TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation
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Obese human and mice were reported to have higher circularity endotoxin (LPS) levels as compared to their lean counter parts. The current study was aimed to reveal the molecular mechanisms underlying the LPS mediated induction of CCL2 in human monocytes/macrophages.
Human monocytic cell line THP-1, THP-1 cells derived macrophages and primary macrophages were treated with LPS and TNF-α (positive control). CCL2 expression was determined with real-time RT-PCR and ELISA. THP-1-XBlue™ cells, THP-1-XBlue™-defMyD cells, TLR4 neutralization antibody, TLR4 siRNA and inhibitors for NF-kB and MAPK were used to study the signaling pathways. Phosphorylation of NF-kB and c-Jun was analyzed by ELISA.
LPS upregulates CCL2 expression at both mRNA (THP-1: 23.40 ± .071 Fold, P < 0.0001; THP-1-derived macrophages: 103 ± 0.56 Fold, < 0.0001; Primary macrophages: 48 ± 1.41 Fold, P < 0.0005) and protein (THP1 monocytes:1048 ± 5.67 pg/ml, P < 0.0001; THP-1-derived macrophages; 2014 ± 2.12, P = 0.0001; Primary macrophages: 859.5 ± 3.54, P < 0.0001) levels in human monocytic cells/macrophages. Neutralization of TLR4 blocked LPS-induced CCL-2 secretion (P < 0.0001). Silencing of TLR4 by siRNA also significantly reduced LPS-induced CCL-2 production. Furthermore, MyD88-Knockout cells treated with LPS did not produce CCL-2. NF-kB and c-Jun phosphorylation was noted in LPS treated cells.
Overall, our data reveal that LPS induces CCL-2 in monocytes/macrophages via TLR4/MyD88 signaling which leads to the activation of NF-kB/AP-1 transcription factors.
American Type Culture Collection
Chemokine (C-C motif) ligand −2
Enzyme-linked immunosorbent assay
Mitogen-activated protein kinase
Myeloid differentiation factor 88
Peripheral blood mononuclear cells
Polymerase chain reaction
Secreted embryonic alkaline phosphatase
A human monocytic cell line
Tumor necrosis factor-alpha
This study was financially supported by Kuwait Foundation for the Advancement of Sciences (KFAS). Grant # RC14016001 (RA-AM-2017-007).
NA performed experiments, analyzed the data and participated in writing the paper. AW, SS, and SK participated in performing experiments. AH contributed in scientific discussions and critically reviewed/edited the manuscript. SA participated in arranging data and preparing graphs. JT critically reviewed and commented the manuscript. SS participated in designing experiments. RA conceived the idea, designed the experiments, analyzed the data, and wrote the manuscript.
This study was supported by funds from Kuwait Foundation for Advancement of Sciences (KFAS), Grant # RC14016001 (RA-AM-2017-007).
Compliance with ethical standards
The authors declare that they have no competing interests.
- 1.Ferrier L, Berard F, Debrauwer L, Chabo C, Langella P, Bueno L, et al. Impairment of the intestinal barrier by ethanol involves enteric microflora and mast cell activation in rodents. Am J Pathol. 2006;168:1148–54.Google Scholar
- 3.Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007;56:1761–72.Google Scholar
- 5.Frost RA, Nystrom GJ, Lang CH. Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle. Am J Phys Regul Integr Comp Phys. 2002;283:R698–709.Google Scholar
- 6.Gustot T, Lemmers A, Moreno C, Nagy N, Quertinmont E, Nicaise C, et al. Differential liver sensitization to toll-like receptor pathways in mice with alcoholic fatty liver. Hepatology. 2006;43:989–1000.Google Scholar
- 7.Song MJ, Kim KH, Yoon JM, Kim JB. Activation of toll-like receptor 4 is associated with insulin resistance in adipocytes. Biochem Biophys Res Commun. 2006Mori M;346:739–45.Google Scholar
- 9.Ahmad R, Al-Mass A, Atizado V, Al-Hubail A, Al-Ghimlas F, Al-Arouj M, et al. Elevated expression of the toll like receptors 2 and 4 in obese individuals: its significance for obesity-induced inflammation. J Inflamm (Lond). 2012;9:48.Google Scholar
- 17.Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest. 2006;116:1494–505.Google Scholar
- 18.Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM, et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes. 2008;57:1470–81.Google Scholar
- 19.Kamei N, Tobe K, Suzuki R, Ohsugi M, Watanabe T, Kubota N, et al. Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance. J Biol Chem. 2006;281:26602–14.Google Scholar
- 20.Lassenius MI, Pietilainen KH, Kaartinen K, Pussinen PJ, Syrjanen J, Forsblom C, et al. Bacterial endotoxin activity in human serum is associated with dyslipidemia, insulin resistance, obesity, and chronic inflammation. Diabetes Care. 2011;34:1809–15.Google Scholar