Inflammatory Effect of Enteric Bacterial-Mediated Intestinal Permeability

描述(申请人提供)：炎症性肠病(IBD)在美国是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存有关。目前，美国与IBD相关的治疗费用估计为63亿美元。IBD的高复发率和缺乏安全和治愈的治疗突出了对这些复杂疾病的替代治疗方法的必要性。细菌蛋白水解酶通过蛋白水解酶激活受体(PARs)诱导肠道通透性，为IBD提供了一个新的治疗靶点。这项建议的目的是：(1)阐明肠道细菌引起肠道通透性的具体机制；(2)评估产酶和抑制肠道细菌对肠道通透性的生物学效应；(3)评估产酶和抑制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。在特定的目标1中，我们将在体外测定产酶和抑制肠道细菌株调节人上皮细胞PARs和通透性的能力。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型粪肠球菌OG1RF菌株，以及缺乏丝氨酸酶活性的野生型和突变型长双歧杆菌ATCC15707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的粪肠球菌OG1RF和长杆菌ATCC15707菌株，并测量细胞通透性和PAR活性。在特定的目标2中，我们将确定产蛋白酶和抑制肠道细菌株调节灵芝小鼠依赖PAR的肠道通透性的能力。为了实现这一目标，我们将无菌野生型和PAR缺陷小鼠与亲本和突变细菌菌株进行单一和双重关联，并测量肠道通透性和PAR激活。在具体目标3中，我们将确定产蛋白酶和抑制肠道细菌株诱导IL-10缺陷(IL-10-/-)小鼠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-/-小鼠与亲本和突变细菌菌株进行单一和双重关联，并测量肠道通透性、PAR激活和炎症。这项拟议的研究的贡献是重大的，因为它将定义肠道细菌改变肠道通透性和促进炎症的精确机制。这项拟议的研究也将具有重要意义，因为研究结果将有助于更广泛地了解肠道微生物区系的失调影响IBD的机制。此外，在我们看来，拟议的研究是创新的，因为它将允许设计合理的以病理生理学为导向的胃肠道疾病益生菌治疗。

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.