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

Chaohui Dai,Shenglong Wu,Guoqiang Zhu,Li Sun,Lihuai Yu,Wenbin Bao

doi:10.1042/bsr20160170

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.

Highlights

Toll-like receptors (TLRs) were found from Drosophila Toll proteins, in addition to participating in regulating the polarity formation of embryo flies back ventral, the receptors directly mediate flies’ natural immune response to microbial infection
Toll-like receptor 4 (TLR4) signalling pathway is the essential member in TLRs family, which plays an important role in a variety of inflammatory reaction especially in diarrhoea and hydropsy of weaned piglets infected by Abbreviations: CD14, cluster of differentiation antigen 14; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; IFN-α, interferon-α; IFN-β, interferon-β; IL, Interleukin; IRAK, Interleukin receptor associated kinase; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; MyD88, myeloid differentiation protein 88; negative control (NC), Negative control; NF-κB, Nuclear factor-κB; qPCR, quantitative real-time PCR; suppressor of cytokine signaling 3 (SOCS3), Suppressor of cytokine signaling 3; signal transducer and activator of transcription 3 (STAT3), Signal transducer and activator of transcription 3; SYBR, Synergy Brands, Inc; TIR, Toll/Interleukin-1 receptor; TLR, Toll-like receptor; TLR4, Toll-like receptor 4; TNF-α, tumour necrosis factor-α; TRAF-6, TNF receptor associated factor-6
We found that the interference efficacy of pcDNA6.2-GW/EmGFPshRNA-1 was the highest whose relative expression level of porcine MyD88 of 0.36 with an interference efficacy of 0.64 and the Western blot results confirmed the interference effect (Figure 1), so we used this vector for lentivirus packaging

Summary

Introduction

Toll-like receptors (TLRs) were found from Drosophila Toll proteins, in addition to participating in regulating the polarity formation of embryo flies back ventral, the receptors directly mediate flies’ natural (inherent) immune response to microbial infection. There are 13 members in the TLRs family identified in mammals, which formed a huge type I transmembrane receptor protein family. Research showed that TLRs played an important role in defence mechanism of activating innate immunity and inducting adaptive immune, especially in innate immunity congenital immunity [1,2]. Toll-like receptor 4 (TLR4) signalling pathway is the essential member in TLRs family, which plays an important role in a variety of inflammatory reaction especially in diarrhoea and hydropsy of weaned piglets infected by Licence 4.0 (CC BY). The TLR4 signalling pathway transmits signals through both myeloid differentiation protein 88 (MyD88)-dependent and -independent pathways. The functional analysis of the MyD88 gene is conductive to characterize the molecular mechanism that leads to diarrhoea and hydropsy of weaned piglets infected by pathogenic E. coli

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Conclusion