Macrophage Circadian Clock Disruption and Inflammation in Heart Failure

单核细胞和巨噬细胞主要来源于骨髓和脾-促炎 小鼠的Ly6chi单核细胞(人CD14hi细胞)和C-C趋化因子受体2(CCR2)+单核细胞 巨噬细胞(MDM)--在慢性心脏组织损伤和左心室(LV)重构中起重要作用 衰竭(HF)，提示这些细胞可能是免疫调节的治疗靶点。重要的是 单核细胞和MDM在多个功能参数中表现出昼夜变化，这在很大程度上是由于 细胞自主分子时钟，由Clock/BMAL1转录复合体和受试者调控 连接到一个涉及Rev-Erba的自动调节回路。值得注意的是，单核细胞的内在生物钟和 MDMS在HF中被破坏，其与炎症和疾病进展的机制联系完全未知。 我们的初步研究表明，单核细胞和MDM时钟中断是HF的特征，并且 单核/巨噬细胞BMal1(以及随后的Rev-erba/b)丢失与免疫上调有关 激活剂E4bp4与加重左室重构有关。根据这些数据，我们假设 心衰时单核细胞/MDM时钟功能紊乱，导致病理性炎症和心脏重构。 REV-ERBA连锁和E4BP4依赖方式，时钟校正是 免疫调节。三个目标将检验这一假设。在目标1中，我们将定义单核细胞的变化 用小鼠冠脉结扎模型建立心衰的生物钟，并测试单核/巨噬细胞的生物钟 髓系特异性BMal1基因敲除(MBK)小鼠的破坏加剧了炎症，先天性免疫扩张， 心衰时左室重构。我们还将测量CD14hi单核细胞中的炎症和时钟基因 患有心力衰竭的人类。在目标2中，我们将描述单核细胞定位的E4bp4的作用，它是一种时钟控制的 炎症转录因子和Rev-erba在心力衰竭中的直接靶点 髓系特异性E4bp4基因敲除(MEK)小鼠冠状动脉结扎后的重塑。我们还将评估是否 单核细胞E4BP4抑制挽救单核/巨噬细胞加重的左室重构 通过诱导髓系特异性BMal1/E4bp4双基因敲除小鼠的HF来扰乱时钟。在《目标3》中，我们将 通过测试，机械地确定以生物钟为靶点作为心衰治疗方法的可能性 SR9009，一种合成的REV-ERBA/b激动剂，是否有利于调节单核细胞和MDM 并改善或逆转已建立心衰的野生型小鼠的左室重构，然后在MEK心衰小鼠中 确定SR9009的作用是否需要单核/巨噬细胞E4bp4。我们还将评估前 人心衰CD14hi单核细胞对激动剂的体内激活反应这些研究将进一步推动我们的 了解巨噬细胞生物钟的变化如何调节炎症和疾病 心力衰竭的研究进展，并测试利用遗传和药理学进行免疫调节的新方法 纠正时钟扰乱引起的病理变化的策略。

Monocytes and macrophages fundamentally originating from the bone marrow and spleen - pro-inflammatory Ly6Chi monocytes in mice (CD14hi cells in humans) and C-C chemokine receptor 2 (CCR2)+ monocyte-derived macrophages (MDMs) - contribute importantly to tissue injury and left ventricular (LV) remodeling in chronic heart failure (HF), suggesting that these cells may represent therapeutic targets for immunomodulation. Importantly, monocytes and MDMs exhibit circadian variation in multiple functional parameters, in large part due to a robust cell-autonomous molecular clock, which is regulated by the CLOCK/BMAL1 transcriptional complex and subject to an autoregulatory loop involving Rev-erba. Notably, whether the intrinsic circadian clock in monocytes and MDMs is disrupted in HF and its mechanistic link to inflammation and disease progression is entirely unknown. Our pilot studies suggest that monocyte and MDM clock disruption is characteristic of HF, and that monocyte/macrophage Bmal1 (and subsequent Rev-erba/b) loss is associated with upregulation of the immune activator E4bp4, and aggravation of LV remodeling. Based on these data, we hypothesize that the monocyte/MDM clock is dysfunctional in HF, leading to pathological inflammation and cardiac remodeling in a REV-ERBa-linked and E4BP4-dependent manner, and that clock correction is a key molecular target for immunomodulation. Three Aims will test this hypothesis. In Aim 1, we will define alterations in the monocyte circadian clock in HF using a murine coronary ligation model, and test whether monocyte/macrophage clock disruption in myeloid-specific Bmal1 knockout (MBK) mice exacerbates inflammation, innate immune expansion, and LV remodeling during HF. We will also measure inflammatory and clock genes in CD14hi monocytes from humans with HF. In Aim 2, we will delineate the role of monocyte-localized E4bp4, a clock-controlled inflammatory transcription factor and direct target of Rev-erba, in HF by assessing inflammation and LV remodeling after coronary ligation in myeloid-specific E4bp4 knockout (MEK) mice. We will also evaluate whether monocyte E4BP4 suppression rescues the aggravated LV remodeling observed with monocyte/macrophage clock disruption by inducing HF in myeloid-specific Bmal1/E4bp4 double knockout mice. In Aim 3, we will mechanistically establish the potential of targeting the circadian clock as a therapeutic approach in HF, by testing whether treatment with SR9009, a synthetic REV-ERBa/b agonist, favorably modulates monocytes and MDMs and ameliorates or reverses LV remodeling in wild-type mice with established HF, and then in MEK HF mice to determine whether the effects of SR9009 require monocyte/macrophage E4bp4. We will also evaluate the ex vivo activation responses of human HF CD14hi monocytes to the agonist. These studies will further our understanding of how changes in the macrophage circadian clock modulate both inflammation and disease progression in HF, and test novel approaches to immunomodulation using genetic and pharmacological strategies to correct the pathological changes induced by clock disruption.