Objectives: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density-Lipoproteins (HDL) has potent anti-inflammatory... Show moreObjectives: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density-Lipoproteins (HDL) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain ill defined.Methods: Human and murine macrophages were pre-incubated with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL.Results: HDL pre-incubation significantly decreased LPS-induced anti-inflammatory IL-10 production, while the opposite was observed for the pro-inflammatory mediators IL-12 and TNF. We show that these effects are mediated by passive cholesterol depletion and lipid raft disruption, without involvement of ABCA1, ABCG1, SR-BI or CD36. These pro-inflammatory effects are confirmed in vivoin peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. Native and reconstituted HDL enhances Toll-Like-Receptor-induced signaling by activating protein kinase C (PKC), since inhibition of PKC ablated the observed HDL effects. Using macrophages from NF-κB luciferase mice, we observed that HDL induces NF-κB activation. Western blot analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice, we show that the observed HDL effects are independent of IKK, NIK and CKII. Furthermore, we observed that STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12. On the other hand, we show that HDL enhances ADAM protease activity, thereby mediating TNF-α release.Conclusions: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC-NF-kB/STAT1. These pro-inflammatory activities on macrophages could at least partly underlie the disappointing therapeutic potential of HDL raising therapy in current cardiovascular clinical trials. Show less
Vorst, E.P.C. van der; Theodorou, K.; Hoeksema, M.A.; Wu, Y.; Goossens, P.; Eck, M. van; ... ; Donners, M.M.P. 2016
Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various... Show moreMembrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.KEYWORDS: bacterial infection; high-density lipoproteins; immune response; inflammation; inflammatory signaling; macrophages; passive cholesterol depletion Show less
