Inflammation Resolving Lipid Mediators: Novel Therapy for Alcohol AssociatedLiver Disease

酒精相关性肝病(ALD)在美国是一个主要的健康问题(特别是在退伍军人中 在COVID大流行期间，酒精滥用/酒精性酒精性痴呆急剧增加。尽管有 尽管这个问题很严重，但目前还没有FDA批准的治疗ALD任何阶段的药物。此外，这些机制 疾病进展和严重程度的调节因素也不完全清楚。膳食脂肪起着重要的作用 酒精摄入在ALD发病中的交互作用，然而，特定的n3多不饱和蛋白的作用 ALD中的脂肪酸(PUFA)定义不明确。我们的中心假设是n3多不饱和脂肪酸有益于 ALD部分通过n3-多不饱和脂肪酸衍生的促分解介质，促进炎症溶解， 改善肠道-肝脏轴，随后减轻肝脏损伤。我们建议n3多不饱和脂肪酸- RvD1是一种潜在的ALD治疗剂，它通过RvD1-FPR2-NEAT1信号途径发挥作用 抑制促炎细胞因子和促进肝细胞损伤的修复，部分是通过增强 促进恢复功能的巨噬细胞。我们推测，由于RvD1受损而导致的炎症消退受损 产生/信号是导致ALD/酒精进展和严重程度的关键营养因素- 人类的关联性肝炎(AH)。该提案的具体目标是：目标1：确定作用和 乙醇肝损伤中n3多不饱和脂肪酸和rvd1有益的分子机制(S) 与ALD相关的炎症的消退。我们将确定是否通过 N3-多不饱和脂肪酸衍生的专门化促分解介质(SPM)以及Rvd1-FPR2和Neat1介导的增加 抑制促炎细胞因子信号转导和将促炎巨噬细胞重新编程为促炎- 恢复性表型。野生型(WT)、Fpr2-/-、Neat1-/-和转基因FAT-1小鼠(能够 内源性地将n6转化为n3多不饱和脂肪酸)将用于这一目的。目标2：研究n3-PUFAs和Rvd1的作用 在维持肠道屏障完整性和消退实验性ALD肠道炎症方面有重要作用。我们会： I)在体内、动物模型和体外肠道器官培养中进行测试，无论是n3多不饱和脂肪酸还是rvd1都有改善 通过减轻肠道免疫功能紊乱损伤肠屏障；ii)体内试验是否干扰了 RvD1-FPR2轴加重，而阻断Neat1信号减弱，肠道炎症和改变 肠道屏障完整性；以及iii)确定肠道微生物区系对n3有益效果的贡献 多不饱和脂肪酸和RvD1在实验性ALD中的表达。目标3：将我们在动物模型中的发现翻译并推广到人类 ALD.利用从退伍军人AH患者身上获得的人体样本，我们将：i)检测血浆SPM水平和 血浆SPM水平与肝损伤、全身炎症和肠道通透性的标记物的相关性；ii) 评估基础和内毒素诱导的全血(体外系统)和单核细胞产生SPM；以及 Iii)检测Rvd1是否能提高单核细胞的吞噬/吞噬能力，并确定 RvD1-FPR2轴的破坏将增强，而NEAT1的沉默将减少，促炎症 人单核细胞中细胞因子的表达。这三个目标将定义ALD的两种未经测试的新机制 启动/进展以及潜在的新疗法，可以迅速为患有ALD的退伍军人启动。

Alcohol-associated liver disease (ALD) is a major health problem in the US (especially in the Veteran population) and alcohol abuse/ALD has increased dramatically during the COVID pandemic. In spite of the magnitude of this problem, there is no FDA-approved therapy for any stage of ALD. In addition, the mechanisms and regulators of disease progression and severity are incompletely understood. Dietary fats play an important interactive role with alcohol consumption in ALD pathogenesis, however, the role of specific n3 polyunsaturated fatty acids (PUFAs) in ALD are not well defined. Our central hypothesis is that n3 PUFAs are beneficial in ALD, in part, via n3-PUFA-derived pro-resolving mediators, which facilitate inflammation resolution, improvement in the gut-liver axis, and subsequent attenuation of liver injury. We propose that n3 PUFA- derived resolvin D1 (RvD1) is a potential therapeutic agent in ALD acting via RvD1-FPR2-NEAT1 signaling to suppress pro-inflammatory cytokines and to promote repair of hepatocellular damage, in part, via enhancement of pro-restorative macrophages. We postulate that compromised inflammation resolution due to impaired RvD1 production/signaling is a critical nutritional factor contributing to the progression and severity of ALD/alcohol- associated hepatitis (AH) in humans. The Specific Aims of the proposal are: Aim 1: determine the role and molecular mechanism(s) underlying n3 PUFA and RvD1 beneficial effects in EtOH-induced liver injury and resolution of inflammation associated with ALD. We will determine whether beneficial effects occur through an increase in n3-PUFA-derived specialized pro-resolving mediators (SPMs), and RvD1-FPR2 and Neat1-mediated suppression of pro-inflammatory cytokine signaling and reprogramming pro-inflammatory macrophages to a pro- restorative phenotype. Wild Type (WT), Fpr2-/-, Neat1-/-, and transgenic fat-1 mice (which are able to endogenously convert n6 to n3 PUFAs) will be used in this Aim. Aim 2: examine the role n3-PUFAs and RvD1 in maintaining gut barrier integrity, and in the resolution of intestinal inflammation in experimental ALD. We will: i) test in vivo, in animal models, and in vitro, in intestinal organoid culture, whether n3 PUFAs or RvD1 improve intestinal barrier damage by attenuating intestinal immune dysregulation; ii) test in vivo whether disruption of the RvD1-FPR2 axis exacerbates, while blocking Neat1 signaling attenuates, intestinal inflammation and alterations in the gut barrier integrity; and iii) determine the contribution of the gut microbiota to the beneficial effects of n3 PUFAs and RvD1 in experimental ALD. Aim 3: translate and extend our findings in animal models to human ALD. Utilizing human samples obtained from Veteran AH patients, we will: i) examine plasma SPM levels and correlate plasma SPM levels with markers of liver injury, systemic inflammation, and intestinal permeability; ii) evaluate basal and LPS-inducible SPM production in whole blood (ex-vivo system) and blood monocytes; and iii) test whether RvD1 improves the phagocytosis/efferocytosis capacity of blood monocytes, and determine if disruption of the RvD1-FPR2 axis will enhance, while silencing of NEAT1 will decrease, pro-inflammatory cytokine expression in human monocytes. These three aims will define both untested novel mechanisms for ALD initiation/progression as well as potential novel therapy that could be rapidly initiated for Veterans with ALD.