Route of Nutrition and Enteric Nervous System Effects on Innate Mucosal Defense

描述(由申请人提供)： 医院获得性感染大大增加了危重病人的费用和发病率。许多炎症和感染并发症似乎是由患者肠道内细菌过度生长和毒力增加引起的。两个免疫系统--后天免疫和先天免疫--提供了对细菌的免疫保护，以控制它们。营养途径和营养类型都会影响这两种系统。该实验室定义了当肠外营养(PN)期间肠道不喂养时获得性免疫防御系统的缺陷。最近，我们发现了先天性粘膜防御系统的缺陷，并将这些缺陷追踪到未能刺激肠道神经系统(ENS)。我们发现，提供ENS神经肽可以维持获得性免疫和先天免疫。有4个特定目标：特定目标1：确定在PN喂养的小鼠中，增加ENT刺激或给予神经肽蛙皮素(BBS)是否会增加腔内和Paneth细胞内的天然免疫分子，包括溶菌酶和防御素水平。潘氏细胞是一种重要的先天性免疫分子--分泌型磷脂酶A2(SPLA2)的来源。我们的数据显示：1)与饲料喂养的小鼠相比，PN喂养的小鼠的Paneth细胞颗粒和sPLA2的数量和大小减少；2)给予PN喂养的小鼠的BBS增加了这些颗粒的大小。我们假设ENS控制着先天免疫。我们将改变营养途径，并用BBS治疗PN喂养的小鼠来证实我们的数据。由于Paneth颗粒中含有sPLA2以外的其他分子，因此除了sPLA2外，我们还将研究神经肽对溶菌酶和防御素的影响。具体目的2：研究胆碱能刺激与不刺激条件下BBS对PN喂养小鼠小肠液杀菌功能的影响。我们的初步数据显示，在PN喂养的小鼠中，BBS增加了Paneth细胞颗粒的大小，但不增加受抑制的sPLA2肠道水平。我们的初步数据显示，从Paneth细胞释放颗粒需要胆碱能刺激。为了验证我们的假设，神经肽(用于颗粒生产)和胆碱能刺激(用于释放)都是必要的，我们将确定神经肽在有和没有胆碱能刺激的情况下对天然免疫分子释放的影响，并在体外确定它们在PN喂养的小鼠中的杀菌能力。具体目的3：确定有或无BBS刺激和有或无苯乙醇组的PN对一种致病细菌侵袭培养的肠上皮细胞的影响。 其他研究人员表明，压力、PN和其他因素刺激肠道细菌增加毒力并攻击宿主组织。我们将研究神经肽和胆碱能 利用研究肠道上皮细胞在与大肠杆菌的培养中的侵袭的模型进行刺激以防止细菌病原体毒力的发展。具体目标4：检测BBS对获得性免疫(GalT细胞群和SIgA)、天然免疫(sPLA2、溶菌酶)、腔分泌物杀菌功能和培养上皮细胞侵袭的影响 由一种毒力病原体)；细菌定植；以及缺乏ENS的基因敲除小鼠的结肠炎发展。为了确认特定目标1和3的有效性，这项工作将检查神经肽在ENS缺陷动物中的使用，以确定服用ENS产品是否可以消除或缓解发生的致命性结肠炎。这项拟议的研究将提供有关ENS在先天性粘膜防御中的作用的机制细节，并将确定新的药物来维持非肠道喂养的应激动物的正常肠道稳态。我们期望这些研究将为我们对粘膜防御的理解提供进步，并与危重受伤和危重疾病的退伍军人高度相关。

DESCRIPTION (provided by applicant): Hospital-acquired infections significantly increase the cost and morbidity of critically ill and critically injured patients. Many of the inflammatory and infection complications appear to be caused by overgrowth and increased virulence of bacteria within the patient's intestine. Two immunologic systems - acquired and the innate immunity - provide immune protection against bacteria to control them. Route and type of nutrition influence both systems. This laboratory defined defects in acquired immune defenses when the gut is not fed during parenteral nutrition (PN). Recently, we discovered defects in innate mucosal defenses and tracked these defects to failure to stimulate the enteric nervous system (ENS). We showed that providing ENS neuropeptides maintains aspects of both acquired and the innate immunity. There are 4 specific aims: Specific Aim 1: To determine whether increasing ENT stimulation or the administration of the neuropeptide, bombesin (BBS), in PN-fed mice increases intra-luminal and intra-Paneth cell innate immune molecules including lysozyme and ¿ defensin levels compared with PN feeding alone. The Paneth cells are the source of an important innate immune molecule, secretory phospholipase A2 (sPLA2). Our data show 1) a decrease in number and size of Paneth cell granules and sPLA2 in PN-fed mice compared with chow-fed mice and 2) that BBS given to PN-fed mice increases size of these granules. We hypothesize that ENS controls innate immunity. We will vary route of nutrition and treat PN-fed mice with BBS to confirm our data. Since Paneth granules contain other molecules besides sPLA2 which play a role in innate immunity, we will also investigate the effects of neuropeptides on lysozymes and ¿ defensins in addition to sPLA2. Specific Aim 2: To determine the effect of BBS with and without cholinergic stimulation on the bactericidal function of small intestinal fluids in PN-fed mice. Our preliminary data show that BBS increases the size of Paneth cell granules but does not increase the depressed sPLA2 gut levels in PN-fed mice. Our preliminary data show that cholinergic stimulation is required for granule release from Paneth cells. To test our hypothesis that both neuropeptides (for granule production) and cholinergic stimulation (for release) are necessary, we will determine the effect of neuropeptides with and without cholinergic stimulation on release of innate immune molecules and will determine their bactericidal potency in vitro in PN-fed mice. Specific Aim 3: To determine the effect of PN with or without BBS stimulation and with and without bethanecol stimulation on invasion of intestinal epithelial cells in culture by a virulent bacterial pathogen. Other investigators showed that stress, PN, and other factors stimulate gut bacteria to increase virulence and attack host tissues. We will investigate the ability of neuropeptides and cholinergic stimulation to prevent development of bacterial pathogen virulence using a model that studies the invasion of intestinal epithelial cells in culture with E. coli. Specific Aim 4: To examine th effect of BBS on acquired immunity (GALT cell populations & sIgA); innate immunity (sPLA2, lysozymes, bactericidal function of luminal secretions, and invasion of epithelial cells in culture by a virulent pathogen); bacterial colonization; and development of colitis in knockout mice lacking an ENS. To confirm validity of Specific Aims 1 & 3, this work will examine the use of neuropeptides in an ENS-deficient animal to determine if administering ENS products eliminates or palliates lethal colitis that develops. The proposed studies will provide mechanistic details about the role of the ENS in innate mucosal defenses and will identify novel agents to maintain normal gut homeostasis in stressed animals that are not fed enterally. We expect these studies will provide advances in our understanding of mucosal defenses and be highly relevant to critically injured and critically ill Veterans.