Specific-sized hyaluronan: a dual targeted therapy for ALD

摘要 酒精性肝病（ALD）在大约20%的酗酒者中发展。ALD的发展是一个复杂的 涉及肝脏中实质和非实质细胞以及免疫细胞募集的过程 以应对损伤和炎症。先天免疫和器官间的相互作用导致了乙醇- 诱导肝损伤，肠和肝脏之间的相互作用特别重要。肠功能受损 在人和啮齿动物模型中，屏障功能与乙醇诱导的肝损伤有关。增加 慢性乙醇中毒时肝巨噬细胞枯否细胞暴露于肠源性LPS 激活TLR 4依赖性炎症介质的产生。慢性乙醇暴露也会使 枯否细胞对LPS的反应，导致炎症介质的产生增加。Hytolonan（HA），一种丰富的 细胞外基质组分以大小特异性方式与细胞通讯。特定尺寸HA 片段是促炎或抗炎的，这取决于HA受体和所涉及的细胞类型 in the response响应.我们已经发现，特定大小的HA 35（平均MW~ 35 kD）使TLR 4正常化。 介导的信号传导，也保护小鼠免受乙醇- 导致肠道和肝脏损伤。通过下一代测序，我们已经确定了一个microRNA的子集， 是由HA 35调节，提供了新的见解的作用机制，乙醇和 HA 35在调节TLR 4信号传导和巨噬细胞极化中的作用 HA 35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肠道免疫。 形成紧密连接。基于HA 35的多能功能，在这里我们将测试以下假设： HA 35是ALD中的双重靶向治疗剂，在慢性炎症后使枯否细胞信号转导正常化。 乙醇暴露和保护肠上皮屏障。我们将在三个具体的假设中解决这个问题。 目标。具体目的1：研究HA 35使大鼠Kupffer中TLR 4信号正常化的机制 细胞和人PBMC。利用生物信息学和基于细胞的测定，我们将1）研究 miRNA调控途径被乙醇和HA 35靶向，这反过来又调控a）控制 核质穿梭和B）巨噬细胞极化。具体目标2：探讨HA 35的影响 维持肠道屏障功能。我们将使用培养的Caco-2细胞和小鼠肠 类器官，以确定HA 35对保护紧密连接免受乙醇影响的直接作用的机制。 具体目标3：测试HA 35预防和治疗慢性乙醇诱导的肠和肝损伤的能力 对小鼠重要的是，用于设备使用的医用级HA是市售的，从而增强了医疗器械的安全性。 我们对HA 35在慢性乙醇中的双重保护功能的研究的快速翻译的可能性 在临床研究中暴露于ALD。

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.