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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-1? (IL-1?), tumour necrosis factor-? (TNF-?), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1? and MIP-1?-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-1? were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-? (IFN-?) or TNF-? 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 ?g/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1?, TNF-?, 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.

SUBMITTER: Dai C 

PROVIDER: S-EPMC5293583 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines.

Dai Chaohui C   Sun Li L   Yu Lihuai L   Zhu Guoqiang G   Wu Shenglong S   Bao Wenbin W  

Bioscience reports 20161117 6


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 an  ...[more]

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