Red Blood Cell Released ATP Regulates Systemic Immune Response in Atherosclerosis

• Published in: The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.03268
• Main Authors: Sun, Haoyu, Zhang, Yunpei, Du, Yong, He, Pingnian
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2021.35.S1.03268

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‐/‐/