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Treatment of experimental colitis by endometrial regenerative cells through regulation of B lymphocytes… Xu, Xiaoxi; Wang, Yong; Zhang, Baoren; Lan, Xu; Lu, Shanzheng; Sun, Peng; Li, Xiang; Shi, Ganggang; Zhao, Yiming; Han, Hongqiu; Du, Caigan; Wang, Hao May 22, 2018

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RESEARCH Open AccessTreatment of experimental colitis byendometrial regenerative cells throughregulation of B lymphocytes in miceXiaoxi Xu1,2,3†, Yong Wang4†, Baoren Zhang1,2†, Xu Lan1,2, Shanzheng Lu5, Peng Sun6, Xiang Li1,2, Ganggang Shi7,Yiming Zhao1,2, Hongqiu Han1, Caigan Du8,9 and Hao Wang1,2*AbstractBackground: Endometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cell derived frommenstrual blood, have been recently evaluated as an attractive candidate source in ulcerative colitis (UC); however,the mechanism is not fully understood. The present study was designed to investigate the effects of ERCs,especially on B-cell responses in UC.Methods: In this study, colitis was induced by administering 3% dextran sodium sulfate (DSS) via free drinkingwater for 7 days to BALB/c mice. In the treated group, mice were injected intravenously with 1 × 106 ERCs on days2, 5, and 8 after DSS induction. Therapeutic effects were assessed by monitoring body weight, disease activity,and pathological changes. Subpopulations of lymphocytes were determined by flow cytometry. IgG deposition inthe colon was examined by immunohistochemistry staining. Cytokine levels were measured by enzyme-linkedimmunosorbent assay (ELISA), Western blot, or polymerase chain reaction (PCR) analysis. Adoptive transfer ofregulatory B cells (Bregs) into colitis mice was performed.Results: Here, we demonstrated that ERC treatment prolonged the survival of colitis mice and attenuated diseaseactivity with fewer pathological changes in colon tissue. ERCs decreased the proportion of immature plasma cells inthe spleen and IgG deposition in the colon. On the other hand, ERCs increased the production of Bregs and theinterleukin (IL)-10 level. Additionally, adoptive transferred Bregs exhibited significant therapeutic effects on colitis mice.Conclusions: In conclusion, our results unravel the therapeutic role of ERCs on experimental colitis through regulatingthe B-lymphocyte responses.Keywords: Ulcerative colitis, Endometrial regenerative cells, B lymphocytes, Immunoregulation, MiceBackgroundUlcerative colitis (UC) is a chronic, relapsing, and non-specific inflammatory disorder of the colon, as shown byan imbalance of the immune system to various factors[1]. It is generally believed that a modified T helper (Th)2-mediated immune response is involved in the patho-genesis of UC, resulting in excessive production of pro-inflammatory cytokines and the destruction of colonictissue. However, B cells also play a pathogenic role inUC by producing autoantibodies leading to damage ofthe intestinal epithelium. The topical deficiency of sIgAand a shift from IgA2 to the less stable IgA1 was re-ported, as well as a significant increase in IgG-producingcells [2]. Systemically, perinuclear anti-neutrophil anti-bodies (p-ANCA) and autoantibodies against tropomyosin-5 are found in the peripheral blood of UC patients, andthe numbers of immature plasma cells overexpressingchemokines was increased [3]. Unfortunately, salvage ther-apy with rituximab (anti-CD20 antibody) caused a severeexacerbation of UC [4]. The failure of B cell-depletiontherapy was also supported by animal models [5]. Re-cently, the regulatory role of B cells has been demon-strated in many immune disorders including UC, with a* Correspondence: hwangca@outlook.com†Equal contributors1Department of General Surgery, Tianjin Medical University General Hospital,154 Anshan Road, Heping District, Tianjin 300052, China2Tianjin General Surgery Institute, Tianjin, ChinaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Xu et al. Stem Cell Research & Therapy  (2018) 9:146 https://doi.org/10.1186/s13287-018-0874-5decrease in regulatory B cells (Bregs) [6]. In the TCRα−/−spontaneous colitis model, B cells protected the colonfrom severe inflammation by generating CD1d upregulatedBregs capable of producing interleukin (IL)-10 [7].Transferred splenic B cells from parasite infection mice orenteroantigen-pulsed B cells suppress colitis in an IL-10-dependent manner [8, 9]. Furthermore, B cells maintaingut homeostasis by cooperation with regulatory T cells(Tregs) [10] or macrophages [11]. However, the effectivestrategy that targets the B cell system for maintenance incolitis is still not clear.Mesenchymal stem cells (MSCs) have been consideredas an emerging therapeutic method to treat UC by differ-entiating into colonic interstitial cells [12] and producingpleiotropic gut trophic factors [13], as well as exerting im-munomodulatory effects [14, 15] through inhibiting the re-sponses of macrophages [15] and T cells [16]. According toprevious studies, MSCs directly modulate B cell function,such as suppressing terminal differentiation, inhibiting pro-liferation and antibody production [17–20]. Meanwhile, re-cent studies have found that MSCs induce the expansionof IL-10-producing Bregs [21]. However, because of theinvasive procedures for obtaining MSCs, and their fewernumber and limited proliferation capacity due to aging orchronic diseases [22], the application of MSCs fromcurrent sources (i.e., the bone marrow, adipose tissue, andumbilical cord) as a cell therapy are restricted.Endometrial regenerative cells (ERCs) derived from men-strual blood, a novel source of adult stem cells, resembleMSCs with similar phenotypic surface markers, multi-differentiation potential, and immunomodulatory proper-ties [23, 24]. Peculiarly, ERCs possess the uniqueadvantages of a noninvasive procedure for harvesting, theirease of abstraction, and their abundant source. ERCs canbe rapidly expanded large scale in vitro with a doublingtime of about 20 h while maintaining karyotypic normalityup to 68 doublings [25]. Meanwhile, ERCs produce largeamounts of growth factors and matrix metalloprotease infavor of tissue repair [23, 25]. We and others have reportedthe therapeutic effects of ERCs in many disease models,such as critical limb ischemia [23], acute liver injury [26],premature ovarian failure [27], and experimental colitis[28]. Recently, we have demonstrated the immunosuppres-sive activities against B cell functions in a cardiac trans-plantation mouse model [29]. However, whether ERCs arecapable of regulating the B cell system to protect mice fromcolitis is unclear. Thus, the present study was undertakento clarify the immunomodulatory effects of ERCs on B cellsin a dextran sodium sulfate (DSS)-induced colitis model.MethodsAnimalsSix- to eight-week-old male BALB/c mice weighing18–22 g (Aoyide Co., Tianjin, China) were used in thepresent study (24 mice were randomly assigned to thethree groups, n = 8 per group). All experiments were con-ducted in accordance with the protocols approved by theAnimal Care and Use Committee of Tianjin MedicalUniversity (Tianjin, China) according to the ChineseCouncil on Animal Care guidelines.Preparation of ERCsERCs were collected and isolated according to a protocoldescribed previously [30, 31]. Briefly, menstrual bloodwas collected from 20- to 40-year-old healthy women onthe first day of the menstruation cycle using Diva cupsafter informed consent was obtained. Menstrual blood(5 ml) was transferred into a 15-ml centrifuging tubecontaining 0.2 ml penicillin/streptomycin and 0.2 mlfetal bovine serum (FBS) in 5 ml phosphate-bufferedsaline (PBS). Mononuclear cells were fractionated byFicoll-Paque density gradient centrifugation. The inter-layer cells were suspended in Dulbecco’s modified Eagle’smedium (DMEM)-F12 supplemented with 10% FBS and1% penicillin/streptomycin and cultured in a T-25 flank.The culture medium was changed the next day. Theadherent cells were then subcultured and passaged twicea week. The morphology of the ERCs was examined bymicroscopy and the expression of the cell surfacemarkers CD34, CD45, CD90, and CD105 were analyzedby flow cytometry as previously described [25, 28].Third- and fourth-passage cells were used for treatment.Splenic B cell isolationAt necropsy, BALB/c mice spleens were collected and madeinto a single cell suspension. After magnetic labeling withbiotin-conjugated CD19 antibody and anti-biotin microbe-ads (Miltenyi Biotech, Germany), B cells were isolated bypositive selection according to the manufacturer’s protocol.Coculture with ERCsPurified CD19+ B cells were cultured in RPMI-1640medium supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 1 × 106 cells/ml with orwithout ERCs. Lipopolysaccharide (LPS) was added tothe medium at a concentration of 10 μg/ml as astimulation and, 72 h later, culture supernatant and Bcells were collected for further analysis.To study the effects of ERCs on B-cell proliferation, somepurified CD19+ cells were stained with carboxyfluoresceinsuccinimidyl ester (CFSE; Invitrogen, Inc., Carlsbad, CA,USA) in PBS/bovine serum albumin (BSA) 0.01% at 37 °C.After 10 min, the reaction was quenched with FBS mediumand B cells were collected.Colitis modelColitis was induced in BALB/c mice by administrationof 3% (wt/vol) DSS (MP Biochemicals) in free drinkingXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 2 of 12water for 7 days. In the ERC-treated group, ERCs(1 × 106 cells/mouse) were suspended in 200 μl PBS andinjected intravenously at days 2, 5, and 8 of DSSadministration. In the untreated group, mice were injectedwith an equal amount of PBS as controls. The body weightwas monitored and clinical signs were recorded daily. TheDisease Activity Index (DAI) was scored according to thefollowing criteria: a) body weight loss = 0 (no change), 1(1–5%); 2 (5–10%), 3 (10–20%), and 4 (> 20%); b) stoolconsistency or diarrhea = 0 (normal), 1 (some soft), 2(soft), 3 (unformed/mild diarrhea), and 4 (severe waterydiarrhea); c) hemoccult positivity and the presence ofgross stool blood = 0 (negative fecal occult blood), 1(negative/positive fecal occult blood), 2 (certain positivefecal occult blood), 3 (visible rectal bleeding), and 4(severe rectal bleeding). The DAI is the sum of the scoresfor body weight loss, stool consistency, and grossbleeding, divided by 3. At day 10, mice were sacrificedafter euthanization and 5 ml cold PBS was injected intothe peritoneal cavity and collected by 3 ml Pasteur tubeafter the abdomen had been massaged for 3 min. Thespleen, mesenteric lymph nodes (MLN), and colon weredissected carefully, and the colon length was measured.Flow cytometry analysis of T and B cellsThe spleen and MLN were minced gently on ice and fil-tered through a 40-μm filter. Cells were suspended instaining buffer at a final concentration of 1 × 107/ml andwashed twice before blocking with anti-CD16/CD32Fc-Block (eBioscience, San Diego, CA, USA). Cellswere stained with primary monoclonal antibodies at4 °C in the dark for 30 min. Fluorescein isothiocyan-ate (FITC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-CD4, and peridinin chlorophyll pro-tein complex (PerCP)-conjugated anti-CD8 were usedto detect T cells, and FITC-conjugated anti-CD19,PerCP-Cy5.5-conjugated anti-CD5, allophycocyanin(APC)-conjugated anti-CD1d, PE-conjugated anti-CD83,and PE-conjugated anti-CD138 were used to detect B cells(eBioscience, San Diego, CA, USA) following the manu-facturer’s instructions. FITC-conjugated anti-CD4, PE-conjugated anti-CD25, and PE-Cy5-conjugated anti-Foxp3were used for the detection of Tregs with a Mouse Regula-tory T Cell Staining Kit after fixation and permeabilization.To detect the proportion of IL-10+ B cells, intracellularstaining was performed. In brief, splenocytes were culturedin complete RPMI-1640 medium and stimulated with CellStimulation Cocktail (plus protein transport inhibitors)(eBioscience, San Diego, CA, USA) at a concentration of2 μl/ml for 5 h. Cells were blocked with anti-CD16/CD32Fc-Block (eBioscience, San Diego, CA, USA) on ice for 15min and stained with FITC-conjugated anti-CD19, PerCP-Cy5.5-conjugated anti-CD5, and APC-conjugated anti-CD1d for 20 min. After fixation and permeabilization(Cytofix/Cytoperm kit, BD), intracellular staining was per-formed on ice with PE-conjugated anti-IL-10 monoclonalantibody for 30 min.Enzyme-linked immunosorbent assay (ELISA)The levels of IL-10 in cell culture supernatants and IL-10,tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IgG incolon tissue homogenous were determined using anELISA kit (eBioscience, San Diego, CA, USA) accordingto the manufacturer’s recommended protocol.Histology and immunohistochemistryColon tissues were fixed in 10% formaldehyde, embed-ded in paraffin, and sectioned into 5-μm section.Sections were prepared for hematoxylin and eosin(H&E) and immunohistochemistry staining as previouslydescribed. IgG deposition in the colon was quantified byimmunohistochemical staining with a primary antibodyspecific for mouse IgG (Abcam). Negative controls wereperformed using PBS instead of the primary antibody.Sections were examined in a double-blinded fashion bytwo pathologists.Reverse transcription polymerase chain reaction (RT-PCR)and Western blot analysis of IL-10To determine the splenic IL-10 levels in the perspective ofgene transcription, total RNA was extracted from thespleen followed by generation of cDNA and detection withflorescent real-time quantitative RT-PCR. The PCR primerwas designed as follows: IL-10, upstream 5’-AGAAGCATGGCCCAGAAATCA-3′, downstream 5’-GGCCTTGTAGACACCTTGGT-3′. To examine the protein ex-pression of IL-10 in the spleen, spleens were homogenizedin lysis buffer on ice. After centrifugation at 13,000 rpm for15 min, supernatants were collected to perform Westernblotting. A primary antibody specific for mouse IL-10or β-actin (Santa Cruz Biotechnology) was used inthe experiments.Adoptive transferPurified CD19+ B cells from colitis mice treated with orwithout ERCs were stained with CD1d and CD5monoclonal antibody. CD1dhiCD5+ B cells were selectedusing a flow cytometer (FACSAria, BD) and injectedinto mice (2 × 106 cells in 250 μl PBS) followed byadministration of DSS.Statistical analysisSurvival data are presented as mean survival time (MST)and were analyzed using the log-rank test. One-wayanalysis of variance (ANOVA) and two-tailed, pairedt tests were used to analyze differences between experi-mental groups. Differences with p values ≤ 0.05 wereconsidered significant.Xu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 3 of 12ResultsCharacterization of ERCsERCs exhibited spindle-shaped, fibroblast-like morph-ology after passage 3 (Fig. 1A) and colony-forming abil-ity. The doubling time was about 24 h, indicating a highproliferative rate. At passage 4, ERCs were detached andstained with the MSC surface markers CD34, CD45,CD90, and CD105. As reported previously, ERCs dem-onstrated high expression of CD90 and CD105, whilelacking CD34 and CD45 expression (Fig. 1B).ERCs attenuated DSS-induced experimental colitisAcute experimental colitis was induced by oral adminis-tration of 3% DSS in free drinking water, resulting in se-vere colitis characterized by body weight loss, bloodydiarrhea, and lethargy (Fig. 2a–c). ERC treatmentdelayed the occurrence of colitis and attenuated itsseverity, exhibited less body weight loss, and reducedmortality significantly. The general condition, stoolconsistency, and bloody stool were also improved byERC treatment (Fig. 2a–c). Consistently, DSS adminis-tration lead to the shortening and rigidity of the colonwith severe injurious hyperemia and ulceration, whichwere ameliorated by ERCs (Fig. 2d). Under the micro-scope, ERCs decreased the pathological changes causedby DSS, including damaged epithelium and crypt struc-ture, glandular disorders, and massive inflammatory cellinfiltration into the mucosa and submucosa (Fig. 2e).Meanwhile, the concentration of TNF-α, IL-1β, and IL-6were analyzed by ELISA. ERC treatment significantlyreduced the elevated level of these proinflammatorycytokines caused by DSS administration (Fig. 2f ). Theseresults demonstrated that the benefits of ERCs on colitiswere probably mediated by anti-inflammatory effects.ERCs inhibited Th cells while inducing Tregs duringDSS-induced colitisTo explore the immunomodulatory effects of ERCs, flowcytometry was performed to analyze the subpopulationof lymphocytes in the spleen. The proportion of CD3+ Tcells in lymphocytes as well as CD3+CD4+ andCD3+CD8+ T cells was increased in the DSS-treatedgroup, and was inhibited by ERCs (Fig. 3a, b). Import-antly, ERCs downregulated the expanded Th1 and Th17Fig. 1 Characterization of ERCs. A The morphology of ERCs. a P4 passage of ERCs 2 days after subculturing. b P4 passage of ERCs 4 days aftersubculturing. B FACS analysis of ERCs using hematopoietic and immunophenotypic markers. Surface expression of CD34, CD45, CD90, and CD105was detected by flow cytometry. Data shown represent three separate experiments, with similar effects observed in eachXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 4 of 12cells in colitis, which are shown by production of theproinflammatory mediators interferon (IFN)-γ andIL-17, respectively, to cause tissue injury (Fig. 3c). On theother hand, ERC treatment elevated the proportion ofTregs in lymphocytes compared with DSS-treated mice,and this was even higher than those from normal mice,which play an important role in the maintenance ofhomeostasis in colitis (Fig. 3d) [14].ERCs inhibited B-cell activation, differentiation, and IgGproduction in colitisAlthough DSS-induced colitis is commonly believed toresult from the imbalance of T cells, dysregulation of theB cell system is also involved in the pathogenesis [2]. Inthe present study, CD83 expression was obviouslyincreased in splenic B cells in DSS-treated mice, indicatingthe relative active states, which was downregulated byFig. 2 The therapeutic effects of endometrial regenerative cell (ERC) treatment on dextran sodium sulfate (DSS)-induced colitis. BALB/c mice inthe ERC-treated group were injected i.v. with ERCs (1 × 106) in 200 μl PBS at days 2, 5, and 8 after DSS induction. Mice in the untreated groupwere injected i.v. with 200 μl PBS instead. a ERCs prolong the survival of DSS-induced colitis mice. Survival rates were monitored daily. P value wasdetermined by log-rank survival test. b, c Body weight, general condition, stool condition, and the appearance of bloody stool were monitored daily.ERCs b attenuated the body weight loss and c alleviated the clinical severity of DSS-induced colitis mice. P value was determined by one-way ANOVA.d, e Mice were sacrificed at day 10 after DSS induction. Colons were dissected and the distal part was paraffin sectioned and H&E staining wasperformed. d Representative photo showing the colon dissected from mice and e the histological sections in each group. f ERCs modulated thebalance of proinflammatory cytokines in the colon. Colon samples were homogenized and the supernatants were harvested. The concentration oftumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 was measured by ELISA. Graphs represent mean ± SEM of triplicate experiments. P value wasdetermined by one-way ANOVA. *P < 0.05Xu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 5 of 12ERC treatment (Fig. 4a). Next, as B cells mainly take partin humoral immunity by differentiating into antibody-producing plasma cells, the proportion of CD138+ cellswere analyzed. Accordingly, colitis mice exhibited moreCD138+ immature plasma cells in the spleen, which werereduced by ERC treatment (Fig. 4b). Furthermore, toexclude that these changes were merely caused by thealtered ratio of T cells and B cells after DSS induction, theproportion of CD138+ cells in CD19+ cells was evaluatedand showed a similar trend (Fig. 4b). Meanwhile,immunohistochemistry staining suggested that the obviousdeposition of IgG in the inflamed colon was also attenuatedby ERCs (Fig. 4c), which was confirmed by ELISA assayshowing a lower level of IgG in the homogenous colonafter ERC treatment (Fig. 4d).Treatment with ERCs upregulated the level of IL-10 andpromoted the expansion of BregsIL-10 is a potent anti-inflammatory cytokine, and theresults of IL-10 signaling are present in inflammatory boweldisease (IBD) patients and animal models [32]. In thepresent study, ERC treatment enhanced the concentrationFig. 3 The regulatory effects of endometrial regenerative cell (ERC) treatment on T lymphocytes. The spleen was dissected and made into a single-cellsuspension. Cells were stained with fluorescently labeled CD3, CD4, CD8, CD25, IFN-γ, IL-17, and Foxp3, and detected by flow cytometry. The proportionof a CD3+ T cells in lymphocytes, b CD4+ and CD8+ T cells in CD3+ T cells, c IFN-γ+ and IL-17+ T cells in lymphocytes, and d CD25+Foxp3+ Tregs inCD3+CD4+ T cells was detected by flow cytometry. Graphs represent mean ± SEM of triplicate experiments. P value was determined by one-way ANOVA.*P < 0.05. DSS, dextran sodium sulfate; Th, T helperXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 6 of 12of IL-10 in the colon and spleen (Fig. 5a–c). Based on theresults from flow cytometry, we observed that largenumbers of IL-10-producing cells were derived from theB-cell pool (Fig. 5d). Thus, we focused on the immunoregu-lation of ERCs on the balance of the B-cell system in colitis,especially IL-10-competent B cells, and found thatERC-treated mice showed increased IL-10+ B cells inthe spleen (Fig. 5e).ERCs modulated the CD1dhiCD5+ Bregs in the spleen,MLN, and peritoneal cavityAs CD1dhiCD5+ B cells are the most well-characterizedphenotype of Bregs in mice, we further explored theeffects of ERCs on the proportion of CD1dhiCD5+ B cellsin different tissues. As shown in Fig. 5f, the proportion ofCD1dhiCD5+ B cells in the spleen, peritoneal cavity, andMLN was increased by ERC treatment. These resultsFig. 4 Endometrial regenerative cells (ERCs) inhibited B-cell activation, differentiation, and antibody deposition in colitis mice. The spleen wasdissected and made into a single-cell suspension. Cells were stained with fluorescently labeled CD19, CD83, and CD138. a The expression ofCD83 on CD19+ B cells and the proportion of b CD19+CD138+ cells in lymphocytes and CD138+ cells in CD19+ B cells was detected by flowcytometry. c IgG deposition in the colon. Colons were dissected and the distal part was paraffin sectioned with immunohistochemistry stainingperformed thereafter. Representative photographs of histological sections of colon from normal, untreated, and ERC-treated groups. Antibodydeposition was observed by immunohistochemistry specific for IgG (×400). d The concentration of IgG in the colon was measured by ELISA. Graphsrepresent mean ± SEM of triplicate separate experiments. P value was determined by one-way ANOVA. *P < 0.05. DSS, dextran sodium sulfateXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 7 of 12indicated that ERC probably induced the expansion ofBregs to systemically exert protective effects.ERCs promoted the production of Bregs in vitroTo investigate the direct effects of ERCs on B cells, puri-fied CD19+ cells were isolated and cocultured withERCs. As shown in Fig. 6a, in the presence of LPS asstimulation, ERCs inhibited B-cell proliferation in adose-dependent manner without inducing apoptosis aswe have previously reported (data not shown). On theother hand, ERCs further enhanced the production ofCD1dhiCD5+ B cells (Fig. 6b) and IL-10+ B cells (Fig. 6c), with a higher concentration of IL-10 in the culturemedia (Fig. 6d).ERC-induced Bregs ameliorated experimental colitis by anadoptive transfer methodAs shown in Fig. 6e, the IL-10 competence ofCD1dhiCD5+ B cells from ERC-treated mice was verifiedby intracellular staining compared with those of theFig. 5 Endometrial regenerative cells (ERCs) upregulated the level of interleukin (IL)-10 and promoted the expansion of Bregs. a Colon sampleswere homogenized and the supernatants were harvested. The concentration of IL-10 in the homogenous colon was measured by ELISA. Theb protein level and c mRNA level of IL-10 in the spleen were examined by Western blot and PCR analysis, respectively. d The proportion of CD19+cells in IL-10+ cells and e IL-10+CD19+ cells in lymphocytes was detected by flow cytometry. f The proportion of CD1dhiCD5+ Bregs in the spleen,peritoneal cavity, and mesenteric lymph nodes (MLN) was detected by flow cytometry. Graphs represent mean ± SEM of triplicate experiments. P valuewas determined by one-way ANOVA. *P < 0.05. DSS, dextran sodium sulfateXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 8 of 12CD1dlowCD5− phenotype. Next, to confirm thetherapeutic benefits of ERC treatment on colitis, splenicCD1dhiCD5+ B cells from ERC-treated colitis mice wereisolated by flow cytometer and adoptively transferredinto mice before DSS administration. We found thatERC-induced Bregs exhibited obvious therapeutic effectson colitis and attenuated the body weight loss of colitismice (Fig. 6f ).Fig. 6 The effects of endometrial regenerative cells (ERCs) on regulatory B cells (Bregs) in vitro and the adoptive transfer of ERC-induced Bregsinto colitis mice. Purified recipient B cells (106/well) were stimulated with 10 μg/ml lipopolysaccharide (LPS) for 72 h. a CFSE-labeled B cells wereharvested thereafter and analyzed by flow cytometry. The proliferation was expressed as a percentage of proliferative B cells versus untreatedLPS-stimulated B cells. b The proportion of CD1dhiCD5+ and c IL-10+ cells in CD19+ B cells was measured by flow cytometry. d Supernatants wereharvested and IL-10 production was measured by ELISA. e The expression of IL-10 in CD1dhiCD5+ and CD1dlowCD5− B cells from ERC-treated colitis mice.f Adoptive transfer of Bregs attenuated body weight loss and disease activity in colitis mice. Graphs represent mean ± SEM of triplicate experiments.P value was determined by one-way ANOVA. *P< 0.05. DSS, dextran sodium sulfate; PBS, phosphate-buffered salineXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 9 of 12DiscussionAs UC is mainly mediated by an atypical Th2 immunereaction, the imbalance of the B-cell system alsocontributes to the occurrence and aggravation of UC.Autoantibody production and deposition resulted in thedestruction of intestinal epithelium and tissue injury [2].On the other hand, IL-10-producing Bregs, which arebelieved to serve as an immune modulator, wereimpaired in UC patients [6]. MSCs modulated B-cellfunction both in vivo and in vitro; however, severalshortcomings limit the application of MSCs [17, 33].ERCs are a novel type of adult MSC derived frommenstrual blood; they share similar surface markers andimmunomodulatory properties to MSCs, but possess thespecific advantages of a noninvasive procedure ofharvesting, ease of extraction, and an abundant source[23, 24]. In this study, we investigated the therapeuticeffects of regulating B cells in DSS-induced colitis. ERCsinhibited B-cell activation, differentiation, and antibodydeposition in the colon, while they induced Breg expansionand promoted IL-10 generation. Additionally, adoptivetransferred Bregs from ERC-treated mice alleviated colitis,indicating the therapeutic role of ERCs on experimentalcolitis through regulating the B-lymphocyte responses.In recent years, MSCs have been considered as anattractive tool in the treatment of UC. In addition totheir multidifferentiation potential [12] and trophicfactor secretion [13], MSCs exert immunomodulatoryeffects on macrophages, dendritic cells, and T cells toattenuate colitis [14–16]. Of note, the immunomodulatoryeffect is not MHC-restricted, since colitis mice can receivea benefit from xenogeneic MSCs as well as syngeneic andallogeneic MSCs [16]. Human MSCs from differentsources [14, 34] are well tolerated by mice and have effectson other diseases [35], underling the possibility of theapplication of xenogeneic ERCs.As reported previously, several types of autoantibodieshave been detected in the serum of UC patients, such asxANCAs and hTM5-specific IgG [36], and local infiltra-tion of IgG-producing plasma cells in the inflamedmucosa increases [2]. We have demonstrated ERC-mediated inhibitory effects on B-cell proliferation andantibody production in a cardiac transplantation model[37]. In this study, ERCs inhibited the overactivated Bcells of colitis mice. Meanwhile, since the activationsurface marker CD83 is the costimulatory molecule,ERCs may suppress the antigen-presenting function of Bcells and subsequent T-cell response indirectly.ERC treatment downregulated local IgG depositionand the proportion of splenic immature plasma cells,suggested that ERCs may protect the colon from thehumoral response directly by means of inhibiting denovo production of autoantibody or B-cell terminaldifferentiation and chemotaxis to the colon in a similarmanner to MSCs. This is supported by previous findingsthat human bone marrow-derived MSCs downregulatedthe expression of chemokines on B cells, such as CXCR4and CXCR5, which are important for B-cell migrationand positioning in secondary lymphoid organs such asthe spleen [20].However, B-cell depletion therapy with anti-CD20monoclonal antibody results in the exacerbation ofcolitis [5]. In contrast, ERCs could functionally inhibit Bcells without inducing apoptosis [37], but furtherpromote the expansion of IL-10-producing Bregs bothin vitro and in vivo. Bregs are capable of skewing thedifferentiation of Tregs and maintaining a Treg pool [38],the role of which has been extensively investigated inmany immune-related diseases such as systemic lupuserythematosus (SLE), experimental autoimmune enceph-alomyelitis (EAE), and arthritis [39]. The suppressiveeffects of B cells in colitis have been demonstrated byTCR-α−/− × Igμ−/− mice lacking B cells spontaneouslydeveloping more severe colitis than TCR-α−/− mice [40]and adoptive transfer of IL-10-producing CD1d+CD5+Bregs inhibiting the DSS-induced intestinal injury in amouse model [41].IL-10 competence is the key marker of Bregs. IL-10-deficient mice are more susceptible to chronic colitis[42] while IL-10-transgenic spleen cells prevent colitisinduction [43]. IL-10 maintains the expression of Foxp3on Tregs [44], an important source of IL-10 in colitismice [45]. In this study, we found the upregulation ofIL-10 and Tregs after ERC treatment in colitis mice.Thus, it is tempting to think that ERC may induce theproduction of IL-10-producing B cells, which results inand from the expansion of Tregs to form a complex loopto regulate the gut homeostasis and suppress colitis, asstated previously [10].Recently, the induction of Bregs by MSCs has beenreported. Qin et al. found that MSCs derived frommouse bone marrow induce Bregs via the stromal cell-derived factor (SDF)-1α–CXCR7 axis [21]. Interestingly,as we reported recently, SDF-1α also mediates theinduction of regulatory immune cells by ERCs, includingCD1d+CD5+ Bregs [29]. In the present study, we foundthat ERCs increased the fraction of CD1d+CD5+ Bregsin the spleen and MLN, as well as in the peritonealcavity. ERCs may migrate to these lymphoid organs toinduce the expansion of Bregs directly, or may promotethe circulation of Bregs among gut-associated lymphoidtissues. To date, it has been suggested that stem cellsmight contribute as tropic suppliers by stimulating tissuerepair through the secretion of paracrine factors.According to Legaki et al., conditioned medium derivedfrom the spindle-shaped amniotic fluid MSCs is able toameliorate DSS-induced colitis [46], underlining thepossibility that ERCs may modulate the immune systemXu et al. Stem Cell Research & Therapy  (2018) 9:146 Page 10 of 12in colitis mice in a similar manner. More in-depthstudies are warranted to clarify how ERCs are involved inthis procedure.To examine the direct effect of Bregs in ERC-basedtherapy, we isolated CD1d+CD5+ Bregs from the spleenof ERC-treated colitis mice to perform adoptive transfer,which is an important IL-10 source. Interestingly, it islikely that ERC-induced Bregs exhibited obvious thera-peutic effects on colitis (being even more effective thanthose from colitis mice without ERC treatment), indicat-ing that the upregulation of Bregs was not a simpleresponse to inflammation or the result of Treg reactions,but was also part of the immunomodulatory effects ofERC treatment.ConclusionsIn summary, this study for the first time demonstrates thatERCs regulate the balance of the B-cell response in experi-mental colitis mice. ERCs inhibited splenic B-cell differen-tiation and reduced the deposition of IgG antibodies inthe intestinal tissue of colitis mice. On the other hand,ERCs increased the production of IL-10 and the propor-tion of CD1d+CD5+ Bregs from colitis mice, an importantsource of IL-10. ERC-pulsed Bregs effectively alleviatedthe symptoms of colitis and promoted intestinal recovery,indicating that ERCs may exert protective effects throughBregs. Thus, it is possible that ERC-mediated B-cellregulation, at least in part, contributes to protection of thecolon from severe inflammation in this preclinical model.Again, ERCs with their unique features of ease of collec-tion, a relatively unlimited source, the immunomodulatoryeffect, and hypoimmunogenicity, as well as the lack oftumorigenesis or tumor acceleration, could make them anattractive novel source of stem cells for cytotherapy forthe prevention and/or treatment of ulcerative colitis.AbbreviationsBreg: Regulatory B cell; DAI: Disease activity index; DSS: Dextran sodiumsulfate; ERC: Endometrial regenerative cell; IBD: Inflammatory bowel disease;LPS: Lipopolysaccharides; MSC: Mesenchymal stem cell; Treg: Regulatory Tcell; UC: Ulcerative colitisAcknowledgementsThe authors are grateful to Dr. Tao Shi for his technical support.FundingThis work was supported by grants to HW from the National Natural ScienceFoundation of China (nos. 81273257 and 81471584), the Tianjin ApplicationBasis and Cutting-Edge Technology Research Grant (no. 14JCZDJC35700), theLi Jieshou Intestinal Barrier Research Special Fund (no. LJS_201412), and theTianjin Medical University Talent Fund.Authors’ contributionsAll authors have made substantial contributions to the preparation of thismanuscript. XX, YW, and BZ: conception and design of the study, data analysisand interpretation, manuscript drafting; XLa, SL, PS, XLi, GS, and YZ: performingthe research, data acquisition, and analysis; HH: data analysis and interpretation;CD: data interpretation and manuscript revision; HW: conception and design,financial support, administrative support, manuscript writing, final approval ofmanuscript. All authors read and approved the final manuscript.Ethics approvalAll experiments were conducted in accordance with the protocols approvedby the Animal Care and Use Committee of Tianjin Medical University (Tianjin,China) according to the Chinese Council on Animal Care guidelines.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Department of General Surgery, Tianjin Medical University General Hospital,154 Anshan Road, Heping District, Tianjin 300052, China. 2Tianjin GeneralSurgery Institute, Tianjin, China. 3Department of Endocrinology andMetabolism, Tianjin Medical University General Hospital, Tianjin, China.4Department of Ultrasound, National Cancer Center/Cancer Hospital, ChineseAcademy of Medical Sciences and Peking Union Medical College, Beijing,China. 5Department of Anorectal Surgery, People’s Hospital of HunanProvince, First Affiliated Hospital of Hunan Normal University, Changsha,Hunan Province, People’s Republic of China. 6Department of General Surgery,Affiliated Hospital of Weifang Medical University, Weifang, Shandong, China.7Department of Colorectal Surgery, The Second Hospital of Tianjin MedicalUniversity, Tianjin, China. 8Department of Urologic Sciences, the University ofBritish Columbia, Vancouver, British Columbia, Canada. 9Immunity andInfection Research Centre, Vancouver Coastal Health Research Institute,Vancouver, British Columbia, Canada.Received: 17 February 2018 Revised: 3 April 2018Accepted: 13 April 2018References1. 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