TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation

  • Nadeem Akhter
  • Amal Hasan
  • Steve Shenouda
  • Ajit Wilson
  • Shihab Kochumon
  • Shamsha Ali
  • Jaakko Tuomilehto
  • Sardar Sindhu
  • Rasheed Ahmad
Research Article



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.



Activating protein-1


American Type Culture Collection


Chemokine (C-C motif) ligand −2


Enzyme-linked immunosorbent assay


Glyceraldehyde-3-phosphate dehydrogenase




Mitogen-activated protein kinase


Myeloid differentiation factor 88


Nuclear factor-kappaB


Peripheral blood mononuclear cells


Polymerase chain reaction


Secreted embryonic alkaline phosphatase


A human monocytic cell line


Toll-like receptors


Tumor necrosis factor-alpha



This study was financially supported by Kuwait Foundation for the Advancement of Sciences (KFAS). Grant # RC14016001 (RA-AM-2017-007).

Author contributions

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

Competing interests

The authors declare that they have no competing interests.


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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Nadeem Akhter
    • 1
  • Amal Hasan
    • 1
  • Steve Shenouda
    • 1
  • Ajit Wilson
    • 1
  • Shihab Kochumon
    • 1
  • Shamsha Ali
    • 1
  • Jaakko Tuomilehto
    • 1
  • Sardar Sindhu
    • 1
  • Rasheed Ahmad
    • 1
  1. 1.Immunology UnitDasman Diabetes Institute (DDI)DasmanKuwait

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