Oxidized phospholipid oxPAPC induces a Th1-like phenotype in regulatory T cells and inhibits their protective function in atherosclerosis

Appleton, B. D.; Palmer, S. A.; Smith, H.; Stephens, L. E.; Major, A. S.

doi:10.1101/2023.02.08.527689

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bioRxiv · 10.1101/2023.02.08.527689

Oxidized phospholipid oxPAPC induces a Th1-like phenotype in regulatory T cells and inhibits their protective function in atherosclerosis

Appleton, B. D.·Palmer, S. A.·Smith, H.·Stephens, L. E.·Major, A. S.

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Abstract

BackgroundRegulatory 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 there is limited information regarding their impact on T cells. Given Treg loss during atherosclerosis progression and oxPL levels in the plaque microenvironment, we sought to determine whether oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), an oxPL associated with atherosclerotic plaques, alters Treg differentiation and function. MethodsNaive CD4+ T cells were cultured under Treg, Th1, and Th17 polarizing conditions 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 CD8+ cells in vitro. In vivo suppression of atherosclerosis was evaluated by adoptively transferring control or oxPAPC-treated Tregs to hyperlipidemic Ldlr-/- mice. ResultsCompared to controls, oxPAPC-treated Tregs were less viable but expressed higher levels of the Th1-associated markers T-bet, CXCR3, and IFN-{gamma}. Th1 and Th17 skewing cultures were unaltered by oxPAPC. IFN-{gamma} is linked to Treg instability, thus Treg polarization experiments were repeated using Ifngr1-/- CD4+ T cells. IFN{gamma}R1 deficiency did not improve cell viability in oxPAPC-treated Tregs, however, T-bet and IFN-{gamma} expression was not increased suggesting a role for IFN-{gamma} 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. ConclusionsOxPAPC elicits Treg-specific changes that induce a Th1-like phenotype dependent on IFN-{gamma} signaling. This is biologically relevant as oxPAPC-treated Tregs are unable to reduce atherosclerosis progression in Ldlr-/- mice. This study supports a role for oxPLs in negatively impacting Treg differentiation and atheroprotective function.

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Appleton, B. D., Palmer, S. A., Smith, H., Stephens, L. E., Major, A. S.. 2023-02-11. Oxidized phospholipid oxPAPC induces a Th1-like phenotype in regulatory T cells and inhibits their protective function in atherosclerosis. https://doi.org/10.1101/2023.02.08.527689

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