High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling

Abstract

Background: Membrane cholesterol is known to modulate a variety of cell signaling pathways and functions. While cholesterol depletion by High-Density Lipoproteins (HDL) has potent antiinflammatory effects in various cell types, its effect on inflammatory responses in macrophages remain ill defined. Methods & Results: Pre-incubation of human and murine macrophages in vitro with human reconstituted (apolipoproteinA-I/phosphatidylcholine) or native HDL 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-B1 or CD36. These pro-inflammatory effects are confirmed in vivo in peritoneal macrophages from ApoA-I transgenic mice, which have high circulating HDL levels. In line, innate responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low 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 microarray analysis and macrophages from NF-kB luciferase mice, we observed that HDL induces NF-kB activation. Western blot and ChIP-PCR analyses showed that in particular the p65 subunit was activated. Using specific knock-out mice for the upstream activation pathways, we show that the observed HDL effects are independent of the upstream kinases IKK, NIK and CKII. Furthermore, using STAT1 knock-out mice we observed that also STAT1 is involved in the pro-inflammatory HDL effects on IL-10 and IL-12 secretion. On the other hand, using pharmacological inhibitors, we show that HDL enhances ADAM protease activity, thereby mediating TNF release. Conclusion and Clinical Relevance: HDL exerts pro-inflammatory effects on macrophages via passive cholesterol depletion by activation of PKC, NF-kB and 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.

Disease- or Storage-Associated Structural Modifications Are Unlikely to Explain HDL Pro-inflammatory Effects on Macrophages

“…(1) Macrophage model: Suzuki et al -111), which was attributed to Atf3 upregulation, albeit at much higher HDL concentrations than used in our study (up to 2,000 mg/mL). We confirmed that rHDL composed of soybean-derived phospholipids reduced IL-12 and IL-10 production, without affecting TNF-a secretion and Atf3 expression in macrophages (0-300 mg/mL HDL range) (van der Vorst et al, 2017). As it has already been shown by Kang et al (2005) that several soybean components have anti-inflammatory activities in macrophages, the observed antiinflammatory effects observed by De Nardo et al may be derived from soybean constituents of their rHDL preparation.…”

“…High-density lipoproteins (HDLs) have been attributed to overt anti-inflammatory functions in a range of cell types, including endothelial cells (Bursill et al, 2010) and smooth muscle cells (van der Vorst et al, 2013). In our recent publication, we have shown that HDLs exert pro-inflammatory actions on macrophages, both in vitro and in vivo, and unraveled the underlying mechanisms and signaling pathways (van der Vorst et al, 2017). These findings swirled up discussions about HDL, particularly as these findings, although concordant with other reports (Smoak et al, 2010), seemed at odds with studies showing largely anti-inflammatory effects of HDL on innate immune cells.…”

Lipid and Non-lipid Factors Affecting Macrophage Dysfunction and Inflammation in Atherosclerosis

“…MyD88 −/− pMOs were also used to show ApoA-I induces release of Il-1α, Il-1β, Il-6, Tnf-α, Cxcl1, Cxcl2, and Ccl2 (De Nardo et al, 2014 ). The second, very recent assessment of HDL revealed that its pro-inflammatory capacity is cell-type-dependant (Van Der Vorst et al, 2017 ). In endothelial cells and smooth muscle cells, the same group previously demonstrated that HDL is anti-inflammatory (Bursill et al, 2010 ; Van Der Vorst et al, 2013 ).…”

“…BMDMs stimulated with a range of nHDL concentrations then a low fixed [LPS] dose-dependently released Tnfα & Il-12. Intriguingly, BMDMs stimulated with a fixed concentration of either nHDL, reconstituted HDL (rHDL; ApoA-I complexed with 1-palmitoyl-2-linoleoyl-PC) or commercially purchased HDL (cHDL) then a low fixed [LPS] released Tnfα & Il-12 but failed to do so without the LPS present, suggesting HDL and LPS synergistically regulate cytokine release (Van Der Vorst et al, 2017 ). These findings were broadly replicated in vivo .…”

“…In line with this finding, minimally modified LDL (mmLDL) and a low dose of LPS synergistically increase chemokine release in MO cell lines (Wiesner et al, 2010 ). Some of the experiments carried out by De Nardo et al, were replicated using soybean phosphatidylcholine rHDL, but got contrasting data (Van Der Vorst et al, 2017 ). They saw a similar reduction in Il-12 and IL-10 release, but Tnfα release and Atf3 expression were unaffected.…”