Mesenchymal stem cell transplantation improves chronic colitis-associated complications through inhibiting the activity of toll-like receptor-4 in mice

The hUC-MSCs were kindly provided by Alliancells Bioscience Co, Ltd. of Tianjin, China. The hUC-MSCs were isolated and expanded by the company, based on a published method [23, 24]. Flow cytometry was used to analyze and identify the MSC phenotype, as previously described [23]. Briefly, cells were stained with phycoerythrin (PE)-conjugated antibodies against CD11b, CD73, CD90, CD45, CD105 and HLA-DR (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and fluorescein isothiocyanate (FITC)-conjugated antibodies against CD19 and CD34 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse isotypic antibodies served as the control. Cells were stained in single label and then analyzed by flow cytometry with a FACS scan (BD Biosciences, Franklin Lake, NJ, USA).

All animal work was approved by the Experimental Animal Center in Hebei Province, (Shijiazhuang, China) and carried out under its procedural and ethical guidelines. Male C57BL/6 J mice (6–8 weeks of age) were randomly allocated to three experimental groups (n = 10). Chronic colitis was induced by multiple-cycle administration of dextran sodium sulfate (DSS; 40, 000–50, 000 MW; Sigma) drinking water. Mice received either regular distilled water (control) or 2% (w/v) DSS drinking water on days 1–5, 8–12, 15–19, 22–26, 29–33, and 36–40. In DSS + MSCs group, 1 × 10⁶/200 μL hUC-MSCs were injected into the caudal vein of each mouse, while the same volume of PBS was given to the control and DSS + vehicle groups. Mice are euthanized by CO2 inhalation followed by cervical dislocation at day 43.

The serum samples were collected from the femoral artery to subsequently evaluate alanine aminotransferase (ALT; Sigma, St Louis, MO, USA), aspartate aminotransferase (AST; Sigma, St Louis, MO, USA), albumin (ALB; Abcam, Cambridge, United Kingdom), total bilirubin (TBIL; Sigma, St Louis, MO, USA) and direct bilirubin (DBIL; Sigma, St Louis, MO, USA) using commercial kits and strictly according to the manufacturers’ instructions.

A limulus amebocyte lysate (LAL-Test, GenScript, USA) was used, according to the manufacturer’s instructions.

DAI and colonic damage were assessed blindly according to a standard scoring system, as previously described [25]. Macroscopic inflammation was scored using the established classification. Samples were processed routinely and stained with hematoxylin and eosin (H&E). Histopathological scores of colons were assigned in a blinded manner by two trained animal pathologists. A scoring system that focused on inflammatory cell infiltration and mucosal damage was used, as previously described [24].

Germiculture of mesenteric lymph nodes (MLN) is an assay of incidence of bacterial translocation. All macroscopically visible MLN were collected under sterile conditions in ice cold Eppendorf tubes and mechanically homogenized in 200 μL of sterile 1× PBS. Then, 0.1 mL of tissue homogenate was cultured on ordinary agar medium at 37 °C for 24 h. Then, the number of bacteria was counted using colony forming-unit (CFU). The rate of bacteria translocation was calculated (germiculture positive specimen number/training specimen total number).

Postmortem, the right liver lobe was removed and liver sections were processed routinely. Hematoxylin-eosin (H&E) staining was performed to assess inflammation, and Masson’s trichrome (MT) staining was performed to assess collagen deposition. Liver histopathological scores were assigned independently and in a blind manner by two well-trained pathologists. Inflammation was graded using the histology activity index (HAI-Knodell score) by Knodell [26].

Immunohistochemistry was done following the protocol the Dako protocol (Dako, Glostrup, Denmark). The samples were incubated with mouse polyclonal anti-TNF-α antibody (1:200, Biolegend, San Diego, CA, USA), rat monoclonal anti-IFN-γ antibody (1:100, Biolegend), rabbit polyclonal anti-IL-1β antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-IL-17A antibody (1:200, Santa Cruz), rabbit polyclonal anti-TLR4 antibody (1:200, Santa Cruz), rabbit polyclonal anti-TRAF6 antibody (1:200, Santa Cruz) and mouse polyclonal anti-NF-κB antibody (1:200, Santa Cruz). Peroxidase-conjugated secondary antibody was used with diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The cells stained brown were considered positive, and the ratio of stained cells to total cells was determine from 10 different fields and calculated using the Image J software (National Institutes of Health, Bethesda, MD, USA).

Western blotting was performed as described previously [27]. Briefly, the tissues were lysed in ice-cold RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0 and protease inhibitors) for 2 h at 4 °C, under agitation. Samples were centrifuged at 12,000 rpm for 20 min at 4 °C and the supernatant was decanted. Equal amounts of proteins (50 μg) were separated by 8% polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA). The proteins were transferred to PVDF membranes (Millipore corp., Billerica, MA, USA) and the membranes were blocked using nonfat powdered milk. The membranes were incubated with the primary antibodies overnight at 4 °C: mouse anti-TNF-α polyclonal (1:500, Biolegend, San Diego, CA, USA), rat anti-IFN-γ monoclonal (1:1000, Biolegend), rat anti-IL-1β polyclonal (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-IL-17A polyclonal (1:500, Santa Cruz), rat anti-TLR4 polyclonal (1:500, Santa Cruz), rat anti-TRAF6 polyclonal (1:500, Santa Cruz) and rat anti-NF-κB polyclonal (1:500, Santa Cruz) antibodies. Peroxidase-conjugated secondary antibody was used with diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The cells stained brown were considered positive, and the ratio of stained cells to total cells was determine from 10 different fields and calculated using the Image J software (National Institutes of Health, Bethesda, MD, USA).

Total RNA was isolated and purified from liver samples using TRIpure Reagent (Aidlab Biotechnologies Co. Ltd., China). cDNA synthesis and real-time PCR were performed as described before [26]. Primers were purchased from Saibaisheng Gene Co. Ltd., China. The primers were designed as: TNF-α-forward 5′-GGA AAG GAC GGA CTG GTG TA-3′, TNF-α-reverse 5’-TGC CAC TGG TCT GTA ATC CA-3′; IFN-γ-forward 5’-GGC AAG TTC AAC GGC ACA G-3′, IFN-γ-reverse 5’-CGC CAG T AG ACT CCA CGA CAT-3′; IL-1β-forward 5′-G AGC ACC TTC TTT TCC TTC ATC TT-3′, IL-1β-reverse 5’-TCA CAC ACC AGC AGG TTA TCA TC-3′; IL-17A-forward 5′-GGA AAG GAC GGA CTG GTG TA-3′, IL-17A-reverse 5′-TG C CAC TGG TCT GTA ATC CA-3′; TLR4-forward 5′-TTT ATT CAG AGC CGT TGG- 3′ TLR4-reverse 5’-CCC ATT CCA GGT AGG TGT-3′; TRAF6-forward 5′-GTA TC C GCA TTG AGA AGC-3′, TRAF6-reverse 5’-GCA GTG AAC CAT CCG TGT-3′; NF-κB-forward 5′-AAG GAT TCG AGC AGT TAG-3′, NF-κB-reverse 5′-AAG AGT TGG TGA TAG GCT-3′; GAPDH-forward 5’-GGC AAG TTC AAC GGC ACA G-3′, and GAPDH-reverse 5’-CGC CAG TAG ACT CCA CGA CAT-3′. Data were analyzed using the 2^-ΔΔCt method [28].

The hUC-MSCs wereisolated from umbilical cords and pooled from a group of four individual donors. The cells were cultured and expanded in vitro in the ultimate conditions. At the end of culture, flow cytometric analysis was performed with a panel of surface markers, which confirmed that the cells were positive for CD73, CD90 and CD105, and negative for CD11b, CD45, HLA-DR, CD19 and CD34 (Fig. 1a). To further confirm the multipotentiality of UC-MSCs after in vitro culture, we assessed their abilities to differentiate into cells of osteogenic and adipogenic lineages. As previously described, UC-MSCs were cultured in the appropriate inducing media for 2 or 3 weeks, and the lipid vacuoles were stained with Oil Red O in the differentiated adipocytes (Fig. 1b), whereas the osteogenic differentiation of MSCs was stained with AlizarinRed (Fig. 1c). These data demonstrate that the UC-MSCs maintained their stem cell characteristics after in vitro culture and expansion.

Compared with the control group, mice in the DSS + vehicle group all developed consistent clinical signs of colitis after DSS administration, including significant loss of BW (22.5% ± 1.90% vs. 0.00% ± 0.00, P < 0.05) (Fig. 2a, c), stool blood, lassitude, and increased DAI (4.00 ± 0.65 vs. 0.00 ± 0.00, P < 0.05) (Fig. 2b, d). MSCs infusion significantly reduced the extent of loss of BW (4.30% ± 1.38% vs. 14.6% ± 1.18%, P < 0.05) and DAI as well (0.30 ± 0.46 vs. 3.05 ± 0.25, P < 0.05) compared to the DSS + vehicle group.

Increased macroscopic gut inflammation was observed in the DSS + vehicle group compared to the control group (Fig. 2e). Shorter colon length and more severe inflammation were also observed in the DSS + vehicle mice compared to the control group (3.93 ± 0.22 vs. 6.30 ± 0.55, P < 0.05), which were improved by MSCs administration (5.18 ± 0.73 vs. 3.93 ± 0.22, P < 0.05) (Fig. 2f). In addition, the degrees of congestion, edema of the colon wall and infiltration of inflammatory cell into the mucosa in the DSS + vehicle group were significantly increased compared with the control group (2.75 ± 0.50 vs. 0.00 ± 0.00, P < 0.05), which were reduced by MSCs compared to the DSS + vehicle group (1.11 ± 0.45 vs. 2.75 ± 0.50, P < 0.05) (Fig. 2g). Histological score of the DSS + vehicle group was significantly higher than that of the control group (11.80 ± 0.96 vs. 0.00 ± 0.00, P < 0.05), which was significantly lowered in the DSS + MSCs group (5.00 ± 0.62 vs. 11.80 ± 0.96, P < 0.05) (Fig. 2h, i).

H&E staining and MT staining were performed to evaluate the effect of MSCs administration on liver pathology (Fig. 3a). Cholangitis, as shown by proliferating bile duct, bile thrombus, necrosis and infiltrated inflammatory cells in sinusoids and centrilobular regions, was found in the DSS + vehicle group. At the same time point, extensive histomorphological signs of chronic hepatitis in DSS mice was observed. MT staining revealed that mice exposed to DSS displayed non-significant perisinusoidal fibrosis, and the degree of collagen deposition did not differ between groups, but differences in inflammatory infiltration were detected. The hepatic histological injury was mainly seen in the central vein and portal area. After MSCs transplantation, histological analyses of liver tissue sections revealed that DSS + MSCs mice appeared to be less alleviated than the DSS + vehicle group, indicating the decreasing inflammatory process. Higher score was found in the DSS + vehicle group compared with the control group (4.70 ± 0.62 vs. 0.00 ± 0.00, P < 0.05), based on the HAI-Knodell score system, which was decreased by MSCs treatment (2.56 ± 0.40 vs. 4.70 ± 0.62, P < 0.05) (Fig. 3b).

Mice in the DSS + vehicle group showed significantly higher levels of ALT and AST (56.82 ± 10.40 vs. 34.50 ± 8.23, P < 0.05; 104.25 ± 17.26 vs. 78.00 ± 10.40, P < 0.05), and lower levels of ALB than the control group (30.33 ± 1.21 vs. 38.60 ± 1.18, P < 0.05). Treatment with MSCs can improve the liver function by reduction of ALT and AST (46.00 ± 8.70 vs. 56.82 ± 10.40, P < 0.05; 87.94 ± 13.51 vs. 104.25 ± 17.26, P < 0.05) and increase of ALB compared to the DSS + vehicle group (35.43 ± 2.43 vs. 30.33 ± 1.21, P < 0.05) (Fig. 3c, d).

Bacterial translocation can be used for measuring intestinal permeability. Large amounts of E. coli were observed in MLN homogenates from the DSS + vehicle group compared to the control group (bacterial translocation rate, 90% vs. 0%). MSCs significantly reduced bacteria translocation to MLN in the DSS + MSCs group compared to the DSS + vehicle group (bacterial translocation rate, 15% vs. 90%) (Fig. 4a, b). Consistent with bacterial translocation, mice in the DSS + vehicle group showed significantly increased level of LPS compared with the control group (0.19 ± 0.03 vs. 0.11 ± 0.01, P < 0.05), which was significantly decreased by MSCs treatment (0.15 ± 0.01 vs. 0.19 ± 0.03, P < 0.05) (Fig. 4c).

TNF-α, IFN-γ, IL-1β and IL-17A were expressed by inflammatory cells close to bile ducts area in inflamed portal tracts, and distributed as spotty or diffuse yellow or brown particles in the cell membrane and cytoplasm. We determined the mRNA expressions of TNF-α, IFN-γ, IL-1β and IL-17A using real-time PCR, finding significantly increased mRNA expression in the DSS + vehicle group (1.94 ± 0.14 vs. 1.00 ± 0.00, P < 0.05; 2.11 ± 0.23 vs. 1.00 ± 0.00, P < 0.05; 2.03 ± 0.19 vs. 1.00 ± 0.00, P < 0.05; 1.91 ± 0.16 vs. 1.00 ± 0.00, P < 0.05) compared to the control group, and all of them were significantly reduced by MSCs administration (1.39 ± 0.17 vs. 1.94 ± 0.14, P < 0.05; 1.72 ± 0.20 vs. 2.11 ± 0.23, P < 0.05; 1.33 ± 0.17 vs. 2.03 ± 0.19, P < 0.05; 1.56 ± 0.15 vs. 1.91 ± 0.16, P < 0.05) (Fig. 5a).

We next determined the expressions of TNF-α, IFN-γ, IL-1β and IL-17A using western blot. Compared to the control group, expressions of TNF-α, IFN-γ, IL-1β and IL-17A were increased in the DSS + vehicle group (0.79 ± 0.07 vs. 0.20 ± 0.02, P < 0.05; 0.82 ± 0.08 vs. 0.15 ± 0.01, P < 0.05; 0.75 ± 0.08 vs. 0.10 ± 0.01, P < 0.05; 0.66 ± 0.07 vs. 0.12 ± 0.01, P < 0.05) (Fig. 6b, c), but all of them were reduced by MSCs administration (0.27 ± 0.03 vs. 0.79 ± 0.08, P < 0.05; 0.41 ± 0.05 vs. 0.82 ± 0.08, P < 0.05; 0.35 ± 0.04 vs. 0.75 ± 0.08, P < 0.05; 0.35 ± 0.04 vs. 0.66 ± 0.07, P < 0.05) (Fig. 5a, b). Relative expressions of TNF-α, IFN-γ, IL-1β and IL-17A proteins by western blot were all significantly higher in the DSS + vehicle group compared to the control group (1.50 ± 0.13 vs. 1.05 ± 0.05, P < 0.05; 1.86 ± 0.10 vs. 1.30 ± 0.06, P < 0.05; 1.79 ± 0.14 vs. 1.11 ± 0.07, P < 0.05; 1.51 ± 0.07 vs. 1.01 ± 0.05, P < 0.05) (Fig. 5c, d), and all of them were decreased by MSCs administration (1.24 ± 0.09 vs. 1.50 ± 0.13, P < 0.05; 1.43 ± 0.15 vs. 1.86 ± 0.10, P < 0.05; 1.27 ± 0.10 vs. 1.79 ± 0.14, P < 0.05; 1.28 ± 0.07 vs. 1.51 ± 0.07, P < 0.05) (Fig. 5d, e).

Compared to the control group, relative expressions of TLR4, TRAF6 and NF-κB mRNA of liver tissue were increased in the DSS + vehicle group compared to the control group (2.46 ± 0.23 vs. 1.00 ± 0.00, P < 0.05; 1.63 ± 0.20 vs. 1.00 ± 0.00, P < 0.05; 1.84 ± 0.13 vs. 1.00 ± 0.00, P < 0.05), and all of them were reduced by MSCs administration (1.61 ± 0.17 vs. 2.46 ± 0.23, P < 0.05; 1.46 ± 0.11 vs. 1.63 ± 0.20, P < 0.05; 1.49 ± 0.19 vs. 1.84 ± 0.13, P < 0.05) (Fig. 6a). Relative expressions of TLR4, TRAF6 and NF-κB proteins from western blot were all significantly higher in the DSS + vehicle group compared to the control group (2.10 ± 0.26 vs. 1.48 ± 0.17, P < 0.05; 1.77 ± 0.18 vs. 1.13 ± 0.11, P < 0.05; 1.86 ± 0.10 vs. 1.42 ± 0.09, P < 0.05) (Fig. 6b, c), and all of them were lowered by MSCs administration (1.40 ± 0.13 vs. 2.10 ± 0.26, P < 0.05; 1.36 ± 0.15 vs. 1.77 ± 0.18, P < 0.05; 1.59 ± 0.09 vs. 1.86 ± 0.10, P < 0.05). Positive expressions of TLR4, TRAF6 and NF-κB by immunohistochemistry in the DSS + vehicle group were much higher compared with the control group (0.72 ± 0.08 vs. 0.15 ± 0.02, P < 0.05; 0.61 ± 0.06 vs. 0.10 ± 0.01, P < 0.05; 0.67 ± 0.08 vs. 0.13 ± 0.02, P < 0.05) (Fig. 6d, e), and all of them were reduced by MSCs administration(0.38 ± 0.04 vs. 0.72 ± 0.08, P < 0.05; 0.29 ± 0.03 vs. 0.61 ± 0.06, P < 0.05; 0.33 ± 0.04 vs. 0.67 ± 0.08, P < 0.05).

Pearson’s correlation analysis showed that serum LPS levels depicted positive correlation with TLR4 mRNA expression in liver tissues and HAI-Knodell score (Fig. 7a, b). The r values were 0.901 and 0.825 respectively, P < 0.05.