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

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.

Fig. 1

Effect of LPS on CCL2 expression in human monocytic cells and macrophages. THP-1 cells were treated with LPS (10 ng/ml), TNF-α (10 ng/ml; positive control) and PBS Control = Ctrl) for 24 h. Cells and culture media were collected. Total RNA was isolated and CCL2 mRNA was quantified by real time PCR. Relative mRNA expression was expressed as fold expression over average of gene expression in BSA treated cells. LPS significantly increased the expression of CCL2 in THP-1 (A). Secreted CCL2 protein was increased in culture media was determined by ELISA. LPS induced high production of CCL2 compared to control (B). THP-1 cells were converted into macrophages and were treated with LPS or TNF- α for 24 h. Cells and culture media were collected. Real time PCR data showed increased CCL2 mRNA expression in LPS treated macrophages compared to control (C). LPS induced CCL2 protein in culture media (D). Primary macrophages were treated with LPS orTNF- α for 24 h. Cells and culture media were collected. Real time PCR data show that LPS increased expression of CCL2 (E). Secreted CCL2 in culture media was determined by ELISA and which was significantly upregulated by LPS (F). Data are shown as mean ± SEM of three independent experiments

Fig. 2

Inhibition of TLR4 down-regulates the LPS induced CCL2. Antibody-treated cells were stimulated with LPS and incubated for 24 h. Cells and culture media were collected. Real time PCR data showed that neutralization of TLR4 significantly suppress LPS induction of CCL2 (A) and reduced CCL2 protein was determined in the culture media by ELISA (B). TLR4 siRNA transfected THP-1 cells showed reduced expression of TLR4 mRNA compared to the cells transfected with control siRNA (C). LPS induced CCL2 expression was significantly inhibited in TLR4 deficient cells at both mRNA (D) and protein (E) levels

Fig. 3

MyD88 deficiency reduced the LPS induced production of CCL2. THP-1 XBlue defMyD cells (cells deficient in MyD88 activity) were treated with LPS (10 ng/ml) or PBS (Vehicle; control) or TNF-α (10 ng/ml; MyD88 independent stimulus). Cells and culture media were collected after 24 h. Real time PCR and ELISA data showed that LPS failed to induce CCL2 at both mRNA (A) and secreted protein (B) levels. XBlue defMyD cells derived macrophages and were treated with LPS (10 ng/ml) or TNF-α (10 ng/ml). CCL2 gene expression was not induced by LPS in MyD88 deficient cells (C and D)

Fig. 4

LPS activates NF-kB and AP-1 transcription factors. THP- 1 cells were treated with LPS for different time points and cell lysates were prepared as described in methods. Samples were run for Quantikine assay on PathScan Sandwich Elisa kit. Our data showed that phosphorylated NF-kB and c-Jun was seen in LPS treated cells (4A and 4B). THP-1-XBlue cells (THP-1 cells stably expressing a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-κB and AP-1) were treated with LPS or PBSor TNF-α for 24 h. Culture media were collected. Cell culture media were assayed for SEAP reporter activity (degree of NF-κB / AP-1 activation) along with CCL2 production. LPS increased NF-kB/AP-1 activity as compared to control (C and D). Similarly, THP- 1-XBlue™-defMyD cells (Cells deficient in MyD88 activity) were treated with LPS (10 ng/ml) or TNF-α (10 ng/ml) for 24 h. SEAP reporter activity (degree of NF-κB /AP-1 activation) was determined in the cell culture media. MyD88 deficiency inhibits the LPS induced activation of NF-kB/AP-1 (E). The results obtained from three independent experiments are shown. The data are presented as mean ± SD