Nadeem Akhter 1 , Ashraf Madhoun 2 , Hossein Arefanian 1 , Ajit Wilson 1 , Shihab Kochumon 1 , Reeby Thomas 1 , Steve Shenouda 1 , Fahd Al-Mulla 1,2 , Rasheed Ahmad 1 , Sardar Sindhu 2 Show Affiliations »
Abstract
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. METHODS: PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H2O2 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 H2O2 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. RESULTS: 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). H2O2-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. CONCLUSION: 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. © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
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. METHODS: PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H2O2 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 H2O2 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. RESULTS: 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). H2O2 -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. CONCLUSION: 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 . © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
Entities: Chemical
Disease
Gene
Species
Keywords:
Hydrogen peroxide; Metabolic inflammation; Oxidative stress; PBMC; Reactive oxygen species; TLR2; TLR4
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Year: 2019
PMID： 31162913 DOI： 10.33594/000000117
Source DB: PubMed Journal: Cell Physiol Biochem ISSN： 1015-8987