Modulation of the innate immune-related genes expression in H9N2 avian influenza virus-infected chicken macrophage-like cells (HD11) in response to Escherichia coli LPS stimulation

•E. coli LPS markedly induced proinflammatory-related genes in H9N2 AIV prior infected HD11 cells.•E. coli LPS stimulation may play a key role in regulating IL-6 gene expression in HD11 cells.•TLR4 expression respond more pronounced to H9N2 AIV sole infection compared to virus infection plus LPS stimulation.•Prior or simultaneously H9N2 AIV infection rapidly induces the iNOS expression in HD11 stimulated with LPS.

Macrophages play important roles in mediating virus-induced innate immune responses and are thought to be involved in the pathogenesis of bacterial superinfections. The innate immune response initiated by both low pathogenicity AIV and bacterial superinfection in their avian host is not fully understood. We therefore determine the transcripts of innate immune-related genes following avian H9N2 AIV virus infection and E. coli LPS co-stimulation of avian macrophage-like cell line HD11 cells. More pronounced expression of pro-inflammatory cytokines (IL-6 and IL-1β) as well as the inflammatory chemokines (CXCLi1 and CXCLi2) was observed in virus infected plus LPS treated HD11 cells compared to H9N2 virus solely infected control. For two superinfection groups, the levels of genes examined in a prior H9N2 virus infection before secondary LPS treatment group were significantly higher as compared with simultaneous virus infection plus LPS stimulation group. Interestingly, similar high levels of IL-6 gene were observed between LPS sole stimulation group and two superinfection groups. Moreover, IL-10 and TGF-β3 mRNA levels in both superinfection groups were moderately upregulated compared to sole LPS stimulation group or virus alone infection group. Although TLR4 and MDA5 levels in virus alone infection group were significantly lower compared to that in both superinfection groups, TLR4 upregulation respond more rapid to virus sole infection compared to LPS plus virus superinfection. Collectively, innate immune-related genes respond more pronounced in LPS stimulation plus H9N2 virus infection HD11 cells compared to sole virus infection or LPS alone stimulation control cells.