Novel Role of DRA/SLC26A3 Downregulation in Gut Dysbiosis, Inflammation and Infection

在腺瘤中下调(DRA)或SLC26A3是关键的肠道氯离子转运体，参与肠道对电解质和水的吸收。DRA功能突变的丧失会导致“先天性氯化物腹泻”或CLD疾病。我们小组的广泛研究现已证实，DRA的下调是感染性和IBD相关性腹泻的关键事件之一。来自我们小组和其他研究人员的新证据以及GWAS研究现在也证实了DRA基因是IBD的易感基因。DRA基因敲除(KO)小鼠表现为CLD表型，粘液层较薄，结肠炎易感性增加。我们最近发现DRA KO小鼠表现出IBD的一些关键特征，包括生物失调和屏障完整性受损。我们的初步研究进一步表明，DRA KO小鼠更容易受到病原体感染，并表现出免疫细胞调节失调。然而，在本研究中观察到的生物失调的详细机制及其在粘液-上皮屏障缺陷中的作用 Drako小鼠及其对肠道炎症和病原体感染的易感性增加还没有得到充分的调查。我们令人兴奋的数据进一步表明，DRA KO小鼠的生物失调与产生短链脂肪酸(SCFA)的类群减少、粪便SCFA水平和粘蛋白降解菌的丰富有关。对DRA KO与野生型小鼠的共居研究表明，部分受损的屏障完整性是微生物生态失调的次要原因。此外，在初步研究中，DRA KO小鼠的固有淋巴样细胞-3和IL-22显著减少，这是对抗病原体感染的关键。初步数据还显示，关键粘蛋白(MUC2和4)在杯状细胞中的表达显着增加，但似乎被困在细胞中，表明分泌异常。基于这些发现，我们的总体假设是：肠道对DRA功能丧失的反应与肠道代谢失调有关 损害粘液和上皮屏障的完整性，导致对肠道炎症和病原体感染的易感性增加，上调DRA表达的策略可能会缓解这些影响。本R56提案的这一假说的关键部分将通过下列特定目的进行检验：目标1a：检查DRA KO小鼠微生物群和粘液以及上皮屏障缺陷的年龄相关性动态变化；目标1b：研究与WT小鼠共居是否调节了上皮性相关因子，包括TJs、粘蛋白的合成和分泌；目标1c：通过深入的代谢组学和元基因组分析检查DRA KO小鼠体内细菌种类和代谢物的作用；目标1d：通过粪便微生物从WT和DRA KO小鼠移植到GF小鼠，评估改变的微生物群在影响粘液层和上皮完整性方面的作用。这些拟议的研究将为DRA诱导的粘液和上皮屏障完整性缺陷以及对炎症和感染的易感性所起的作用提供机械性的见解，并可能有助于解开新的治疗靶点。

Down regulated in adenoma (DRA) or SLC26A3 is the key intestinal chloride transporter involved in electrolyte and water absorption in the gut. Loss of function mutations in DRA result in “Congenital Chloride Diarrhea” or CLD disease. Extensive studies from our group have now established that downregulation of DRA is one of the key events in infectious and IBD-associated diarrhea. Emerging evidence from our group and others and GWAS studies have now also established DRA gene to be an IBD susceptibility locus. DRA knockout (KO) mice exhibit a CLD phenotype, thinner mucus layer and increased susceptibility to colitis. We recently showed that DRA KO mice exhibit some of the key features of IBD, including dysbiosis and compromised barrier integrity. Our preliminary studies further showed that DRA KO mice are more susceptible to pathogen infection and exhibit immune cell dysregulation. However, the detailed mechanisms underlying dysbiosis and its role in defective mucus-epithelial barrier observed in DRAKO mice and their increased susceptibility to gut inflammation and pathogen infection have not been fully investigated. Our exciting data further showed that the dysbiosis in DRA KO mice was associated with decreased short chain fatty acid (SCFA) producing taxa, fecal SCFA levels and enrichment in mucin-degrading bacteria. Cohousing studies of DRA KO with wildtype mice showed that part of the compromised barrier integrity was secondary to microbial dysbiosis. Further, in preliminary studies, DRA KO mice showed a marked reduction in Innate Lymphoid Cells-3 and IL-22, critical for combating pathogen infection. Preliminary data also showed that the expression of key mucins (MUC2 and 4) was significantly higher in goblet cells but appeared to be trapped in cells suggesting abnormal secretion. Based upon these findings, our overall hypothesis is that “Gut dysbiosis in response to the loss of DRA function contribute to compromised mucus and epithelial barrier integrity leading to increased susceptibility to gut inflammation and pathogen infection and strategies to upregulate DRA expression may alleviate these effects”. The key component of this hypothesis for this R56 proposal will be tested by the following Specific Aims: Aim 1a: Examine the age-dependent dynamic changes in the microbiome and mucus and epithelial barrier defects in DRA KO mice; Aim 1b: Investigate if cohousing with WT mice modulates the epithelium-associated factors including TJs, mucin synthesis and secretion; Aim 1c: Examine the role of altered bacterial species and metabolites by in-depth metabolomic and metagenomic analysis in DRA KO mice; Aim 1d: Evaluate the role of altered microbiome in affecting mucus layer and epithelial integrity via fecal microbial transplant approaches from WT and DRA KO mice to GF mice. The proposed studies will provide mechanistic insights into the role of DRA induced dysbiosis in defective mucus and epithelial barrier integrity and susceptibility to inflammation and infection and may aid in unraveling novel therapeutic targets.