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.