Specific-sized hyaluronan: a dual targeted therapy for ALD

摘要 酒精性肝病(ALD)发生在大约20%的酗酒者中。ALD的发展是一个复杂的过程 涉及肝脏实质细胞和非实质细胞的过程以及免疫细胞的募集 以应对损伤和炎症。与生俱来的免疫和器官间的串扰导致了酒精- 诱导性肝损伤，尤其是肠和肝之间的相互作用。受损的肠道 在人类和啮齿动物模型中，屏障功能与乙醇诱导的肝损伤有关。增加了 慢性乙醇作用下驻留肝巨噬细胞枯否细胞对肠源性内毒素的暴露 激活依赖TLR4的炎症介质的产生。长期接触酒精也会使人敏感 库普弗细胞转化为内毒素，导致炎性介质的产生增加。透明质酸(HA)，一种丰富的 细胞外基质成分，以特定大小的方式与细胞通信。特定大小的HA 片段要么是促炎的，要么是抗炎的，这取决于所涉及的HA受体和细胞类型 在回应中。我们发现，特定大小的HA35(平均分子量~35kD)使TLR4- 慢性酒精暴露后Kupffer细胞中介导的信号转导，也保护小鼠免受乙醇- 引起肠道和肝脏损伤。通过下一代测序，我们已经确定了一组microRNAs 是由HA35相互调节的，为乙醇和 HA35在调节TLR4信号和巨噬细胞极化中的作用此外，我们的团队发现，特定大小的 HA35促进肠道健康，至少部分是通过增加β-防御素的表达和促进 形成紧密的连接。基于HA35的多功能功能，这里我们将检验这一假设 HA35是ALD的双靶向治疗剂，在慢性阻塞性肺疾病后使Kupffer细胞信号转导正常化。 酒精暴露和保护肠道上皮屏障。我们将从三个具体的方面来阐述这一假设 目标。特异目的1：探讨HA35对大鼠枯否细胞TLR4信号的正常化机制 细胞和人外周血单核细胞。利用生物信息学和基于细胞的分析，我们将1)调查 乙醇和HA35相互靶向的miRNA调节通路，这反过来又调节a)控制 核质穿梭和b)巨噬细胞极化。具体目标2：审问HA35的影响 论肠道屏障功能的维持。我们将使用培养的Caco-2细胞和小鼠肠道 有机化合物以确定HA35在保护紧密连接免受乙醇伤害方面的直接作用机制。 具体目标3：检测HA35对慢性乙醇性肠道和肝脏损伤的防治能力 在老鼠身上。重要的是，用于设备的医用级HA在商业上是可用的，从而提高了 快速翻译我们关于HA35在慢性乙醇中的双重保护功能的研究的可能性 暴露于ALD的临床研究。

ABSTRACT Alcoholic liver disease (ALD) develops in approximately 20% of alcoholics. The development of ALD is a complex process involving both parenchymal and non-parenchymal cells in the liver, as well as recruitment of immune cells in response to damage and inflammation. Innate immunity and inter-organ cross talk contribute to ethanol- induced liver injury with interactions between intestine and liver of particular importance. Impaired intestinal barrier function is associated with ethanol-induced liver injury in both humans and rodent models. Increased exposure of Kupffer cells, the resident hepatic macrophages, to gut-derived LPS during chronic ethanol activates TLR4-dependent production of inflammatory mediators. Chronic ethanol exposure also sensitizes Kupffer cells to LPS, resulting in increased production of inflammatory mediators. Hyaluronan (HA), an abundant extracellular matrix component, communicates with cells in a size-specific manner. Specific-sized HA fragments are either pro-inflammatory or anti-inflammatory, depending on the HA receptor and cell type involved in the response. We have discovered that specific-sized HA35 (average MW~35kD) normalizes TLR4- mediated signaling in Kupffer cells after chronic ethanol exposure and also protects mice from ethanol- induced gut and liver injury. By Next Generation Sequencing, we have identified a subset of microRNAs that are reciprocally regulated by HA35, providing novel insights into the mechanism of action for both ethanol and HA35 in regulating TLR4 signaling and macrophage polarization Furthermore, our team finds that specific-sized HA35 promotes intestinal health, at least in part by increasing expression of β-defensins and promoting the formation of tight junctions. Based on the multi-potent functions of HA35, here we will test the hypothesis that HA35 is a dually targeted therapeutic agent in ALD, normalizing Kupffer cell signal transduction after chronic ethanol exposure and protecting the intestinal epithelial barrier. We will address this hypothesis in three Specific Aims. Specific Aim 1: Investigate the mechanism for normalization of TLR4 signaling by HA35 in rat Kupffer cells and human PBMCs. Making use of both bioinformatics and cell based assays, we will 1) investigate the miRNA regulatory pathways reciprocally targeted by ethanol and HA35, which in turn regulate a) the control of nuclear-cytoplasmic shuttling and b) macrophage polarization. Specific Aim 2: Interrogate the impact of HA35 on maintenance of intestinal barrier function. We will use both cultured Caco-2 cells and mouse intestinal organoids to determine mechanisms for the direct effect of HA35 on protecting tight junctions from ethanol. Specific Aim 3: Test the ability of HA35 to prevent and treat chronic ethanol-induced intestinal and liver injury in mice. Importantly, medical grade HA for device-use is commercially available, thus enhancing the likelihood for a rapid translation of our studies on the dually protective functions of HA35 in chronic ethanol exposure into clinical investigations in ALD.