Regulation Of IL-1? By The Lipopolysaccharide Of Haemophilusparasuis

Haemophilus parasuis?HPS?,a small Gram-negative bacteria,exists in the upper respiratory tract of swines and often causes symptoms such as multiple serositis,arthritis and meningitis in swines.HPS usually coinfection with porcine reproductive and respiratory syndrome virus?Porcine reproductive and respiratory syndrome virus,PRRSV?in swine production,leading to higher morbidity and high mortality.Some highly virulent HPS strains can break through the host's innate and acquired immune defenses and evade cellular and humoral immune systems.HPS's own molecular structure?such as lipopolysaccharide?LPS??and produced protein substances can be used as virulence factors,which play an important role in adhering and invading the host and evading the host's natural immunity and inflammation process,causing pig disease even death.Therefore,the study of HPS pathogenic and inflammatory related signaling pathways is very important.The P2X7 receptor is a membrane protein receptor and is an ATP-gated cation channel,after ATP binds to the P2X7 receptor,the opening of the cation channel causes Ca²⁺,Na⁺inflow and K⁺outflow,and promotes the formation of cell membrane channels,which in turn leads to the release of inflammatory cytokines such as IL-1?.NLRP3 inflammasome mainly includes NLRP3 protein,Caspase-1 protein and apoptosis-associated speck-like protein containing CARD?ASC?.After the NLRP3 inflammasome is launched,activated Caspase-1 cleaves pro-IL-1?into biologically active IL-1?and secretes it extracellularly.At present,there is no report on whether HPS affects the organism by activating the ATP/P2X7 receptor signaling pathway.TOLL-like receptors?TLRs?belong to pattern recognition receptors?PRRs?that can recognize various pathogen-associated molecular patterns?PAMPs?to drive natural immune responses.TLR4 belongs to the TLRs family and can activate two signaling pathways including MyD88/TIRAP and TRIF/TRAM to induce an inflammatory response.PAMP is a highly conserved structure in microorganisms.The more representative PAMPs include lipopolysaccharides?LPS?of Gram-negative bacteria.LPS of Gram-negative bacteria can recognize TLR4,causing the expression of inflammatory factors,and leading to inflammation.1.Epidemiological analysis of Haemophilus parasuisIn this study,HPS was isolated and identified from 103 suspectedsamples from diseased pig farms in 12 regions of Shandong Province to survey the prevalence of HPS.The results showed that the isolation rate of Haemophilus parasuis in the suspectedsamplesin Shandong Province was 28.16%.Among them,type 4 and 5serotypes were the most prevalent,followed by types 1 and 12.The disease can occur year-round,but in spring?March,April?and autumn?October,November?,it is more likely to outbreak.The study found that most susceptible piglets are from 31 to 50days old,which is related to the early weaning reproduction model and the change regulation of maternal antibodies of piglets.A total of 447 suspected serum samples of PRRSV were tested?31 dual suspected samples of HPS and PRRSV were also tested?.The results showed that 166 samples were positive for PRRSV and 16samples were positive for both HPS and PRRSV,indicating that clinical HPS and PRRSV coinfections often occur.2.Mechanism of ATP/P2X7 receptor signaling pathway in HPS infected host cellsAfter stimulating PAM cells with HPS?1×10⁷ CFU/mL?,HPS LPS?500 ng/mL?,and E.coli LPS?500 ng/mL?,respectively,PAM cells were treated with ATP?3 mM?,Apyrase?10?M?,and Nigericin?10?M?,respectively.The ELISA,fluorescence quantitative PCR and Western Blot were used to detect the transcription and expression of IL-1?and related proteins from the level of gene transcription and protein expression.The test results indicate that ATP triggers the expression and secretion of IL-1?through P2X7 receptor-mediated signaling pathway,and this process is related to the formation of NLRP3 inflammasome.A-740003 is a competitive P2X7 receptor antagonist that can inhibit the function of P2X7 receptor.After stimulating PAM cells with HPS LPS?500 ng/mL?,PAM cells were treated with different concentrations of A-740003?1?M,10?M,100?M?,and the expressions of IL-1?and related proteins were detected by ELISA and Western Blot.The test results further indicated that ATP could trigger the expression and secretion of IL-1?through P2X7 receptor-mediated signaling pathway,and this process was closely related to the activation of NLRP3 inflammasome but not in a concentration-dependent manner,indicating that ATP/P2X7 receptor-mediated signaling pathway can cause IL-1?expression in HPS infected host cells.3.The relationship between TLR4 and HPS LPS up-regulation of IL-1?After TLR4 was interfered by siRNA,PAM cells were stimulated with LPS of HPS,and the transcription and expression of IL-1?and related proteins were detected from the gene transcription level and protein expression level by fluorescence quantitative PCR,ELISA and Western Blot.The results showed that the expression and secretion of IL-1?were affected by the activation or inhibition of TLR4.After TLR4 was interfered by its siRNA,the expressions of P2X7 receptor and NLRP3were down-regulated,and then the expression of IL-1?was down-regulated.There may be an interaction between TLR4 and the ATP/P2X7 receptor signaling pathway.After the TLR4 gene was silenced,the release of ATP was down-regulated,indicating that TLR4 plays a role in the process of ATP release.The release of ATP triggered by TLR4 indirectly activates the ATP/P2X7 receptor signaling pathway,so the activation of TLR4 also promotes the activation of the ATP/P2X7 receptor signaling pathway.This study provides clinical and theoretical basis for the diagnosis and prevention of HPS,new ideas and evidence for clarifying the signal pathway triggered by HPS,and a basis for drug target of HPS.