Regulation of proinflammatory cytokiness by map kinase phosphapatase in avian influenza virus infected chicken macrophages

Abstract

Avian influenza virus (AIV) causes an economically important disease called avian influenza in poultry which pose potential threat to humans worldwide. Mitogen activated protein (MAP) kinases such as extracellular signal regulated kinase (ERK), c-Jun N terminal kinase (JNK) and p38 play an important role in Low pathogenicity Avian Influenza Virus (LPAIV)-infected chicken macrophages to induce host inflammatory responses in controlling viral replication as well as production of proinflammatory cytokines. Activation of MAP kinases is regulated by a group of protein phosphatases known as MAP Kinase phosphatase (MKP) or Dual specificity phosphatases (DUSP). Hence the study of the role of MAPK in the regulation of proinflammatory cytokine secretion from macrophages and the role of MKP in regulating MAP kinase pathway is vital for understanding the pathogenesis of AIV in birds. The first objective of this study was to investigate the role of MKP in the regulation of proinflammatory cytokine in AIV infected chicken macrophages. Chicken macrophage cell line (HTC) was infected with a LPAIV H9N2 and proinflammatory response post infection was investigated using western blotting, real time RT PCR using SYBR green and siRNA transfection to understand the role of MKP in the regulation of proinflammatory response. Our results showed that MKP1 and MKP5 are upregulated in response to AIV infection at 10 h and 20 h p.i. indicating their involvement in the inflammatory response. Subsequent knockdown of MKP1 siRNA in HTC cells followed by realtime RT PCR showed that at 10 h and 20 h post infection the realtime RT PCR data showed that upregulation of proinflammatory cytokine IL-1β was moderate for all three siRNA specific for MKP1 used. Our second objective was to obtain detailed knowledge of the role of MAPK kinases and to differentiate their function in H9N2 infected chicken macrophages through the study of differential gene expression and regulation in HTC cells treated with specific inhibitors against MAP kinase ERK (U0126), p38 (SB203580), JNK (InSolution JNK Inhibitor II) followed by infection with H9N2 virus using Agilent Chicken expression microarray. Microarray data demonstrated differential regulation of a large number of genes among the various study groups. Ingenuity Pathway Analysis (IPA) analysis revealed that in response to H9N2 infection with ERK inhibitor treatment had more effect on the expression of many of the upstream and downstream molecules involved in MAPK signaling pathway, pathway analysis of ERK, STAT3 and NFkB and network of molecules in inflammatory response.