Oxidized Phospholipid oxPAPC Alters Regulatory T-Cell Differentiation and Decreases Their Protective Function in Atherosclerosis in Mice

Abstract

Background: Regulatory T cells (T_regs) are protective in atherosclerosis but reduced during disease progression due to cell death and loss of stability. However, the mechanisms of T_reg dysfunction remain unknown. Oxidized phospholipids are abundant in atherosclerosis and can activate innate immune cells, but little is known regarding their impact on T cells. Given T_reg loss during atherosclerosis progression and oxidized phospholipid levels in the plaque microenvironment, we investigated whether oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), an oxidized phospholipid associated with atherosclerotic plaques, alters T_reg differentiation and function.

Methods: CD4⁺ T cells were polarized to T_reg, T helper (Th) 1, and Th17 cells with or without oxPAPC and assessed by flow cytometry. Gene expression in oxPAPC-treated T_regs was analyzed by bulk RNA sequencing. Functional studies of oxPAPC-induced T_regs were performed by coculturing T_regs with CellTrace Violet-labeled cells in vitro, and by adoptively transferring T_regs to hyperlipidemic Ldlr^-/- mice to measure atherosclerosis progression.

Results: Compared with controls, oxPAPC-treated T_regs were less viable, but surviving cells expressed higher levels of the Th1-associated markers T-bet, CXCR3, and IFN (interferon)-γ. Th1 and Th17 skewing cultures were unaltered by oxPAPC. IFN-γ is linked to T_reg instability, thus T_reg polarization experiments were repeated using Ifngr1^-/- CD4⁺ T cells. IFNγR1 (INF gamma receptor 1) deficiency did not improve cell viability in oxPAPC-treated T_regs; however, T-bet and IFN-γ expression was not increased in surviving cells suggesting a role for IFN-γsignaling. OxPAPC-treated T_regs were less suppressive in vitro, and adoptive transfer studies in hyperlipidemic Ldlr^-/- mice showed that oxPAPC-induced T_regs possessed altered tissue homing and were insufficient to inhibit atherosclerosis progression.

Conclusions: OxPAPC elicits T_reg-specific changes altering T_reg differentiation and inducing a Th1-like phenotype in surviving cells partially through IFN-γ signaling. This is biologically relevant as oxPAPC-treated T_regs do not reduce atherosclerosis progression in Ldlr^-/- mice. This study supports the role of oxidized phospholipids in negatively impacting T_reg differentiation and atheroprotective function.

Keywords: atherosclerosis; cell death; disease progression; interferon; phospholipid.