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

Metabolic inflammation is associated with increased expression of saturated free fatty acids, proinflammatory cytokines, chemokines, and adipose oxidative stress. Macrophage inflammatory protein (MIP)-1 alpha recruits the inflammatory cells such as monocytes, macrophages, and neutrophils in the adipose tissue; however, the mechanisms promoting the MIP-1 alpha expression remain unclear. We hypothesized that MIP-1 alpha co-induced by palmitate and tumor necrosis factor (TNF)-alpha in monocytic cells/macrophages could be further enhanced in the presence of reactive oxygen species (ROS)-mediated oxidative stress. To investigate this, THP-1 monocytic cells and primary human macrophages were co-stimulated with palmitate and TNF-alpha and mRNA and protein levels of MIP-1 alpha were measured by using quantitative reverse transcription, polymerase chain reaction (qRT-PCR) and commercial enzyme-linked immunosorbent assays (ELISA), respectively. The cognate receptor of palmitate, toll-like receptor (TLR)-4, was blunted by genetic ablation, neutralization, and chemical inhibition. The involvement of TLR4-downstream pathways, interferon regulatory factor (IRF)-3 or myeloid differentiation (MyD)-88 factor, was determined using IRF3-siRNA or MyD88-deficient cells. Oxidative stress was induced in cells by hydrogen peroxide (H2O2) treatment and ROS induction was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. The data show that MIP-1 alpha gene/protein expression was upregulated in cells co-stimulated with palmitate/TNF-alpha compared to those stimulated with either palmitate or TNF-alpha (P< 0.05). Further, TLR4-IRF3 pathway was implicated in the cooperative induction of MIP-1 alpha in THP-1 cells, and this cooperativity between palmitate and TNF-alpha was clathrin-dependent and also required signaling through c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B). Notably, ROS itself induced MIP-1 alpha and could further promote MIP-1 alpha secretion together with palmitate and TNF-alpha. In conclusion, palmitate and TNF-alpha co-induce MIP-1 alpha in human monocytic cells via the TLR4-IRF3 pathway and signaling involving c-Jun/NF-kappa B. Importantly, oxidative stress leads to ROS-driven MIP-1 alpha amplification, which may have significance for metabolic inflammation.