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

Journal of Diabetes & Metabolic Disorders volume 17, pages 77–84 (2018)Cite this article

Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

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.

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Abbreviations

AP-1:

Activating protein-1

ATCC:

American Type Culture Collection

CCL-2:

Chemokine (C-C motif) ligand −2

ELISA:

Enzyme-linked immunosorbent assay

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

LPS:

Lipopolysaccharide

MAPK:

Mitogen-activated protein kinase

MyD88:

Myeloid differentiation factor 88

NF-κB:

Nuclear factor-kappaB

PBMC:

Peripheral blood mononuclear cells

PCR:

Polymerase chain reaction

SEAP:

Secreted embryonic alkaline phosphatase

THP-1:

A human monocytic cell line

TLRs:

Toll-like receptors

TNF-α:

Tumor necrosis factor-alpha

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Acknowledgments

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

Funding

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

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Affiliations

  1. Immunology Unit, Dasman Diabetes Institute (DDI), Al-Soor Street, P.O. Box 1180, 15462, Dasman, Kuwait

    Nadeem Akhter, Amal Hasan, Steve Shenouda, Ajit Wilson, Shihab Kochumon, Shamsha Ali, Jaakko Tuomilehto, Sardar Sindhu & Rasheed Ahmad

Authors
  1. Nadeem Akhter
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  2. Amal Hasan
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  3. Steve Shenouda
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  4. Ajit Wilson
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  5. Shihab Kochumon
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  6. Shamsha Ali
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  7. Jaakko Tuomilehto
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  8. Sardar Sindhu
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  9. Rasheed Ahmad
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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.

Corresponding author

Correspondence to Rasheed Ahmad.

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Competing interests

The authors declare that they have no competing interests.

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Akhter, N., Hasan, A., Shenouda, S. et al. TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation. J Diabetes Metab Disord 17, 77–84 (2018). https://doi.org/10.1007/s40200-018-0341-y

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  • DOI: https://doi.org/10.1007/s40200-018-0341-y

Keywords

  • CCL2 Production
  • Metabolic Inflammation
  • TLR4 siRNA
  • CCL2 Gene Expression
  • Primary Macrophages