MIS416 Enhances Therapeutic Functions of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Against Experimental Colitis by Modulating Systemic Immune Milieu

Human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), have recently been considered a promising alternative treatment for inflammatory bowel disease (IBD) due to their unique immunomodulatory properties and ability to promote tissue regeneration. However, despite many years of research and pre-clinical studies, results from clinical trials using these cells have been diverse and conflicting. This discrepancy is caused by several factors, such as poor engraftment, low survival rate, and donor-dependent variation of the cells. Enhancement of consistency and efficacy of MSCs remains a challenge for the feasibility of cell-based therapy. In this study, we investigated whether administration of MIS416, a novel microparticle that activates NOD2 and TLR9 signaling, could enhance the therapeutic efficacy of hUCB-MSCs against Crohn’s disease, using dextran sulfate sodium (DSS)-induced colitis model. Colitis was experimentally induced in mice by using 3% DSS, and mice were administered a retro-orbital injection of MIS416 and subsequent intraperitoneal injection of hUCB-MSCs. Mice were examined grossly, and blood, spleen, and colon tissues were subsequently collected for further ex vivo analyses. To explore the effects of MIS416 on the therapeutic process, hUCB-MSCs and primary isolated immune cells were cultured with MIS416, and in vitro assays were performed. Compared to the single administration of hUCB-MSCs, co-administration with MIS416 improved the therapeutic efficiency of the stem cells by significantly alleviating the symptoms of IBD. Interestingly, MIS416 did not exert any direct effect on the immunomodulatory capacity of hUCB-MSCs. Instead, systemically injected MIS416 altered the immune milieu in the colon which caused hUCB-MSCs to be more readily recruited toward the lesion site and to suppress inflammation more efficiently. In addition, considerable numbers of regulatory immune cells were stimulated as a result of the cooperation of MIS416 and hUCB-MSCs. These findings indicate that co-administration with MIS416 enhances the therapeutic potential of hUCB-MSCs by systemically regulating the immune response, which might be an effective strategy for overcoming the current obstacles to stem cell therapy in clinical practice.

inTrODUcTiOn Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis, are chronic relapsing disorders characterized by excessive intestinal inflammation. Although the precise etiology of IBDs remains unclear, it is known that several causes are involved in the disease onset. The concerted action of genetic susceptibility, environmental risk factors, and alterations of the microbiota triggers dysregulated immune responses, resulting in the impairment of mucosal barrier functions. Conventional treatments for IBDs, including antibiotics, anti-inflammatory drugs, and immunosuppressive medicines have limitations such as drug resistance and low therapeutic responses in certain groups of patients. To overcome these limitations, alternative therapies, such as probiotics, anti-tumor necrosis factor (TNF) therapy, and trans plantation of MSCs have recently emerged (1,2). Among these remedies, MSCs have been studied for IBD treatment because of their immunomodulatory properties, tissue regenerative capacity, and ability to migrate toward damaged areas. Key immunomodulatory function of MSCs that have been demonstrated both in vitro and in vivo is their ability to inhibit the excessive proliferation and maturation of immune cells (3).
Although the therapeutic use of human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) has been investigated for decades, standardization issues remain to be overcome. For example, reduced productivity of MSCs caused by replicative senescence and donor-to-donor variations make it difficult to maintain consistent therapeutic effects for each recipient (4). Several strategies have recently been investigated for enhancement of the therapeutic potential of MSCs. Previously, we reported that NOD2 activation through muramyl dipeptide (MDP) priming upregulated prostaglandin E2 (PGE2) secretion from hUCB-MSCs and increased anti-inflammatory effects in experimental models of IBD (5). Similarly, priming of MSCs with growth factors or cytokines has also been reported (6). However, these methods have not been fully verified with regards to safety or optimization. Although many investigations have been performed to elaborate these strategies, other simplified methods are still needed for convenient application.
MIS416 is a novel immunomodulatory microparticle derived from Propionibacterium acnes, which consists of MDP and bacterial DNA. Phagocytic cells, key responders to MIS416, internalize MIS416, resulting in the activation of cytoplasmic receptors, NOD2 and TLR9 (7). NOD2 and TLR9-dependent pathways have been highlighted as therapeutic targets in IBDs, as NOD2 and TLR9 dysfunctions have been shown to play a central role in disease pathophysiology. For example, a frame-shift mutation of NOD2 was associated with the development of Crohn's disease in a study of over 400 unrelated subjects (8), and in experimental colitis, a NOD2 deficiency exacerbated disease severity due to an uncontrolled immune response leading to immune hyperresponsiveness to intestinal antigens mediated by IL-12-producing antigen-presenting cells (9). In contrast, overexpression of the NOD2 gene rescued mice from peptidoglycan-induced colitis (10), and mice deficient in TLR9 and MyD88 no longer demonstrated probiotic-mediated inhibitory effects on intestinal inflammation in experimental colitis (11). Furthermore, TLR9-induced type I interferon (IFN) resolved intestinal inflammation, and this effect was abolished by type I IFN neutralizing antibodies (12). As a NOD2 and TLR9 agonist, MIS416 has the potential to immune modulate IBDs by distinct and complimentary mechanisms. As well as inducing expansion of the peripheral pool of splenic myeloid-derived suppressor cells (13), MIS416 treatment has been shown to induce innate IFN-γ, nitric oxide (NO), and IL-10 in healthy animal and human studies. As a result of the altered immune milieu, MIS416 treatment also promoted expansion of splenic regulatory T (Treg) cells, and in a model of neuroinflammation, this was associated with suppression of the inflammatory response mediated by T helper (Th) 1, Th2, and Th17 cells (14). As a result of its efficacy in experimental autoimmune encephalomyelitis, MIS416 is currently in clinical trials for multiple sclerosis (13,14).
The mechanisms of action of MSCs in the therapeutic setting include both direct cell-to-cell contact as well as the secretion of soluble factors which modulate diverse immune cell subsets (15)(16)(17)(18). hUCB-MSCs were detected in inflamed colons and alleviated the severity in mouse colitis model (5,19), suggesting that their immunomodulatory effects occur in the localized environment, and that localization of MSCs at the inflammatory sites is a key factor for their therapeutic effects (20,21). Accordingly, many studies have demonstrated the distribution of injected MSCs at the inflamed colon in experimental colitis model by various methods, such as luciferase, green fluorescent protein (GFP), or indocyanine green labeling (19,22,23). Several molecules, such as monocyte chemotactic protein-1 (MCP-1/ CCL2), stromal cell-derived factor-1 (SDF-1/CXCL12), integrins, and matrix metalloproteinases, are involved in the recruitment of MSCs into inflamed tissues (24). Among these factors, MCP-1 is produced by various cells including monocytes/macrophages which are a major source of MCP-1 (25). It has also been reported that secreted MCP-1 stimulates migration of MSCs to the target region (26), and this has been demonstrated in an experimental rat model of stroke, where MCP-1 from ischemically damaged tissue was shown to facilitate migration of the transplanted human MSCs toward the site of injury (27).
As MIS416 co-administration might be a novel method for improving hUCB-MSC-based therapies against IBDs, in the present study, we investigated whether MIS416 co-administration could accelerate the therapeutic efficacy of hUCB-MSCs in a dextran sulfate sodium (DSS)-induced colitis model.

Mice
All experimental processes were approved by the Seoul National University Institutional Animal Care and Use Committee (IACUC No. SNU-170523-3) in accordance with the guidelines of the committee. C57BL/6J mice (male, 6-8 weeks old) were obtained from Orientbio (Sungnam, Republic of Korea). Mice were housed in a temperature-and humidity-controlled room in the animal facility of Seoul National University. Colitis was experimentally induced by administration of 3% DSS (MP Biochemicals, Solon, FigUre 1 | Simultaneous administration of MIS416 and human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) enhances therapeutic effects of the cells against experimental colitis. Mice were exposed to 3% dextran sulfate sodium (DSS) in their drinking water for 7 days and injected intraperitoneally hUCB-MSCs at day 1, and MIS416 at day 1 and 8 through retro-orbital route.  OH, USA) in drinking water supplied ad libitum for 7 days unless the application of humane endpoint was needed, DSS treatment was replaced by normal drinking water after day 7. MIS416 (Innate Immunotherapeutics, Auckland, New Zealand) was injected into the retro-orbital sinuses on day 1 and day 8 as described in Figure 1A. Sub sequently, hUCB-MSCs were suspended in phosphate-buffered saline (PBS) (2 × 10 6 cells/200 μl per head) and infused into mice intraperitoneally on day 1. Body weight and survival rate were monitored over 12 days. On day 7, the therapeutic potential of the treatments was measured by evaluating the disease activity index (DAI), including body weight loss (0-4), stool consistency (0-4), bleeding (0-4), general activity (0-2), and coat roughness (0-4), with a maximum DAI score of 18 and the humane endpoint was established at DAI = 13.5. On day 11, colon, serum, and spleen samples were collected from sacrificed mice for further ex vivo examinations. To define the systemic influence of MIS416, mice were sacrificed a day after injection (day 2), and colon, serum, and spleen samples were collected for analyses.

histopathological evaluation
The collected colon samples were fixed with 10% formalin. The tissues were embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin (H&E) and picrosirius red (PSR). The histopathological score was obtained by evaluating H&E stained slides based on the following five indexes: loss of goblet cells, infiltration of immune cells, crypt abscesses, hyperemia and edema, and loss of epithelium, with a maximum score of 15 (graded from 0-3 for the severity of each index). Generation of fibrotic tissue in the colon was assessed by PSR staining, followed by counterstaining with fast green (Sigma-Aldrich, St. Louis, MO, USA). The ratio of the fibrotic (PSR-positive) area was assessed using ImageJ software version 1.51j8 (National Institutes of Health, Bethesda, MD, USA).
cytokine Production IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 in the serum of mice were measured by flow cytometry using the CBA Mouse isolation and culture of hUcB-Mscs Human adult stem cells, including hUCB-MSCs were isolated as previously described (30,31). Isolated hUCB-MNCs were cultured with KSB-3 complete media (Kangstem Biotech, Seoul, Korea) containing 10% FBS (Gibco BRL) and antibiotics. After 3 days, non-adherent cells were removed, and adherent cell colonies were consistently cultured to establish sharp and spindle-shaped hUCB-MSCs. The cells possessed the characteristics of MSCs (30,31) and were verified by expression of surface markers by flow cytometric analysis ( Figure S1 in Supplementary Material). We used the cells at passage 8 for in vivo experiments and passage 8-10 for in vitro experiments.

cell cycle assay
After indicated treatment and harvest, hUCB-MSCs were washed in PBS twice prior to fixation with ice-cold 70% ethanol (over 30 min, −20°C). Fixed cells were washed in PBS and resuspended in 400 µl PBS, containing RNase A (6.25 µg/ml) and propidium iodide (50 µg/ml), and incubated at 37°C for 30 min. Cell cycle analysis was performed using a FACSCalibur flow cytometer and evaluated using Cell Quest software (BD Bioscience).

Western Blot
Whole-cell lysates were prepared with the protein lysis buffer Pro-prep (Intron Biotechnology Co.), and the concentration was measured via the Bradford method using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), with bovine serum albumin (BSA) as the standard. For each protein sample, a 10 µg aliquot was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking with 3% BSA in Tris-buffered saline, the blots were probed overnight at 4°C with the following primary antibodies: IDO-1 (BioLegend, San Diego, CA, USA), iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX-2 (Abcam, Cambridge, MA, USA), RIP2, MyD88 (Millipore, Billerica, MA, USA), IKK-α, IκB-α (Cell Signaling Technology, Beverly, MA, USA), phospho-NF-κB p65, total NF-κB p65 (Santa Cruz Biotechnology), phospho-JNK, total JNK, phospho-p38 mitogenactivated protein kinase (MAPK), total p38 MAPK, phospho-ERK, total ERK, and GAPDH (Cell Signaling Technology) as the housekeeping control. The membranes were blotted with secondary antibodies at room temperature for 1 h, and the proteins were detected with enhanced chemiluminescence reagent (GE Healthcare Life Science, Buckinghamshire, UK). Detailed information for all antibodies is provided in Table S1 in Supplementary Material.

cell Proliferation assay
Proliferation of hUCB-MSCs and hUCB-MNCs was determined after treatment with MIS416. In addition, to identify the suppression ability of hUCB-MSCs, isolated CD4 + T cells were co-cultured with MIS416 pre-treated hUCB-MSCs, followed by a proliferation assay. Cell proliferation was measured using a bromodeoxyuridine (BrdU) ELISA kit (Roche, Indianapolis, IN, USA). After the indicated treatment, BrdU labeling solution (100 µM) was added into the culture medium in 96-well plate. The culture medium containing BrdU solution was removed after overnight incubation at 37°C, and the cells were fixed with FixDenat solution for 30 min at room temperature. The cells were then incubated with an anti-BrdU-POD working solution for 90 min at room temperature. Following three rinses with washing solution, a substrate solution was added to the cells and incubated for 5~30 min at room temperature. After sufficient color development, the OD values were read at 450 nm on a microplate reader (Tecan).

cell Migration assay
Human adult stem cells, including hUCB-MSCs were suspended in culture medium, and 500 µl of the cell suspension (1 × 10 4 cells/ ml) was added to transwell inserts (8 µm pore size). Subsequently, 500 µl of MSC conditioned medium was added to the lower chambers. After 24 h of incubation, hUCB-MSCs that migrated to the underside of the membrane were fixed, and the remaining cells in the upper chamber were carefully swiped with a cotton swab. The membranes of the transwell were stained with DAPI and sealed on slides. A confocal microscope (Nikon, Eclipse TE200, Japan) was used to count the number of cells on the underside of the insert for each group.

Flow cytometry
To confirm the expression of cell surface markers, isolated primary cells were stained with a fluorochrome-conjugated antibody and analyzed. After cell surface staining, the cells were stained with antibodies against intracellular protein, as necessary. For intracellular staining, we used transcription factor buffer set (BD Biosciences, #562725) according to manufacturer's instruction. Briefly, the cells were fixed by 1× Fix/Perm Buffer and permeabilized by 1× Perm/Wash Buffer.  Table S1 in Supplementary Material. Fluorescence was detected with a FACSCalibur flow cytometer and evaluated using Cell Quest software (BD Biosciences).

Quantitative Pcr
Total RNA was extracted from tissues and cultured cells by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis was performed using Superscript™ III reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using SYBR-Green PCR Master Mix with an ABI 7300 sequence detection system. The mRNA levels of each gene were normalized using GAPDH as the housekeeping gene.
immunohistochemistry 4-μm-thick paraffin-embedded sections of colon were deparaffinized and rehydrated. For permeabilization, the samples were incubated with 0.05% Triton X-100 solution at room temperature for 10 min and blocked with 5% normal goat serum at room temperature for 1 h. Then, the cells were stained with rat antimouse Foxp3 monoclonal antibody (eBioscience, San Diego, CA, USA). Before imaging, nuclei were counterstained with DAPI. The images were captured by a confocal microscope (Nikon).
Detailed information for all antibodies is provided in Table S1 in Supplementary Material.

In Vivo cell Tracking
For in vivo cell trafficking, hUCB-MSCs were transduced with a GFP-encoding retroviral vector. For retrovirus preparation, the pMX-GFP vector and retrovirus packaging vectors were cotransfected into 293FT cell (Invitrogen) using FuGENE 6 transfection reagent (Promega). Viral supernatants were collected at 48 h post-transfection, filtered through a 0.45 µm PVDF membrane filter, and then directly used to infect hUCB-MSCs. Transduction efficiency was monitored by fluorescence microscopy and flow cytometric analysis ( Figure S2 in Supplementary Material). These cells (2 × 10 6 cells/head) were intraperitoneally injected into mice on day 1. On day 2 and day 11, mice were sacrificed, and colon samples were collected. The distribution of hUCB-MSCs was imaged by confocal imaging (Nikon, Eclipse TE200, Japan) using 4-μm-thick paraffin-embedded sections. Further, colon samples were chopped and filtered to obtain a single-cell suspension, on day 2 and day 11. Then, samples were stained with antibody of hUCB-MSCs surface markers, anti-human CD29, and fluorescence was detected by flow cytometric analysis.

statistical analysis
The mean values of all data are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA

Mis416 enhances the Therapeutic effect of hUcB-Mscs against Dss-induced colitis in Mice
The ligands of pattern-recognition receptors, as components of innate immune systems, affect the diverse functions of hUCB-MSCs, including immunomodulation, migration, proliferation, differentiation, and cytokine secretion (16,(32)(33)(34)(35). To investigate whether MIS416, a microparticle comprising NOD2 and TLR9 agonists, could enhance the therapeutic effects of hUCB-MSCs, we co-injected hUCB-MSCs and MIS416 in DSS-induced colitis mice. The survival rate was further increased by co-treatment compared to hUCB-MSC or MIS416 single treatments (

Mis416 improves the anti-inflammatory Function and Tissue regenerative capacity of hUcB-Mscs in the colon
We next examined H&E-stained colon samples to investigate the histopathological changes in the colons of DSS colitis mice on day 11. In the same context as previous results, colonic inflammation was more effectively resolved by co-treatment with MIS416 and hUCB-MSCs than each single treatment (Figure 2A)  In addition, fibrosis-associated mucosal and submucosal collagen depositions were quantified by PSR staining. Treatment of hUCB-MSCs or MIS416 alone did not show significant changes, on the other hand, only co-treatment markedly decreased fibrosis and enhanced tissue regeneration ( Figure 2B)  To examine whether MIS416 could regulate the immunomodulatory ability of hUCB-MSCs, we evaluated the expression levels of COX-2, iNOS, and IDO-1, which are enzymes related to immunomodulation by hMSCs (36)(37)(38). Western blot analysis revealed that MIS416 treatment had no significant effect on the expressions of COX-2, iNOS, and IDO-1 (Figure 3D)  In addition, key downstream adaptor molecules, RIP2 for NOD2 and MyD88 for TLR9 were not changed by MIS416 treatment. The phosphorylation levels of signaling cascades, NF-κB and MAPK were also not altered (Figure 3E; Figure S3B in Supplementary Material). Consistent with previous studies (5,30), the proliferation of hMNCs co-cultured with hUCB-MSCs was markedly inhibited. However, hUCB-MSCs primed with MIS416 did not show a significant difference from unstimulated cells ( Figure 3F) [(+) vs U p < 0.0001; (+) vs U + M p < 0.0001; U vs U + M p > 0.9999]. We next evaluated the migratory ability of hUCB-MSCs primed with MIS416 in vitro. The number of migrated cells remained unchanged after MIS416 treatment ( Figure 3G) (U vs U + M p = 0.9489). Overall, these findings suggest that MIS416 augments the therapeutic abilities of hUCB-MSCs by indirect mechanism, but not by direct influence on the cells.

exposure to Mis416 causes increases in the number of immune cells via activation of innate immune cells such as cD14 + Macrophages
We observed that co-treatment with hUCB-MSCs and MIS416 was better than either treatment alone in DSS-induced colitis, although MIS416 had no direct effect on hUCB-MSCs. Thus, we hypothesized that MIS416 would indirectly upregulate the therapeutic effects of hUCB-MSCs by targeting NOD2 or TLR9 on other cells. We investigated changes in the spleen, the largest secondary lymphoid organ which contains various immune cells, after systemic administration of MIS416 on day 11. Significant enlargement of the spleen was identified in both mice treated with MIS416 alone and co-treated with hUCB-MSCs ( Figure 4A). The length and weight of the spleen were considerably increased in MIS416-treated groups (Figures 4B,C) [for length, (+) vs M p = 0.0109; (+) vs U + M p = 0.0254/for weight, (+) vs M p = 0.0002; (+) vs U + M p = 0.0049]. To determine whether MIS416 elicited a detectable splenic response at an earlier time point, we investigated the spleen of mice a day after MIS416 treatment (on day 2). Although there was no significant change in the length (Figures 4D,E), the weight of the spleen was increased ( Figure 4F) [for length, (+) vs M p > 0.9999/for weight, (+) vs M p = 0.0458]. We mimicked this phenomenon in vitro by treating hUCB-MNCs with MIS416 and observed that cell proliferation was dose-dependently elevated (Figures 4G,H)

Mis416 and hUcB-Mscs collaborate in the Modulation of intestinal immune Balance by regulating Polarization of Th cell lineages
We further investigated whether MIS416 treatment altered the proportions of immune cells in hUCB-MNCs using flow cytometric analysis. Consistent with previous studies (7,14), the proportion of proinflammatory effector cells, including CD3 + , CD4 + , and CD8 + cells, were decreased whereas CD19 + cells were increased ( Figure S5  In the same context, the colonic infiltration of Foxp3 + Treg cells alteration of the immune environment by Mis416 improves the immunosuppressive effect of hUcB-Mscs The immune system in the peritoneal cavity dynamically interacts with both the lymphatic system and general circulation by exchange of the fluids and cells, and intraperitoneally infused hMSCs remain in peritoneal cavity while interacting with the peritoneal immune system (39,40). To address whether MIS416mediated immune change affected immunomodulation of hUCB-MSCs, we analyzed the cytokine profiles in the serum of MIS416-treated mice on day 2. Although the level of TNF was not significantly elevated, it was observed that secretion of IFN-γ, IL-6, and IL-12 was markedly increased by MIS416 infusion on day 2 ( Figure 6A

Mis416-induced secretion of McP-1 Promotes the Migration of hUcB-Mscs
The engraftment to inflamed sites is crucial for therapeutic potential of hUCB-MSCs (19,22,23). Therefore, we investigated whether MIS416 administration could upregulate the mobiliza-

DiscUssiOn
In this study, we have proposed co-administration of MIS416 and MSCs as an enhancement strategy for cell therapy. This strategy may be more convenient for clinical application compared to previous methods (44)(45)(46) that require additional preparatory steps, such as cell priming or genetic manipulation. Also, this approach could reduce the associated risks with such additional manipulations, which include tumor formation and contamination of a heterogeneous population. Through gross, histologic, and serologic assessments, we demonstrated that administration of MIS416 distinctly increased the therapeutic effects of hUCB-MSCs in experimental colitis model. Of note, the improved therapeutic efficiency was not likely mediated by direct interaction between MIS416 and hUCB-MSCs, as we demonstrated that MIS416 treatment could not alter the proliferative, immune cell suppressive, and migratory capacity of hUCB-MSCs. In fact, this finding is not unexpected, due to the physical properties of MIS416, which restrict cell uptake and subsequent sensing of MIS416 ligands to phagocytic innate immune cells, such as plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages (7,14). Consistent with hUCB-MSCs, CD4 + T cells were not directly influenced by MIS416. And MIS416-mediated expansion of immune cells was observed only when innate immune cells were present (7,14). In the present study, we also demonstrated that MIS416-mediated increased proliferation of hUCB-MNCs was relatively impeded when CD14 + macrophage-like cells were depleted.
We showed that application of MIS416 microparticle contributed to increased numbers of immune cells in the spleen. In addition, we examined the effects of MIS416 on the composition of splenic immune cells. Interestingly, proinflammatory cells, including Th1 cells and Th17 cells, were suppressed, and conversely, the proportions of Th2 cells, B cells, and Treg cells were increased. Stimulation of innate immune receptors and their downstream signaling pathways modulated the balance between various immune cells (47,48). The innate ligands within MIS416 each have well documented immune regulatory activities. NOD2 ligand has been shown to regulate Th1 responses and simultaneously, play a crucial role in the induction of Th2 immune responses and Treg cells (49)(50)(51). In addition to this, TLR9 stimulation drives maturation and proliferation of B cells (52,53). Based on these results, it may be delineated that MIS416 favors the development of regulatory immune cell subsets, although MIS416 induced increases in the quantity of both pro-and anti-inflammatory immune cells. The DSS-induced increase of proinflammatory cytokines, except for IL-6 was attenuated in the serum of MIS416-treated mice on day 11. This suggests that alternative immune cells induced by MIS416 treatment are preferentially able to suppress inflammation and gradually restore immunologic balance in the body.
Imbalance between Th1/Th17 cells and Treg cells in intestine leads to dysregulated inflammation and consequently onset of IBDs (54,55). MIS416 is known to promote myeloid cell activity and increase the innate IFN-γ to modify disease activity in an auto-immune model (13,14). In the same context with the previous article (7), the level of IFN-γ was elevated in the serum of MIS416-infused mice. In agreement, Girvan et al. and White et al. have reported that MIS416-mediated innate IFN increased the secretion of TGF-β1 and IL-10, MIS416 also increased the expression of PDL-1 on myeloid cells known to induce the formation of Treg cells (7,14). Moreover, the type I IFN induced by TLR9 activation suppressed the differentiation of Th1 and Th17 cells, whereas the expansion of Treg cells was promoted (12,(56)(57)(58). Consistent with these reports, we observed that treatment with MIS416 decreased the proportion of Th17 cells in hUCB-MNCs and the levels of IL-17A and IL-23 in the serum of mice. In addition, MIS416 increased the colonic infiltration of Treg cells by upregulating IL-10 secretion. Importantly, the levels of IL-17A and IL-23 were mostly reduced in hUCB-MSCs and MIS416 cotreated mice, and the infiltration of Treg cells and IL-10 secretion were notably augmented by co-treatment. Our findings indicate that MIS416 and hUCB-MSCs cooperated to resolve an intestinal inflammation and attenuate the severity of experimental colitis by controlling the balance in Th1, Th17, and Treg cells.
In addition to IFN-γ, the level of IL-6 and IL-12 secretion in the serum of mice was also elevated by MIS416 treatment. Reciprocal interaction between peritoneal immune system and general immune system is accomplished by exchanging the fluids and cells. In addition, peritoneal immune response affects the functions of infused hMSCs, which is attached to specific peritoneal sites, such as mesentery and omentum (39,40). The combination of these cytokines enhanced the immunosuppressive ability of hUCB-MSCs by upregulating the COX-2-and IDO-1-related pathways (5,59). Although MIS416 has been reported to increase the level of NO in the serum (14), MIS416-induced cytokines could not alter NO production by hUCB-MSCs. Based on these data, it seems that upregulation of these cytokines plays a role in increased immunomodulation and tissue regeneration by hUCB-MSCs in vivo.
Mesenchymal stem cells suppress activated immune cells through direct cell-to-cell contact inhibition as well as environmental change mediated by soluble factors (17,60). Thus, mobilization of MSCs into inflamed sites is important for suppression of activated immune cells placed in the adjacent lesion. Consistent with previous studies (19,22,23), hUCB-MSCs migrated to inflamed colon of mice. More interestingly, this study showed that systemic infusion of MIS416 immediately generated robust production of MCP-1. MCP-1 is known to induce the migration of various types of cells involved in the recovery process, including MSCs (26,27,61). MIS416-induced MCP-1 enhanced the migratory capacity of hUCB-MSCs in vitro and in vivo; thus, many more hUCB-MSCs mobilized to the inflamed colon in response to MCP-1.
Through these findings, we reveal that application of MIS416 ameliorated DSS-induced colitis compared to a single application of hUCB-MSCs and that this effect was mediated through three different ways. First, by inhibition of Th1 and Th17 cells, polarization of Th2 cells, and enhancement of Treg and B cells. In particular, MIS416 and hUCB-MSCs cooperated to shift the balance from Th1/Th17 to the Treg-directed responses. Second, MIS416mediated changes in immune milieu facilitated the increase of cytokines, such as IFN-γ, IL-6, and IL-12. The hUCB-MSCs stimulated by these cytokines subsequently suppress proinflammatory cells in the inflamed colon. Last, MIS416-induced MCP-1 enhanced the migratory capacity of hUCB-MSCs, resulting in an increase in colonic infiltration. In summary, MIS416 enhances the therapeutic efficacy of hUCB-MSCs against experimental colitis by improving the immunosuppressive capacity of the cells and regulating immune homeostasis in the gut.

eThics sTaTeMenT
All animal experimental processes were approved by Seoul National University Institutional Animal Care and Use Committee (IACUC No. SNU-170523-3) in accordance with the guidelines of the committee. All experiments using human UCB or UCBderived cells were approved by Institutional Review Board (IRB) of the Boramae Hospital and Seoul National University (IRB No. 1707/001-008) with informed maternal consent. All subjects gave written informed consent in accordance with the Declaration of Helsinki.
aUThOr cOnTriBUTiOns B-CL and NS designed the study, collected and analyzed the data, and wrote the manuscript. JL, IK, and J-JK collected and analyzed the data. SL collected the data. SC collected and analyzed the data. GW analyzed data and contributed to the writing of the paper. K-SK designed and supervised the study, analyzed the data, and wrote the manuscript.