Inflammatory Effect of Enteric Bacterial-Mediated Intestinal Permeability

描述（由申请人提供）：炎症性肠病（IBD）是美国高度流行的肠道疾病，影响140万人。这些疾病与生活质量下降和心理并发症有关。目前美国IBD相关治疗费用估计为63亿美元。IBD的高复发率和缺乏安全和治愈性治疗强调了对这些复杂疾病的替代治疗方法的需求。能够通过蛋白酶激活受体（PAR）诱导肠道通透性的细菌蛋白酶为IBD提供了一种新的治疗靶点。本提案的目的是（I）阐明肠道细菌诱导肠道通透性的具体机制，（II）评估产生和抑制蛋白酶的肠道细菌对肠道通透性的生物学效应，以及（III）评估产生和抑制蛋白酶的肠道细菌对肠道的炎症效应。为了实现这些目标，我们提出了三个具体目标。在具体目标1中，我们将确定产生蛋白酶和抑制蛋白酶的肠道细菌菌株在体外调节人上皮细胞中PAR和渗透性的能力。为了实现这一目标，我们将使用缺乏明胶酶或丝氨酸蛋白酶活性的野生型和突变体粪肠球菌OG 1 RF菌株，以及缺乏丝氨酸蛋白酶抑制剂活性的野生型和突变体长双歧杆菌ATCC 15707菌株。我们将形成紧密连接的T-84上皮细胞单层暴露于亲本和突变体E。faecalis OG 1 RF和B. longum ATCC 15707菌株，并测量细胞渗透性和PAR活化。在具体目标2中，我们将确定产生蛋白酶和抑制蛋白酶的肠道细菌菌株调节无菌小鼠中PAR依赖性肠通透性的能力。为了实现这一目标，我们将单和双关联无菌野生型和PAR缺陷小鼠与亲本和突变菌株，并测量肠道通透性和PAR激活。在具体目标3中，我们将确定产生蛋白酶和抑制蛋白酶的肠道细菌菌株在无菌IL-10缺陷（IL-10-/-）小鼠中诱导肠道通透性和炎症的能力。为了实现这一目标，我们将无菌野生型和IL-10-/-小鼠与亲本和突变菌株单和双相关，并测量肠通透性、PAR活化和炎症。这项研究的贡献是重要的，因为它将定义肠道细菌改变肠道通透性并导致炎症的精确机制。拟议的研究也将具有重要意义，因为其结果将有助于更广泛地了解肠道微生物群生态失调影响IBD的机制。此外，我们认为，拟议的研究是创新的，因为它将允许设计合理的病理生理学导向的益生菌治疗GI疾病。 公共卫生相关性：拟议的项目与公共卫生有关，因为它将通过调节肠道微生物群来确定炎症性肠病（IBD）的新治疗方法。本提案中概述的目的将（I）阐明肠道细菌诱导肠道通透性的具体机制，（II）研究产生蛋白酶和抑制蛋白酶的肠道细菌对肠道通透性的生物学效应，以及（III）研究产生蛋白酶和抑制蛋白酶的肠道细菌对肠道的炎症效应。该建议将确定细菌蛋白酶和肠通透性作为IBD的新的病理生理学导向的治疗靶点。此外，该提案将确定丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）生产的细菌作为益生菌制剂的IBD的管理。

DESCRIPTION (provided by applicant): Inflammatory bowel diseases (IBD) are highly prevalent intestinal diseases in the United States affecting 1.4 million individuals. These diseases are associated with reduced quality of life and psychological co-morbidity. Current estimates for IBD associated treatment costs in the US are $6.3 billion. The high rate of recurrence and lack of safe and curative treatments for IBD underscore the need for alternate therapeutic approaches for these complex diseases. Bacterial proteases capable of inducing intestinal permeability via protease-activated receptors (PARs) present a novel therapeutic target for IBD. The goals for this proposal are to (I) elucidate the specific mechanism by which enteric bacteria induce intestinal permeability, (II) assess the biological effect of protease producing and inhibiting enteric bacteria on intestinal permeability, and (III) assess the inflammatory effect of protease producing and inhibiting enteric bacteria on the intestine. To address these goals we have proposed three specific aims. In specific aim 1 we will determine the ability of protease-producing and - inhibiting enteric bacterial strains to regulate PARs and permeability in human epithelial cells in vitro. To achieve this aim we will use wild-type and mutant Enterococcus faecalis OG1RF strains that lack gelatinase or serine protease activity, and wild-type and mutant Bifidobacterium longum ATCC15707 strains that lack serpin activity. We will expose tight junction forming T-84 epithelial cell monolayers to parental and mutant E. faecalis OG1RF and B. longum ATCC15707 strains and measure cell permeability and PAR activation. In specific aim 2 we will determine the ability of protease-producing and inhibiting enteric bacterial strains to regulate PAR- dependent intestinal permeability in gnotobiotic mice. To achieve this aim we will mono- and dual-associate germ-free wild-type and PAR deficient mice with parental and mutant bacterial strains and measure intestinal permeability and PAR activation. In specific aim 3 we will determine the ability of protease-producing and inhibiting enteric bacterial strains to induce intestinal permeability and inflammation in gnotobiotic IL-10 deficient (IL-10-/-) mice. To achieve this aim we will mono- and dual-associate germ-free wild-type and IL-10-/- mice with parental and mutant bacterial strains and measure intestinal permeability, PAR activation, and inflammation. The contribution of the proposed research is significant as it will define a precise mechanism by which enteric bacteria alter intestinal permeability and contribute to inflammation. The proposed research will also be of significance because the outcome will contribute to the broader understanding of mechanisms by which dysbiosis in the intestinal microbiota affects IBD. Additionally, the proposed research is innovative in our opinion as it will allow for the design of rational pathophysiology-directed probiotic treatment of GI disease. PUBLIC HEALTH RELEVANCE: The proposed project is relevant to public health as it will identify a novel therapeutic approach for Inflammatory Bowel Diseases (IBD) via modulation of the intestinal microbiota. The aims outlined in this proposal will (I) elucidate the specific mechanism by which enteric bacteria induce intestinal permeability, (II) investigate the biological effect of protease-producing and -inhibiting enteric bacteria on intestinal permeability, and (III) investigate the inflammatory effect of protease-producing and -inhibiting enteric bacteria on the intestine. This proposal will identify bacterial proteases and intestinal permeability as novel pathophysiology- directed therapeutic targets for IBD. Additionally, this proposal will identify serine protease inhibitor (serpin)- producing bacteria as probiotic agents for the management of IBD.