Regulatory T cells license macrophage pro-resolving functions in atherosclerosis regression

Regression of atherosclerosis is an important clinical goal, however the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, however numbers of these immunosuppressive cells decrease with disease progression. Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. To test if Tregs are required for the resolution of atherosclerotic inflammation and plaque regression during lipid-lowering therapy, we combined CD25 monoclonal antibody (PC61 mAb)-mediated Treg depletion with single-cell RNA-sequencing of immune cells in the plaque and conventional analyses of atherosclerosis. Single cell RNA-sequencing revealed that Tregs from aortic plaques shared some similarity with splenic Tregs, but were distinct from skin and colon Tregs supporting recent findings of tissue-dependent Treg heterogeneity. Furthermore, Tregs from progressing plaques expressed markers of natural Tregs derived from the thymus, whereas Tregs in regressing plaques lacked Nrp1 and Helios expression, suggesting that they are induced in the periphery during lipid lowering. Treatment of atherosclerotic mice with PC61 mAb effectively depleted Tregs in the blood and peripheral tissues, including plaques, and blocked the regression of atherosclerosis induced by apoB anti-sense oligonucleotides. Morphometric analyses revealed that control antibody-treated mice showed a 40% decrease in plaque burden and macrophage content under regression conditions, whereas PC61 mAb-treated mice showed no change in plaque size or inflammatory cell content compared to baseline. Moreover, Treg depletion enhanced inflammatory signaling and blocked tissue reparative functions of macrophages in the regressing plaque, including M2-polarization, efferocytosis and sensing of specialized pro-resolving lipid mediators. Together, these data establish essential roles for Tregs in the resolution of atherosclerotic inflammation and plaque remodeling during regression. Single cell RNA sequencing of aortic sorted CD45+ cells from atherosclerotic C57BL/J6 mice (n=12, 1012 AAV-PCSK9 at 8wk old mice, then WD for 20 wks). Then 4 mice on WD were used as progression group, 2979 cells identified, Using Illumina HiSeq4000. The other 8mice were switched into chow diet for further 3 wks and also i.p. treated with ApoB-ASO (50 mg/kg, total of 2 doses in first week) as regression groups. 4 mice were either treated with IgG control antibody and 4 mice were treated with anti-CD25 antibody. 1782 cells from control and 2357 cells from treated groups were identified, using Illumina HiSeq4000.