Background & Aims

Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice.

Methods

We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2′,7′-dichlorofluorescein diacetate and flow cytometry.

Results

The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis).

Conclusions

Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

中文翻译：

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肠内再生的再生家庭成员3 alpha改变肠道菌群并控制炎症的结肠炎小鼠。

背景与目标

Paneth细胞功能障碍会导致肠道C型凝集素和抗菌肽缺乏，从而导致肠道菌群功能失调，改变粘膜屏障并促进炎症性肠病的发展。我们调查了人类再生家族成员3 alpha基因（REG3A）的转基因（TG）表达是否改变了粪便微生物群并影响了小鼠结肠炎的发展。

方法

我们对通过白蛋白基因启动子在肝细胞中表达人类再生家族成员3 alpha（hREG3A）的C57BL / 6小鼠进行了研究。在这些小鼠中，hREG3A通过胆汁到达肠腔。一些小鼠被给予右旋糖酐硫酸钠（DSS）诱导结肠炎。从小鼠收集粪便，并通过16S核糖体RNA测序分析微生物群的组成。还从仅表达1个人REG3A拷贝的小鼠中分析了粪便微生物组转基因，但喂养了新生的对照小鼠（不表达hREG3A）的粪便。从对照组小鼠喂食或喂食粪便后，监测表达hREG3A的小鼠的DSS诱导的结肠炎。通过给予三硝基苯磺酸在另一组对照和hREG3A-TG小鼠中诱发结肠炎。一些小鼠经直肠内注射了hREG3A蛋白。从小鼠收集结肠组织，并通过组织学和免疫组织化学分析以检测粘蛋白2，以及通过16S核糖体RNA荧光原位杂交，转录分析和定量聚合酶链反应。我们使用2'，7'-dichlorofluorescein diacetate和流式细胞仪测量了细菌培养物和粪便微生物群中的活性氧（ROS）水平。

结果

与对照小鼠相比，表达hREG3A的小鼠的粪便微生物组成发生了明显变化，梭状芽孢杆菌（Ruminococcaceae，Lachnospiraceae）的富集和拟杆菌属（Prevotellaceae）的耗竭。与对照组相比，TG小鼠在给予DSS后发展为较不严重的结肠炎，这与保留的肠道屏障完整性，减少的细菌易位，上皮炎症和氧化损伤有关。在REG3A杂合的TG小鼠中，几个月后，粪便微生物群的组成发生了类似的变化，而REG3A在出生时带有野生型母体微生物群。这些小鼠在施用DSS后发展为严重程度较轻的结肠炎。饲养和无菌小鼠饲喂REG3A-的粪便与未饲喂REG3A -TG小鼠粪便的小鼠相比，TG小鼠和给予DSS的结肠炎发展程度较轻，并减少了toll-like受体4的脂多糖活化，并延长了存活时间。与对照小鼠相比，REG3A TG小鼠在暴露于2,4,6-三硝基苯磺酸后仅发生了轻度结肠炎。与未接受直肠内hREG3A的小鼠相比，接受直肠内hREG3A并暴露于2,4,6-三硝基苯磺酸的对照小鼠显示出更少的结肠损伤和炎症。在DSS给药过程中，来自REG3A- TG小鼠的粪便样品的ROS水平低于来自对照小鼠的粪便。向细菌培养物中添加hREG3A可降低ROS的水平并提高对氧敏感的共生细菌的存活率（Faecalibacterium prausnitzii和Roseburia intestinalis（）。

结论

携带表达hREG3A的肝细胞的小鼠（其进入肠道内腔）对结肠炎的敏感性低于对照小鼠。我们发现hREG3A通过降低ROS水平来改变结肠菌群。REG3A -TG小鼠的粪便微生物群可保护非TG小鼠免于诱发结肠炎。这些发现表明，在保持肠道菌群及其减少炎症的能力方面，氧化应激的减轻作用。