Alcohol Intoxication and Postburn Intestinal Immunity

描述（由申请人提供）：酒精（EtOH）中毒仍然是烧伤后发病的主要因素。我们实验室最近的研究表明，烧伤前急性EtOH中毒导致：1)肠道淋巴细胞[佩耶氏斑（PP）和肠系膜淋巴结（MLN）] T细胞增殖、IL-2和IFN-y产生减少；2) MLN IL-12产量降低；3) PP和MLN IL-18产量增加；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）抑制剂Ac-YVAD- CHO （5 mg/kg）治疗大鼠，可防止EtOH和烧伤介导的MLN IFN-y产生减少和肠道水肿的发生。这些初步研究结果共同表明，IL-18的上调和IL-12产生的减少在EtOH中毒和烧伤后肠道免疫和屏障功能的改变中起作用。先前的研究表明，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重要作用。因此，拟议研究的总体目标是描述EtOH中毒和烧伤后肠道免疫和屏障功能受损的机制。我们推测，EtOH中毒和烧伤后，肠道淋巴器官IL-18在IL-12缺乏的情况下上调，导致肠道免疫力下降和屏障功能受损。利用急性EtOH中毒和烧伤大鼠模型，Specific Aim 1的研究将表征IL-18和IL-12改变的来源（巨噬细胞/树突状细胞），并确定通过重组IL-12单独恢复IL-12或通过中和抗IL-18抗体联合抑制IL-18是否可以防止：(1)EtOH中毒和烧伤后肠道免疫抑制，以及(2)肠道屏障功能受损。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、IFN-y、IL-4和IL-10的产生）来确定。肠道屏障功能将通过测定肠通透性和量化MLN中的易位细菌来测量。在Specific Aim 2中，在EtOH中毒和烧伤前使用抗生素选择性去污肠道的研究将确定肠道细菌是否对IL-18/ il -12介导的肠道免疫和屏障功能的改变至关重要。最后，Specific Aim 3的研究将确定丝裂原活化蛋白激酶（Erk-1/2, p-38， JNK）和/或信号传感器和激活蛋白（STAT-4和-6）是否参与了IL-18/IL- 12介导的PP和MLN T细胞功能抑制的信号传导。我们的研究描述了肠道免疫和屏障功能受损的机制，将有助于设计烧伤患者在EtOH中毒影响下持续损伤的治疗方法。

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.