Haemophilus Parasuis Infection Activates NOD1/2-RIP2 Signaling Pathways In PK-15 Cells

Haemophilus parasuis is the causative agent of Gl?sser’s disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis, causing a signaling response and subsequent the secretion of a series of inflammatory cytokines. In recent decades, Gl?sser’s disease has become one of the most important epidemic diseases of pigs around the world, which causes great economic losses. It is known that H. parasuis is able to invade PK-15 cells, which may be related to the ability of H. parasuis serovar 5 to cause the septicemic spread of Gl?sser’s disease, but the mechanism is poor understood. NOD1/2 signaling pathways play an important role in detecting the intracellular microorganisms, and it could activate the NF-κB and MAPKs pathways through recruiting receptor interacting protein 2(RIP2), causing inflammatory reactions within inflammatory cytokines. We have identified H. parasuis infection contributed to the activation of NF-κB and MAPKs pathway linked to inflammation. In this study, we analysed the relationship between NOD1/2-RIP2 signal pathways and the inflammatory response in PK-15 cells infected by H. parasuis as well as its potential mechanism. The main research works were as following: 1. H. parasuis enhances NOD2 and phospho-RIP2 protein expression in PK-15 cells Western blot assay showed that H. parasuis infection up-regulated the NOD2 protein expression in a time and does dependent manner but the NOD1 protein level is constant. It suggested that NOD2 is involved in the infection by H. parasuis in PK-15 cells. However, NOD1 also have the potential effect in the progress. In addition, H. parasuis enhanced the expression of phospho-RIP2 which is an important adapter molecular of NOD1/2. These results indicated that H. parasuis infection could activate the NOD2-RIP2 sinaling pathway in PK-15 cells, probably as well as NOD1-RIP2 pathway. 2. NOD1/2-RIP2 axis is involved in H. parasuis-induced NF-κB but not MAPKs activation We synthetized certain siRNAs targeting porcine NOD1, NOD2 and RIP2. The results of western blot and luciferase reporter assay showed that knockdown of NOD1, NOD2 and RIP2 significantly decreased H. parasuis-induced phospho-P65 protein level and NF-κB promoter activity, while no change of phospho-P38, phospho-JNK or phospho-ERK protein level could be observed. Theses results suggested that H. parasuis-induced NOD1/2-RIP2 activation could trigger downstream NF-κB signal transduction but not MAPKs. 3. NOD1/2-RIP2 axis is involved in H. parasuis-induced CCL4, CCL5 and IL-8 expression in PK-15 cells PK-15 cells were infected with H. parasuis, followed by transfected with Si-NOD1, Si-NOD2 and Si-RIP2. Real-time PCR results showed that knockdown of NOD1, NOD2 and RIP2 significantly down-regulated the mRNA expression of CCL4, CCL5 and IL-8. It indicated that H. parasuis-induced CCL4, CCL5 and IL-8 secretion in PK-15 cells is related to the activation of NOD1/2-RIP2 pathways. Collectively, in this study, we demonstrate that NOD1/2-RIP2 axis is involved in H. parasuis-induced NF-κB activation and CCL4, CCL5 and IL-8 expression in PK-15 cells. All these results may provide a new mechanism cytologically by which H. parasuis induces a series of inflammatory cytokines, leading to the high mortality of infected pigs.