School of Biomedical Sciences, Faculty of Medicine & Dentistry, University of Plymouth, Drake Circus, Plymouth, United Kingdom.
College of Medicine, University of Hail, Hail, Kingdom of Saudi Arabia.
PLoS One. 2019 May 23;14(5):e0214681. doi: 10.1371/journal.pone.0214681. eCollection 2019.
Macrophages (Mϕs) play a central role in mucosal immunity by pathogen sensing and instruction of adaptive immune responses. Prior challenge to endotoxin can render Mφs refractory to secondary exposure, suppressing the inflammatory response. Previous studies demonstrated a differential subset-specific sensitivity to endotoxin tolerance (ET), mediated by LPS from the oral pathogen, Porphyromonas gingivalis (PG). The aim of this study was to investigate ET mechanisms associated with Mφ subsets responding to entropathogenic E. coli K12-LPS. M1- and M2-like Mφs were generated in vitro from the THP-1 cell line by differentiation with PMA and Vitamin D3, respectively. This study investigated ET mechanisms induced in M1 and M2 Mφ subsets, by measuring modulation of expression by RT-PCR, secretion of cytokines by sandwich ELISA, LPS receptor, TLR4, as well as endogenous TLR inhibitors, IRAK-M and Tollip by Western blotting. In contrast to PG-LPS tolerisation, E. coli K12-LPS induced ET failed to exhibit a subset-specific response with respect to the pro-inflammatory cytokine, TNFα, whereas exhibited a differential response for IL-10 and IL-6. TNFα expression and secretion was significantly suppressed in both M1- and M2-like Mφs. IL-10 and IL-6, on the other hand, were suppressed in M1s and refractory to suppression in M2s. ET suppressed TLR4 mRNA, but not TLR4 protein, yet induced differential augmentation of the negative regulatory molecules, Tollip in M1 and IRAK-M in M2 Mφs. In conclusion, E. coli K12-LPS differentially tolerises Mφ subsets at the level of anti-inflammatory cytokines, associated with a subset-specific divergence in negative regulators and independent of TLR4 down-regulation.
巨噬细胞（Mϕs）通过病原体感应和指导适应性免疫反应，在黏膜免疫中发挥核心作用。先前的内毒素挑战可以使 Mφ 对二次暴露产生抗性，从而抑制炎症反应。先前的研究表明，口腔病原体牙龈卟啉单胞菌（PG）的 LPS 介导了对内毒素耐受（ET）的不同亚群特异性敏感性。本研究旨在研究与肠道致病菌大肠杆菌 K12-LPS 反应的 Mφ 亚群相关的 ET 机制。通过用 PMA 和维生素 D3 分别分化，从 THP-1 细胞系中体外生成 M1 和 M2 样 Mφ。本研究通过 RT-PCR 测量表达的调节、夹心 ELISA 测量细胞因子的分泌、LPS 受体、TLR4 以及内源性 TLR 抑制剂 IRAK-M 和 Tollip 的 Western 印迹来研究 M1 和 M2 Mφ 亚群中诱导的 ET 机制。与 PG-LPS 耐受化相反，大肠杆菌 K12-LPS 诱导的 ET 未能表现出对促炎细胞因子 TNFα 的亚群特异性反应，而对 IL-10 和 IL-6 则表现出不同的反应。TNFα 的表达和分泌在 M1 和 M2 样 Mφ 中均受到显著抑制。另一方面，IL-10 和 IL-6 在 M1 中受到抑制，而在 M2 中则不受抑制。ET 抑制 TLR4 mRNA，但不抑制 TLR4 蛋白，但诱导负调节分子 Tollip 在 M1 和 IRAK-M 在 M2 Mφ 中的差异扩增。总之，大肠杆菌 K12-LPS 在抗炎细胞因子水平上对 Mφ 亚群进行差异耐受，与负调节分子的亚群特异性差异以及 TLR4 下调无关。
