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Background 3-dimensional (3D) cultured tonsil-derived mesenchymal stem cells (TMSCs) showed higher therapeutic efficacy in murine colitis model than conventional 2D-cultured TMSCs via increased anti-inflammatory cytokine expression. Here, we evaluated the in-vivo localization and formation of 3D-cultured TMSCs using electron microscopy. Methods Chronic mouse colitis was induced by oral administration of 1.5% dextran sulfate sodium for 30 days. 3D-cultured TMSCs and 2D-cultured TMSCs was injected intraperitoneally on day 6 and day 16 after inducing colitis in mice. TMSC was 3D-cultured as a spheroid for 24 hours before intraperitoneal injection. On day 31, mice were euthanized and the intraperitoneal localization of 3D-cultured TMSCs was evaluated using immunofluorescence analysis. Moreover, the in-vivo characterization of 3D-cultured TMSCs was evaluated using electron microscopy. Results In vitro 3D-cultured TMSC spheroids were measured to have an average size of 182.4 μm on day 1, 169.4 μm on day 2, and 154.1 μm on day 3; however, the size of spheroids gradually decreased after 2 days of culture. In mice treated with 2D-cultured TMSCs, there were no detectable TMSCs at the time of sacrifice. In mice treated with 3D-cultured TMSCs, white variable sized spherical aggregates were observed. These aggregates were attached to peritoneal cavity including omentum and mesentery. In fluorescence microscopy, these aggregates were stained with human nuclear antigen antibody, indicating that the transplanted 3D-cultured TMSCs formed a cluster in the peritoneum even 14 days after intraperitoneal injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. In the inside of spheroids, suspected mesenchymal cell and mesothelial cell were distributed. Conclusion Intraperitoneally injected 3D-cultured TMSCs were found as aggregates in the peritoneal cavity of mouse even 14 days after injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. Based on the results of current study, functional studies evaluating the role of TMSC as a cell therapy, including cell-to-cell communication between TMSC and immune cells, are needed.

Background Although associations between various single nutrients and exacerbation of inflammatory bowel disease (IBD) have been reported, more recent attention has focused on overall dietary patterns and quality rather than a single nutrient. This study was to investigate the association of dietary inflammatory potential and dietary quality with disease severity in young adult Crohn’s disease (CD) patients. Methods Non-consecutive 3-day food records were investigated in 25 patients with CD aged 19-40 years. Patients with moderate or severe active disease were excluded to investigate their usual dietary habits. A survey on dietary behavior was conducted and clinical data including the disease progress were collected. To investigate potential pro-inflammatory dietary habits in a patient's usual diet, food-based index of dietary inflammatory potential score (FBDI), glycemic index (GI), and glycemic load (GL) were calculated. Diet quality was evaluated using a tool called Diet Quality Index-international (DQI). Results Although most patients showed adequate caloric intake, 72% of the enrolled patients had a potential pro-inflammatory pattern identified as FBDI. The FBDI was significantly associated with intake of mixed coffee and sweeten drinks, beef, and pork (P < 0.05). Patients who had a serious clinical course such as history of surgery or the use of biologics, tended to have a higher FBDI (6.45 vs 2.44, P = 0.08), and showed a higher proportion with a low DQI (53% vs 20%, P = 0.06). Only 30% of the high FBDI group showed deep remission after 12 to 18 months of follow-up endoscopic or image study, whereas 72.7% of the low FBDI group showed deep remission (P =0.05). A significantly positive correlation was derived between FBDI and GL (r =0.452, P = 0.02). Conclusion For the care of patients with CD, it is necessary to evaluate the dietary quality as well as the total nutrient intake, and we need to educate the patients that dietary habits can affect the disease prognosis.

Background It has been suggested that changes in gut microbiota have an important effect on the development of inflammatory bowel disease (IBD). In studies using germ-free mice, it has been reported that the absence of microbiota rarely causes dextran sulfate sodium (DSS)-colitis. This study was to investigate whether the changes in DSS-colonic inflammation in mice treated with antibiotics were comparable to the results in germ-free mice. Methods Ampicillin + enrofloxacin were treated in six 9-week-old C57BL/6 mice for initial 3 days and stools were sampled daily to measure the total amount of bacterial 16S rRNA. The time of loss and restoration of gut microbiota was investigated after repeated antibiotics. The severity of colitis and barrier damage were compared between the following groups: (i) control group, (ii) DSS group, and, and (iii) DSS + antibiotics group. The DSS group was allowed to drink 3% DSS from day 5 and maintained for 7 days, and colon tissues were obtained from all groups on day 12. The inflammatory markers (IL-1α, IL-6, IL-17, tumor necrosis factor (TNF)-α) and gut barrier markers (Zonular occludens (ZO)-1, occludin, claudin-1, claudin-4) in colon tissues were comparatively analyzed between groups. Results Bacterial 16S rRNA was not detected from day 4 after the antibiotics, and restoration started from day 11. After repeated antibiotics, microbial depletion was re-confirmed on day 14, but restoration appeared on day 18, confirming faster recovery. The expression levels of IL-1α, IL-17, and TNF-α in the DSS + antibiotics group did not differ from the control but were significantly lower than the DSS group (all p < 0.01). However, the expression of ZO-1 and claudin-4 was significantly lower in the DSS + antibiotics treatment group than in the control group (p < 0.05). Conclusion The antibiotic cocktail could dramatically reduce gut microbiota over a period, but the effect would decrease as it is repeated. Depletion of gut microbiota after antibiotics would dramatically reduce DSS-colonic inflammation but could damage the gut barrier or at least not prevent barrier dysfunction.

Background The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway has not been fully evaluated in inflammatory bowel disease. We evaluated the level of PD-1/PD-L1 in the mucosa of patients with ulcerative colitis and revealed the role of PD-1/PD-L1 in the immunomodulatory mechanism of tonsil-derived mesenchymal stem cells (T-MSCs). Methods We used chronic murine colitis model induced by dextran sodium sulfate (DSS). We measured the PD-1 and PD-L1 levels in inflamed colonic tissues before and after the treatment with T-MSC. We also measured the levels of PD-1 and PD-L1 in the colonic tissues from patients with ulcerative colitis and compared with those from normal controls. Results In chronic colitis model, the level of PD-L1 was decreased than normal controls (fold change) (1.0 vs. 0.46±0.08, P=0.05). However, the level of PD-1 was increased than normal controls (1.0 vs. 6.84±8.16, P=0.142). After treatment with T-MSC which showed significant improvement in body weight, disease activity index and colon length, the levels of PD-1 and PD-L1 were recovered; PD-L1 was significantly increased (1.04±0.77 vs. 0.46±0.08, P=0.031) and the level of PD-1 was decreased (3.63±1.94 vs. 6.84±8.16, P=0.537). When measuring the level of PD-1 and PD-L1 in both soluble medium and cell lysate of T-MSC, we found that the PD-1 and PD-L1 were expressed in both forms (182.63 pg/ml in soluble medium and 11.85 pg/protein(ug) in cell lysate after 7 days of differentiation period). In the analysis using human colonic tissues, a significantly increase in the levels of PD-1 and PD-L1 was observed in the colonic mucosa of patients with UC compared with normal controls (PD-1: 2.09±3.95 vs. 7.93±14.21, P=0.034 and PD-L1: 2.24±2.91 vs. 10.08±18.13, P=0.044). Conclusion The altered expression of PD-1 and PD-L1 in colonic mucosa may a possible mechanism of ulcerative colitis and T-MSC-derived PD-L1 could repress the colitis.