Activated cholesterol metabolism is integral for innate macrophage responses by amplifying Myd88 signaling
Sumio Hayakawa, … , Ichiro Manabe, Yumiko Oishi
Sumio Hayakawa, … , Ichiro Manabe, Yumiko Oishi
Published November 22, 2022
Citation Information: JCI Insight. 2022;7(22):e138539. https://doi.org/10.1172/jci.insight.138539.
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Research Article Inflammation Vascular biology

Activated cholesterol metabolism is integral for innate macrophage responses by amplifying Myd88 signaling

Abstract

Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein’s self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr–/– mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.

Authors

Sumio Hayakawa, Atsushi Tamura, Nikita Nikiforov, Hiroyuki Koike, Fujimi Kudo, Yinglan Cheng, Takuro Miyazaki, Marina Kubekina, Tatiana V. Kirichenko, Alexander N. Orekhov, Nobuhiko Yui, Ichiro Manabe, Yumiko Oishi

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Figure 7

PRX inhibits the TLR4/Myd88/NF-κB pathway.

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PRX inhibits the TLR4/Myd88/NF-κB pathway. (A) RAW cells transfected wit...
(A) RAW cells transfected with NFκB-Luc were cultured in the presence or absence of PRX for 20 hours and treated with LPS or vehicle for 4 hours. Luciferase activities are shown relative to the activity in cells transfected with NFκB-Luc with no treatment. *P < 0.05 vs. LPS-untreated cells, #P < 0.05 vs. PRX-untreated cells, Tukey-Kramer post hoc test. (B) RAW cells were treated for 20 hours with or without PRX and then for 4 hours with or without LPS. Nuclear and cytoplasmic fractions were prepared from cell lysates and subjected to Western blotting for the NF-κB p65 subunit. Lamin A/C was used as an internal control for nuclear protein. Representative blots from 3 individual experiments are shown. (C) PRX decreased interaction between CFP-Myd88 and Flag-Myd88. HEK293T cells were transfected with CFP-Myd88 expression plasmids with or without Flag-Myd88. The cell lysates were subjected to immunoprecipitation using anti-Flag antibody. Representative blots from 3 individual experiments are shown. (D) Whole-cell lysates from RAW cells were subjected to native PAGE. Bands with slow migration correspond to the oligomerized forms of Myd88 protein, as indicated. A photograph of the same protein run on an SDS-PAGE gel is shown as a control at the bottom. Relative band intensity corresponding to the oligomerized Myd88 compared with that of LPS-untreated control cells are shown in the bar graph. n = 3 in each group. *P < 0.05 vs. unstimulated cells, #P < 0.05 vs. LPS only–treated cells. Tukey-Kramer post hoc test. Data shown as mean ± SD in all panels where P values are shown.