Pre-Treatment with IL-1Β Enhances the Efficacy of MSC Transplantation In

RESEARCH ARTICLE

Pre-treatment with IL-1b enhances the efficacy of MSC transplantation in DSS-induced colitis

Hongye Fan1, Guangfeng Zhao1, Liu Liu1, Fei Liu1, Wei Gong1, Xianqin Liu1, Liu Yang1, Jianjun Wang1 and Yayi Hou1,2

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly due to their immunosuppressive properties. Although interleukin-1b (IL-1b) is one of the most important inflammatory mediators, growing evidence indicates that IL-1b signaling elicits the immunosuppressive properties of MSCs. However, it remains unclear how IL-1b signaling accomplishes this activity. Here, we focus on the therapeutic efficacy of IL-1b-primed MSCs in the dextran sulfate sodium (DSS)-induced colitis model, in addition to the underlining mechanisms. We first found that IL-1b-primed MSCs, without any observable phenotype change in vitro, significantly attenuated the development of DSS-induced murine colitis. Moreover, IL-1b-primed MSCs modulated the balance of immune cells in the spleen and the mesenteric lymph nodes (MLNs) through elevating cyclooxygenase-2 (COX-2), IL-6 and IL-8 expression and influencing the polarization of peritoneal macrophages. Importantly, IL-1b-primed MSCs possessed an enhanced ability to migrate to the inflammatory site of the gut via upregulation of chemokine receptor type 4 (CXCR4) expression. In summary, IL-1b-primed MSCs have improved efficacy in treating DSS-induced colitis, which at least partly depends on their increased immunosuppressive capacities and enhanced migration ability. Cellular & Molecular Immunology (2012) 9, 473–481; doi:10.1038/cmi.2012.40; published online 22 October 2012

Keywords: IL-1b; mesenchymal stem cells; ulcerative colitis

INTRODUCTION However, growing evidence has indicated that IL-1b signaling sup- Mesenchymalstemcells(MSCs)arederivedfromavarietyoftissuesand ports colonic homeostasis and attenuated colonic inflammation. have the capacity for self-renewing and differentiation.1,2 MSC-based IL-1b signaling can protect mice from intestinal damage after therapies have provided convincing evidence for their potential anti- Citrobacter rodentium infection16 and from DSS-induced colitis.17 inflammatory and immunomodulatory effects in treating a variety of Mice that are deficient in IL-1 receptor 1 (IL-1R1) exhibit exacerbated inflammatory and autoimmune diseases, including ulcerative colitis DSS-induced colitis, which is indicated by a higher disease activity (UC),3 rheumatoid arthritis4 and sepsis.5 MSCs can migrate to sites of index score and an increased mortality compared with wild-type mice. tissue damage,6,7 where they are activated by inflammatory cytokines Moreover, blocking IL-1b activity with anakinra, a recombinant form (such as IFN-c) and several chemokines, which can recruit or suppress of IL-1R antagonist, enhances the severity of DSS-induced colitis in lymphocytes.8–11 wild-type mice.18 IL-1b induces the production of cyclooxygenase-2 Interleukin-1b (IL-1b) is an important inflammatory factor that is (COX-2), which promotes the expression of prostaglandin E2 expressed abundantly during the active phase of UC.12–14 The UC (PGE2).19,20 PGE2, in turn, has been reported to inhibit mucosal mice induced by DSS treatment exhibit many symptoms similar to inflammation during DSS-induced colitis in mice and rats.21,22 human UC, such as diarrhea, bloody feces, body weight loss, mucosal Similarly, pre-treatment with a low dose of IL-1b has also been shown ulceration and shortening of the large intestine.13 The mechanism to suppress colonic inflammation in rabbits via the production of behind DSS-induced UC can be attributed to abundant IL-1b secre- PGE2.23 tion from macrophages in lamina propria following DSS stimulation, Notably, several studies have indicated that MSCs activated by which initiates an intestinal inflammatory cascade.15 Moreover, IL-1b can exhibit immunosuppressive properties. First, IL-1R1, administration of anti-IL-1b antibodies improves DSS-induced co- required for IL-1b signal transduction, is abundantly expressed in litis,14 and mice that are deficient in the NOD-like receptor family, human MSCs. Second, Groh et al.24 showed that T-cell activation pyrin domain containing 3 (NLRP3) inflammasome, a caspase-1- could be inhibited by the cell-free supernatant from MSCs treated activating complex which regulates IL-1b maturation, are relatively with IL-1b for 24 h. Third, IL-1b secreted from CD14 monocytes resistant to intestinal inflammation.15 has been reported to activate MSCs. In addition, IL-6, a hallmark of

1Immunology and Reproductive Biology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China and 2Jiangsu Key Laboratory of Molecular Medicine, Nanjing, China Correspondence: Dr Yayi Hou, Immunology and Reproductive Biology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China. E-mail: [email protected] Received 3 June 2012; revised 27 August 2012; accepted 28 August 2012 Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 474

MSC activity, is vigorously secreted by MSCs following stimulation stimulated with 10 ng/ml murine IL-1b for another 48 h and harvested with IL-1b in a time- and dose-dependent manner, which re- for reverse transcription-PCR and western blotting. In vitro models quires the activation of the p38 MAPK and ERK pathways.25 All were divided into five groups (Table 1). of these data suggest that IL-1b could elicit the immunosuppressive properties of MSCs through IL-1R1. However, the mechanism DSS-induced mice colitis through which IL-1b elicits these immunosuppressive properties Acute colitis was induced in C57BL/6 male mice (8-week-old) by is still unclear. administering 3% DSS (molecular weight 40 000 Da; Sigma) from In the present study, our results demonstrated that pre-treatment day 0 to day 7 in the drinking water ad libitum and changing to regular with IL-1b enhanced the efficacy of MSC transplantation in DSS- water from day 8 onward, as previously described.20,30 induced colitis. Pre-treating MSCs with IL-1b also modulated the C57BL/6 mice were randomly divided into the following five groups balance of immune cells through elevating COX-2 and IL-8 expres- (n510 mice/group): (i) control group; (ii) DSS treatment; (iii) DSS sion. IL-1b also promoted MSC migration to inflammatory sites with MSC treatment; (iv) DSS with IL-1b-primed MSC treatment; (v) through upregulating CXCR4 expression. DSS with IL-1R1 siRNA-transfected MSC treatment (Table 2). At day 1, mouse tails were intravenously injected with 23106 MSCs (MSCs, b m MATERIALS AND METHODS IL-1 -primed or IL-1R1 siRNA-transfected MSCs) diluted in 200 l PBS or a vehicle control (PBS alone). Mice were killed at day 14, except Isolation and culture of umbilical cord-derived MSCs 26 for the mice in the DSS treatment group, which were killed at day 11. MSCs were isolated using previously described methods. Briefly, Colitis severity was assessed daily by scoring the clinical disease human umbilical cords were obtained from full-term deliveries with activity (0–4), including evaluating stool consistency, presence of fecal informed consent of the mother after caesarian section. The cords blood and weight loss. The entire colon was removed from the cecum were aseptically stored at 4 uC in sterile phosphate-buffered saline to the anus, and colon length and weight were measured as indirect (PBS) supplemented with 200 U/ml penicillin-streptomycin (Sigma inflammation markers. For histopathological analysis, colon segments Aldrich, St. Louis MO, USA). Tissue collection for research was were fixed in 10% buffered formalin phosphate, and paraffin-embedded approved by the Ethics Committee of the Drum Tower Hospital of sections were prepared for hematoxylin and eosin (H&E) staining. Nanjing University Medical School, China. Fresh human umbilical Histological scores were blindly determined. For tissue inflammation, cords were collected and rinsed in PBS at 4 uC, and umbilical arteries increased numbers of inflammatory cells in the lamina propria were and veins were removed and cut into 1- to 2-mm pieces in DMEM/F- scored as 1, confluence of inflammatory cells extending into the sub- 12 medium (Gibco, Grand Island, NY, USA). The pieces of cords were mucosa was scored as 2 and transmural extension of the infiltrate was digested with mixed enzymes, including type II collagenase, neutral scored as 3. For tissue damage, discrete lymphoepithelial lesions were protease and hyaluronidase at 37 uC for 3 h. After three washes with scored as 1, mucosal erosions were scored as 2 and extension through PBS, umbilical cord-derived MSCs were plated into a culture flask. deeper structures of the bowel wall was scored as 3. The combined The medium was replaced every 3 days. Colonies of fibroblast-like cells histological colitis severity score ranged from 0 to 6. formed approximately 7 days later, and the cultures were trypsinized and passaged into a new flask for further expansion. In vivo bioluminescence and CM-DiI fluorescent imaging For bioluminescence imaging and fluorescent imaging, luciferase- MSC treatment expressing MSCs were labeled with CM-DiI dye (Molecular Probes, MSCs were pre-treated with 10 ng/ml murine IL-1b (Peprotech, Eugene, Oregon, USA) according to the manufacturer’s protocol. Rocky Hill, NJ, USA) for 48 h to activate IL-1b signaling. IL-1b signal- C57BL/6 mice were randomly divided into the following groups ing in MSCs was inhibited by downregulating IL-1R1 expression with (n55 each group): (i) DSS with MSC treatment; and (ii) DSS with small RNA interference (siRNA). Specific sequence for human IL-1R1 IL-1b-primed MSC treatment. At day 1, C57BL6 mice were treated siRNA was designed and synthesized by Ribobio (GuangZhou, with 3% DSS. At day 2, mice were injected 2.03106 MSCs via tail vein. China). Two suitable sequences were selected (IL-1R1–siRNA#1, At day 4, mice were given an intraperitoneal injection of 0.2 mg D- GAACACAAAGGCACUAUAAdTdT, and IL-1R1–siRNA#2, GCA- Luciferin Potassium Salt (Caliper, Hopkinton, USA) and killed 10 min GCAUA UAUCCAGUUAA dTdT) and mixed as siRNA for the fol- later. Spleen, mesenteric lymph nodes (MLNs), and colon were lowing experiments. MSCs were cultured to 40% confluency and obtained and imaged with an IVIS Lumina XR System (Caliper) and transfected with the siRNA duplex and negative control siRNA using then frozen for cryosectioning. For visualizing the localization of Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according labeled MSCs based on fluorescence of CM-DiI dye (red), cryosec- to the manufacturer’s recommended protocol. Then, 10 nM siRNA tioned tissue was counter-stained with 49-6-diamidino-2-phenylin- was used for 10-cm plates. After 24 h of transfection, the cells were dole, and fluorescent images were collected with a confocal scanning

Table 1 Grouping of MSCs used in vitro experiment Table 2 Grouping of mice used in vivo experiment Group (MSCs) Treatment Group (mice) DSS treatment MSC treatment

Con No treatment Control – – siRNA Con Negative control siRNA transfection DSS 1 – IL-1b 10 ng/ml IL-1b DSS1MSC 1 MSCs IL-1R1 siRNA si-IL-1R1 transfection DSS1IL-1b MSC 1 IL-1b-primed MSCs IL-1R1 siRNA1IL-1b After IL-1R1 siRNA transfection, 10 ng/ml IL- DSS1IL-1R1 siRNA MSC 1 IL-1R1 siRNA-transfected 1b was added MSCs

Abbreviations: IL-1b, interleukin-1b; IL-1R1, IL-1 receptor 1; MSC, mesenchymal Abbreviations: DSS, dextran sulfate sodium; IL-1b, interleukin-1b; IL-1R1, IL-1 stem cell; siRNA, small RNA interference. receptor 1; MSC, mesenchymal stem cell; siRNA, small RNA interference.

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 475 laser system (Olympus FluoView 1000; Olympus Corp., Lake Success, from the upper side of the filter were scraped off with a cotton swab, NY, USA) attached to an inverted microscope (IX81; Olympus Corp., and filters were stained with crystal violet. The number of cells that had Tokyo, Japan). migrated to the lower side of the filter was counted under a light microscope at 34 magnification in five randomly selected fields. Flow cytometric analysis Monoclonal antibodies against human CD11b, CD106, CD14, CD19, Statistical analysis CD31, CD34, CD45, HLA-DR, CD29, CD44, CD73, CD90 and Results were presented as mean6s.e.m. Student’s t-test was used to isotype-matched controls, as well as monoclonal antibodies against compare between two groups. One-way ANOVA analysis of variance mouse CD206, CD11c, F4/80, CD3, CD4, IL-17A, IL-4, IFN-c, Foxp3 was used to compare among three or more groups. Differences were and isotype-matched controls, were obtained from eBiosciences, San considered to be significant with P values of ,0.05. Statistical calcula- Diego, CA, USA. For the detection of intracellular cytokines, cells were tions and survival curves were performed with GraphPad Prism soft- incubated with phorbol myristate acetate (50 ng/ml) and ionomycin ware (San Diego, CA, USA). (750 ng/ml) for 4 h. Labeled cells were analyzed on FACS Calibur flow cytometer BD Biosciences, San Diego, USA and data were analyzed RESULTS using CellQuest software (BD Biosciences). The effect of IL-1b signaling on the morphology, phenotype and viability of MSCs Quantitative PCR (Q-PCR) analysis MSCs were generated from human umbilical cord tissue, and their Q-PCR for GAPDH, COX-2, IL-6, IL-8 and CXCR4 was performed on colonies visually appeared as a homogenous population of cells after an Applied Biosystems 7300 Sequence Detection System (Applied four passages (Figure 1a). The MSC cultures were then assayed routi- Biosystems, Foster city, CA, USA) using SYBR green dye (Bio-red). nely for the presence of MSC-related cell surface antigens by flow For quantification, the relative mRNA level of specific gene expression cytometry analysis. The results showed that the phenotype of MSCs 2DCt was obtained using the 2 method. was positive for CD29, CD44, CD73, CD90 and HLA-ABC (MHC-I) and was negative for CD11b, CD106, CD14, CD19, CD31, CD34, Transwell migration assay CD45 and HLA-DR (MHC-II) (Figure 1b). The migratory ability of MSCs was evaluated using Transwell plates IL-1b, one of the most important soluble inflammatory mediators, (Corning Costar, Tewksbury, USA), which were 6.5 mm in diameter has been found to be enhanced in UC. IL-1R1, the major receptor for with 8-mm pore filters. In brief, MSCs were suspended in serum-free IL-1b, is expressed in MSCs. To study the impact of IL-1b signaling on medium and seeded into the upper well, and 10 ng/ml murine IL-1b MSCs, we first downregulated the expression of IL-1R1 on MSCs using containing medium was placed in the lower well of a Transwell plate. siRNA. The two siRNAs mixtures targeted against different regions of Following incubation for 12 h at 37 uC, cells that had not migrated IL-1R1 mRNA were cotransfected into MSCs, and 48 h later the cells

a c Con si Con IL-1β ** 1.0 0.8 0.6 0.4 0.2 si IL-1R1 si IL-1R1+IL-1β Relative mRNA

expression of IL1R1 0.0 Con si Con si IL-1R1

Con si Con si IL-1Rl IL-1R1

b GAPDH

isotype d 3 *** control CD11b CD106 CD14 CD19 IL-1β 2 si IL-1R1 si IL-1R1+IL-1β OD450 1 CD31 CD34 CD45 HLA-DR 0 β β Con IL-1 si Con si IL-1R1

CD29 CD44 CD73 CD90 HLA-ABC si IL-1R1+IL-1

Figure 1 The effects of IL-1b signaling on MSC phenotype, morphology and vitality. (a) The morphology of MSCs was observed by microscopy. (b) The effect of IL-1b signaling on the phenotype of MSC. Expression of phenotypic surface antigens on MSCs was detected using FACS. There was no significant difference between the different treatment groups of MSCs. (c) Knockdown of IL-1R1 with siRNA. MSCs were either mock transfected or transfected with the mixture of two different siRNAs against IL-1R1. The cells were then harvested at day 2 and analyzed at the mRNA and protein level. (d)IL-1b signaling affects the viability of MSCs. The viability of MSCs was measured using the Cell Counting Kit-8 method. IL-1b, interleukin-1b; IL-1R1, IL-1 receptor 1; MSC, mesenchymal stem cell; siRNA, small RNA interference.

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 476

were harvested for Q-PCR and western blot analysis. The results MSC-treated mice. Moreover, M2 macrophages were significantly showed that IL-1R1 was significantly downregulated at both the more prevalent in the IL-1b-primed MSC-treated mice than in the mRNA and protein levels (Figure 1c). IL-1R1 siRNA transiently transfected MSC-treated mice (Figure 3a). Importantly, after being cultured for 2 days with or without IL-1b Moreover, IL-1b-primed MSC-treated colitis mice displayed sig- (10 ng/ml), the five experimental conditions for MSCs (control, si nificantly increased numbers of Treg cells and Th2 cells, in addition control, IL-1b, IL-1R1 siRNA, IL-1R1 siRNA1IL-1b) retained their to decreased numbers of Th1 and Th17 cells in their spleens and MLNs homogeneous fibroblastoid cell morphology, with a smooth border compared with those in DSS-treated mice (Figure 3b–e). Notably, and unaffected expression of surface markers (Figure 1a and b). there were significantly elevated Treg cell numbers in IL-1b-primed Moreover, IL-1b did not affect the viability of MSCs, but IL-1R1 MSC-treated mice compared to MSC-treated mice. siRNA significantly reduced viability of MSCs either in the absence MSCs can inhibit T-cell activation and proliferation in vitro or presence of IL-1b (Figure 1d). These data indicate that IL-1b sig- through soluble factors. Recent studies suggest that these soluble fac- naling does not affect the morphology and phenotype of MSCs, but IL- tors, including PGE2, IL-6 and IL-8, are critical for the immunomo- 1b signaling is important to maintain the viability of MSCs. dulatory function of MSCs. COX-2 (a key enzyme in the synthesis of PGE2),31 IL-832 and IL-633 are known to be downstream transcripts of IL-1b-primed MSCs attenuate the development of DSS-Induced the IL-1b signaling pathway, but it is unclear whether this pheno- colitis menon is also the case for MSCs. We found that IL-1b could signifi- Similar to previous reports,27,28 we successfully induced colitis in cantly elevate COX-2, IL-6 and IL-8 mRNA expression and COX-2 C57BL/6 mice by oral administration of 3% DSS. The mouse colitis protein expression, while IL-1R1 siRNA-transfected MSCs showed was characterized by sustained bloody diarrhea, weight loss, 100% abrogated IL-1b signaling effects (Figure 4). mortality at day 11 (Figure 2a, b and d), shorter and lighter colons Our results indicated that the marked attenuation of IL-1b-primed (Figure 2c), histological changes with severe colonic transmural MSCs on the development of DSS-induced colitis may be related to inflammation, increased wall thickness, localized inflammatory cell modulating the immune cell profile though elevated COX2, IL-6 and infiltration, epithelial ulceration with degeneration of crypt archi- IL-8 expression. tecture and loss of goblet cells (Figure 2e and f). The next day, following oral administration with 3% DSS, the mice were intrave- IL-1b signaling promotes the migration of MSCs nously injected through the tail vein with PBS, MSCs, IL-1b-primed Given the migratory ability of MSCs to travel to inflammatory or MSCs, or IL-1R1 siRNA transiently transfected MSCs. At day 14, injured sites, we assessed whether the IL-1b-primed MSCs had the mice showed ameliorated colitis and improved survival rates to improved migration in vivo. The day after being treated with DSS, different degrees compared with those in the PBS group (Figure 2a– the mice were intravenously injected with luciferase-expressing f). Strikingly, compared to the mice treated with MSCs or IL-1R1 MSCs, which were dual-labeled with CM-DIL and IL-1b-primed. siRNA transiently transfected MSCs, IL-1b-primed MSC-treated On the fifth day, Luciferase substrate was injected into the peritoneal mice had a significantly improved survival rate, lower weight loss cavity. The mice were killed after 10 min, and the organs were imme- and clinical disease scores in addition to longer length and heavier diately collected to image bioluminescence. IL-1b-primed MSCs colons (Figure 2a–e). Histologically, IL-1b-primed MSCs exhibited showed stronger bioluminescence signal in the spleen, MLN and colon significantly ameliorated colonic transmural inflammation and than unprimed MSCs (Figure 5a). decreased wall thickness, restored goblet cells and suppressed To confirm the localization of luminescent MSCs in each organ, mucosal ulceration and focal loss of crypts, all of which was as- frozen sections of the organs were imaged. Representative fluorescent sociated with a decrease in disease score and histological score microscopic images showed that IL-1b-primed MSCs labeled with (Figure 2e and f). CM-DIL (red) were readily detected in the spleen, MLN and colon, These data consistently demonstrated that IL-1b-primed MSCs whereas less bioluminescence signal was detected in MSCs labeled with were more effective in the prevention of DSS-induced colitis than CM-DIL and injected into mice, and no signal was detected in PBS unprimed MSCs or the IL-1R1 siRNA transiently transfected MSCs. injected mice (Figure 5b). Our results indicated that pretreating MSCs This finding suggests that IL-1b signaling is important for the thera- with IL-1b could improve the in vivo localization of MSCs to inflam- peutic efficacy of MSCs in treating DSS-induced colitis. matory sites during DSS induced colitis.

IL-1b-primed MSCs alters the balance of immune cells The enhanced migratory ability of IL-1b-primed MSCs is related to MSCs have been previously shown to have immunomodulatory upregulation of CXCR4 expression effects. They can both suppress the proliferation and activation of Given the greater migratory ability of IL-1b-primed MSCs to the innate and adaptive immune cells and activate regulatory T cells inflammatory or injured sites and that overexpression of CXCR4 (Tregs). Because of these properties, we next investigated the effects receptor could promote the migration of MSCs to the injured tis- of IL-1b-primed MSCs on the profile of immune cells in DSS-induced sues,34 we investigated whether IL-1b could elevate MSC migration colitis mice. in vitro and enhance CXCR4 expression. We used a transwell migra- Flow cytometry revealed that CD11c1 M1 macrophages were sig- tion assay to answer this question. As expected, our results showed that nificantly increased in the peritoneal cavity of DSS-induced colitis the migratory response of MSCs to IL-1b was more pronounced than mice and were significantly decreased in IL-1b-primed MSC-treated those in the culture medium without IL-1b, while the IL-1R1 siRNA- mice, but there was no notable difference among the mice treated with transfected MSCs showed unchanged migration ability either in the MSCs, IL-1b-primed MSCs, or IL-1R1 siRNA transiently transfected absence or presence of IL-1b (Figure 6a). Moreover, Q-PCR and wes- MSCs (Figure 3a). tern blot analysis showed that both CXCR4 mRNA and protein However, CD2061 M2 macrophages were decreased in DSS-treated expressions were significantly elevated in MSCs cultured with IL-1b mice but significantly increased to the baseline level in IL-1b-primed (10 ng/ml) for 48 h (Figure 6b and c), and the IL-1R1 siRNA-trans-

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 477

a 10 b 100 Control 0 80 DSS DSS+MSC

-10 * *** 60 DSS+IL-1β MSC DSS+si IL-1R1 MSC -20 * 40

-30 Percent survival 20 Body Weight Loss (%) Body Weight -40 0 0 987654321 1011 1312 14 0 51015 Day Day

1 2 c d 8 * *** 1.0 0.8 6 * 0.6 4 0.4 2 colon weight (g) 0.2 colon length (cm) 0 0.0

DSS MSC βMSC DSS MSC MSC control control β IL-1 IL-1

Day 11 si IL-1R1 MSC si IL-1R1 MSC

1 3 45 e 5 6 * ** 4 4 3 * **

2 2

1 Histological Score Clinical Disease Score 0 0

DSS MSC MSC DSS MSC MSC β β Day 14 IL-1 IL-1 si IL-1R1 MSC si IL-1R1 MSC

Normal DSS+PBS DSS+MSC f

DSS+IL-1β MSC DSS+siIL-1R1 MSC

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 478

Figure 2 IL-1b-primed MSCs attenuate the development of DSS-induced colitis. (a) The body weight changes were calculated as a percentage of initial body weight on day 0. (b) Survival rates were compared among normal mice, DSS-treated mice, DSS with MSC-treated mice, DSS with IL-1b-primed MSC-treated mice and DSS with IL-1R1 siRNA transiently transfected MSC mice. (c) Macroscopic images of representative mouse colons harvested on days 11 and 14. The numbers on the top of the picture indicated as follows: 1, normal mouse; 2, DSS-treated mouse; 3, DSS with MSC-treated mouse; 4, DSS with IL-1b-primed MSC-treated mouse; 5, DSS with IL- 1R1 siRNA transiently transfected MSC mice. (d) Assessment of colonic weight and length upon killing. (e) Colitis scores and Histology scores indicated that IL-1b primed MSCs prevented DSS-induced pathology. Colitis scores were based on the presence of loose stool, bleeding, and macroscopic inflammation. Histology scores were derived from microscopic analyses of longitudinal colon sections from each mouse. (f) Photomicrographs (magnification 310) of an H&E-stained paraffin section of a representative mouse colon from each treatment group. DSS, dextran sulfate sodium; H&E, hematoxylin and eosin; IL-1b, interleukin-1b; IL-1R1, IL-1 receptor 1; MSC, mesenchymal stem cell; siRNA, small RNA interference.

fected MSCs could not promote CXCR4 expression even after IL-1b unclear whether the therapeutic efficacy of MSCs during UC is stimulation. Together, these results suggested that the influence of IL- related to IL-1b. Here, we determined that IL-1b-primed MSCs 1b on the migratory ability of MSCs may be attributed to inducing had better efficacy in treating DSS-induced mice colitis, which enhanced CXCR4 expression. might be due to the altered properties of MSCs following stimulation of IL-1b or one of several other proteins. DISCUSSION MSCs have the inherent function of repairing injured tissue. Our Increasingly more studies have revealed the beneficial effects of results showed that all of the surviving mice treated with MSCs (MSCs, MSC therapy in treating many diseases, including UC. MSCs can IL-1b-primed MSCs, or IL-1R1 siRNA transiently transfected MSCs) be derived from different human tissues and organs and, in the last exhibited therapeutic effects following treatment. The IL-1b-primed several years, the human umbilical cord has gained increasing MSCs displayed the best efficacy, while the IL-1R1 siRNA transiently attention as a possible clinical alternative source for MSCs other transfected MSCs displayed the worst efficacy. These results suggest than bone marrow.2 IL-1b is not only the key inflammatory factor that activation of the IL-1b signaling pathway in MSCs is beneficial for in UC, but also mediates the development of DSS-induced UC. IL- their therapeutic efficacy during murine colitis. We hypothesize that 1b can elicit the immunosuppressive properties of MSCs, but it is the MSCs that were primed by IL-1b were already activated in vitro

Figure 3 IL-1b-primed MSCs decrease systemic and local inflammatory responses in acute colitis. (a) M1 and M2 macrophage levels in mice peritoneal cavity. (c–e) Th1, Th2, Th17 and Treg cell levels in spleens and MLNs of mice. IL-1b, interleukin-1b; MSC, mesenchymal stem cell; Treg, regulatory T cell; MLN, mesenteric lymph node.

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 479

a 15 b *** ***

10 b Con si Con IL-1 si IL-1R1 si IL-1R1+IL-1 GAPDH 5 COX-2 Relative mRNA

expression of COX-2 0 b b Con IL-1 si Con si IL-1R1

si IL-1R1+IL-1 b c 200 *** *** 30 ** **

150 20 100 10 50 Relative mRNA Relative mRNA expression of IL-8 expression of IL-6 0 0 b b b b Con Con IL-1 IL-1 si Con si Con si IL-1R1 si IL-1R1

si IL-1R1+IL-1 si IL-1R1+IL-1

Figure 4 The effect of IL-1b signaling on the immunomodulation of MSCs. (a) After treatment with 10 ng/ml IL-1b for 24 and 48 h, the COX-2 expression of MSCs was evaluated by Q-PCR and western blot. (b, c) IL-1R1 siRNA-transfected MSCs were treated with or without IL-1b for 24 h, after which IL-8 and IL-6 expression was analyzed. COX-2, cyclooxygenase-2; IL-1b, interleukin-1b; IL-1R1, IL-1 receptor 1; MSC, mesenchymal stem cell; Q-PCR, quantitative PCR; siRNA, small RNA interference. before tail intravenous injection, but unprimed or IL-1R1 siRNA tran- showed that IL-1b-primed MSCs were recruited to the inflamed site siently transfected MSCs are activated by in vivo IL-1b following tail (spleen, MLN and colon) in a greater numbers than other MSCs. This vein injection, as IL-1b is present in UC lesions. finding indicated that IL-1b could facilitate the recruitment of MSCs IL-1b can drive the enhanced invasiveness of MSCs (derived from to the inflamed sites. Furthermore, IL-1b could also promote the ovarian endometrioma),29 and MSCs are preferentially recruited by migration of MSCs in vitro. The SDF-1/CXCR4 axis has been impli- lymphoid organs and the inflamed colon in the UC mice.2 Our results cated in the migration of MSCs to the impaired brain after hypoglossal

PBS IL-1b MSC abMSC IL-1b MSC MSC Luminescence Spleen 2500 Spleen Spleen

MLN 2000 Node MLN

1500

Colon

Colon 1000 Colon

Counts Color Scale Min =614 Max = 2594

Figure 5 In vivo bioluminescence imaging of fluc-Mscs delivery. (a) Representative bioluminescence images. IL-1b-primed MSC-injected mice showed stronger signal in spleens, MLNs and colons than unprimed MSCs. (b) Ex vivo imaging of CM-DIL dye-labeled MSCs in spleens, MLNs and colons. Arrows indicate MSCs in organs of MSC-injected mice. IL-1b, interleukin-1b; MLN, mesenteric lymph node; MSC, mesenchymal stem cell.

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 480

a Con si Con IL-1b 800 *** *** 600

400

200 si IL-1R1 si IL-1R1+IL-1b 0 b b Number of Migrated Cells Con si Con IL-1 si IL-1R1

si IL-1R1+IL-1

20 **** b c b 15

10 b Con si Con IL-1 si IL-1R1si IL-1R1+IL-1

Relative mRNA 5 GAPDH expression of CXCR4 CXCR4 0 b b Con IL-1 si Con si IL-1R1

si IL-1R1+IL-1

Figure 6 Activation of IL-1b signaling promotes the migration of MSCs. (a) Here, 10 ng/ml IL-1b was added into the lower chamber of the Transwell plates, and magnification photographs of representative stained filters were taken at 12 h; 34 darker areas are due to higher cell density. The average number of migrating cells per field was assessed by counting at least four high-power random fields per filter. (b, c) The gene and protein levels of CXCR4 in MSCs were checked after IL-1b treatment by Q-PCR and western blot, respectively. Results are mean6s.d. from three experiments each performed in duplicate. IL-1b, interleukin-1b; MSC, mesenchymal stem cell; Q-PCR, quantitative PCR.

nerve injury in a rat model, resulting in tissue regeneration,30 and over may facilitate MSCs survival, neutrophil recruitment and immuno- expression of CXCR4 improves MSC migration to infracted myocar- suppressive capacity. dium and ameliorates cardiac performance.31 Our results showed that Although most of the immunocytes detected above did not show IL-1b-primed MSCs significantly increased expression of CXCR4 at significant difference between IL-1b-primed MSC mice and those both the mRNA and protein levels in vitro. Altogether, these results treated with other MSCs, IL-1b induced elevated expression of suggest that IL-1b could enhance the ability of MSCs to target and COX-2, IL-8 and IL-6 in MSCs. This result may potentially be localize to inflammatory sites and elevate their migration ability attributed to the timing of immunocyte collection. We collected through increasing CXCR4 expression, all of which could render immunocytes when all of the surviving mice were recovered from MSCs more efficacious in cell-based repair therapy. colitis, a time when the three types of MSCs may have already MSCs have extensive immunomodulatory functions on activated carried their immunomodulatory function, so that the immune immunocytes. Our results showed that MSCs could both shape the profilecouldbethesameamongthethreetypesofMSC-treated host response to effectively polarize macrophage from M1 to M2 and mice. switch T cells from a Th1/Th17 immune profile towards a Th2/Treg In summary, in the present study, we showed that systemically immune profile. All of these results are consistent with previous infused IL-1b-primed MSCs could effectively attenuate DSS-induced reports. However, the ability of MSCs to modulate the activation of murine colitis. IL-1b-primed MSCs could mediate their immunosup- immunocytes is not entirely innate. For example, the immunosuppressive effect via two modes of action. First, IL-1b enhanced the pressive ability of MSCs is induced by inflammatory cytokines such as ability of MSCs to migrate to the inflamed spleen, MLN and colon IFN-c, TNF-a and IL-1b.32 We also found that IL-1b could signifi- of DSS-induced colitis mice by elevating CXCR4 expression, where cantly elevate COX-2, IL-8 and IL-6 expression in MSCs, which was MSCs could repair the injured intestinal mucosa and perform their abrogated following knockdown of IL-1R1. COX-2 is a key enzyme in immunosuppressive functions. Second, IL-1b promoted MSC secre- the synthesis of PGE2, and MSC-derived PGE2 is involved in skewing tion of IL-8, which is necessary for MSCs to recruit neutrophils and the inflammatory environment towards an anti-inflammatory profile, macrophages. IL-1b-pimed MSCs can then more efficiently suppress altering the cytokine secretion profile of T-cell subsets (Th1, Th2, or the excessive inflammatory and immune reactions by direct cell–cell Tregs).27,33 IL-8 is known as a specific neutrophil-attracting chemo- contact or by secretion of PGE2. kine, and activated MSCs can mediate neutrophil recruitment through IL-8.34 IL-6 could promote MSC secretion of PGE235 as well as maintain the proliferative and undifferentiated state of bone marrow- 36 1 Tsagias N, Koliakos I, Karagiannis V, Eleftheriadou M, Koliakos GG. Isolation of derived MSCs. Altogether, these data suggest that IL-1b signaling mesenchymal stem cells using the total length of umbilical cord for transplantation enhances the ability of MSCs to produce COX-2, IL-8 and IL-6 and purposes. Transfus Med 2011; 21: 253–261.

Cellular & Molecular Immunology Pre-treatment with IL-1b enhances the efficacy of MSC transplantation HY Fan et al 481

2 Liang L, Dong C, Chen X, Fang Z, Xu J, Liu M et al. Human umbilical cord mesenchymal 20 Mizel SB, Dayer JM, Krane SM, Mergenhagen SE. Stimulation of rheumatoid synovial stem cells ameliorate mice trinitrobenzene sulfonic acid (TNBS)-induced colitis. Cell cell collagenase and prostaglandin production by partially purified lymphocyte- Transplant 2011; 20: 1395–1408. activating factor (interleukin 1). Proc Natl Acad Sci U S A 1981; 78: 2474–2477. 3Gonza´lezMA,Gonzalez-ReyE,RicoL,Bu¨ scher D, Delgado M. Adipose-derived 21 Kabashima K, Saji T, Murata T, Nagamachi M, Matsuoka T, Segi E et al.The mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell and autoimmune responses. Gastroenterology 2009; 136: 978–989. activation in the gut. J Clin Invest 2002; 109: 883–893. 4Gonza´lez MA, Gonzalez-Rey E, Rico L, Bu¨scher D, Delgado M. Treatment of 22 Nitta M, Hirata I, Toshina K, Murano M, Maemura K, Hamamoto N et al. Expression of experimental arthritis by inducing immune tolerance with human adipose-derived the EP4 prostaglandin E2 receptor subtype with rat dextran sodium sulphate colitis: mesenchymal stem cells. Arthritis Rheum 2009; 60: 1006–1019. colitis suppression by a selective agonist, ONO-AE1-329. Scand J Immunol 2002; 5Ne´meth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K et al. Bone marrow 56: 66–75. stromal cells attenuate sepsis via prostaglandin E2-dependent reprogramming of host 23 Cominelli F, Nast CC, Llerena R, Dinarello CA, Zipser RD. Interleukin 1 suppresses macrophages to increase their interleukin-10 production. Nat Med 2009; 15: 42–49. inflammation in rabbit colitis. Mediation by endogenous prostaglandins. J Clin Invest 6 Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H et al.Nonhematopoietic 1990; 85: 582–586. mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after 24 Groh ME, Maitra B, Szekely E, Koc¸ ON. Human mesenchymal stem cells require myocardial infarction. Blood 2004; 104: 3581–3587. monocyte-mediated activation to suppress alloreactive T cells. Exp Hematol 2005; 7 Wojakowski W, Tendera M. Mobilization of bone marrow-derived progenitor cells in 33: 928–934. acute coronary syndromes. Folia Histochem Cytobiol 2005; 43: 229–232. 25 Liu CH, Hwang SM. Cytokine interactions in mesenchymal stem cells from cord blood. 8 Chamberlain G, Fox J, Ashton B, Middleton J. Mesenchymal stem cells: their Cytokine 2005; 32: 270–279. phenotype, differentiation capacity, immunological features and potential for 26 Lasiglie`D, Traggiai E, Federici S, Alessio M, Buoncompagni A, Accogli A et al. Role of homing. Stem Cells 2007; 25: 2739–2749. IL-1 beta in the development of human TH17 cells: lesson from NLPR3 mutated patients. PLoS One 2011; 6: e20014. 9 Dazzi F, Ramasamy R, Glennie S, Jones SP, Roberts I. The role of mesenchymal stem 27 Zhang Q, Shi S, Liu Y, Uyanne J, Shi Y, Shi S et al. Mesenchymal stem cells derived cells in haemopoiesis. Blood Rev 2006; 20: 161–171. from human gingiva are capable of immunomodulatory functions and ameliorate 10 Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic Z et al. MCP-3 is a inflammation-related tissue destruction in experimental colitis. J Immunol 2009; myocardial mesenchymal stem cell homing factor. Stem Cells 2007; 25: 245–251. 183: 7787–7798. 11 Ryan JM, Barry F, Murphy JM, Mahon BP. Interferon-gamma does not break, but 28 Gonzalez-Rey E, Anderson P, Gonza´lez MA, Rico L, Bu¨scher D, Delgado M. Human promotes the immunosuppressive capacity of adult human mesenchymal stem adult stem cells derived from adipose tissue protect against experimental colitis and cells. Clin Exp Immunol 2007; 149: 353–363. sepsis. Gut 2009; 58: 929–939. 12 Kwon KH, Murakami A, Hayashi R, Ohigashi H. Interleukin-1b targets interleukin-6 in 29 Kao AP, Wang KH, Long CY, Chai CY, Tsai CF, Hsieh TH et al. Interleukin-1b induces progressing dextran sulfate sodium-induced experimental colitis. Biochem Biophys cyclooxygenase-2 expression and promotes the invasive ability of human mesenchymal Res Commun 2005; 337(2): 647–654. stem cells derived from ovarian endometrioma. Fertil Steril 2011; 96: 678–684. 13 Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method 30 Ji JF, He BP, Dheen ST, Tay SS. Interactions of chemokines and chemokine receptors in the induction of reliable experimental acute and chronic ulcerative colitis in mice. mediate the migration of mesenchymal stem cells to the impaired site in the brain after Gastroenterology 1990; 98: 694–702. hypoglossal nerve injury. Stem Cells 2004; 22: 415–427. 14 Arai Y, Takanashi H, Kitagawa H, Okayasu I. Involvement of interleuking-1 in the 31 Cheng Z, Ou L, Zhou X, Li F, Jia X, Zhang Y et al. Targeted migration of mesenchymal development of ulceratie colitis inducted by dextran sulfate sodiium in mice. stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac Cytokine 1998; 10: 890–896. performance. Mol Ther 2008; 16: 571–579. 15 Bauer C, Duewell P, Mayer C, Lehr HA, Fitzgerald KA, Dauer M et al. Colitis induced in 32 Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI et al. Mesenchymal stem cell- mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome. mediated immunosuppression occurs via concerted action of chemokines and nitric Gut 2010; 59: 1192–1199. oxide. Cell Stem Cell 2008; 2: 141–150. 16 Lebeis SL, Powell KR, Merlin D, Sherman MA, Kalman D. Interleukin-1 receptor 33 Najar M, Raicevic G, Boufker HI, Fayyad Kazan H, de Bruyn C, Meuleman N et al. signaling protects mice from lethal intestinal damage caused by the attaching and Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of effacing pathogen Citrobacter rodentium. Infect Immun 2009; 77: 604–614. lymphocyte subsets: Combined comparison of adipose tissue, Wharton’s Jelly and 17 Kojouharoff G, Hans W, Obermeier F, Ma¨nnel DN, Andus T, Scho¨lmerich J et al. bone marrow sources. Cell Immunol 2010; 264: 171–179. Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation 34 Romieu-Mourez R, Franc¸ois M, Boivin MN, Bouchentouf M, Spaner DE, Galipeau J. in chronic dextran sulphate sodium-induced colitis in mice. Clin Exp Immunol 1997; Cytokine modulation of TLR expression and activation in mesenchymal stromal cells 107: 353–358. leads to a proinflammatory phenotype. J Immunol 2009; 182: 7963–7973. 18 Gonza´lez-Navajas JM, Law J, Nguyen KP, Bhargava M, Corr MP, Varki N et al. Interleukin 35 Bouffi C, Bony C, Courties G, Jorgensen C, Noe¨l D. IL-6-dependent PGE2 secretion by 1 receptor signaling regulates DUBA expression and facilitates Toll-like receptor 9-driven mesenchymal stem cells inhibits local inflammation in experimental arthritis. Plos antiinflammatory cytokine production. JExpMed2010; 207: 2799–2807. One 2010; 5: e14247. 19 Martin M, Neumann D, Hoff T, Resch K, DeWitt DL, Goppelt-Struebe M. Interleukin-1- 36 Pricola KL, Kuhn NZ, Haleem-Smith H, Song Y, Tuan RS. Interleukin-6 maintains induced cyclooxygenase 2 expression is suppressed by cyclosporin A in rat mesangial bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent cells. Kidney Int 1994; 45: 150–158. mechanism. J Cell Biochem 2009; 108: 577–588.

Cellular & Molecular Immunology