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Oxidized phospholipid oxPAPC alters regulatory T cell differentiation and decreases their protective function in atherosclerosis

ABSTRACT: Background: Regulatory T cells (Tregs) are protective in atherosclerosis but reduced during disease progression due to cell death and loss of stability. However, the mechanisms of Treg dysfunction remain unknown. Oxidized phospholipids (oxPLs) are abundant in atherosclerosis and can activate innate immune cells, but little is known regarding their impact on T cells. Given Treg loss during atherosclerosis progression and oxPL levels in the plaque microenvironment, we investigated whether oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), an oxPL associated with atherosclerotic plaques, alters Treg differentiation and function. Methods: CD4+ T cells were polarized to Treg, Th1, and Th17 cells with or without oxPAPC and assessed by flow cytometry. Gene expression in oxPAPC-treated Tregs was analyzed by bulk RNA sequencing. Functional studies of oxPAPC-induced Tregs were performed by co-culturing Tregs with CTV-labeled cells in vitro, and by adoptively transferring Tregs to hyperlipidemic Ldlr-/- mice to measure atherosclerosis progression. Results: Compared to controls, oxPAPC-treated Tregs were less viable, but surviving cells expressed higher levels of the Th1-associated markers T-bet, CXCR3, and IFN-. Th1 and Th17 skewing cultures were unaltered by oxPAPC. IFN- is linked to Treg instability, thus Treg polarization experiments were repeated using Ifngr1-/- CD4+ T cells. IFNR1 deficiency did not improve cell viability in oxPAPC-treated Tregs, however, T-bet and IFN- expression was not increased in surviving cells suggesting a role for IFN-signaling. OxPAPC-treated Tregs were less suppressive in vitro, and adoptive transfer studies in hyperlipidemic Ldlr-/- mice showed that oxPAPC-induced Tregs possessed altered tissue homing and were insufficient to inhibit atherosclerosis progression. Conclusions: OxPAPC elicits Treg-specific changes altering Treg differentiation and inducing a Th1-like phenotype in surviving cells partially through IFN- signaling. This is biologically relevant as oxPAPC-treated Tregs do not reduce atherosclerosis progression in Ldlr-/- mice. This study supports the role for oxPLs in negatively impacting Treg differentiation and atheroprotective function.

ORGANISM(S): Mus musculus

PROVIDER: GSE236226 | GEO | 2023/07/04

REPOSITORIES: GEO

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Project description: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.

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Oxidized phospholipid oxPAPC alters regulatory T cell differentiation and decreases their protective function in atherosclerosis

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