Alternate high fat diet aggravates atherosclerosis through an IL1b-dependent reprogramming of neutrophil progenitors

Systemic immune responses induced by chronic hypercholesterolemia contribute to the initiation, progression and complications of atherosclerosis. However, the consequences of an alternate high fat diet and the associated variations in plasma cholesterol levels on atherosclerosis are unknown. To address this relevant issue concerning common situations where dietary habits change over time, we developed a novel protocol in athero-prone mice that allowed us to compare the effects of alternate versus continuous high fat diet (HFD) on atherosclerosis, the overall period of time of exposure to HFD being similar between groups. We showed that alternate HFD lead to accelerated atherosclerosis in Ldlr-/- and Apoe-/- mice compared to continuous HFD. The pro-atherogenic effect of alternate HFD was also found in Apoe-/-Rag2-/- mice lacking T, B, and NKT cells, ruling out a role for the adaptive immune system in the observed phenotype. Using different complementary in vivo approaches, we found that lipid-rich diet discontinuation in alternate group downregulated Runx1, a negative regulator downstream of TLR-4 pathways. As a result, after re-exposure to HFD, myeloid progenitors were more sensitive to hypercholesterolemia leading to acute differentiation into IL-1b- producing immature neutrophils, which caused a state of emergency myelopoiesis. Consecutively, neutrophils markedly increased in the peripheral blood, infiltrated atherosclerotic lesions and locally released neutrophil extracellular traps. Anti-Ly6G neutrophil depletion abolished the pro-atherogenic effects of alternate HFD. Specific deletion of Il1b and Il1b receptor confirmed that the IL-1b signaling pathway was a major driver of the pro-inflammatory and pro-atherogenic neutrophil signature. Finally, treatment with anti-IL-1b neutralizing antibody or inflammasome inhibitor during re-exposure to HFD reversed the pro-atherogenic effects of alternate HFD. In summary, we showed that alternate HFD accelerates atherosclerosis through IL-1b-dependent neutrophil progenitor reprogramming. Lin- cells from femur bones of HFD exposed and non-exposed Ldlr-/- mice were dissected and single cell RNA Seq libraries were prepared with ParseBio Evercode WT Mega v2 kit.

慢性高胆固醇血症诱导的全身性免疫应答，参与动脉粥样硬化（atherosclerosis）的起始、进展及并发症形成。然而，交替式高脂饮食及其伴随的血浆胆固醇水平波动对动脉粥样硬化的影响尚不明确。为探究饮食结构随时间改变这一常见场景下的相关科学问题，我们在动脉粥样硬化易感小鼠（athero-prone mice）中构建了全新实验方案，得以比较交替式与持续性高脂饮食（high fat diet, HFD）对动脉粥样硬化的影响，且两组小鼠的高脂饮食总暴露时长保持一致。我们发现，与持续性HFD相比，交替式HFD可加速低密度脂蛋白受体敲除（Ldlr-/-）小鼠与载脂蛋白E敲除（Apoe-/-）小鼠的动脉粥样硬化进程。在缺失T细胞、B细胞及自然杀伤T（NKT）细胞的载脂蛋白E与Rag2双敲除（Apoe-/-Rag2-/-）小鼠中，同样观察到交替式HFD的致动脉粥样硬化效应，由此排除适应性免疫系统在该观测表型中发挥作用的可能。通过多种互补性体内实验方法，我们发现交替组的高脂饮食中断可下调Runt相关转录因子1（Runx1）的表达——后者是Toll样受体4（TLR-4）通路下游的负调控因子。在此基础上，再次暴露于HFD时，髓系祖细胞对高胆固醇血症的敏感性增强，进而快速分化为分泌白细胞介素1β（IL-1β）的未成熟中性粒细胞，引发紧急髓系造血状态。随后，外周血中性粒细胞大幅增多，浸润动脉粥样硬化病变部位并局部释放中性粒细胞胞外陷阱。使用抗Ly6G抗体耗竭中性粒细胞，可完全消除交替式HFD的致动脉粥样硬化效应。对Il1b及其受体进行特异性敲除实验证实，IL-1β信号通路是促炎性与致动脉粥样硬化性中性粒细胞特征的主要驱动因素。最后，在再次暴露于HFD期间，使用抗IL-1β中和抗体或炎症小体抑制剂进行干预，可逆转交替式HFD的致动脉粥样硬化效应。综上，我们证实交替式HFD通过依赖IL-1β的中性粒细胞祖细胞重编程加速动脉粥样硬化进程。本研究分离了暴露于HFD与未暴露于HFD的Ldlr-/-小鼠股骨中的Lin-细胞，并使用ParseBio Evercode WT Mega v2试剂盒构建单细胞RNA测序（single cell RNA Seq）文库。