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 6

PRX decreases endolysosomal cholesterol.

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PRX decreases endolysosomal cholesterol. (A) RAW cells were first treate...
(A) RAW cells were first treated with or without PRX (2 mM βCD) for 20 hours then with LPS for 4 hours. Cellular cholesterol was quantified with GC/MS (n = 6). *P < 0.05. Tukey-Kramer post hoc test. (B) Effects of PRX on cholesterol efflux. n = 4 in each group. *P < 0.05 vs. untreated cells, #P < 0.05 vs. LPS only–treated cells. Tukey-Kramer post hoc test. (C) Effects of PSC-833 on cholesterol efflux. n = 4. *P < 0.05 vs. untreated cells, #P < 0.05 vs. PRX plus LPS-treated cells, Tukey-Kramer post hoc test. (D) Expression of Il6, Il1b, and Tnf mRNA in RAW cells treated with PRX for 20 hours, followed by LPS stimulation for 4 hours. *P < 0.05 vs. LPS-untreated cells, #P < 0.05 vs. LPS only–treated cells, Tukey-Kramer post hoc test. (E) RAW cells were treated with or without PRX and LPS for the indicated times. IL-6 protein in the conditioned medium was quantified using an ELISA. n = 3 in each group. Data shown are representative of 3 independent experiments. **P < 0.01 vs. PRX-untreated cells at each time point. Student’s 2-tailed t test. (F) RAW cells were treated for 20 hours with PRX and/or PSC-833 prior to 4 hours with LPS. Expression of Il6 mRNA was assessed with qPCR. *P < 0.05 vs. LPS-untreated cells, #P < 0.05 vs. PRX and LPS-treated cells. (G) RAW cells were treated for 20 hours with PRX or N-PRX prior to 4 hours with LPS. Expression of Il6 mRNA was assessed with qPCR. *P < 0.05 vs. LPS-untreated cells, #P < 0.05 vs. PRX and LPS-treated cells, Tukey-Kramer post hoc test. (H) Expression of Abca1 mRNA in RAW cells treated with PRX for 20 hours, followed by stimulation with LPS for 4 hours. *P < 0.05 vs. control cells. Tukey-Kramer post hoc test. Data shown as mean ± SD in all panels where P values are shown.