Specific-pathogen-free C57BL/6J mice (6–8-week-old) were sourced from Charles River Laboratories, Japan. The mice were maintained under standard conditions (24°C, 50–60% humidity, and a 12 h light/dark cycle) with ad libitum access to standard mouse chow diet (LabDiet Autoclavable Rodent Diet 5010, #0006524, PMI Nutrition International). We monitored body and stool conditions and measured the body weight of the mice each day. The mice were euthanized via cervical dislocation after being anesthetized with intraperitoneal administration of ketamine (75 mg/kg) and medetomidine (1 mg/kg), and colonic tissues were collected following euthanasia. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Kagoshima University (Permit no: MD19057), and the experiments were performed in accordance with the committee guidelines for animal experiments.

The DSS-induced colitis mouse model is an established experimental model that enables the investigation of various colitis symptoms, including diarrhea, weight loss, bloody stool, mucosal ulceration, and shortening of the large intestine. In this study, acute experimental colitis was induced in mice using 3% DSS (FUJIFILM Wako Pure Chemical Industries, Ltd.; average molecular weight: 5,000). Control non-colitis mice were administered distilled water. Colon tissue was collected 5 days after DSS treatment for pathological and histological analyses.

After intraperitoneal administration of 200 µg (10 mg/kg) HGF or phosphate-buffered saline (PBS), as a vehicle for HGF, to DSS-induced colitis mice or untreated control mice for 5 day, body weight, colon length, and disease activity index (DAI) based on clinical scores for weight loss, stool consistency, and bleeding (each score from 0 to 4, and the parameter values were summed, maximum score 12) were measured (23), and the colon tissues were extracted for the assessment of pathological score and gene expression assay. This colitis model is characterized by the absence of severe epithelial defects in the colon.

Colon tissue was fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The stages of colitis were evaluated via blinded histopathological analysis, according to previously described morphological criteria, namely epithelial properties, depth of inflammation, and degree of ulceration (24). Epithelial properties were scored as follows: 0, intact crypt; 1, loss of the basal one-third of the crypt; 2, loss of the basal two-thirds of the crypt; 3, loss of the entire crypt, with intact surface epithelium; and 4, loss of the entire crypt and surface epithelium (erosion). The depth of inflammation was scored as follows: 0, no infiltration; 1, crypt base; 2, mucosa; 3, submucosa; 4, submucosa (extensive). The degree of ulceration was scored as follows: 0, none; 2, positive; 4, positive (extensive). A histological score ranging from 0 to 12 was assigned to each histological specimen.

LPMCs were isolated from the large intestine of the mice according to a previous report (23). Briefly, the colon samples were cut into small pieces in a Ca²⁺/Mg²⁺-free 1.5% Hank's balanced salt solution (1.5% HBSS), washed three times at 37°C with PBS, and incubated with HBSS containing 1 mM dithiothreitol (#15-508-031, Invitrogen; Thermo Fisher Scientific, Inc.) and 5 mM EDTA (#15-575-020, Invitrogen) for 30 min at 37°C to remove the epithelial layer. The samples were then placed in a digestion solution containing 1.5% HBSS, 1.0-3.0 mg/ml collagenase A (#11-088-793-001, Roche Diagnostics GmbH), and 0.1 mg/ml DNase I (#11-284-932-001, Roche Diagnostics GmbH) for 1–2 h at 37°C. The samples were passed through a 100-µm strainer, transferred to a 50-ml Falcon tube, and centrifuged 200 × g, for 5 min at 4°C. The supernatant was washed, and the pellets were resuspended in 40% Percoll and overlaid on a 75% Percoll fraction. Mononuclear cells were collected at the interphase, washed, and resuspended in RPMI-1640 medium (#11875-093, Life Science Products Inc.) containing 10% FBS and 1% penicillin/streptomycin. After isolation, the LPMCs were seeded in 12-well plates (1×10⁶ cells per well) and treated with 10 ng/ml human HGF (E3112; EA Pharma Co., Ltd.) for 24 h in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin.

Colonic LPMCs were separated using MACS. LPMCs with specific CD (cluster of differentiation) antibodies were magnetically labeled with their respective magnetic beads (CD11b; #130-049-601, Miltenyi Biotec). The cell suspension was loaded onto a MACS LS Column (#130-042-401, Miltenyi Biotec), which was placed in the magnetic field of a MACS separator (#130-042-301, Miltenyi Biotec). The MACS separation buffer contained BSA (#A9576, Miltenyi Biotec) at a final concentration of 0.5%. After the cells were separated, they were analyzed for gene expression.

Total RNA was extracted from the colon tissues or LPMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using SYBR (Applied Biosystems). After detection of the threshold cycle for each mRNA in each sample, relative mRNA concentrations were calculated and normalized to that of β-actin. PCR conditions included an initial holding period of 95°C for 30 sec, followed by a 2-step PCR program consisting of 40 cycles of 95°C for 5 sec and 60°C for 34 sec. All reactions were performed in duplicate. The following genes were analyzed (forward, reverse primers: Takara Bio Inc.): β-actin, inducible nitric oxide synthase (iNOS), CD86, arginase-1 (Arg-1), CD206, TNF-α, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, and transforming growth factor-β1 (TGF-β1). The primers used in this experiment are listed in Table I.

LPMCs were washed with PBS and then blocked with anti-mouse CD16/32 Antibody (Clone:93, BioLegend) in a fluorescence-activated cell sorter (FACS) buffer for 10 min. After Fc receptor blockade, the LPMCs were stained with AF700-conjugated anti-CD11b (Clone: M1/70, BioLegend), PE-conjugated anti-c-MET (Clone: eBioclone7, Invitrogen; Thermo Fisher Scientific, Inc.), APC-A750-conjugated anti-CD86 (Clone: FA-11, Invitrogen; Thermo Fisher Scientific, Inc.), PC7-conjugated anti-iNOS (Clone: CXNFT, Invitrogen; Thermo Fisher Scientific, Inc.), eFluor-conjugated anti-Arg-1 (Clone: A1exF5, Invitrogen; Thermo Fisher Scientific, Inc.), and APC-conjugated anti-CD206 (Clone: C068C2, BioLegend) for 1 h on ice. Cells were washed and then resuspended in 4% paraformaldehyde PBS (Fujiwako), 2% bovine calf serum (Sigma-Aldrich; Merck KGaA), 0.1% sodium azide (Sigma-Aldrich; Merck KGaA), and 0.1% HEPES. They were then assayed on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo (Becton Dickinson & Company).

Segments of the distal colon were removed from each animal on day 5 after DSS administration, sectioned longitudinally, and washed in PBS containing penicillin and streptomycin. Segments of length 1 cm each were then placed in 24-well flat-bottom culture plates (Asahi Glass) containing 1 ml fresh RPMI-1640 medium supplemented with penicillin and streptomycin and incubated at 37°C for 24 h. This experiment was performed on 13 mice from each group. The culture supernatants were stored at 30°C until further analysis.

The concentrations of TNF-α, IL-6, IL-10, and TGF-β1 in the culture supernatants were measured using a Mouse Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions and analyzed in duplicate using a microplate reader (Bio-Rad Laboratories) at 450 nm.

Differences between two or three groups were appropriately analyzed using Kruskal-Wallis test followed by Dunn test and Mann-Whitney U test (IBM SPSS version 28). P<0.05 indicated statistical significance. The in vitro experiments were performed at least twice independently.

First, we investigated whether LPMCs of mice with DSS-induced colitis expressed c-MET. The expression of c-Met of LPMC was higher in mice with DSS-induced colitis than in non-colitis mice. In addition, the percentage of c-MET-positive LPMCs was higher in mice with DSS-induced colitis than in non-colitis mice (Fig. 1A). Next, we investigated whether HGF affected the cytokine expression from LPMCs in mice with DSS-induced colitis or non-colitis. The LPMCs isolated from both mice groups were cultured under both conditions of presence and absence of HGF for 24 h. The expression of several cytokines and the genes encoding them was evaluated. A significant increase was observed in the expression of IL-10, TGF-β, TNF-α, and IL-6 in LPMCs cultured with HGF compared to that in the untreated group (P=0.002, 0.004, 0.003, 0.005, respectively). Furthermore, the expression of IL-10 and IL-6 was numerically increased in cell culture supernatant with HGF-treated LPMCs compared to that in the untreated LPMCs (P=0.05), whereas no difference was observed in TNF-α and TGF-β1 expression in both groups (Fig. 1B and C).

We investigated whether HGF affected the phenotype of macrophages, which are CD11b cells derived from the LPMCs of mice with DSS-induced colitis by MACS. No difference was observed in iNOS mRNA expression between HGF-treated and untreated CD11b cells, whereas Arg-1 and CD206 expression numerically increased in the CD11b cells treated with HGF compared to that in the untreated cells, and CD86 expression decreased. The levels of M2 markers Arg-1 and CD206 increased in HGF-treated CD11b cells (Fig. 2A). Moreover, we analyzed M1 and M2 protein expression of CD11b cells treated with HGF using flow cytometry (Fig. 2B). In HGF-treated cells, iNOS and CD86-positive cell percentages were higher, whereas Arg-1 and CD206-positive cell percentages were lower than in untreated cells. These results suggest that HGF could induce transformation from the M1 phenotype to an M2-like phenotype in CD11b cells.

Furthermore, IL-10 expression was significantly increased in CD11b cells treated with HGF compared to that in the untreated group (P=0.038), whereas IL-6 expression decreased (P<0.001). We observed a significant increase in TGF-β expression in CD11b cells treated with HGF compared to that in the untreated group. However, no difference was observed in TNF-α expression between the HGF-treated and untreated CD11b cells. Moreover, the secretion of TNF-α was significantly increased in the cell culture supernatant of CD11b cells treated with HGF (P=0.029), and the secretion of IL-10 was numerically increased in the cell culture supernatant of CD11b cells treated with HGF, whereas the secretion of IL-6 was significantly decreased in the cells treated with HGF compared to that in the untreated cells (P=0.029). In addition, no difference was observed in TGF-β concentration between the two groups (Fig. 2C and D).

The DSS-induced colitis mouse model was evaluated on day 5 after DSS administration. The body weight of the mice in the vehicle group was significantly decreased compared to that in the HGF-treated group, and the DAI in the vehicle group was significantly increased compared to that in the HGF-treated group (P<0.05, Fig. 3A and B). Although no significant differences were observed in colon length and pathological score between the vehicle and HGF-treated groups, the DAI and colon length in the HGF-treated group were lower and longer than those in the vehicle group, respectively (Fig. 3C-E).

Moreover, gene expression in CD11b cells separated from LPMCs of HGF-treated and vehicle groups was evaluated by RT-qPCR. IL-6 expression was significantly decreased in the HGF-treated group compared to that in the vehicle group (P=0.029), whereas the expression levels of TNF-α, IL-10, and TGF-β1 were not significantly different between the vehicle and HGF-treated groups (Fig. 3F). IL-6 secretion numerically decreased in the supernatant from the tissue culture of the HGF-treated group compared to that of the supernatant from the vehicle group; however, no significant difference was observed in cytokine secretion between the two groups (Fig. 3G).