Alcohol Intoxication and Postburn Intestinal Immunity

描述(由申请人提供)：酒精(乙醇)中毒仍然是烧伤后发病的主要因素。本实验室最近的研究表明，烧伤前急性乙醇中毒导致：1)肠淋巴组织[Peyer‘s patches(PP)和肠系膜淋巴结(MLN)]T细胞增殖、IL-2和IFN-γ产生减少；2)MLN IL-12产生减少；3)PP和MLN IL-18产生增加；4)肠道通透性增加；5)MLN细菌聚集增加。用Caspase-1(一种将前-IL-18转化为成熟IL-18的酶)的抑制剂Ac-YVAD-CHO(5 mg/kg)治疗大鼠，可以防止乙醇和烧伤导致的mln干扰素-γ产生的减少和肠水肿的发生。这些初步发现表明，IL-18的上调和IL-12的产生减少在乙醇中毒和烧伤后肠道免疫和屏障功能的改变中发挥了作用。以往的研究表明，肠道屏障功能受损以及随后细菌及其产物的释放在烧伤后的发病机制中起着重要作用。因此，本研究的总体目标是阐明乙醇中毒和烧伤后肠道免疫功能和屏障功能受损的机制。我们推测，在乙醇中毒和烧伤后，肠道淋巴器官中IL-18的表达上调，而IL-12的产生缺失，导致肠道免疫功能下降和屏障功能受损。利用急性乙醇中毒和烧伤大鼠模型，针对特定目标1的研究将确定改变的IL-18和IL-12的来源(巨噬细胞/树突状细胞)，并确定单独应用重组IL-12恢复IL-12，或联合中和抗IL 18抗体抑制IL-18是否可防止：1)抑制肠道免疫，以及2)乙醇中毒和烧伤后肠屏障功能受损。肠道免疫将通过测量PP和MLN T细胞增殖和细胞因子谱(IL-2、干扰素-γ、IL-4和IL-10产生)来确定。肠屏障功能将通过测定肠道通透性和对MLN中移位的细菌进行量化来衡量。针对特定目的，在乙醇中毒和烧伤前使用抗生素对肠道进行选择性净化的研究将确定肠道细菌是否在IL-18/IL-12介导的肠道免疫和屏障功能改变中起关键作用。最后，针对特定目标3的研究将确定丝裂原活化蛋白激酶(Erk-1/2，p-38，JNK)和/或信号转导和激活蛋白(STAT-4和-6)是否参与了IL-18/IL-12介导的乙醇中毒和烧伤后抑制PP和MLN T细胞功能的信号转导。本研究阐明了烧伤患者肠道免疫功能和屏障功能受损的机制，有助于设计烧伤患者乙醇中毒致伤的救治方案。

DESCRIPTION (provided by applicant): Alcohol (EtOH) intoxication remains a major factor in post-burn pathogenesis. Recent studies from our laboratory have shown that acute EtOH intoxication before burn injury results in: 1) decreased intestinal lymphoid [Peyer's patches (PP) and mesenteric lymph nodes (MLN)] T cell proliferation, IL-2 and IFN-y production; 2) decreased MLN IL-12 production; 3) increased PP and MLN IL-18 production; 4) increased intestinal permeability; and 5) increased bacterial accumulation in MLN. Treatment of rats with Ac-YVAD- CHO, (5 mg/kg), an inhibitor of Caspase-1 (an enzyme that converts pro-IL-18 to mature IL-18), prevents the EtOH and burn-mediated decrease in MLN IFN-y production and the development of intestinal edema. These preliminary findings collectively suggest that IL-18 up-regulation together with a decrease in IL-12 production plays a role in altered intestinal immunity and barrier function following EtOH intoxication and burn injury. Previous studies have suggested that impaired intestinal barrier function and subsequent release of bacteria and their products play significant roles in post-burn pathogenesis. Therefore the overall goal of the proposed studies is to delineate mechanism responsible for impaired intestinal immunity and barrier function following EtOH intoxication and burn injury. We hypothesize that IL-18 up-regulation in the absence of IL-12 production in intestinal lymphoid organs following EtOH intoxication and burn injury results in decreased intestinal immunity and impaired barrier function. Using a rat model of acute EtOH intoxication and burn injury, studies in Specific Aim 1 will characterize the source (macrophage/dendritic cell) of altered IL-18 and IL-12 and determine whether IL-12 restitution alone by administering recombinant IL-12 or in combination with IL-18 inhibition by neutralizing anti-IL18 antibody prevents the: 1) suppression of intestinal immunity, and 2) impaired intestinal barrier function following EtOH intoxication and burn injury. Intestinal immunity will be determined by measuring PP and MLN T cell proliferation and cytokine profile (IL- 2, IFN-y, IL-4 and IL-10 production). Intestinal barrier functions will be measured by determining intestinal permeability and by quantifying translocated bacteria in MLN. In Specific Aim 2 studies using approaches of selective decontamination of intestine with antibiotics prior to EtOH intoxication and burn injury will determine whether intestinal bacteria are critical to IL-18/IL-12-mediated alterations in intestinal immunity and barrier function. Finally, studies in Specific Aim 3 will determine whether Mitogen Activated Protein Kinase (Erk-1/2, p-38, JNK) and/or Signal Transducer and Activator Proteins (STAT-4 and -6) are involved in signaling IL-18/IL- 12-mediated suppression of PP and MLN T cell functions following EtOH intoxication and burn injury. Our studies delineating mechanism of impaired intestinal immunity and barrier function would help in designing approaches for treatment of burn patients who sustained injury under the influence of EtOH intoxication.