Matthew J Butcher1, Adam R Filipowicz1, Tayab C Waseem1, Christopher M McGary1, Kevin J Crow1, Nathaniel Magilnick1, Mark Boldin1, Patric S Lundberg1, Elena V Galkina2. 1. From the Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk (M.J.B., A.R.F., T.C.W., C.M.M., K.J.C., P.S.L., E.V.G.); and Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA (N.M., M.B.). 2. From the Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk (M.J.B., A.R.F., T.C.W., C.M.M., K.J.C., P.S.L., E.V.G.); and Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA (N.M., M.B.). galkinev@evms.edu.
Abstract
RATIONALE: Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. OBJECTIVE: Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. METHODS AND RESULTS: We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. CONCLUSIONS: Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.
RATIONALE: Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. OBJECTIVE: Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. METHODS AND RESULTS: We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atheroscleroticmice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. CONCLUSIONS:Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.
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