Lactobacillus maintains healthy gut mucosa by producing L-Ornithine

Gut mucosal layers are crucial in maintaining the gut barrier function. Gut microbiota regulate homeostasis of gut mucosal layer via gut immune cells such as RORγt (+) IL-22(+) ILC3 cells, which can influence the proliferation of mucosal cells and the production of mucin. However, it is unclear how gut microbiota execute this regulation. Here we show that lactobacilli promote gut mucosal formation by producing L-Ornithine from arginine. L-Ornithine increases the level of aryl hydrocarbon receptor ligand L-kynurenine produced from tryptophan metabolism in gut epithelial cells, which in turn increases RORγt (+)IL-22(+) ILC3 cells. Human REG3A transgenic mice show an increased proportion of L-Ornithine producing lactobacilli in the gut contents, suggesting that gut epithelial REG3A favors the expansion of L-Ornithine producing lactobacilli. Our study implicates the importance of a crosstalk between arginine metabolism in Lactobacilli and tryptophan metabolism in gut epithelial cells in maintaining gut barrier.

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Policy information about availability of computer code Data collection 1)Gut microbiota were analyzed by Majorbio Biotechnology Company (Shanghai, China) using primers that target to the V3-V4 regions of 16S rRNA; 2)Expression of coding mRNA and miRNA were analyzed by Beijing Capitalbio Technology Co., Ltd. using Affymetrix 3 IVT Genechip arrays; The quality of isolated RNA samples was evaluated with an Agilent Bioanalyzer 2100 (Agilent technologies) and the purified RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher).
3)Single-cell suspensions of Peyer's patches (PP) and spleen of mice were analyzed by flow cytometry (BD FACS Calibur or BD LSRFortessa); 4) Hematoxylin/eosin (H&E) staining and Immunostaining were analyzed by Leica DMI 6000B and/or Zeiss LSM 700 confocal microscope. 5) Immunostaining of mucus layers and localization of bacteria by Fluorescent in situ hybridization by Leica DMI 6000B and/or Zeiss LSM 700 confocal microscope. 6)HPLC-MS and HPLC-MS/MS analyses were performed by ProfLeader Biotechnology Company (Shanghai, China) through a Waters Xevo-TQXS system or water acquity UPLL system. 7) ELISAs were analysed using TECAN infinite M200PRO.

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October 2018 Data analysis 1) For Gut microbiota analyses, Operational Taxonomic Unit (OTU) analysis was performed as follows: Sequences were processed (trimmed) using the Mothur software; For each clustering, Morisita-Horn dissimilarity was used to compute a sample distance matrix from the initial count matrix, and the distance matrix was subsequently used to generate a hierarchical clustering using Ward's minimum variance method; 2) Expression of coding mRNA and miRNA were analyzed using Cluster 3.0 and MAS 3.0; 3) Flow cytometry data were analyzed by FlowJo-vio 4) Hematoxylin/eosin (H&E) staining and Immunostaining were analyzed by Image J. 5) Graphbad Prism was used in other analyses such as qRT-PCR, ELISA etc.. 5) Immunostaining of mucus layers and localization of bacteria by Fluorescent in situ hybridization by Image J. 6) HPLC-MS and HPLC-MS/MS analyses of L-kynurenine were performed by ProfLeader Biotechnology Company (Shanghai, China) through Analyst (V.1.5.2), Masslynx 4.1 and SIMCA-P13.0.
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability 1)Raw 16S rRNA gene sequence data for the feces microbiota were deposited in the NCBI Short Read Archive under BioProject Accession Number PRJNA326574, Which is related to Figure 3A and 2B, 2)Microarray data: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111111, which is related to Figure 4A.
3) Data of flow cytometry , histological and immunostaining, immunostaining of mucus layers and localization of bacteria by Fluorescent in situ hybridization, Lkynurenine HPLC-MS and HPLC-MS/MS analyses, HPLC/MASS analyses of gut contents, qRT-PCR, immunoblotting and ELISA, which are related to Figure 1 to Figure  6, and supplementary data may be found in 317 Lab, Nankai university school of medicine, Nankai university.

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Methodology
Sample preparation 1)Single-cell suspensions of Peyer's patches (PP) and spleen of mice were prepared by mashing in a cell strainer (70 mm), stained and analyzed by flow cytometry; 2)For the staining of lamina propria (LP) lymphocytes, colon or small intestine were isolated, cleaned by shaking in ice-cold PBS four times before tissue was cut into 1 cm pieces. The epithelial cells were removed by incubating the tissue in HBSS with 2 mM EDTA for 30 min at 37 with shaking. The LP cells were isolated by incubating the tissues in digestion buffer (DMEM, 5% fetal bovine serum, 1 mg/ ml Collagenase IV and DNase I for 40 min. The digested tissues were then filtered through a 40-mm filter. Cells were resuspended in 10 ml of the 40% fraction of a 40: 80 Percoll gradient and overlaid on 5 ml of the 80% fraction in a 15 ml Falcon tube. LP cells were collected at the interphase of the Percoll gradient, washed and resuspended in medium, and then stained and analyzed by flow cytometry. Dead cells were eliminated through 7-AAD staining. 3)For intracellular staining, the cells were cultured and stimulated for 6 hrs with 50ng/ml phorbol 12-myristate 13-acetate and 1 μg/ml ionomycin (Sigma) in the presence of GolgiStop. After incubation for 6 hrs, cells were washed in PBS, and then fixed in Cytofix/Cytoperm, permeabilized with Perm/Wash buffer, and stained with FITC-, PE-, APC-APC/cy7-, PerCP/Cy5.5-or PE/cy7conjugated antibodies. Meanwhile, dead cells were eliminated through 7-AAD staining. 4)For IL-22 production, cells were stimulated directly ex vivo by incubating for 6 hr with 20 ng/ ml rIL-23 in the presence of cell stimulation cocktail for the final 3 hr of culture. Cells were fixed and permeabilized by using Foxp3 fix/perm buffer set, as described by manufacturers and stained with IL-22 and RORγt antibodies.