Transcription Analysis On Response Of Porcine Alveolar Macrophage Cells To Haemophilus Parasuis

Haemophilus parasuis is an opportunistic pathogen that colonizes in the upper respiratory tract of the pig and can cause Gl?sser disease,under certain conditions,such as stress,and coinfection with other immunosuppressive pathogens,characterized by fibrinous polyserositis,arthritis and meningitis and bronchopneumonia,which are leading to severe economic losses in the swine production.Porcine alveolar macrophages as an essential component of innate immune cells,which often mediate phagocytosis,produce reactive oxygen species and nitric oxide to kill pathogens,and release some cytokines and chemokines to promote recruitment of neutrophils and other immune cells to the site of infection for playing immune function.In this study,from the perspective of host cell infected with Haemophilus parasuis,RNA-seq technique was firstly used to analyze the transcriptional profiles of macrophages infected with Haemophilus parasuis.The main research results are as follows:1.The comparison of H.parasuis SH0165 and HPS1712 resistance to phagocytes and induction cytokine expressionH.parasuis SH0165 and HPS1712 strain were interacted with the porcine alveolar macrophages cell 3D4/21 for 8h.The results showed that the phagocytosis rate of HPS1712 was significantly higher than in SH0165,which indicated that resistant phagocytic capacity of SH0165 was stronger than HPS1712.The 3D4/21 cells were incubated with SH0165 and HPS1712 with a multiplicity of infection(MOI)of 10 for different time,and then detected the m RNA expression of cytokines.The expression of IL-1?,IL-6,IL-8 and IL-10 in SH0165 infected group were higher than that in HPS1712 infected group of 8h.The expression of IL-1? and IL-6 in HPS1712 infected group were greater than in SH0165 infected group at 12 h and 24 h.We found the infectious dose of SH0165 and HPS1712 were at the moi of 10 by phagocytosis and the detection expression of cytokines.2.Transcription analysis on response of porcine alveolar macrophage 3D4/21 cells infected with H.parasuisIn this study,porcine alveolar macrophages 3D4 / 21 were infected with H.parasuis SH0165,Nagasaki,SW114 and HPS1712 at the moi of 10 in vitro.The results of whole transcription expression analysis showed that SH0165,Nagasaki,SW114 and HPS1712 infected-groups were 542,502,662 and 37 differentially expressed genes,including 283,336,359,34 up-regulated genes and 259,166,303,3 down-regulated genes respectively,and further functional clustering analysis of these differentially expressed genes.3.The biological processes gene ontology and KEGG pathway enrichment analysis of differentially expressed genesThe annotation of differentially expressed genes and the biological process gene ontology,KEGG pathway enrichment analysis revealed that these genes were mainly associated with transcriptional factors,gene expression,cell apoptosis,cell differentiation,inflammatory response,immune response,signal transduction.Pathway analysis showed that the main pathways include: cytokine-cytokine receptors interaction,apoptosis,chemokine signaling pathway,NF-k B and MAPK signal pathway,the p53 signal pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway and so on.By analyzing the differential expression genes in these pathways,we found that the host took different strategies to activate the inflammatory response and the immune response against Haemophilus parasuis infection.Particularly,the differentially expressed genes of the virulent strains are mainly related to the biological processes of macrophage biological functions such as cytokines and chemokines production,transcriptional regulators,zinc ion binding,arginase activity,and the like.The differential expressed genes of the attenuated strains were mainly related to transcriptional regulators,signal transduction,phagosome formation and other functions.4.q RT-PCR validate the RNA-seq dataIn order to verify the accuracy of RNA-seq data,we used fluorescence quantitative PCR to validate the genes in diverse signaling pathways,especially about cytokine and cytokine receptors interaction,which contain 22 up-regulated genes and 6 down-regulated genes.Fluorescence Quantification PCR results are consistent with RNA-seq results in the trend,indicating that RNA-seq results were reliable.5.Biological function of IL-19The detection of expression of IL-19 m RNA and protein level in 3D4/21 cells infected with Haemophilus parasuis and si RNA and over expression experiments and piglet infection.It was discovered and confirmed that IL-19 was associated with Haemophilus parasuis infection and could inhibit IL-1? and IL-8.