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Negative impact of bone-marrow-derived mesenchymal stem cells on dextran sulfate sodium-induced colitis.

AIM
To investigate the effects of mesenchymal stem cells (MSCs) on dextran sulfate sodium-induced inflammatory bowel disease (IBD).


METHODS
C57BL/6 mice were fed 3.5% (g/L) dextran sulfate sodium. On day seven, the mice received intraperitoneal injections of 1×10(6) MSCs. The survival rate, disease activity index values, and body weight, were monitored daily. On day ten, colon lengths and histopathologic changes were assessed. In addition, immunoregulatory changes following MSC administration were evaluated by determining the levels of effector T cell responses in the spleen and mesenteric lymph nodes, and the expression levels of inflammatory cytokines in homogenized colons.


RESULTS
Intraperitoneal administration of MSCs did not prevent development of colitis and did not reduce the clinicopathologic severity of IBD. No significant difference was evident in either survival rate or disease activity index score between the control and MSC-treated group. Day ten-sacrificed mice exhibited no significant difference in either colon length or histopathologic findings. Indeed, the MSC-treated group exhibited elevated levels of interleukin (IL)-6 and transforming growth factor-β, and a reduced level of IL-10, in spleens, mesenteric lymph nodes, and homogenized colons. The IL-17 level was lower in the mesenteric lymph nodes of the MSC-treated group (P=0.0126). In homogenized colons, the IL-17 and tumor necrosis factor-α (P=0.0092) expression levels were also lower in the treated group.


CONCLUSION
MSC infusion provided no significant histopathologic or clinical improvement, thus representing a limited therapeutic approach for IBD. Functional enhancement of MSCs is needed in further study.

Some clinical data on the use of MSCs to treat IBD have appeared [13][14][15][16][17][18][19][20] . MSCs were infused intralesionally or through a fistula, thus not systemically. The work confirmed that MSCs improved fistular pathogenesis. Recently, allogeneic MSCs have been used to treat IBD; the cells were delivered via intralesional or intrafistular injection. de la Portilla et al [15] considered that local injection was preferable to systemic infusion when IBD was to be treated. As MSCs exhibit immunomodulatory effects, we hypothesized that MSCs per se would exert therapeutic effects in IBD models. Although MSCs are used to treat autoimmune diseases such as graftversus-host disease (GvHD), rheumatoid arthritis (RA) [21][22][23][24][25][26] , and possible rejection of skin allografts [27] ; any potential role for MSCs in IBD treatment remains unclear. Thus, in the present study, we explored the anti-inflammatory actions of mouse bone-marrowderived MSCs used to treat dextran sulfate sodium (DSS)-induced IBD.

Mice
Eight-to-ten-week-old female C57BL/6 (B6, H-2k b ) mice were purchased from OrientBio (Sungnam, Korea) and were maintained under specific pathogenfree conditions in an animal facility in which the humidity was controlled at 55% ± 5%, with a 12 h light/dark cycle and a temperature of 22 ℃ ± 1 ℃. The air in the facility was HEPA-filtered to exclude bacteria and viruses. Mouse chow and tap water were available ad libitum. All protocols used were approved by the Animal Care and Use Committee of the Catholic University of Korea.

Induction of DSS-induced colitis
IBD was induced by feeding mice 3.5% DSS (molecular weight 36-50 ku; MP Biomedicals LLC, Santa Ana, CA, United States) dissolved in UV-sterilized tap water, available ad libitum for seven days. On day seven, all animals were returned to plain water. Survival after DSS administration was monitored daily, and disease activity index scores, which evaluate weight loss, stool consistency, and rectal bleeding, were assessed.

Isolation, culture, and administration of MSCs
Donor (C57BL/6, H-2kb) bone marrow cells were collected by flushing mouse femurs and tibias with Dulbecco's modified Eagle's medium (Gibco of Thermo Fisher Scientific Inc., Waltham, MA, United States) supplemented with 15% heat-inactivated fetal bovine serum (Gibco). Suspended cells were plated in 95-mm-diameter culture dishes in 1 mL of complete medium, at a density of 2 × 10 7 cells/mL.
Cultures were incubated at 37 ℃ under 5% CO2 in

INTRODUCTION
The inflammatory bowel diseases (IBDs) are principally Crohn's disease and ulcerative colitis. Although the developmental mechanism remains poorly understood, IBD pathogenesis is characterized by the development of an exaggerated immune response to intestinal bacteria in genetically-susceptible individuals [1,2] . The current therapies for IBD include anti-tumor necrosis factor (TNF)-α and anti-interferon (IFN)-γ antibodies, and anti-α-integrin drugs. Although symptoms are thus relieved, there is no cure. Furthermore, relapse is always a risk. Thus, it is essential to develop more effective therapeutic approaches to treat IBD. Recently, the utility of mesenchymal stem cells (MSCs) has been suggested, because MSCs exert immunosuppressive effects and aid in tissue repair [3][4][5][6][7][8] . Some preclinical studies explored the utility of MSCs in treatment of IBD [9][10][11][12] . MSCs were injected subcutaneously, intravenously, or intraperitoneally, and attenuated IBD. However, the MSCs used had been pretreated; the cells used included interleukin (IL)-12p40-transduced MSCs, autoimmune regulator knock-out MSCs, and MSCs coated with mucosal vascular addressin cell adhesion molecule-1 and a humidified chamber. After 3 h, nonadherent cells were removed by changing the medium. Cells at 80% confluency were trypsinized by incubation in 0.5 mL of 0.25% trypsin/1 mmol/L EDTA for 2 min at room temperature. Trypsin was neutralized by addition of 1.5 mL of complete medium. Cells were harvested and expanded in 75-T flasks; cultures were maintained at 37 ℃ under 5% CO2 in a humidified chamber and subcultured before confluence was attained. After ten passages, the MSCs were surface-stained for c-kit, CD11b, CD34, CD106, CD45, CD31, Sca-1, CD44, and CD29, and were characterized by flow cytometry. Prior to surface staining, the cells were Fc-blocked with 1 µg of mouse spleen and mesenteric lymph nodes 1 × 10 5 cells for 15 min at room temperature. After blocking, 1 µL amounts of antibody solutions were added and incubation for 30 min at room temperature followed.

Histopathologic evaluation
Mice were euthanized for blinded histopathologic analysis of IBD target tissue (the large intestine). Organs were harvested, cryo-embedded, and sectioned. The sections were fixed in 10% buffered formalin (Sigma-Aldrich) and stained with hematoxylin (Sigma-Aldrich) and eosin Y (1% solution; Muto Pure Chemical Co., Ltd, Tokyo, Japan).

ELISAs
Colons were homogenized in buffer [50 mmol/L Tris-HCL (pH 7.4), 250 mmol/L NaCl, 5 mmol/L EDTA] with protease inhibitors (Hoffman La-Roche, Basel, Germany) and centrifuged at 11000 × g for 15 min. The resulting supernatants were collected and subjected to sandwich ELISAs. Solutions of antimouse IFN-γ, IL-17, IL-6, IL-10, TNF-α, and TGF-β (RD Systems, Minneapolis, MN, United States) were added to wells of a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4 ℃. Wells were blocked with blocking solution (PBS with 1% bovine serum albumin and 0.05% Tween-20) for 2 h at room temperature. The test samples and standard recombinant IFN-γ, IL-17, IL-6, IL-10, TNF-α, and TGF-β (R and D Systems) were added to separate wells of the 96-well plate and incubated at room temperature for 2 h. The plate was washed, biotinylated antibodies against IFN-γ, IL-17, IL-6, IL-10, and TNF-α, and an anti-TGF-β polyclonal antibody (RD Systems) were added, and reactions allowed to proceed for 2 h at room temperature. The plate was washed, a 2000-fold dilution of ExtrAvidinalkaline phosphatase (Sigma-Aldrich) was added, and the reaction allowed to proceed for a further 2 h. The plate was washed and 50-µL amounts of P-nitrophenyl phosphate disodium salt (Pierce Chemical Company, Rockford, IL) diluted to 1 mg/mL in diethanolamine buffer were added to each well.

Statistical analysis
Data were analyzed by Student's t-tests or analyses of variance using GraphPad Prism software (version 5.01, GraphPad Software Inc., La Jolla, CA, United States). Survival was compared between groups by Kaplan-Meier analysis and using the log-rank test. Data are presented as mean ± SD, and a P < 0.05 was considered as significant.

Clinical outcome of MSC therapy in the DSS-induced IBD model
To explore the effects of MSCs on IBD, we gave single ip injections of donor bone marrow-derived MSCs to DSS-induced IBD mice. C57BL/6 mice were fed 3.5% DSS ad libitum from day zero to day six. On day seven, mice received ip injections of 1 × 10 6 MSCs.
The median survival times were ten days for the control group and eleven days for the MSC-treated group ( Figure 1A). Also, the disease activity index scores ( Figure 1B) and body weight changes from effects on T cells, we measured the levels of proinflammatory cytokines and helper T cell cytokines, and that of regulatory T (Treg) cells, by flow cytometry. The cytokine levels in the MSC-treated group were similar to those in the control group ( Figure 4A). The median percentages of IFN-γ in the control and MSC-treated groups were 1.10 and 1.02%, respectively. The median percentages of IL-4, IL-17, IL-6, IL-10, and Treg cells in the control group were 5.64%, 2.63%, 1.49%, 1.46%, and 22.20%, respectively, and 5.97%, 2.41%, 1.88%, 1.65%, and 21.30%, respectively, in the MSCtreated group. Figure 4B shows the percentages of cytokines in mesenteric lymph nodes; the average percentages of IFN-γ, IL-4, IL-6, IL-10, and Treg cells in the control group were 2.24%, 4.02%, 2.19%, 0.89%, and 22.65%, respectively, and 2.53%, 6.11%, 2.82%, 1.57%, and 21.15%, respectively, in the MSC-treated group. The mean percentage of IL-17 was significantly higher in the control group compared to the MSC-treated group (2.61% vs 1.92%, P = 0.0126). Although the level of IL-17 in mesenteric lymph nodes was slightly higher in the MSC-treated group, the levels of most other cytokines, including IFN-γ, IL-4, IL-6, and IL-10, and Treg cells, did not differ significantly between the groups. Interestingly, in the spleen, the percentage of IL-6 was elevated and that of IL-10 reduced in the MSC-treated group.

Changes in the cytokine profile in homogenized colonic tissue of DSS-treated mice after MSC therapy
Finally, we analyzed homogenized colon supernatants via ELISA to explore the local immunoregulatory effects of MSCs. Mouse colons were harvested on day ten after DSS treatment, feces were removed and the colons homogenized in a buffer containing protease inhibitors. The levels of IFN-γ, IL-17, and TGF-β were lower in the MSC-treated compared to the control group (201.51, 6.99, and 246.21 pg/mL day zero did not differ between the two groups.

Histopathologic changes following MSC therapy in the DSS-induced IBD model
On day ten, large intestines (from the appendix to the anus) were harvested and colon lengths measured. Generally, shorter colon length correlates with the severity of IBD pathogenesis, and the median colon lengths were 3.8 cm in the control group and 3.7 cm in the MSC-treated group ( Figure  2). The median colon length in normal mice not fed DSS was 6.4 cm. Distal colons were harvested on day ten and stained with hematoxylin and eosin. DSS-induced IBD is characterized by goblet cell loss and inflammatory cell infiltration. Unlike normal mice, the control and MSC-treated groups exhibited epithelial disruption, damage to the lamina muscularis mucosae, and submucosal edema (Figure 3).

Reciprocal regulation of IL-6 and IL-10 in spleens of mice with DSS-colitis upon MSC therapy
To confirm that MSCs exerted immunoregulatory vs 4010.58, 14.42, and 269.96 pg/mL, respectively), and TNF-α levels were significantly lower (14.25 pg/mL vs 24.94 pg/mL, P = 0.0092) ( Figure 5). The level of IL-4 in the MSC-treated group was 34.15 pg/ mL and 22.18 pg/mL in the control group; the level thus decreased upon MSC treatment. As also found in the spleen, the level of IL-6 increased and that of IL-10 decreased in the MSC-treated group. In the control group, the level of IL-6 was 387.48 pg/mL and that of IL-10 156.25 pg/mL. In the MSC-treated group, the level of IL-6 was 546.71 pg/mL and that of IL-10 35.89 pg/mL. Interestingly, the level of IL-10 decreased by more than fourfold in the MSCtreated group.

DISCUSSION
Currently, MSCs are used to treat chronic inflammatory diseases, including GvHD, RA, and IBD [28] . However, no clear therapeutic effect of MSCs on IBD has been shown. Considerable progress has been made in understanding the mechanisms by which MSCs exert immunomodulatory functions. MSCs exert immunosuppressive and anti-inflammatory effects and are considered to suppress T cell proliferation [29][30][31] . MSCs suppress T cells in a manner independent of variations in the major histocompatibility complex [32] , and exert effects on lymphocytes involved in both the innate and adaptive immune systems. Our goal was to treat IBD with MSCs; we hypothesized that MSCs would exert immunomodulatory effects that would be of clinical value in improving the pathogenesis of IBD. However, we found, for the first time, that MSCs were not helpful in IBD treatment; the cells were not clinically efficacious. MSCs were unable to restore the immune balance. Both our in vivo and ex vivo data indicate that MSCs were not effective. Also, the ex vivo data indicate that MSCs did not influence the balance between IL-6 and IL-10 levels.
Although MSCs are known to possess immunomodulatory properties, these have recently been shown to not be constitutive, being rather highly dependent on inflammatory conditions. "Licensing" by acute inflammatory Th1-type cytokines [33,34] , especially the proinflammatory cytokine IFN-γ [35,36] , is required. Polchert et al [37] evaluated the roles played by IFN-γ-activated MSCs, and the role of IFN-γ in such activation. MSCs were activated in the presence of IFN-γ. However, in a chronic inflammatory environment, MSCs may aggravate inflammation. It is thought that, in autoimmune environments such as IBD, GvHD, and RA, MSCs secrete cytokines that aggravate the Th17 condition. Examples of MSCs aggravating, or having no effect on, autoimmune diseases such as RA are extant [22,26,38,39] . Also, in a previous study, we showed that MSCs were ineffective for treating Th17-mediated collagen-induced arthritis [25] . Those data, and our present findings, suggest that MSCs are unable to exert immunomodulatory functions when a Th17 response is in play. Also, MSCs are known to produce IL-6 and TGF-β, which play important roles in regulating the differentiation of naïve T cells into Th17 or Treg cells [22,40,41] . MSCs produce TGF-β in the absence of stimulatory cytokines; but synthesize high levels of IL-6 in the presence of proinflammatory cytokines such 2034 February 21, 2015|Volume 21|Issue 7| WJG|www.wjgnet.com  as IFN-γ or TNF-α. Although TGF-β promotes the differentiation of naïve T cells into anti-inflammatory Treg cells, TGF-β and IL-6 (acting together) polarize T cells into proinflammatory Th17 cells [42][43][44] . Several studies, including our previous work, confirmed that MSCs can promote the expansion of Th17 cells under appropriate conditions [25,45] . In our present work, we found that MSCs did not exert significant effects in a DSS-induced IBD model. The high levels of IL-6 and TGF-β developing in the colon after DSS induction may have allowed the MSCs to induce Th17 cell proliferation and expansion.
Some preclinical studies on the use of MSCs for treatment of IBD have appeared (Table 1). However, some authors did not use MSCs alone, rather subjecting the MSCs to IL-12p40 transduction [11] , autoimmune regulator knockout [10] , or coating with antibodies against MAdCAM-1 and VCAM-1 [9] . Other authors did use MSCs alone to treat IBD. However, although one report claimed that multiple MSC infusion improved the clinical symptoms of IBD, the levels of only TNF-α and IL-1β were shown [10] . Some clinical studies have explored the effects of adipose-derived stem cells (ASCs) and MSCs in IBD patients (Table 2). ASCs and MSCs were used to treat fistulas. In all studies, stem cells were injected after tract curettage and closing. Although all studies reported improvements in IBD symptoms, the basic 2035 February 21, 2015|Volume 21|Issue 7| WJG|www.wjgnet.com Thus, MSC infusion alone may not be sufficient to re-establish the immune balance in IBD patients. MSCs may require other factors to exert polarized immunomodulatory functions in an established chronic inflammatory environment [46] . Several studies have shown that MSCs treated in various ways improved IBD pathogenesis [9,11,12] . In our earlier work, we found that TGF-β-transduced MSCs improved collageninduced arthritis symptoms [25] . Also, we evaluated combinatorial cell therapy (MSCs and Treg cells) of skin allografts [27] and GvHD [47] , and explored whether such therapy enhanced solid-organ transplant tolerance [48] . We also found that administration of MSCs with Treg   [10] BALB/c 4% DSS Syngeneic: Mouse (BALB/c) BM 1 × 10 6 iv 2, 5, 8 d Multiple injection Positive Parekkadan et al [12] C57BL/6 RAG-1 -/-CD4 + CD45RB hi 5 × 10 5 Allogeneic: Mouse (Aire-/-) BM 2.5 × 10 5 iv 0, 3 wk Aire -/injection Positive iv injection Ko et al [9] C57BL/6 5% DSS Allogeneic: Mouse (C57BL-10 × CBA/CA) BM 1 × 10 6 iv 2 d MAdCAM-1-VCAM-1-Ab-coated Positive Gonzalez-Rey et al [49] C57BL/6 a: 5% DSS Xenogeneic: Human adipose a: 1-50 × 10 5 hMSC, 1 × 10 6 mMSC c: 1 × cells, or IL-10-producing Tr1 cells, was an efficient therapeutic approach in the DSS-induced IBD model; the IL-10 level in the colon was reduced in MSCtreated groups. Therefore, MSCs in combination with other factors, and/or multiple injections of MSCs, may be effective for treatment of IBD. We suggest that further studies are needed to explore the localization and survival of MSCs administered by intraperitoneal infusion in the murine IBD model.

Background
Inflammatory bowel disease (IBD) is an inflammatory autoimmune disorder of the colon caused by colonic microbes, dietary habits, and/or genetic factors. IBD is common in Western countries. IBD patients present with clinical symptoms of diarrhea, and, histopathologically, immune cell infiltration of the colon is evident. Complete recovery is rare; relapses are common. Recently, mesenchymal stem cells (MSCs) have emerged as a new therapeutic approach for autoimmune diseases accompanied by inflammatory changes.

Research frontiers
MSCs are under preclinical and clinical investigation as a new treatment for autoimmune diseases.

Innovations and breakthroughs
IBD has been treated using MSCs combined with other interventions, such as genetic modification of MSCs. In the present study, the authors report, for the first time, that MSCs alone do not exert significant effects on IBD. MSCs were unable to regulate the balance between interleukin (IL)-6 and IL-10 levels.

Applications
This study provides the first evidence that the use of MSCs alone to treat IBD may be inadequate. If MSCs are to be used, their immunomodulatory function must be improved by addition of other factors; for example Treg cells or IL-10producing Tr1 cells, and/or genetic modification.