BASIC AND TRANSLATIONAL—ALIMENTARY TRACT Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis Marion Darnaud, 1,2, * Alexandre Dos Santos, 1,2, * Patrick Gonzalez, 1,2, * Sandrine Augui, 1,2 Claire Lacoste, 1,2 Christophe Desterke, 2 Gert De Hertogh, 3 Emma Valentino, 1,2 Emilie Braun, 1,2 Jinzi Zheng, 4,5 Raphael Boisgard, 4,5 Christel Neut, 6 Laurent Dubuquoy, 6 Franck Chiappini, 1,2 Didier Samuel, 1,2 Patricia Lepage, 7 Francesca Guerrieri, 8 Joel Doré, 7 Christian Bréchot, 1,2,9 Nicolas Moniaux, 1,2 and Jamila Faivre 1,2,10 1 INSERM, U1193, Paul-Brousse University Hospital, Hepatobiliary Centre, Villejuif, France; 2 University Paris-Sud, Université Paris-Saclay, Faculté de Médecine Le Kremlin-Bicêtre, France; 3 Department of Imaging and Pathology, Unit of Translational Cell and Tissue Research, University of Leuven, Leuven, Belgium; 4 CEA, DSV, Institut d’Imagerie Biomédicale, Orsay, France; 5 INSERM, U1023, Université Paris-Sud, Orsay, France; 6 LIRIC-U995, University Lille, Inserm, CHU Lille, Lille, France; 7 Institut National de la Recherche Agronomique, UMR 1319 MICALIS, Jouy-en-Josas, France; 8 Center for Life NanoScience@Sapienza, Istituto Italiano di Tecnologia, Roma, Italy; 9 Pasteur Institute, Paris, France; and 10 Assistance Publique-Hôpitaux de Paris (AP-HP), Pôle de Biologie Médicale, Paul-Brousse University Hospital, Villejuif, France BACKGROUND & AIMS: Paneth cell dysfunction causes de- ficiencies in intestinal C-type lectins and antimicrobial pep- tides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of in- flammatory bowel diseases. We investigated whether trans- genic (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 per- formed 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 micro- biome 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 co- housing 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, tran- scriptional analyses, and quantitative polymerase chain reac- tion. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2 0 ,7 0 -dichloro- fluorescein 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. Keywords: Mouse Model; IBD; Bacteria; LPS. Gastroenterology 2018;154:1009–1023 BASIC AND TRANSLATIONAL AT