Red Blood Cell Released ATP Regulates Systemic Immune Response in Atherosclerosis

Abstract

Our studies demonstrated that disturbed flow-induced ATP release from red blood cells (RBCs) via pannexin 1 (Panx1) channels plays a significant role in site-specific vascular inflammation and atherosclerosis. This is supported by a 40-60% reduction of atherosclerotic plaque observed in high fat diet (HFD)-fed ApoE-/- mice with RBC panx1 deletion that nearly abolished the release of ATP. Given the interplay of the immune system in atherosclerosis, we hypothesize that disturbed blood flow-induced release of ATP from RBCs at micromolar concentration is a major source of extracellular ATP (orders of magnitude higher than other ATP sources), which plays a crucial role in purinergic signaling-mediated local and systemic immune cell activation, cytokine production, and phenotypic transformation, serving as an important mediator in immune system-mediated initiation and progression of atherosclerosis. To test this hypothesis, we conducted experiments in 3 mouse groups (n = 4 to 12 per group): chow diet-fed wild type control, and 16 weeks HFD-fed ApoE-/- and ApoE-/-/RBC Panx1-/- mice. Plasma inflammatory cytokines were measured via ELISA. Subpopulations of peripheral blood monocytes (PBMCs) were characterized by flow cytometry, and the cytokine secretion capacity of T cells was examined by intracellular interferon-gamma (IFN-γ) staining in isolated splenocytes. Results showed that plasma IFN-γ and interleukin 1 beta (IL-1β) increased 10 and 8 fold, respectively, in HFD ApoE-/- mice when compared to the control group. These increases were reduced to levels close to the control in HFD ApoE-/-/RBC Panx1-/- mice. PBMCs analysis showed a significantly increased pro-inflammatory Ly6Chigh monocytes in HFD ApoE-/- mice from 2.9 ± 0.5% (control) to 19.2 ± 2.0%, while only 7.6 ± 1.0% was found in HFD ApoE-/-/RBC Panx1-/- mice. Intracellular IFN-γ staining of isolated splenocytes from HFD ApoE-/- mice showed a 5-fold increase in IFN-γ+ T cells (%) when compared to the control. In contrast, ApoE-/-/RBC Panx1-/- mice had significantly reduced percentage of IFN-γ+ T cells when compared with ApoE-/- mice (0.12 ± 0.03% vs 1.5 ± 0.48%, p < 0.05), especially in CD4+ T cells (0.06 ± 0.01% vs 0.79 ± 0.21%, p < 0.05). These cells further demonstrated their increased inflammatory potential, as stimulation with phorbol 12-myristate 13-acetate and Ionomycin yielded a markedly higher percentage of IFN-γ+ T cells in the HFD ApoE-/- group (40.4 ± 3.3%) than control (22.7 ± 1.8%) and ApoE-/-/RBC Panx1-/- group (26.6 ± 3.2%), and more prominent differences were found in CD4+ subgroup with values at 29.0 ± 2.5%, 11.1 ± 1.7%, and 14.5 ± 2.9%, respectively (p<0.05). In conclusion, our results indicate that shear stress-induced release of ATP from RBCs via Panx1 channels plays important roles in the immune cell recruitment, activation, and cytokine production, contributing to immune system-mediated vascular inflammation and the initiation and progression of atherosclerosis. Our findings provide novel mechanistic insight into local hemodynamics and systemic risk factor-mediated vascular pathogenesis and disease progression, directly benefiting clinical diagnosis and the prediction of atherosclerosis development.