title: Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines.
abstract: As a critical adapter protein in Toll-like receptor (TLR)/Interleukin (IL)-1R signalling pathway, myeloid differentiation protein 88 (MyD88) plays an important role in immune responses and host defence against pathogens. The present study was designed to provide a foundation and an important reagent for the mechanistic study of MyD88 and its role TLR/IL-1R signalling pathways in porcine immunity. Lentivirus-mediated RNAi was used to generate a porcine PK15 cell line with a silenced MyD88 gene and quantitative real-time PCR (qPCR) and Western blotting were used to detect changes in the expression of critical genes in the Toll-like receptor 4 (TLR4) signalling pathway. ELISA was used to measure the levels of seven proinflammatory cytokines-interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β-in cell culture supernatants after MyD88 silencing. We successfully obtained a PK15 cell line with 61% MyD88 mRNA transcript down-regulated. In PK15 cells with MyD88 silencing, the transcript levels of TLR4 and IL-1β were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-α (IFN-α) or TNF-α The ELISA results showed that the levels of most cytokines were not significantly changed apart from IL-8 without stimulation, which was significantly up-regulated. When cells were induced by lipopolysaccharide (LPS) (0.1 μg/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1β, TNF-α, IL-6, IL-8 and IL-12 of Blank and negative control (NC) group up-regulated more significantly than RNAi group (P<0.05), which revealed that the MyD88 silencing could reduce the TLR4 signal transduction which inhibited the release of proinflammatory cytokines and finally leaded to immunosuppression.
authors.affiliation: Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
authors.forename: Chaohui
authors.initials: C
authors.lastname: Dai
authors.affiliation: Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
authors.forename: Lihuai
authors.initials: L
authors.lastname: Yu
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
authors.forename: Shenglong
authors.initials: S
authors.lastname: Wu
authors.affiliation: Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China wbbao@yzu.edu.cn.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Bao
mesh_terms: D000818:Animals; D002460:Cell Line; D054407:Chemokine CCL4 / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000378:metabolism*; D007249:Inflammation / Q000378:metabolism*; D007378:Interleukins / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D000081207:Primary Immunodeficiency Diseases; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
chemical_list: D054407:Chemokine CCL4; D007378:Interleukins; D018950:Lipopolysaccharide Receptors; D053594:Myeloid Differentiation Factor 88; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha
keywords: Toll-like receptor (TLR)/Interleukin (IL)-1R pathway; gene silencing; myeloid differentiation protein 88 (MyD88); pig; proinflammatory cytokines