Dietary Gluten-Induced Gut Dysbiosis is Accompanied by Selective Upregulation of microRNAs with Intestinal Tight Junction and Bacteria-Binding Motifs in Rhesus Macaque Model of Celiac Disease. Macaca mulatta

The study describes miRNA expression in intact jejunum following feeding of gluten containing monkey chow to gluten sensitive (GS) rhesus macaques. Gluten feeding induced several inflammation associated miRNAs validated and predicted to directly target intestinal tight junction proteins. Upregulation of these microRNAs was accompanied significantly reduced mRNA expression of claudin-1, claudin-3 and occludin and severe gut dysbiosis. These findings suggest that miRNA mediated downregulation of intestinal epithelial tight junction proteins could increase intestinal permeability and facilitate translocation of dysbiotic bacteria into the systemic circulation. Overall design: Four age and weight matched gluten sensitive (GS) Indian rhesus macaques were fed gluten containing monkey chow. Five rhesus macaques served as normal healthy controls. Jejunal segments were collected at necropsy from both groups. All animals were necropsied at 60 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturer’s recommendation. microRNA expression profiling was performed using TaqMan ®OpenArray® Human microRNA panels. Data analysis was performed using ExpressionSuite® software. Data was normalized to two endogenous controls (RNU44 and RNU48). Delta CT values were calculated by subtracting individual miRNA CT values from an average of both endogenous controls. Comparisons were made between gluten sensitive and normal healthy control macaques. Four age and weight matched gluten sensitive Indian rhesus macaques were fed gluten containing monkey chow. Five rhesus macaques served as normal healthy controls. Jejunal segments were collected at necropsy from both groups. All animals were necropsied at 60 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturer’s recommendation. microRNA expression profiling was performed using TaqMan ®OpenArray® Human microRNA panels. Data analysis was performed using ExpressionSuite® software. Data was normalized to two endogenous controls (RNU44 and RNU48). Delta CT values were calculated by subtracting individual miRNA CT values from an average of both endogenous controls. Comparisons were made between gluten sensitive and normal healthy control macaques. Four age and weight matched gluten sensitive Indian rhesus macaques were fed gluten containing monkey chow. Five rhesus macaques served as normal healthy controls. Jejunal segments were collected at necropsy from both groups. All animals were necropsied at 60 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturer’s recommendation. microRNA expression profiling was performed using TaqMan ®OpenArray® Human microRNA panels. Data analysis was performed using ExpressionSuite® software. Data was normalized to two endogenous controls (RNU44 and RNU48). Delta CT values were calculated by subtracting individual miRNA CT values from an average of both endogenous controls. Comparisons were made between gluten sensitive and normal healthy control macaques.

本研究探讨了麸质敏感（gluten sensitive, GS）恒河猴喂食含麸质猴粮后，完整空肠组织中的microRNA（miRNA）表达谱。麸质喂养可诱导多种炎症相关miRNA的表达，此类miRNA经验证及预测可直接靶向肠道紧密连接蛋白。该类microRNA的上调，伴随紧密连接蛋白claudin-1（claudin-1）、claudin-3（claudin-3）及闭合蛋白（occludin）的mRNA表达显著降低，同时伴发严重的肠道菌群失调。本研究结果提示，miRNA介导的肠道上皮紧密连接蛋白下调可增加肠道通透性，促进失调菌群易位进入体循环。
总体实验设计：选取4只年龄与体重匹配的麸质敏感印度恒河猴，喂食含麸质的猴粮；另设5只恒河猴作为正常健康对照组。所有动物均在猴免疫缺陷病毒（Simian Immunodeficiency Virus, SIV）感染后60天进行剖检，收集两组动物的空肠组织片段。取约100 ng总RNA，按照制造商推荐的操作流程进行逆转录及预扩增。采用TaqMan®OpenArray®人类microRNA芯片进行microRNA表达谱分析，使用ExpressionSuite®软件完成数据分析。以两个内参基因（RNU44和RNU48）对数据进行标准化，通过两个内参的平均CT值减去单个miRNA的CT值计算ΔCT值，随后比较麸质敏感组与健康对照组猕猴的相关指标。
选取4只年龄与体重匹配的麸质敏感印度恒河猴，喂食含麸质的猴粮；另设5只恒河猴作为正常健康对照组。所有动物均在SIV感染后60天进行剖检，收集两组动物的空肠组织片段。取约100 ng总RNA，按照制造商推荐的操作流程进行逆转录及预扩增。采用TaqMan®OpenArray®人类microRNA芯片进行microRNA表达谱分析，使用ExpressionSuite®软件完成数据分析。以两个内参基因（RNU44和RNU48）对数据进行标准化，通过两个内参的平均CT值减去单个miRNA的CT值计算ΔCT值，随后比较麸质敏感组与健康对照组猕猴的相关指标。
选取4只年龄与体重匹配的麸质敏感印度恒河猴，喂食含麸质的猴粮；另设5只恒河猴作为正常健康对照组。所有动物均在SIV感染后60天进行剖检，收集两组动物的空肠组织片段。取约100 ng总RNA，按照制造商推荐的操作流程进行逆转录及预扩增。采用TaqMan®OpenArray®人类microRNA芯片进行microRNA表达谱分析，使用ExpressionSuite®软件完成数据分析。以两个内参基因（RNU44和RNU48）对数据进行标准化，通过两个内参的平均CT值减去单个miRNA的CT值计算ΔCT值，随后比较麸质敏感组与健康对照组猕猴的相关指标。