Mucosal Defense Mechanisms in Substance Abuse

描述（由申请人提供）：在肠道中，抵抗病原微生物的第一道防线是排列在其广泛粘膜表面的上皮细胞层。这些细胞为感染提供了物理屏障，并通过检测和消除肠道病原体（包括人类免疫缺陷病毒（HIV）和肠道沙门氏菌）在粘膜免疫部位发挥关键作用。除了上皮细胞外，肠粘膜还含有能够进行预编程行为的广泛神经系统，并且它容纳了体内最大的淋巴细胞群体;这三种细胞类型通过调节炎症和协调粘膜对感染的防御反应的分子信号彼此通信。阿片类药物滥用是宿主对HIV和其他粘膜病原体易感性的重要辅助因素，但其对粘膜宿主防御的影响相对未知。阿片类药物可能会损害肠内免疫诱导和效应部位的神经-免疫-上皮相互作用。拟议的实验将测试的一般假设，阿片类药物通过神经元和神经元阿片受体介导的行动，损害肠粘膜上皮细胞和淋巴细胞的免疫反应，阿片类神经免疫信号增强粘膜炎症。在具体目标1中，我们将通过测量分泌的细胞因子及其mRNA表达，确定并比较阿片类药物对促炎细胞因子释放的影响以及培养的猪肠上皮细胞炎症前后阿片受体表达的变化，以及来自派伊尔集合淋巴结和吸收性粘膜外植体的阿片类药物的影响。我们还将评估阿片类药物的行动对肠上皮细胞伤口愈合的电细胞基质阻抗传感。在具体目标2中，我们将比较阿片受体和阿片肽的上皮细胞，神经元和白细胞的派尔集合淋巴结和吸收粘膜的免疫细胞化学和原位杂交方法的形态学相互关系。我们还将通过测量沙门氏菌感染引起的细胞因子释放和神经元兴奋性的变化，研究阿片类药物对猪肠上皮细胞-神经元共培养物的影响。最后，我们将通过放射性配体结合技术表征神经膜中炎症和未炎症派尔集合淋巴结中特异性阿片样物质结合位点的配体亲和力和G蛋白偶联偶联的变化。在具体目标3中，我们将表征δ-阿片受体介导的趋化因子诱导的肠道T淋巴细胞的趋化性，并确定阿片类药物对神经介导的分泌型免疫球蛋白-A从肠粘膜外植体释放的影响。这个多方面的跨学科项目的结果将提供阿片类药物如何改变粘膜防御功能的基本理解。此外，他们还将推出新的药物靶点，用于调节对HIV和其他肠道病原体的粘膜免疫反应，以及提供保护免受这些微生物侵害的口服疫苗。公共卫生相关性拟议的跨学科研究的目标是阐明阿片类药物滥用破坏粘膜免疫部位肠神经元、上皮细胞和免疫细胞之间相互作用的机制，这些部位是肠病原体如沙门氏菌感染的关键靶点。鼠伤寒和人类免疫缺陷病毒（HIV）。我们的实验将提供新的和重要的信息阿片类药物的能力，影响由鼠伤寒沙门氏菌，肠侵袭性人畜共患病病原体和潜在的HIV疫苗载体引起的粘膜反应。我们在这个多方面的项目中获得的结果将促进我们对阿片类药物如何改变粘膜防御功能的理解。此外，他们将揭示新的药物靶点，用于调节对肠道病原体（包括HIV）的粘膜免疫反应，并提供新的信息，有助于开发基于细菌载体的口服疫苗，这些疫苗旨在保护肠道相关淋巴组织（该病毒的主要体内储存库）免受HIV感染。

DESCRIPTION (provided by applicant): In the intestinal tract, the first line of defense against pathogenic microorganisms is the layer of epithelial cells that line its extensive mucosal surface. These cells provide a physical barrier to infection and play critical roles at mucosal immune sites by detecting and eliminating enteric pathogens, including the human immunodeficiency virus (HIV) and Salmonella enterica. The intestinal mucosa contains, in addition to epithelial cells, an extensive nervous system capable of pre-programmed behavior, and it houses the largest lymphocyte population in the body; these three cell types communicate with each other through molecular signals which modulate inflammation and coordinate mucosal defense responses to infection. Opioid abuse is an important co-factor in host susceptibility to HIV and other mucosal pathogens, but its impact on mucosal host defense is relatively unknown. Opioid drugs may impair neuro-immuno-epithelial interactions at immune inductive and effector sites in the intestine. The proposed experiments will test the general hypotheses that opioids impair the immune responses of epithelial cells and lymphoid cells in the intestinal mucosa through actions mediated by neuronal and extraneuronal opioid receptors, and that opioid neuroimmune signaling is enhanced by mucosal inflammation. In Specific Aim 1, we will determine and compare the effects of opioids on proinflammatory cytokine release and changes in opioid receptor expression before and after inflammation in porcine intestinal epithelial cells in culture and from explants of Peyer's patch and absorptive mucosae through measurements of secreted cytokines and their mRNA expression. We will also assess opioid actions on intestinal epithelial cell wound healing by electric cell-substrate impedance sensing. In Specific Aim 2, we will compare the morphological interrelationships between opioid receptors and opioid peptides in epithelial cells, neurons and leukocytes of Peyer's patch and absorptive mucosae by immunocytochemical and in situ hybridization methods. We will also examine opioid effects on an epithelial-neuronal co-culture from porcine intestine through measurements of cytokine release and changes in neuronal excitability evoked by Salmonella infection. Finally, we will characterize changes in the ligand affinity and G protein coupling coupling of specific opioid binding sites in neural membranes from inflamed and uninflamed Peyer's patches by radioligand binding techniques. In Specific Aim 3, we will characterize delta-opioid receptors mediating chemokine-induced chemotaxis of intestinal T lymphocytes, and determine the effects of opioids on neurally-mediated secretory immunoglobulin-A release from intestinal mucosa explants. The results from this multi-faceted transdisciplinary project will provide a fundamental understanding of how opioid drugs act to alter mucosal defense function. Moreover, they will unveil new drug targets for the modulation of mucosal immune responses to HIV and other gut pathogens as well as oral vaccines offering protection from these microbes. PUBLIC HEALTH RELEVANCE The goal of the proposed transdisciplinary investigation is to elucidate the mechanisms by which opioid drugs of abuse disrupt the interplay between enteric neurons, epithelial cells and immunocytes at sites of mucosal immunity, which are key targets for infection by enteropathogens like S. typhimurium and the human immunodeficiency virus (HIV). Our experiments will provide new and significant information on the ability of opioids to influence mucosal responses evoked by Salmonella typhimurium, an enteroinvasive zoonotic pathogen and potential HIV vaccine vector. The results that we obtain in this multi-faceted project will advance our understanding of how opioid drugs act to alter mucosal defense function. In addition, they will reveal new drug targets for the modulation of mucosal immune responses to enteric pathogens including HIV, and provide new information contributing to the development of oral vaccines based on bacterial vectors that are designed to confer protection against HIV infection in gut-associated lymphoid tissue, the major body reservoir for this virus.