Macrophage small RNA export to HDL protects against inflammation and atherosclerosis

摘要：动脉粥样硬化是缺血性心血管疾病(CVD)的根本原因，是一种最好的病理学方法。 特点是由于动脉壁中持续的脂肪沉积而导致炎症失控。尽管 炎症在动脉粥样硬化进展中的重要性，很少有治疗方法是针对 这一过程是由于对生物活性脂类以外的致动脉粥样硬化刺激物缺乏了解。此前，我们 已报道脂蛋白，即高密度脂蛋白和低密度脂蛋白，运输小的非编码RNA(SRNA)，最近， 我们发现脂蛋白高度富含来自细菌和真菌物种的sRNA。 微生物群与环境。有趣的是，这些外来的微生物sRNAs(MsRNAs)也非常丰富 在动脉粥样硬化病变内的巨噬细胞中，并激活sRNA敏感的Toll样受体(TLR)7/8 损伤巨噬细胞。我们推测，高密度脂蛋白接受病变巨噬细胞的msRNA，从而抑制 TLR7/8激活和下游促炎基因表达。支持这一模型的是巨噬细胞 被发现容易将msRNA输出到高密度脂蛋白，高密度脂蛋白处理被发现阻断了msRNA诱导的 激活。在心血管疾病中，高密度脂蛋白获得与高密度脂蛋白功能障碍相关的反应性二羰基修饰。 在初步研究中，我们发现在高密度脂蛋白上的反应性二羰基修饰，例如异uglandins(IsoLG)， 损害了高密度脂蛋白-msRNA的输出。此外，我们还发现，IsoLG修饰的高密度脂蛋白增加了巨噬细胞的摄取 以及滞留和诱导促炎基因表达。初步研究还发现，治疗老鼠 使用反应性清除剂阻断IsoLG修饰的模型可减少动脉粥样硬化。因此，我们的目标是 使用非靶向锁定核酸(NtLNA)阻断TLR7/8的msRNA激活。令人惊讶的是，我们发现 NtLNA治疗在4周内显著减少了APOE-/-小鼠30%的动脉粥样硬化病变面积 西式饮食。此外，单细胞rna测序方法表明ntLNA治疗受到抑制。 损伤巨噬细胞的促炎基因表达及促炎细胞数量的减少 动脉粥样硬化病变中的巨噬细胞。根据初步研究，我们假设高密度脂蛋白可以去除msrna。 从病变巨噬细胞到逆向内吞作用，这一过程被高密度脂蛋白上的IsoLG修饰所抑制。 此外，我们假设病损巨噬细胞中内切溶酶体tlr7/8的msrna激活很容易。 靶向ntLNAs以减少动脉粥样硬化。为了验证这一假设，我们的目标是I。)确定 巨噬细胞msRNA输出到高密度脂蛋白的机制及后果，高密度脂蛋白-msRNA输出对 巨噬细胞激活，并确定体内高密度脂蛋白介导的反向sRNA运输(RSRT)途径，II。)定义 二羰基修饰和msRNA Cargo对心血管疾病中高密度脂蛋白功能障碍的影响，以及目标 巨噬细胞msRNA受体抑制动脉粥样硬化进展和促进消退。这个项目将 开辟了脂蛋白、细胞外sRNA、宿主/非宿主相互作用以及 炎症，它适用于许多其他疾病，但对心血管疾病尤为关键。

Summary: Atherosclerosis is the underlying cause for ischemic cardiovascular disease (CVD), a pathology best characterized as uncontrolled inflammation in response to continual lipid deposition in the arterial wall. Despite the importance of inflammation in the progression of atherosclerosis, very few therapies are designed to target this process due to a general lack of knowledge of atherogenic stimuli outside of bioactive lipids. Previously, we have reported that lipoproteins, namely HDL and LDL, transport small non-coding RNAs (sRNA), and recently, we discovered that lipoproteins are highly-enriched with sRNAs derived from bacterial and fungal species in the microbiome and environment. Interestingly, these foreign, microbial sRNAs (msRNAs) are also highly-abundant in macrophages within the atherosclerotic lesions and activate sRNA-sensing toll-like receptors (TLR)7/8 in lesion macrophages. We posit that HDL accepts msRNAs from lesion macrophages, and thus, suppresses TLR7/8 activation and down-stream pro-inflammatory gene expression. Supporting this model, macrophages were found to readily export msRNAs to HDL and HDL treatments were found to block msRNA-induced activation. In CVD, HDL acquire reactive dicarbonyl modifications which are associated with HDL dysfunction. In preliminary studies, we found that reactive dicarbonyl modifications on HDL, e.g. isolevuglandins (IsoLG), impair HDL-msRNA export. Moreover, we found that IsoLG-modified HDL had increased macrophage uptake and retention and induced pro-inflammatory gene expression. Preliminary studies also found that treating mouse models with reactive scavengers to block IsoLG modifications decreased atherosclerosis. Therefore, we aimed to block msRNA activation of TLR7/8 using non-targeting locked-nucleic acids (ntLNA). Strikingly, we found that ntLNA treatments significantly reduced atherosclerotic lesion area by 30% over 4 weeks in Apoe-/- mice on western diet. In addition, single-cell RNA sequencing approaches demonstrated that ntLNA therapy repressed pro-inflammatory gene expression in lesion macrophages and reduced the number of pro-inflammatory macrophages in atherosclerotic lesions. Based on preliminary studies, we hypothesize HDL removes msRNA from lesion macrophages through retro-endocytosis, a process that is inhibited by IsoLG modifications on HDL. Furthermore, we posit that msRNA activation of endo-lysosome TLR7/8 in lesion macrophages can be readily targeted with ntLNAs to reduce atherosclerosis. To test this hypothesis, we aim to I.) Determine the mechanism(s) and consequences of macrophage msRNA export to HDL, the impact of HDL-msRNA export on macrophage activation, and define the HDL-mediated reverse sRNA transport (RsRT) pathway in vivo, II.) Define the impact of dicarbonyl modifications and msRNA cargo on HDL dysfunction in CVD, and III.) Target macrophage msRNA receptors to inhibit atherosclerosis progression and promote regression. This project will open entirely new fields of study for lipoproteins, extracellular sRNAs, host/non-host interactions, and inflammation, which have applicability to many other diseases, but is particularly critical for CVD.