Umbilical cord-derived mesenchymal stem cell extracts reduce colitis in mice by re-polarizing intestinal macrophages

Human umbilical cord mesenchymal stem cells (hUC-MSCs), originating in Wharton’s jelly, are multipotent stem cells that home to damaged tissues and can modulate the immune system. We examined whether administering extracts of MSCs (MSC-Ex) instead of MSCs could augment the beneficial effects of MSC therapy by overcoming the low homing efficiency of MSCs systemically administered in inflammatory bowel diseases (IBD). Dextran sodium sulfate-induced colitis model was established in C57BL/6 mice, and MSC-Ex was administered intraperitoneally. MSC-Ex reduced colitis, disease activity index (DAI), and histological colitis scores, and increased the body weight. Treatment with MSC-Ex completely blocked the induction of inflammatory cytokines, which were strongly detected in mice with colitis. MSC-Ex shifted the macrophage functional phenotype from M1 to M2 by decreasing the levels of MCP1, CXCL9, and iNOS, but increasing the levels of IL-10, LIGHT, CCL1, and Arg-1. MSC-Ex recovered the destruction of the epithelial barrier in the differentiated Caco-2 cells in vitro. Treatment with MSC-Ex was more potent than that with MSC in reducing DAI, the histological score, and nitrite levels. These data strongly support that MSC-Ex treatment can be a potent approach to overcome severe refractory IBD.

Stem cell therapies have emerged as novel therapeutic tools for patients with IBD who respond poorly to conventional treatments with anti-inflammatory drugs 6 . In most clinical studies, mesenchymal stem cells (MSCs) are favored over hematopoietic stem cells (HSCs) because of their low immunogenic phenotype 7 . MSCs express defined surface markers, including CD90, CD73, and CD105, and have immuno-regulatory and regenerative properties. They express low levels of type I HLA antigen, but do not express type II HLA antigens or T cell co-activators. MSCs are obtained from the bone marrow, adipose tissue, and the umbilical cord 8 . The numbers of MSCs in the adult bone marrow are very low (0.001~0.01%) and removal of MSCs poses risks for the donor, whereas MSCs from the adipose tissue present a different cytokine profile based on the tissue origin. Human umbilical cord MSCs (hUC-MSCs) are isolated from Wharton's jelly of the umbilical cord and are distinguishable from the MSCs present in UC blood 9 . hUC-MSCs can be easily isolated in large quantities from postnatal cord tissues, which are usually discarded after birth. Moreover, UC-MSCs can be extensively expanded in culture, frozen/thawed, and possess immunomodulatory properties 10,11 .
Many studies have shown that the therapeutic capacity of MSCs is mostly mediated by their paracrine effects. MSCs produce a range of cytokines involved in modulating the immune effects 12,13 . MSCs can suppress T cell proliferation when T cells are stimulated by the pro-inflammatory cytokine INF-γ, and express IL-6 and IL-10 to block macrophage differentiation towards dendritic cells 14 . In mice with induced colitis, MSCs inhibit innate immunity by blocking the differentiation and maturation of monocytes, suppressing T cells, and increasing the populations of T regulatory cells 15 . Recently, Forbes et al. have conducted a phase II study using bone marrow derived MSCs. The administration of these MSCs reduced disease activity in patients with luminal CD, which was refractory to biologic therapy 16 . Numerous studies demonstrate that allogeneic MSCs can be a therapeutic alternative for treating patients with IBD. Although MSCs show long-term efficacy, their efficacy in the early stages, for maintaining disease remission, as well as the dose and frequency of infusion, should be determined. To overcome the low homing efficiency and rapid clearance of MSCs post-administration, we investigated the therapeutic effects of UC-MSC extracts (MSC-Ex) in a mouse model of induced colitis. Treatment with MSC-Ex efficiently blocked the induction of pro-inflammatory cytokines and switched the macrophage functional phenotype from M1 to M2 in the colon and peritoneum of mice with induced colitis. The therapeutic effects of MSC-Ex were more potent than those of MSCs.

MSC-Ex suppresses dextran sodium sulfate (DSS)-induced colitis.
Although MSCs are an attractive treatment for IBD patients, their survival in vivo remains controversial. Many studies have shown that paracrine signaling of MSCs is one of the main mechanisms behind their therapeutic effects 12,14 . Based on this notion, we hypothesized that a robust administration of paracrine factors, using MSC-Ex, may be a promising therapeutic approach. To assess whether administering MSC-Ex ameliorates ulcerative colitis, we established experimental models of DSS-induced chronic colitis after three cycles of 5-day treatments with 2% DSS and a 5-day recovery between each cycle. The colitis-induced mice showed a loss of ~20% body weight on day 25 (Fig. 1A). The colitis-induced group was intraperitoneally administered MSC-Ex (150 μg per mouse) once per day, for 10 days, on days 26 to 35. The administration of MSC-Ex significantly improved clinical parameters such as body weight and disease activity index (DAI) (Fig. 1A,B); however, the MSC-Ex-treated group did not recover the shortened colon length compared with that of the healthy control group. (7.0 ± 0.2 vs 9.2 ± 0.3 cm) (Fig. 1C). Myeloperoxidase (MPO) activity widely correlates with neutrophil content in the tissue 17 . Compared with the healthy control, MPO activity was increased in the colon of colitis-induced mice. In contrast, the colon from the MSC-Ex treated group showed significantly less MPO activity and immunoreactivity compared with that of the healthy control group (Fig. 1D). The MSC-Ex treated group showed greatly decreased histological colitis scores used to indicate the extent of inflammation, crypt damage, and percentage of colitis. The broken colonic mucosal structure and thickening of the mucosal and muscle layers were greatly recovered in the MSC-EX treated group compared with those in the PBS-treated group (Fig. 1D).
Because IBD is a chronic intestinal disease, characterized by the prolonged activation of various immune cells in the intestine, immune responses to MSC-Ex may be different in acute DSS-induced colitis. We next used 4% DSS for 7 days to generate acute DSS-induced colitis, and administered MSC-Ex five times on days 3 to 7 ( Fig. 2A). We observed a marked decrease in the DAI and MPO activity in the MSC-Ex treated group compared with that in the PBS-treated group (Fig. 2B,C). However, we did not detect differences in the histological colitis score between the DSS-PBS group and the DSS-MSC-Ex group (Fig. 2D). Interestingly, the length of the colon was markedly recovered in the MSC-Ex treated group compared with that in the PBS-treated group (6.8 ± 0.46 vs 5.8 ± 0.87 cm) (Fig. 2E). Collectively, these results indicate that administration of MSC-Ex ameliorates the clinical parameters in DSS-induced colitis.

MSC-Ex reduced pro-inflammatory cytokines in the intestine.
To examine the immune response modulation of MSC-Ex, we analyzed cytokine expression in the colon tissues of chronic colitis-induced mice. Whole cell lysates, prepared from the colonic tissues, were analyzed by immunoblotting using the human cytokine antibody array. Cytokine protein arrays showed that the levels of several cytokines, including sICAM-1, IL-1β, IL-16, CXCL10, MCP-1, CXCL9, and TIMP-1, were upregulated (≥50-fold) in the colitis-induced PBS-treated group compared with those in the normal healthy control group. In contrast, the colons of the colitis-induced MSC-Ex-treated group showed greatly reduced levels of these cytokines. The cytokine profile of the MSC-Ex treated group was similar to that of the healthy control group on the background of inflammatory environment (Fig. 3A). We further analyzed the expression of the cytokines IL-17, IL-10, and TGF-β1 using enzyme-linked immunosorbent assay (ELISA) assays. The level of the pro-inflammatory cytokine IL-17 was significantly decreased (Fig. 3B), whereas those of the anti-inflammatory cytokines IL-10 and TGF-β1 were increased in the MSC-Ex-treated group compared with those in the PBS-treated group (Fig. 3C,D). These results indicate that MSC-Ex markedly reduces the inflammatory state in the colitis-induced mice.

MSC-Ex shifted the macrophage functional phenotype in the colitis-induced colon.
To investigate the mechanism by which MSC-Ex exert their therapeutic effects, we next focused on the colonic macrophages because they are one of the most numerous leukocytes, and play a key role in homeostasis through the M1/M2 polarization in the colon 5 . We isolated RNA and examined the expression levels of inflammatory markers using quantitative real-time RT-PCR (qRT-PCR) from the digested colon tissues of chronic colitis-induced mice. The levels of M1 markers, such as MCP-1 and CXCL9, were greatly decreased (Fig. 4A,B), while those of the M2 markers, such as arginase-1 (Arg-1) and IL-10, were significantly increased in the mice treated with MSC-Ex (Fig. 4C,D). M1 and M2 macrophages have marked differences in arginine metabolism. M1 macrophages metabolize arginine to nitric oxide via the inducible nitric oxide synthase (iNOS), while the M2 macrophages metabolize arginine to ornithine via Arg-1 18 . To better define the macrophage phenotypes recruited in colitis, the colons were stained with anti-CD68, a macrophage-specific marker, as well as with anti-iNOS and anti-Arg-1 5 . Increased infiltration of CD68-positive macrophages was detected in the inflamed mucosa of the colitis-induced mice. The iNOS-positive cell infiltrations were predominantly observed in the colitis-induced PBS-treated group compared with the healthy control and colitis-induced MSC-Ex treated groups. Conversely, Arg-1 positive cells were prevalent in the MSC-Ex-treated group, even though inflammation was nearly ameliorated (Fig. 4E). These results were consistently observed in immunoblot analysis, which showed decreased levels of iNOS, and increased levels of the Arg-1 proteins, in MSC-Ex-treated colons (Fig. 4F). These results imply that MSC-Ex shifts the macrophage phenotype to exert therapeutic outcomes.
Phenotype switch of peritoneal macrophages by treatment with MSC-Ex. Large peritoneal macrophages (F4/80 high and CD11 high ) are maintained in the peritoneal cavity, can undergo rapid proliferation, and arrive at the afflicted inflammatory tissues 19 . We investigated whether MSC-Ex treatment influences the peritoneal macrophage phenotype. F4/80 + /CD11b + macrophage cells, from the peritoneum of mice with acute colitis, were isolated by flow cytometry and the M1/M2 status was analyzed by qRT-PCR (Fig. 5A). M1 markers of MCP1 and CXCL9 were decreased, while M2 markers of Arg-1, IL-10, LIGHT, and CCL1 were increased in the MSC-Ex treated group compared with the PBS-treated group (Fig. 5B) 5 . We further measured the levels of IL-17 and IL-10 in the macrophages using ELISA. The level of anti-inflammatory cytokine IL-10 was highly enhanced, however, that of the pro-inflammatory cytokine IL-17 was reduced in the MSC-Ex treated group (Fig. 5C). These results confirmed that MSC-Ex modulates macrophage polarization from the M1 to M2 state in colitis-induced mice.

MSC-Ex recovered damaged intestinal epithelial barrier. The disruption of the intestinal mucosal
barrier is an important indicator in the pathogenesis of IBD. The permeability of the intestinal epithelial barrier is regulated by the adhesion structures of adherens and tight junctions 20 . Adherens junctions, comprised of E-cadherin and nectins, are responsible for initiating cell-cell contacts. To investigate whether MSC-Ex treatment generated the functional recovery of the gut barrier, we developed a co-culture system of intestinal epithelial Caco-2 cells with RAW 264.7 macrophage cells as an intestinal model in vitro (Fig. 6A). After 14 days of culture, Caco-2 cells underwent spontaneous differentiation, leading to the formation of a monolayer with features similar to those of mature enterocytes. We observed a pattern of continuous E-cadherin staining in the monolayer of Caco-2 cells. LPS treatment induced inter-epithelial gaps and destabilization of cell-cell junctions in the differentiated monolayer sheet of Caco-2 cells, mimicking the destruction of the epithelial barrier. Co-administering the MSC-Ex (30 ug/mL) treatment with LPS greatly prevented the destruction of the epithelial barrier (Fig. 6B). Gene expression analysis of the co-cultured RAW 264.7 cells indicated that the expressions of the M1 markers (MCP-1 and CXCL9) were reduced, while those of the M2 markers (Arg-1, IL-10, LIGHT, and CCL1) were increased (Fig. 6C). In agreement with the above results, the expression of IL-17 was reduced, while that of IL-10 was increased, in the RAW 264.7 culture media (Fig. 6D). These results indicate that MSC-Ex exerts therapeutic effects by inhibiting the M1 and stimulating the M2 phenotype, resulting in the protection of adherens junctions and epithelial integrity in the colon.

MSC-Ex is superior to UC-MSCs in chronic IBD models.
To strengthen the evidence for the therapeutic effects of MSC-Ex, we compared the therapeutic efficacy between MSC-Ex and MSCs in chronic IBD models. MSCs and MSC-Ex, prepared from an identical number of MSCs (1 × 10 6 cells), were administrated intraperitoneally in colitis-induced mice (Fig. 1A). Both treatments significantly ameliorated colitis; however, the DAI and histologic colitis scores were lower in the MSC-Ex-treated group compared with those in the MSC-treated group in the absence of differences in the colon length (Fig. 7B-D). In the co-cultured RAW 264.7 cells, MSC-Ex significantly reduced the levels of nitrite compared with those in the MSC-treated cells, in the absence and presence of LPS stimulation (Fig. 7E); nitrite is a major final metabolite of NOS used as a marker of inflammation in IBD. These results strongly support our theory that treatment with MSC-Ex is more efficient than that with MSCs in inducing immunosuppression.

Discussion
MSC therapy has emerged as a promising therapeutic strategy for inflammatory bowel disease. MSC therapy for IBD is well tolerated, but its clinical results with respect to efficacy are inconsistent, possibly because of the different methods used for the isolation and expansion, and MSC types such as bone marrow-, adipose-or umbilical cord-derived MSCs 15, 20-23 . The immuno-modulatory ability of MSCs may be the result of their ability to migrate to inflamed tissue. However, emerging data show that only a limited number of engrafted MSCs reach the inflamed colon, and that the immunosuppressive activity of MSCs is mainly mediated by soluble paracrine factors 24 . Here, we focused on evaluating the therapeutic effects of umbilical cord-derived MSC-Ex and the mechanisms underlying the immunosuppressive activities of MSC-Ex in DSS-induced colitis. Our results show that intraperitoneally injected MSC-Ex significantly improved the inflammatory symptoms in DSS-induced IBD mice. UC-MSCs can produce components that interfere with both the innate and adaptive immune systems. UC-MSCs produce IL-6, required by the dendritic cells to acquire tolerogenic phenotypes, prostaglandin E2 (PGE2), which inhibits the cytotoxicity of NK cells and T cell proliferation, and indoleamine 2,3-dioxygenease (IDO), which represses the differentiation of circulating T follicular helper cells; UC-MSCs also stimulate immunomodulatory cytokines such as TGFβ1 and TNF-stimulated gene 6 proteins (TSG-6) 25-28 . The intraperitoneal administration of MSC-Ex, used in our study, robustly induced the influx of anti-inflammatory factors to the highly inflamed peritoneum of DSS-colitis induced mice. Antibody arrays showed that the cytokine profiles of colonic extracts from MCS-Ex-treated colitis mice were similar to those from normal healthy mice (Fig. 3A). The expressions of ICAM-1, IL-1β, IL-16, CXCL10, MCP-1, CXCL9, CXCL12, and TIMP-1 were greatly reduced in MSC-EX-treated colitis group compared with those in the PBS-treated colitis group. CXCL9, CXCL10, CXCL12, IL-16, and MCP1 are chemo-attractants that recruit monocytes, T cells, and dendritic cells to the inflammatory site; the expression of these cytokines is significantly increased in IBD patients 29,30 . Thus, MSC-Ex contributes to a counter-regulatory response to excessive pro-inflammatory cytokine activity in the colitic colon.
Macrophages are divided into two functional subsets: the classically activated M1 (pro-inflammatory M1) and alternatively activated M2 (anti-inflammatory M2) macrophages 31 . MSC-Ex altered the macrophage phenotype from M1 to M2 by significantly increasing the expression of Arg-1, LIGHT, and CCL1 in the peritoneal macrophages (Fig. 5). The altered macrophage signature was detected in mRNA and protein in both acute and chronic colitis models. Treatment with MSC-Ex, administered to RAW 264.7 macrophages co-cultured with intestinal epithelial Caco-2 cells, led to a distinct shift from the M1 to the M2 phenotype, preserving the epithelial barrier function (Fig. 6). The ratio of iNOS (M1-specific enzyme) to Arg-1 (M2-specific enzyme), indicative of arginine metabolism, was radically decreased in the MSC-Ex-treated colon of chronic colitis-induced mice (Fig. 4E,F). Consistent with our findings, other groups have shown that intraperitoneally injected M2 macrophages enter the circulation, extravasate into the colon, and decrease inflammation, resulting in the scavenging of debris, angiogenesis, and tissue repair. M2 macrophages recruit Th2 lymphocytes, eosinophils, basophils, and Tregs using CCL17, CCL22, and CCL24 30,32 . Thus, the M2 polarization of macrophages by MSC-Ex may result in a favorable environment that accommodates tissue homeostasis and repair. IL-10 plays a major role in the maintenance of intestinal homeostasis. IL10 −/− mice develop spontaneous colitis 33 . Patients with a gene mutation in the IL-10 receptor (IL-10R) develop severe pediatric IBD 34 . In our study, MSC-Ex-treated colonic tissues and peritoneal macrophages produced markedly high levels of IL-10 transcripts. Leung and colleagues 35 reported that bone marrow-derived M2 macrophages from wild type mice reduced dinitrobenzene sulfonic acid (DNBS)-driven colitis more than the M2 macrophages from IL-10 −/− mice. Notably, all macrophages synthesize IL-10; however, the M2 macrophages produce less nitric oxide. IL-10, secreted by the M2 macrophages, may inhibit pro-inflammatory cytokines. The anti-colitic effect of MSC-Ex may be dependent in part on the capacity to stimulate IL-10 production in macrophages.
In IBD, the rationale for MSC therapy is mainly centered on their immunosuppressive activities. MSC cells have been a source of therapeutic tools because of their sustained and slow release of IL-10, in response to the intestinal environment, and trafficking to the damaged colon. We demonstrate that re-polarization of differentiated macrophages by MSC-Ex re-educated macrophages to continuously secret IL-10. In this study, we acknowledge that the therapeutic components of MSC-Ex were not fully characterized. However, we found that MSC-derived conditioned medium (MSC-CM) and MSC-Ex have very distinct cytokine profiles, and that the MSC-Ex prepared from different umbilical cords show consistent cytokine expression patterns (Supplementary Figure 1). In addition, MSC-Ex-mediated therapeutic effects were not observed with extracts from WI-38 fibroblasts (Supplementary Figure 2). The data support the specificity of MSC-Ex for IBD treatment. We also observed local immunologic responses at the site of MSC injection, but not at that of MSC-Ex injection, in the colitis-induced mice (unpublished data). Although further studies are required to determine whether treatment with MSC-Ex can be used safely to treat patients with IBD, our findings suggest that MSC-Ex could provide an alternative therapeutic tool to overcome the low homing efficiency of MSCs in stem cell therapies for IBD patients.

Materials and Methods
Ethics statement. This work was approved by the Institutional Review Board of Asan Medical Center Isolation and expansion of UC-MSCs. The umbilical cord samples were obtained at the time of caesarean delivery from healthy pregnant women. UC-MSCs were prepared according to the method previously described 36 . The cord was rinsed several times with sterile saline and cut into 3-mm-thick pieces. The vessels and amnion were removed from cord segments. Wharton's jelly tissues were minced and digested for 3 h in MEM (11095-080; Invitrogen-Gibco, Carlsbad, CA) with 0.1% collagenase A (10103578001; Roche, Mannheim, Germany) at 37 °C in a shaking incubator. To remove large pieces of tissue, the cells were filtered through a 70 μM mesh (103760; BD Falcon, San Jose, CA) and pelleted using low-speed centrifugation at 200 × g for 10 min. The isolated cells were plated in DMEM supplemented with 10% FBS, 50 μg/ml penicillin and 100 μg/ml streptomycin (Invitrogen-Gibco) at 37 °C in a humidified 5% CO 2 incubator. MSCs were selected using attachment to plastic culture plates after 72 h. MSCs at passages 3, 4, and 5 were pooled for MSC-Ex or MSC therapy.
Preparation of MSC-Ex. UC-MSCs (1 × 10 6 cells) were washed twice with cold phosphate-buffered saline (PBS) and resuspended in five times the packed cell volume of PBS. After 30 min of incubation on dry ice, the cells were transferred to a water bath maintained at 37 °C for 3 min. The freezing and thawing was repeated three times. After centrifuging at 12,000 × g for 10 min, the supernatant was collected and stored at −80 °C.
MPO activity assays. The colon (50 mg/mL) was homogenized in phosphate buffer (50 mM, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide (H5882; Sigma, St. Louis, MO) and centrifuged at 12,000 × g for 10 min to collect the supernatant. The supernatant (7 μL ELISA. The colon was homogenized in PBS containing 1% Triton X-100 and protease inhibitor cocktail and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatants were collected and quantitatively analyzed for IL-17 Cytokine antibody array. Colons were homogenized in PBS containing 1% Triton X-100 and protease inhibitor cocktail, centrifuged, and quantified using the Bradford assay. A normalized protein content was analyzed with the Mouse Cytokine Array Panel A (ARY006; R&D system) according to the manufacturer's instructions. A semi-quantitative analysis of the comparative intensity of the spots was performed with a GS-800 calibrated densitometer image analysis program (GS-800; BIO-RAD, Hercules, CA).
Caco-2 cells were placed on the upper inserts, cultivated to differentiate into polarized monolayers, and then transferred into the same 6-well plate. The inserted membrane, with a pore size of 0.4 μM, was used to allow for the transmission of soluble factors. LPS (1 μg/mL) was applied to RAW 264.7 cells for 4 h, and then the media were changed. Caco-2 cells were then treated with MSC-Ex (30 μg/mL) for 24 h. The levels of IL-17 and IL-10 were evaluated using the RAW 264.7 culture medium. The RAW 264.7 cells were harvested and analyzed using qRT-PCR.
Isolation of peritoneal macrophages. The mouse peritoneal macrophages were collected from the mouse abdomen using a syringe (25 G needle) with 5 mL PBS. The cells were cultured for 4 h to allow attachment in complete RPMI 1640 medium containing 10% FBS and 50 μg/mL penicillin/streptomycin. After removing the floating cells, the attached macrophages were cultured in complete RPMI 1640 medium for 2 days. Statistical analysis. All data are expressed as the mean ± s.d. The statistical significance of the differences was evaluated using the non-parametric Wilcoxon rank sum test. Statistical significance was defined as P < 0.05.