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

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. To investigate oxPAPC-induced alterations in Tregs, we polarized Tregs from mouse naïve CD4+ T cells for three days in vitro in the presence or absence of oxPAPC. We then analyzed differential gene expression using data obtained through RNA-seq. We compared control Tregs (4 replicates) to oxPAPC-treated Tregs (4 replicates).