Inflammatory Ly6Chi monocytes and their conversion to M2 macrophages drive atherosclerosis regression
Karishma Rahman, … , P’ng Loke, Edward A. Fisher
Karishma Rahman, … , P’ng Loke, Edward A. Fisher
Published August 1, 2017; First published June 26, 2017
Citation Information: J Clin Invest. 2017;127(8):2904-2915. https://doi.org/10.1172/JCI75005.
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Research Article Cardiology Vascular biology

Inflammatory Ly6Chi monocytes and their conversion to M2 macrophages drive atherosclerosis regression

Abstract

Atherosclerosis is a chronic inflammatory disease, and developing therapies to promote its regression is an important clinical goal. We previously established that atherosclerosis regression is characterized by an overall decrease in plaque macrophages and enrichment in markers of alternatively activated M2 macrophages. We have now investigated the origin and functional requirement for M2 macrophages in regression in normolipidemic mice that received transplants of atherosclerotic aortic segments. We compared plaque regression in WT normolipidemic recipients and those deficient in chemokine receptors necessary to recruit inflammatory Ly6Chi (Ccr2–/– or Cx3cr1–/–) or patrolling Ly6Clo (Ccr5–/–) monocytes. Atherosclerotic plaques transplanted into WT or Ccr5–/– recipients showed reduced macrophage content and increased M2 markers consistent with plaque regression, whereas plaques transplanted into Ccr2–/– or Cx3cr1–/– recipients lacked this regression signature. The requirement of recipient Ly6Chi monocyte recruitment was confirmed in cell trafficking studies. Fate-mapping and single-cell RNA sequencing studies also showed that M2-like macrophages were derived from newly recruited monocytes. Furthermore, we used recipient mice deficient in STAT6 to demonstrate a requirement for this critical component of M2 polarization in atherosclerosis regression. Collectively, these results suggest that continued recruitment of Ly6Chi inflammatory monocytes and their STAT6-dependent polarization to the M2 state are required for resolution of atherosclerotic inflammation and plaque regression.

Authors

Karishma Rahman, Yuliya Vengrenyuk, Stephen A. Ramsey, Noemi Rotllan Vila, Natasha M. Girgis, Jianhua Liu, Viktoria Gusarova, Jesper Gromada, Ada Weinstock, Kathryn J. Moore, P’ng Loke, Edward A. Fisher

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Figure 4

Macrophage dynamics show Ly6chi monocyte recruitment is the key kinetic change impairing regression in Ccr2–/– recipient mice.

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Macrophage dynamics show Ly6chi monocyte recruitment is the key kinetic ...
Aortic arches from Apoe–/– donors fed WD for 14 weeks were transplanted into recipients. (A) Schematic of timeline for EdU and bead injections into recipient mice to assess recruitment of Ly6Chi and Ly6Clo monocytes, respectively, into transplanted aortic arches under regression conditions. (B) Representative flow cytometry plots of CD45+CD115+ circulating monocytes showing Ly6C versus EdU, and (C) quantification of EdU incorporation in circulating Ly6Chi versus Ly6Clo populations showing that EdU is preferentially incorporated into Ly6Chi monocytes in WT, Ccr2–/–, and Ccr5–/– mice (n = 4–8 per group). (D) Analysis of EdU+ cells/section of atherosclerotic plaques transplanted into WT, Ccr2–/–, or Ccr5–/– recipients showing significantly reduced recruitment into plaques of Ly6ChiEdU+ cells into Ccr2–/– compared with WT recipients. (E) Representative flow cytometry plots of CD45+CD115+ circulating monocytes showing Ly6C versus beads, and (F) quantification of bead incorporation in circulating Ly6Chi versus Ly6Clo monocyte populations showing that beads are preferentially incorporated into Ly6Clo monocytes in WT, Ccr2–/–, and Ccr5–/– mice (n = 8–10 per group). (G) Analysis of bead+ cells/section of atherosclerotic plaques transplanted into WT, Ccr2–/–, or Ccr5–/– recipients showing significantly reduced recruitment of Ly6Clo bead+ cells into Ccr5–/– compared with WT mice. Quantification in plaque sections of (H) Ki67 (to assess proliferation) and (I) cleaved caspase 3 (to assess apoptosis) showed no significant differences in proliferation or apoptosis between transplant recipient groups. Quantifications in D, G, H, and I were done in aortic arch plaques from mice 5 days after transplantation (n = 8–9 per group). #P = 0.05, *P < 0.05 when compared with WT group using 1-way ANOVA with Dunnett’s multiple comparisons testing.