The Effect Of Innate Immune-related Genes Expression In H9N2 Avain Influenza Virus-infected Chicken Macrophage-like Cells(HD-11) In Response To Escherichia Coli LPS Stimulation

H9N2 subtype avian influenza viruses(AIVs) are distributed worldwide, resulting in great economic losses due to reduced egg production or high mortality associated with co-infection with other pathogens. LPS, the principal component of Gram-negative bacteria outer membrane, is an important virulence factor of Escherichia coli. It can activate the innate immune response, mediated mainly by macrophages and dendritic cells. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. This study aims to shed light on the synergistic pathogenesis and underlying mechanisms responsible for the severity of systemic infection and inflammatory damage in low virulent avian H9N2 virus infected avian host caused by pathogenic avian E. coli superinfection. We therefore determine the transcripts of innate immune-related genes following avian H9N2 virus infection and E.coli LPS co-stimulation of avian macrophage-like cell line HD-11 cells.1. For both superinfection groups, similar kinetics of viral RNA expression levels was detected between viruses solely infected control group and viruses infected plus LPS stimulated HD-11 cells group. A marked increase of virus loads was examined from 1 h to 8 h p.i. both in H9N2 virus solely infected cells and in simultaneous virus infection plus LPS stimulation cells.2. More pronounced expression of pro-inflammatory cytokines IL-6, IL-1β and IFN-α as well as the inflammatory chemokines CXCLi1 and CXCLi2 was observed in virus infected plus LPS treated HD-11 cells compared to H9N2 virus solely infected control. Particularly, elevated genes expression was most pronounced for a prior H9N2 infected cells in response to E. coli LPS addition.The genes expression of IL-6 mRNA was observed to be reached at maximum level at 1 h in response to LPS addition as compared to other genes expression that the peak expression levels were seen at 2 h post stimulated with E. coli LPS.3. The maximum expression of IL-10 mRNA and TGF-β3 mRNA in virus infected plus LPS treated HD-11 cells was noticed at 2 hpi. HD-11 cells solely infected with H9N2 AIV showed elevated levels of Anti-inflammatory IL-10. While the genes expression of TGF-β3 mRNA reached maximum at 28 hpi, and then downregulated.4. The mRNA levels of TLR4 and MDA5 in H9N2 infection plus LPS stimulation cells were significantly higher as compared with that either in virus alone or in LPS solely treated cells at most time points examined. Interestingly, in cells infected with H9N2 and stimulated with E. coli LPS, MDA5 mRNA showed maximum expression at 1 h p.i. However,compared to TLR4 gene expression in simultaneously cells infected with H9N2 and stimulated with E. coli LPS that the peak expression levels were seen at 4 h after infection.Collectively, innate immune-related genes respond more pronounced in E. coli LPS treatment of avian H9N2 virus infected HD-11 cells compared to only virus infected or LPS solely stimulated control cells. These results agree with the high mortality observed in the chickens superinfected with low-pathogenicity avian influenza H9N2 virus and pathogenic E. coli.