Akhter Nadeem, Madhoun Ashraf, Arefanian Hossein, Wilson Ajit, Kochumon Shihab, Thomas Reeby, Shenouda Steve, Al-Mulla Fahd, Ahmad Rasheed, Sindhu Sardar
Department of Microbiology & Immunology, Dasman Diabetes Institute (DDI), Dasman, Kuwait.
Animal and Imaging Core Facility, Dasman Diabetes Institute (DDI), Dasman, Kuwait.
Cell Physiol Biochem. 2019;53(1):1-18. doi: 10.33594/000000117.
BACKGROUND/AIMS: Innate immune toll-like receptors (TLRs) are emerging as nutrient sensors. Oxidative stress in the adipose tissue in obesity acts as a critical early trigger of altered pathophysiology. TLR2/TLR4 adipose upregulation has been associated with insulin resistance in humans; however, it remains unclear whether oxidative stress can modulate expression of TLR2/4 and related immune-metabolic regulators (IRF3/5) in immune cells. We, therefore, assessed their expression along with proinflammatory cytokines in the human PBMC following induction of oxidative stress.
PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with HO to induce oxidative stress. ROS was measured by DCFH-DA assay. Target gene and protein expression was determined using real-time RT-PCR and flow cytometry/confocal microscopy, respectively. TLR2/4 expression by HO in presence of ROS-inhibitors or leptin/LPS/fatty acids was also assessed. Expression of phosphorylated/total ERK1/2, c-Jun, p38, and NF-κB was determined by western blotting. The data (mean±SEM) were compared using unpaired student's t-test or ANOVA; all P-values <0.05 were considered significant.
TLR2/4 mRNA/protein expression was elevated by oxidative stress in PBMC compared to controls (P<0.001). This induction was abrogated by apocynin/N-acetyl cysteine treatments (P<0.01). HO-induced TLR2/4 gene expression was further enhanced by leptin, LPS, oleate, or palmitate (P<0.05). Oxidative stress also promoted expression of IRF3/5 and proinflammatory cytokines including IFN-γ, IL-1β, IL-6, TNF-α, and MCP-1/CCL2. This oxidative stress in PBMC involved MAPK/NF-κB dependent signaling.
Taken together, oxidative stress upregulates expression of TLR2/4, IRF3/5 and signature proinflammatory cytokines in PBMC, involving MAPK/NF-κB dependent signaling, all of which may have implications for metabolic inflammation.
背景/目的：天然免疫Toll样受体（TLRs）正逐渐成为营养传感器。肥胖患者脂肪组织中的氧化应激是病理生理学改变的关键早期触发因素。TLR2/TLR4在脂肪组织中的上调与人类胰岛素抵抗有关；然而，氧化应激是否能调节免疫细胞中TLR2/4及相关免疫代谢调节因子（IRF3/5）的表达仍不清楚。因此，我们评估了氧化应激诱导后人外周血单个核细胞（PBMC）中它们的表达以及促炎细胞因子的表达。
采用Ficoll-Paque法从健康供者血液中分离PBMC，并用HO处理细胞以诱导氧化应激。通过DCFH-DA测定法检测活性氧（ROS）。分别使用实时逆转录聚合酶链反应（RT-PCR）和流式细胞术/共聚焦显微镜测定靶基因和蛋白表达。还评估了在存在ROS抑制剂或瘦素/LPS/脂肪酸的情况下HO对TLR2/4的表达。通过蛋白质印迹法测定磷酸化/总细胞外信号调节激酶1/2（ERK1/2）、c-Jun、p38和核因子κB（NF-κB）的表达。使用不成对学生t检验或方差分析比较数据（平均值±标准误）；所有P值<0.05被认为具有统计学意义。
与对照组相比，氧化应激使PBMC中TLR2/4的mRNA/蛋白表达升高（P<0.001）。阿朴吗啡/N-乙酰半胱氨酸处理可消除这种诱导作用（P<0.01）。瘦素、LPS、油酸或棕榈酸可进一步增强HO诱导的TLR2/4基因表达（P<0.05）。氧化应激还促进了IRF3/5和促炎细胞因子的表达，包括干扰素-γ（IFN-γ）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化蛋白-1/CCL2。PBMC中的这种氧化应激涉及丝裂原活化蛋白激酶（MAPK）/NF-κB依赖性信号传导。
综上所述，氧化应激上调PBMC中TLR2/4、IRF3/5和标志性促炎细胞因子的表达，涉及MAPK/NF-κB依赖性信号传导，所有这些可能对代谢性炎症有影响。
