The Mechanism Of Inflammatory Response Induced By Haemophilus Parasuis Serotype 5 Infection In 3D4/21

Haemophilus parasuis(HPS)can cause the swine Glasser’s disease.It is one of the main bacterial pathogens that has seriously harmed the development of the pig industry in recent years and has brought huge economic losses to the pig industry.HPS mainly colonizes in the upper respiratory tract of pigs.However,under certain circumstances,such as stimulate of enviroment and the decline of porcine immunity,HPS can cause pulmonary infections,leading a strong inflammatory response,resulting in lung injury.Then the bacteria can infect other tissues and organs through the blood circulation,and further causing arthritis,polyserositis,and meningitis,eventually leading to septicemia or death.The strong inflammatory response caused by HPS infection of the lungs is the main cause of systemic infection and death of pigs,but the specific mechanism of its causing a strong inflammatory response is unclear.In this study,an in vitro infection model of HPS was established with porcine alveolar macrophages cell line 3D4/21.These studies explored the specific mechanism of severe pulmonary inflammation caused by H.parasuis infection,and laid a foundation for further elucidating the pathogenic mechanism of H.parasuis.1.Establishment of porcine alveolar macrophage model through Haemophilus parasuis infectionIn this study,HPS5-SQ strains can cause lung inflammation and lung injury in piglets;HPS5-SQ strains with different multiplicity of infection(MOI =1,10,100)were used to infect pig alveolar macrophages 3D4/21,respectively.An in vitro infection model of HPS was established through Giemsa staining,adhesion test,invasion assay and lactate dehydrogenase(LDH)cytotoxicity detection test.Giemsa staining experiment showed that HPS5-SQ can invade 3D4/21 cells,the number of bacteria adhered to the cell surface and the intracellular number of bacteria are dose-dependent.In adhesion and invasion tests,we found that when the multiplicity of infection was MOI=10,100,the adhesion and invasion ability of HPS5-SQ to 3D4/21 cells was significantly higher than that of MOI=1;when the infection time was 2h,HPS5-SQ had the strongest adhesion and invasion ability to 3D4/21 cells.Lactate dehydrogenase(LDH)cytotoxicity detection test illustrated that when the MOI of HPS5-SQ was 10,no cytotoxicity is shown to infected cells within 12 hours.When MOI=100,the infected cells showed obvious cytotoxicity,so MOI=10 was selected as the multiplicity of infection for the test.In this study,an in vitro infection model of HPS infected porcine alveolar macrophages 3D4/21 was initially established,which laid the foundation for studying the mechanism of HPS-induced inflammation of alveolar macrophages.2.Effect of Haemophilus parasuis serotype 5 infection on immune response of porcine alveolar macrophages 3D4/21Alveolar macrophages are the earliest immune cells in the lungs that contact with pathogens.They are involved in phagocytosis and killing of various pathogens,processing and presenting exogenous antigens,and secreting a variety of cytokines to mediate inflammation.Porcine alveolar macrophage is one of the target cell of HPS infection,and the large amount of inflammatory cytokines produced during HPS infection is a key factor in lung tissue injury.In this study,HPS5-SQ with MOI=10 was used to infect 3D4/21 cells for3,6,12 and 24 hours.The effect of Haemophilus parasuis type 5 infection on the immune response of porcine alveolar macrophages 3D4/21 was detected by fluorescence quantitative PCR test and DCFH-DA fluorescence staining method.The results showed that there were significantly increases in the transcriptional level of TLR2 receptors and proinflammatory cytokines such as TNF-α,IL-1β and IL-18,while the antigen-presenting molecule MHC Ⅰand the costimulatory molecules CD80,CD86 did not change significantly after HPS5-SQ infection of 3D4/21 cells;the transcription level of the costimulatory molecule CD40 was up-regulated at 3h and 6h after HPS5-SQ infection,but when the infection time was extended to 12 h and 24 h,the transcription level was down-regulated and there was no significant difference compared with the control group.In addition,this experiment found that after HPS5-SQ infection of 3D4/21 cells,the transcription level of antigen-presenting molecule MHC Ⅱ was down-regulated,and with the prolongation of infection time,its transcription level gradually decreased,this phenomenon was extremely significant at 12 h and 24 h.This result indicates that the infection of HPS5-SQ may inhibit the antigen presenting function of macrophages.At the same time,it was found that HPS5-SQ infection can cause a significant increase of ROS in 3D4/21 cells.3.The effect of HPS5-SQ infection on inflammatory signal pathway in porcine alveolar macrophage 3D4/21NLRP3 inflammasome plays an important role in the inflammatory response caused by a variety of pathogen infections.Preliminary experiments found that 12 hours after HPS5-SQ infection of 3D4/21 cells,the transcription levels of inflammatory cytokines and intracellular ROS were up-regulated most obviously.In this study,HPS5-SQ with MOI=10was used to infect 3D4/21 cells for 12 hours,and the effect of HPS5-SQ infection in 3D4/21 cells on inflammatory response-related signal pathways were detected by fluorescence quantitative PCR,Western blot and DCFH-DA fluorescence staining.The results showed that HPS5-SQ infection of 3D4/21 cells can activate the NLRP3 inflammasome signaling pathway and participate in the up-regulation of cytokine IL-1β.At the same time,the pretreatment of 3D4/21 cells with ROS scavenger(NAC)can significantly down-regulate the transcription level of proinflammatory cytokines caused by HPS5-SQ infection.Further studies found that the pretreatment cells with NAC can also inhibit the expression of NLRP3 inflammasomes,significantly down-regulate the transcription of NLRP3,and dramatically reduce the expression of cleaved-caspase-1 protein.The above results indicate that the ROS produced by HPS5-SQ infected 3D4/21 cells can participate in the activation of the NLRP3 inflammasome signaling pathway,up-regulate the expression of inflammatory factors,and aggravate the inflammatory response.