The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways

Abstract

Chronic low-grade inflammation, also known as metabolic inflammation, is a hallmark of obesity and parallels with the presence of elevated circulatory levels of free fatty acids and inflammatory cytokines/chemokines. CCL4/MIP-1β chemokine plays a key role in the adipose tissue monocyte recruitment. Increased circulatory levels of TNF-α, palmitate and CCL4 are co-expressed in obesity. We asked if the TNF-α/palmitate could interact cooperatively to augment the CCL4 production in human monocytic cells and macrophages. THP-1 cells/primary macrophages were co-treated with TNF-α/palmitate and CCL4 mRNA/protein expression was assessed using qRT-PCR/ELISA. TLR4 siRNA, a TLR4 receptor-blocking antibody, XBlue™-defMyD cells and pathway inhibitors were used to decipher the signaling mechanisms. We found that TNF-α/palmitate co-stimulation augmented the CCL4 expression in monocytic cells and macrophages compared to controls (p < 0.05). TLR4 suppression or neutralization abrogated the CCL4 expression in monocytic cells. Notably, CCL4 cooperative induction in monocytic cells was: (1) Markedly less in MyD88-deficient cells, (2) IRF3 independent, (3) clathrin dependent and (4) associated with the signaling mechanism involving ERK1/2, c-Jun, JNK and NF-κB. In conclusion, TNF-α/palmitate co-stimulation promotes the CCL4 expression in human monocytic cells through the mechanism involving a TLR4-MyD88 axis and MAPK/NF-κB pathways. These findings unravel a novel mechanism of the cooperative induction of CCL4 by TNF-α and palmitate which could be relevant to metabolic inflammation.

MIP-1α Expression Induced by Co-Stimulation of Human Monocytic Cells with Palmitate and TNF-α Involves the TLR4-IRF3 Pathway and Is Amplified by Oxidative Stress

“…Thus, several lines of evidence lead to the finding that this cooperative induction of MIP-1α involves the TLR4-mediated signaling. This is in agreement, at least in part, with other reports indicating the involvement of TLR4 in palmitate-induced cytokine/chemokine expression in monocytic cells [ 21 , 40 ].…”

“…On the contrary, MIP-1α transcripts and secreted protein were significantly diminished in IRF3-ablated cells following co-stimulation with palmitate and TNF-α. This implies that the cooperative expression of MIP-1α was IRF3-dependent; which is contrary to MyD88-dependent expression of MIP-1β in THP-1 cells [ 40 ]. The involvement of NF-κB/MAPK signaling pathways was indicated by hyperphosphorylation NF-κB and c-Jun signaling proteins as detected by western blotting.…”

Increased Adipose Tissue Expression of Interferon Regulatory Factor (IRF)-5 in Obesity: Association with Metabolic Inflammation

“…We speculate that the increased expression of these chemokines may play a role in promoting chronic inflammation via the recruitment of activated monocytes/macrophages in the adipose tissue. In support of this argument, TNF-α, IL-6, IL-8, CCL2, CCL4, CCL5, and CCL19 were found to be implicated with metabolic inflammation or insulin resistance in various tissues and organs [21,26,27,28,29,30,31,32]. Our data showing upregulated adipose expression of these chemokines in obese individuals are supported, at least in part, by other studies [28,33,34,35].…”

Elevated adipose tissue associated IL-2 expression in obesity correlates with metabolic inflammation and insulin resistance

“…Relative gene expression to control, lean AT, was calculated using comparative Ct method as we previously described 56,58,59 . Results were normalized to GAPDH, and averages ± standard error of the mean (SEM) are shown relative to controls as indicated 60 .…”