Mannose ameliorates experimental colitis by protecting intestinal barrier integrity | Panda Anku

Participants

All patients with IBD were recruited from the Department of Gastroenterology at Nanfang Hospital, affiliated with Southern Medical University (Guangzhou, China). The diagnosis of CD or UC was based on clinical, radiologic, and endoscopic examinations and histologic findings. The baseline characteristics are described in Supplemental Table 1. All patients provided informed consent, and the study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University.

Mice and treatments

Male IL-10-deficient (IL-10−/−) C57BL/6 J mice (12 weeks old, 22–25 g) were obtained from the Shanghai Research Center for Model Organisms (Shanghai, China). Male C57BL/6 J mice (8 weeks old, 22–25 g) were purchased from the Animal Institute of Southern Medical University (Guangzhou, China). Mice were maintained under SPF conditions. All animal experiments in this study were approved by the Welfare and Ethical Committee for Experimental Animal Care of Southern Medical University.

DSS-induced colitis

Chemically-induced colitis was established by providing the mice with 3.0% (wt/vol) DSS (MP Biomedicals, Santa Ana, CA, USA, molecular weight of 36,000–50,000) dissolved in drinking water for 7 days. Starting from the first day of the DSS challenge, mannose (Sigma-Aldrich) or glucose (Sigma-Aldrich) was administered orally at the indicated dose (5, 50, 500, and 5000 μg/g/d) per day. Furthermore, 8 a.m. and 8 p.m. were chosen as a set time for feeding, with a duration of 7 days.

Mito-tempo treatment

Mice were treated with mito-tempo (Merck, HY-112879) at a dose of 2 mg/kg by i.p injection every day. Mice were randomly divided into eight groups: control group (mice received regular drinking water), mito-tempo group (mice received regular drinking water together with i.p injection of 2 mg/kg of mito-tempo), mannose group (mice received regular water together with administration by gavage of 500 μg/g/d mannose), mito-tempo + mannose group (mice received regular drinking water together with i.p injection of 2 mg/kg of mito-tempo and administration by gavage of 500 μg/g/d mannose), DSS model group (mice received 3% DSS in drinking water), DSS + mito-tempo group (mice received 3% DSS in drinking water together with i.p injection of 2 mg/kg of mito-tempo), DSS + mannose group (mice received 3% DSS in drinking water together with administration by gavage of 500 μg/g/d mannose), DSS + mito-tempo +mannose group (mice received 3% DSS in drinking water together with i.p injection of 2 mg/kg of mito-tempo and administration by gavage of 500 μg/g/d mannose).

Bafilomycin A1 treatment

Mice were treated with bafilomycin A1(Baf A1) (Selleck, S1413) at a dose of 2 mg/kg by i.p injection every day. Mice were randomly divided into eight groups: control group (mice received regular drinking water), Baf A1 group (mice received regular drinking water together with i.p injection of 2 mg/kg of Baf A1), mannose group (mice received regular water together with administration by gavage of 500 μg/g/d mannose), Baf A1 + mannose group (mice received regular drinking water together with i.p injection of 2 mg/kg of Baf A1 and administration by gavage of 500 μg/g/d mannose), DSS model group (mice received 3% DSS in drinking water), DSS + Baf A1 group (mice received 3% DSS in drinking water together with i.p injection of 2 mg/kg of Baf A1), DSS + mannose group (mice received 3% DSS in drinking water together with administration by gavage of 500 μg/g/d mannose), DSS + Baf A1 + mannose group (mice received 3% DSS in drinking water together with i.p injection of 2 mg/kg of Baf A1 and administration by gavage of 500 μg/g/d mannose).

Mesalazine treatment

To evaluate the therapeutic effect of mannose on colitis, mice were given a high dose of DSS (5.0%, wt/vol) for 3 days, followed by mannose (5 mg/kg per day) or glucose (5 mg/kg per day) and mesalazine (Adisha, H20143164, 1 mg/kg per day) alone or in combination for another 4 days. Mice were randomly divided into six groups: DSS model group (mice received 5.0% DSS in drinking water for three days), DSS + glucose group (mice received intragastric (i.g) administration of 500 μg/g/d of glucose after receiving 5.0% DSS in drinking water for three days), DSS + mannose group (mice received intragastric (i.g) administration of 500 μg/g/d of mannose after receiving 5.0% DSS in drinking water for 3 days), DSS + mesalazine group (mice received intragastric (i.g) administration of 1 mg/kg/d of mesalazine after receiving 5.0% DSS in drinking water for 3 days), DSS + glucose+mesalazine group (mice received intragastric (i.g) administration of 500 μg/g/d of glucose plus 1 mg/kg/d of mesalazine after receiving 5.0% DSS in drinking water for three days), DSS + mannose+mesalazine group (mice received intragastric (i.g) administration of 500 μg/g/d of mannose plus 1 mg/kg/d of mesalazine after receiving 5.0% DSS in drinking water for three days).

Spontaneous chronic colitis

To determine the effect of mannose on spontaneous chronic colitis, 15-week-old IL-10−/− mice in the treatment group were treated with glucose (1.0%, wt/vol) or mannose (1.0%, wt/vol) for 4 weeks.

Cells and stimulation conditions

Human colonic epithelial NCM460 cells were cultured in RPMI 1640 medium with no glucose (Invitrogen, Carlsbad, CA, USA, 11879020) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM glucose, and 10% fetal bovine serum (FBS). The cells were treated with 2.0% DSS (MP Biomedicals, Santa Ana, CA, USA, molecular weight of 36,000–50,000) in the presence or absence of mannose (25 mM) for 24 h. To eliminate the effect of the mannose transporter, cells were pretreated with ouabain (1 μM, Selleck, S4016) for 4 h before incubation. Rotenone (5 μM, Merck, HY-B1756) was added simultaneously with DSS and mannose to inhibit mitochondrial respiratory activity. In some cases, protease inhibitors, such as the cathepsin B inhibitor CA-074 (10 μM, Selleck, HY-103350), cysteine protease inhibitor aloxistatin (1 μM, Selleck, S7393), serine protease inhibitor nafamostat mesylate (5 μM, Selleck, S1386), and aspartic protease inhibitor pepstatin A (10 μM, Selleck, S7381), were added simultaneously with DSS and mannose. Bafilomycin A1 (1 μM, Selleck, S1413) was added simultaneously with DSS and mannose for 24 h. mito-tempo (2 μM, Merck, HY-112879), a mitochondrial-targeted antioxidant, was used to stimulate cells along with DSS plus mannose stimulation.

Quantitative RT-PCR

Total RNA was extracted from mouse colon tissue or NCM460 cells using TRIzol reagent (TransGene Biotech, Beijing, China) and transcribed into cDNA using TranScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGene Biotech). Real-time PCR analysis using TransStart Tip Green qPCR SuperMix (TransGene Biotech) was performed on a 7900HT fast real-time PCR system (Applied Biosystems, San Francisco, CA, USA). The target gene expression levels were normalized to the expression of β-actin in the same samples.

Immunoprecipitation and immunoblotting

Protein samples were loaded on SDS-polyacrylamide gels, separated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with bovine serum albumin (BSA, 5%) for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies. Subsequently, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. For immunoprecipitation, whole cell lysates were incubated with 1 μg of antibody and protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The eluted immunoprecipitates were resolved via SDS-PAGE, and the associations between proteins of interest were examined using specific antibodies.

Immunohistochemical and immunofluorescence staining

For immunohistochemical staining, antigen retrieval was performed in citrate buffer (pH 6.0) (Sigma-Aldrich, C2488) at 120 °C for 10 min, and endogenous peroxidase activity was blocked by exposure to 3.0% H2O2 for 15 min. Sections were then incubated with primary antibodies at 4 °C overnight. Immunoreactivity was detected using the corresponding HRP-conjugated secondary antibody and visualized using a PierceTM DAB Substrate kit (Thermo Fisher, Carlsbad, CA, USA).

For immunofluorescence staining, cells were grown in confocal dishes, fixed in 4.0% formaldehyde for 15 min at room temperature, and permeabilized with 0.25% Triton X-100 for 10 min at room temperature. After blocking with 5.0% FBS for 1 h, cells were incubated with primary antibodies overnight at 4 °C, rinsed, and incubated with fluorescently labeled secondary antibodies for 1 h in the dark. Finally, cells were counterstained with Hoechst 33342 (Cell Signaling Technology, 4082). The quantitative colocalization was calculated using the JACoP plugin in single Z-stack sections of deconvolved images. The nonspecific signal correction was performed using ImageJ with the subtract background plugin. All images were subjected to identical post-acquisition processing.

Histopathological assessment

For pathological assessment, the H&E stained sections were evaluated by a blinded pathologist following the criteria of histological score previously reported55. The colitis score (maximum = 8) was the sum of the following two features: Epithelium (0: Normal morphology; 1: Loss of goblet cells, 2: Loss of goblet cells in large areas; 3: Loss of crypts; 4: Loss of crypts in large areas), and infiltration (0: No infiltrate; 1: Infiltrate around crypt basis; 2: Infiltrate reaching to lamina muscularis mucosae; 3: Extensive infiltration reaching the lamina muscularis mucosae and thickening of the mucosa with abundant edema; 4: Infiltration of the lamina submucosa).

Measurement of serum mouse albumin

Serum albumin levels were measured by a mouse-specific albumin ELISA kit (Abcam, ab207620) according to the manufacturer’s instructions. In brief, the sample and the antibody cocktail were added to each well and incubated for 1 h at room temperature After washing the wells three times with washing buffer, 100 μl of TMB development solution was added and incubated for 10 min in the dark. Then, 100 μl of stop solution was added to each well and the OD value was recorded at 450 nm.

Measurement of fecal α1-antitrypsin

Fecal samples were dissolved in PBS to a concentration of ~500–1000 ng/ml. Alpha-1-antitrypsin levels were quantified using a mouse-specific α1-antitrypsin SimpleStep ELISA Kit (Abcam, ab267809) according to the manufacturer’s instructions. Briefly, the sample and the antibody cocktail were added to each well and incubated for 1 h at room temperature. After washing three times with the washing buffer, 100 μl of TMB development solution was added to each well and incubated for 10 min in the dark. Then, 100 μl of stop solution was added and the OD value was recorded at 450 nm.

Measurement of mannose

Measurement of circulating mannose

An aliquot (50 μl) of standard, quality control (QC), a human plasma sample, or mouse serum sample was first mixed with 200 μl methanol-acetonitrile-water (v:v:v, 2:2:1) mixed solvent containing 5 μg/ml d-mannose-1-13C (Sigma-Aldrich) as an internal standard. The mixture was then vortex mixed for 30 s and centrifuged for 30 min at 20,000 × g at room temperature. After centrifugation, an aliquot (10 μl) of the supernatant was taken for LC–MS/MS analysis. The chromatographic separation was achieved by a Shimadzu Nexera UHPLC LC-30A equipped with an AB SCIEX Triple Quad™ 4500, crosslinked HPLC column (3.0mml.D. × 100 mm, 1.7 μm, waters) with a flow rate of 0.35 ml/min. The LC–MS/MS system was controlled, and data was acquired by Analyst software version 1.6.

Measurement of mannose in cells

After incubated with d-mannose-1-13C (Sigma-Aldrich, 25 mM) for 12 and 24 h, the lysosomal fraction, mitochondrial fraction, and the whole cell lysates of NCM460 cells were isolated. The fraction was extracted with 1 ml ethanol: water (1:1). The mixture was then centrifuged for 30 min at 20,000 × g at room temperature. After centrifugation, an aliquot (10 μl) of the supernatant was taken for LC–MS/MS analysis. The same method was used for the determination of glucose in cells.

Metabolic extraction of intracellular metabolites

For 13C-mannose-tracing assays, NCM460 cells were treated in glucose-free RPMI 1640 medium (11879, Thermo Fisher Scientific) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM glucose, and 10% FBS and 25 mM 13C-labeled-mannose. Cells were rapidly washed with ice-cold PBS three times and extracted with 500 µl extraction solvent (50% methanol, 30% acetonitrile, and 20% water) after 24 h. Cells were then centrifuged at 16,000 × g for 30 min at 4 °C, and the supernatants were assessed by LC–MS analysis. Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) was used together with a Thermo Fisher Scientific Accela HPLC system. The HPLC setup consisted of a ZIC-pHILIC column (SeQuant, 150 mm × 2.1 mm, 5 µm, Merck KGaA) with a ZIC-pHILIC guard column (SeQuant, 20 mm × 2.1 mm) and an initial mobile phase of 20% 20 mM ammonium carbonate, pH 9.4 and 80% acetonitrile. Cell extracts (5 µl) were injected, and the metabolites were separated over a 15-min mobile phase gradient. All metabolites were detected across a mass range of 75–1000 m/z using the Exactive mass spectrometer at a resolution of 25,000 (at 200 m/z), with electrospray ionization and polarity switching to enable both positive and negative ions to be determined in the same run over a total analysis time of 23 min. Lock masses were used, and the mass accuracy obtained for all metabolites was below 5 p.p.m. Data were acquired with Thermo LCquan 2.7 (Thermo Fisher Scientific) software. To examine whether mannose is involved in glycoprotein synthesis, protein lysate of NCM460 cells were deglycosylated by Protein Deglycosylation Mix 2 (New England Bio-labs), according to the non-denaturing reaction protocol distributed by the manufacturer. Briefly, 100 μg of protein lysate of NCM460 cells, 5 μl of 10 × deglycosylation mix buffer, and 5 μl of protein deglycosylation mix 2 were mixed to a total volume of 50 μl and incubated at 25 °C for 30 min, followed by overnight incubation at 37 °C. The supernatant was hydrolyzed by 2 M trifluoroacetic acid at 100 °C for 16 h. An aliquot (10 μl) of the supernatant was taken for LC–MS/MS analysis.

FITC-dextran permeability assay

Intestinal permeability was assessed by oral administration of fluorescein isothiocyanate-dextran with a molecular weight of 3000–5000 Da (FD4, Sigma-Aldrich). At the beginning of the experiment, food and water were forbidden for 4 h, and mice were subsequently gavage fed with FITC-dextran solution at 500 μg/g body weight. Serum was collected 4 h post-feeding, and FITC-dextran measurements were performed in duplicate by fluorometry (excitation, 490 nm; emission, 530 nm; Cytofluor 2300, Millipore).

TER measurement

Confluent monolayers of NCM460 cells or isolated primary colonic epithelial cells were grown in 24-well Transwell chambers (polycarbonate membrane, filter pore size 0.4 μm, area 0.33 cm2; Costar) for 24 h, and then TER was measured at 37 °C using an Epithelial Volt Ohm Meter (Millipore, Billerica, MA, USA). TER values were calculated by subtracting the blank filter and by multiplying the surface area of the filter. All the measurements were performed in triplicate.

Isolation of primary colonic epithelial cells

The primary colonic epithelial cells of mice were purified as previously described34. Briefly, the colonic tissues were cut into pieces and trained at 37 °C in Dulbecco’s modified Eagle medium (DMEM) containing 5% FBS and 1 mM dithiothreitol (DTT) for 30 min. The remaining tissue was induced in 30 ml phosphate-buffered saline (PBS) containing 1.5 mM EDTA for an additional 10 min The supernatants were filtered, centrifuged for 5 min at 400 × g, and the cell pellet was resuspended in DMEM containing 5% FBS. Finally, the primary colonic epithelial cell suspension was purified by centrifugation through a 25%/40% discontinuous percoll gradient at 600 × g for 30 min.

Cell viability assay

For apoptosis quantification by annexin V, NCM460 cells were harvested and stained with annexin V-FITC and propidium iodide according to the manufacturer’s instructions (Annexin V-APC/7-AAD apoptosis kit, Multi Sciences, China). In brief, the cells were washed with PBS and subsequently incubated for 5 min at room temperature in the dark in 500 μl of 1× binding buffer containing 5 μl of Annexin V-PE and 10 μl of 7-AAD. The cells were acquired and analyzed in the Dakewe EXFLOW-206.

Mitochondrial respiration measurements

Oxygen consumption rate (OCR) was analyzed using the Seahorse Bioscience Extracellular Flux Analyzer (XF96) (Seahorse Biosciences, MA, USA). After exposure to several inhibitors, the media of NCM460 cells was removed and replaced by XF basic media 30 min prior to the measurement of oxygen consumption rate (OCR). Several drugs were added in sequence, including oligomycin (1 μg/mL), FCCP (1 μM), and rotenone (1 μM) + antimycin A (10 μM). Basal OCR was determined before any following operations, while non-mitochondrial OCR was defined as the value after the injection of the last drug. The rate of respiration-driven ATP synthesis and proton leak-driven respiration was measured after the addition of oligomycin (1 μg/ml). The difference between this rate and basal OCR was defined as ATP-linked OCR, and similarly, Proton leak OCR was defined as the gap between this rate and non-mitochondrial OCR. After two measurement cycles, 1 μM of the FCCP was added, and the rate was determined. The difference between which and non-mitochondrial OCR was defined as maximal OCR in the same way. After a further two measurement cycles, 1 μM rotenone was added to block complex I in addition to 10 μM antimycin A to inhibit complex III, and thus non-mitochondria was determined. Each value of OCR was normalized by the amount of cellular protein content in each well.

Mitochondrial function assays

The cells were washed twice with PBS and labeled at 37 °C for 30 min with the following fluorescent probes: 400 nM MitoTracker Green (Ex490 nm/Em516 nm), 2.5 μM MitoSOX Red (specifically detects mitochondrial O2), and 10 μM JC-1 (a sensitive marker of mitochondrial membrane potential). Fluorescence was detected on a Nikon A1R scanning laser confocal microscope (Nikon Corporation, Tokyo, Japan). Quantification was performed in images after appropriate thresholding using the ImageJ software (NIH Image).

Lysosomal function assays

LysoTracker staining

NCM460 cells were incubated with LysoTracker Green DND-26 (1 µM) in culture media at 37 °C for 30 min. Hoechst 33342 (Cell Signaling Technology) was used to stain the cells for another 20 min.

Acridine orange staining

Cells were washed three times with PBS and stained with 2.5 μg/ml acridine orange (Sigma-Aldrich) for 15 min.

PDMPO staining

After being washed by PBS three times, NCM460 cells were incubated with PDMPO (1 µM, AAT Bioquest, 21204) in culture media at 37 °C for 30 min and then fixed in 4.0% formaldehyde for 15 min at room temperature and permeabilized with 0.25% Triton X-100 for 10 min at room temperature. After blocking with 5.0% FBS for 1 h, cells were incubated with anti-LAMP2 antibody overnight at 4 °C, rinsed, and incubated with PE-labeled secondary antibodies for 1 h in the dark.

FITC-dextran measurement

After being washed by PBS three times, NCM460 cells were incubated with fluorescein isothiocyanate-dextran (70 kDa, Merck, 46945) for 4 h and then fixed in 4.0% formaldehyde for 15 min at room temperature and permeabilized with 0.25% Triton X-100 for 10 min at room temperature. After blocking with 5.0% FBS for 1 h, cells were incubated with anti-LAMP2 antibody overnight at 4 °C, rinsed, and incubated with PE-labeled secondary antibodies for 1 h in the dark. For immunofluorescence and direct fluorescence quantifications of colocalization, numbers of puncta per cell and the ratio were performed blinded. Quantification was performed in images after appropriate thresholding using the ImageJ software. The quantitative colocalization was calculated using the JACoP plugin in single Z-stack sections of deconvolved images. The nonspecific signal correction was performed using ImageJ software with the subtract background plugin. All images were subjected to identical post-acquisition processing.

Lysosome and mitochondria isolation

Lysosomes were isolated by a lysosome isolation kit (Abcam, ab234047) according to the manufacturer’s protocol. In brief, the cells were isolated in ice-cold lysosome isolation buffer for 2 min and homogenized. The supernatant was collected by centrifugation (500 × g, 10 min) at 4 °C and layered onto a discontinuous density gradient. The lysosomes were further isolated using an ultracentrifuge for 2 h at 145,000 × g at 4 °C.

Mitochondria were isolated by a mitochondria isolation kit for the cells (Abcam, ab110170) according to the manufacturer’s instruction. Briefly, the cells were collected and suspended in Reagent A. Then, the cells were homogenized and centrifuged at 1000 × g at 4 °C for 10 minutes. The supernatant was saved as SN1. The pellet was resuspended with Reagent B. After homogenization and centrifugation, the supernatant was collected as SN2. SN1 and SN2 were combined and centrifuged at 12,000 × g at 4 °C for 15 min. Subsequently, the pellet was resuspended in Reagent C supplemented with protease inhibitors and used for further analysis.

Measurement of cathepsin B activity

Cathepsin B activity was measured in the culture medium or in tissue homogenate using the fluorogenic substrate N-Suc-Leu-Leu-Val-Tyr7-AMC. Excitation at 380 nm and emission at 440 nm were used for activity determinations.

Generation of gene knockout cells with CRISPR/Cas9

NCM460 cells were transfected with 2 µg of PDH CRISPR/Cas9 KO plasmid or cathepsin B CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology) using UltraCruz® transfection reagent (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Twenty-four hours after transfection, the expression levels of PDH and cathepsin B in the cells were assessed by immunoblotting analysis.

Statistical analysis

All results were expressed as mean ± SD. Statistical significance between two groups was evaluated using the Student’s t-test, while comparisons of multiple groups were assessed by two-way analysis of variance (ANOVA), followed by Student-Newman–Keul’s test. p < 0.05 was considered significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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