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 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 5

CCR2 deficiency in recipient leukocytes impairs their recruitment to atherosclerotic plaques, where they normally display M2 characteristics.

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CCR2 deficiency in recipient leukocytes impairs their recruitment to ath...
(A) Schematic of CD45.1 (donor) to CD45.2 (recipient) aortic transplantation experiments with (B) quantification of immunohistochemical staining of CD45.1 and CD45.2 in aortic arch plaques from Apoe–/– mice on 14-week WD (BL; n = 7) or 5 days after transplant into WT mice (Regr; n = 8), or chemokine receptor–KO recipient mice (Ccr2–/– or Ccr5–/–; n = 8); ***P < 0.001 for the indicated comparisons group using 1-way ANOVA with Dunnett’s multiple comparisons testing. (C) Representative images of aortic plaques stained for CD45.1 and CD45.2, imaged at ×40 quantification. (D) Quantification of immunohistochemical staining of MR+ with CD45.2 or CD45.1 showing 80.72% ± 3.597% MR+ cells originate from recipient CD45.2 mice (n = 3) with representative images at ×40 magnification. (E) Schematic of transplantation experiment using CD68-GFP reporter mice to mark recipient monocytes/macrophages. (F) Representative flow cytometry plots showing that a majority of CD45+CD11b+F4/80+MR+ macrophages are GFP+ in aortic arches harvested 5 days after transplantation (n = 3). (G) Schematic of transplant experiment for single cell RNA-seq experiments, with (H) scatter plots showing, at a single-cell level, that cells expressing high levels of Apoe (i.e., recipient) are also positive for high levels of M2 macrophage markers Cd206 (MR) and Arg1 in the population of Cd11b+F4/80+ macrophages isolated from aortic arches 3 days after transplantation.