Human Umbilical Cord Blood Derived-Mesenchymal Stem Cells Alleviate Dextran Sulfate Sodium-Induced Colitis by Increasing Regulatory T Cells in Mice

Ying Li, Ke Ma, Luping Zhang, Hong Xu, Nan Zhang, Ying Li, Ke Ma, Luping Zhang, Hong Xu, Nan Zhang

Abstract

Inflammatory bowel disease (IBD), which main clinical manifestations include abdominal pain and diarrhea occurring repeatedly, is a kind of autoimmune disease. It has been reported in preceding studies that mesenchymal stem cells (MSCs) can reduce inflammation by regulating the function of immune cells. But studies about the interaction between MSCs and adaptive immune cells, especially in IBD models, are insufficient. Therefore, the objective of this research was to estimate the therapeutic effects of MSCs from human umbilical cord blood (hUCB-MSCs) in an IBD model of rodent and to clarify the therapeutic mechanisms of hUCB-MSCs. Dextran sulfate sodium (DSS) was used to induce colitis in rodent. Mice with colitis were treated with intraperitoneal infusions of hUCB-MSCs and evaluated for mortality and diverse disease symptoms containing weight reduction, diarrhea, and bloody stools. The levels of histopathologic severity and generation of regulatory T cells (Treg) were also determined. Treatment with hUCB-MSCs ameliorated the clinical and histopathologic severity of acute and chronic colitis in mice. Furthermore, T cell infiltration into the inflamed colon was significantly decreased (p = 0.0175), and Foxp3+ cells were substantially higher in the hUCB-MSC group than that of the DSS group. Our results suggest that hUCB-MSCs are able to alleviate inflammation via adding Foxp3+ Tregs in an IBD model of mouse. As a result, these findings suggest the opportunity of hUCB-MSC being applied to patients with IBD.

Keywords: colitis; dextran sulfate sodium; human umbilical cord blood; mesenchymal stem cells; regulatory T cells.

Copyright © 2020 Li, Ma, Zhang, Xu and Zhang.

Figures

FIGURE 1
FIGURE 1
Characterization of hUCB-MSCs. (A) Representative micrograph showing the typical cell morphology of hUCB-MSC colonies of proliferating fibroblast-like mesenchymal cells. (B) The phenotype of hUCB-MSCs and the percentage of cells with positive cell surface markers (blue line was negative staining control, red line was specific staining of indicated antibody).
FIGURE 2
FIGURE 2
hUCB-MSCs are capable of suppressing T cell proliferation in vitro. (A) Inhibitory effects of hUCB-MSCs on PHA-stimulated PBMC proliferation. In the presence or absence of PHA at 5 μg/ml, 1 × 105 PBMCs were cultured alone or in combination with hUCB-MSCs for 4 days, and then counted using CCK-8. *p < 0.05, ***p < 0.001 and ****p < 0.0001. The results showed are from more than three independent experiments. (B,C) 1 × 105 CFSE-labeled CD4 or CD8 cells were stimulated with 10 μg/ml plate-bound CD3 antibodies and 1 μg/ml CD28 antibodies for 4 days. At the beginning of the stimulation period, add half number of hUCB-MSCs to the relevant wells. Flow cytometry was used to observe proliferation of CD4 and CD8 cells. **p < 0.01, ***p < 0.001 and ****p < 0.0001. The results showed are from more than three independent experiments.
FIGURE 3
FIGURE 3
hUCB-MSCs show hyper-immunomodulatory potency with the stimulation of IFN-γ. (A) qPCR for IDO, TNF-α, PGE2, and TGF-β1 mRNA expression level in hUCB-MSCs with or without IFN-γ stimulation. (B) hUCB-MSCs were cultured in the presence of IFN-γ for 2 days, after which IDO, PGE2, and TGF-β1 levels in the supernatant were determined using ELISA. The results shown are from duplicate cultures performed in parallel. *p < 0.05, **p < 0.01, and ns, no significant difference.
FIGURE 4
FIGURE 4
Treatment with hUCB-MSCs protects against DSS-induced acute colitis. Weight loss (A), survival rate (B), and disease activity index (C) were determined daily. MPO activity in colonic protein extracts were used to show neutrophil infiltration (D). Colon length (E,F) and histopathological signs (G,H) were determined on day 10. The control group used tap water. n = 6–8 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001. The magnification of HE staining images is 10 × 10.
FIGURE 5
FIGURE 5
hUCB-MSC ameliorated DSS-induced chronic colitis. Body weight (A), colitis score (B), and survival rate (C) were determined daily. Colon length (D,E) and histopathological signs (F,G) were determined on day 25. n = 5–6 mice per group. *p < 0.05 and ns, no significant difference. The magnification of HE staining images is 10 × 10.
FIGURE 6
FIGURE 6
hUCB-MSC treatment reduced T cell infiltration in colon and induced Tregs in MLN, SPL, and colon. (A) Evaluation of T cell (%) infiltration to the colon. Flow cytometry analysis of CD3+ T cells gated in CD45+ population. (B,C) MLNs and splenocytes were analyzed for expression of Treg (staining for Foxp3 and CD25 in gated CD4+ T cells) via flow cytometry. *p < 0.05 and **p < 0.01. (D) Immunohistochemical staining (10 × 40) of CD4+ cells and Foxp3+ cells in colon tissues were showed. (E) Statistical results of immunohistochemical staining images for CD4+ cells and Foxp3+ cells. **p < 0.01 and ***p < 0.001.

References

    1. Anderson P., Souza-Moreira L., Morell M., Caro M., O’Valle F., Gonzalez-Rey E., et al. (2013). Adipose-derived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis. Gut 62 1131–1141. 10.1136/gutjnl-2012-302152
    1. Bouma G., Strober W. (2003). The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3 521–533. 10.1038/nri1132
    1. Cao W., Cao K., Cao J., Wang Y., Shi Y. (2015). Mesenchymal stem cells and adaptive immune responses. Immunol. Lett. 168 147–153. 10.1016/j.imlet.2015.06.003
    1. Chamberlain G., Fox J., Ashton B., Middleton J. (2007). Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 25 2739–2749. 10.1634/stemcells.2007-0197
    1. Chang P., Zhang B., Shao L., Song W., Shi W., Wang L., et al. (2018). Mesenchymal stem cells over-expressing cxcl12 enhance the radioresistance of the small intestine. Cell Death Dis. 9:154. 10.1038/s41419-017-0222-1
    1. Chen K., Wang D., Du W. T., Han Z. B., Ren H., Chi Y., et al. (2010). Human umbilical cord mesenchymal stem cells hUC-MSCs exert immunosuppressive activities through a PGE2-dependent mechanism. Clin. Immunol. 135 448–458. 10.1016/j.clim.2010.01.015
    1. Gonzalez M. A., Gonzalez-Rey E., Rico L., Buscher D., Delgado M. (2009). Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 136 978–989. 10.1053/j.gastro.2008.11.041
    1. Gonzalez-Rey E., Anderson P., González M. A., Rico L., Büscher D., Delgado M. (2009). Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. Gut 58 929–939. 10.1136/gut.2008.168534
    1. Heidari M., Pouya S., Baghaei K., Aghdaei H. A., Namaki S., Zali M. R., et al. (2018). The immunomodulatory effects of adipose-derived mesenchymal stem cells and mesenchymal stem cells-conditioned medium in chronic colitis. J. Cell. Physiol. 233 8754–8766. 10.1002/jcp.26765
    1. Himmel M. E., Yao Y., Orban P. C., Steiner T. S., Levings M. K. (2012). Regulatory T-cell therapy for inflammatory bowel disease: more questions than answers. Immunology 136 115–122. 10.1111/j.1365-2567.2012.03572.x
    1. Kadle R. L., Abdou S. A., Villarreal-Ponce A. P., Soares M. A., Sultan D. L., David J. A., et al. (2018). Microenvironmental cues enhance mesenchymal stem cell-mediated immunomodulation and regulatory T-cell expansion. PLoS One 13:e0193178. 10.1371/journal.pone.0193178
    1. Kim H. S., Shin T. H., Lee B. C., Yu K. R., Seo Y., Lee S., et al. (2013). Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2. Gastroenterology 145 1392–1403. 10.1053/j.gastro.2013.08.033
    1. Le Blanc K., Mougiakakos D. (2012). Multipotent mesenchymal stromal cells and the innate immune system. Nat. Rev. Immunol. 12 383–396. 10.1038/nri3209
    1. Lee E. S., Lim J. Y., Im K. I., Kim N., Nam Y. S., Jeon Y. W., et al. (2015). Adoptive transfer of treg cells combined with mesenchymal stem cells facilitates repopulation of endogenous treg cells in a murine acute GVHD model. PLoS One 10:e0138846. 10.1371/journal.pone.0138846
    1. Madrigal M., Rao K. S., Riordan N. H. (2014). A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J. Transl. Med. 12:260. 10.1186/s12967-014-0260-8
    1. Maynard C. L., Harrington L. E., Janowski K. M., Oliver J. R., Zindl C. L., Rudensky A. Y., et al. (2007). Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3- precursor cells in the absence of interleukin 10. Nat. Immunol. 8 931–941. 10.1038/ni1504
    1. Melief S. M., Schrama E., Brugman M. H., Tiemessen M. M., Hoogduijn M. J., Fibbe W. E., et al. (2013). Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells 31 1980–1991. 10.1002/stem.1432
    1. Nauta A. J., Fibbe W. E. (2007). Immunomodulatory properties of mesenchymal stromal cells. Blood 110 3499–3506. 10.1182/blood-2007-02-069716
    1. Qiu Y., Guo J., Mao R., Chao K., Chen B. L., He Y., et al. (2017). TLR3 preconditioning enhances the therapeutic efficacy of umbilical cord mesenchymal stem cells in TNBS-induced colitis via the TLR3-Jagged-1-Notch-1 pathway. Mucosal Immunol. 10 727–742. 10.1038/mi.2016.78
    1. Sakaguchi S., Miyara M., Costantino C. M., Hafler D. A. (2010). FOXP3+ regulatory T cells in the human immune system. Nat. Rev. Immunol. 10 490–500. 10.1038/nri2785
    1. Selmani Z., Naji A., Zidi I., Favier B., Gaiffe E., Obert L., et al. (2008). Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4+CD25highFOXP3+ regulatory T cells. Stem Cells 26 212–222. 10.1634/stemcells.2007-0554
    1. Tanoue T., Atarashi K., Honda K. (2016). Development and maintenance of intestinal regulatory T cells. Nat. Rev. Immunol. 16 295–309. 10.1038/nri.2016.36
    1. Turner J. R. (2009). Intestinal mucosal barrier function in health and disease. Nat. Rev. Immunol. 9 799–809. 10.1038/nri2653
    1. Ueno A., Jeffery L., Kobayashi T., Hibi T., Ghosh S., Jijon H. (2018). Th17 plasticity and its relevance to inflammatory bowel disease. J. Autoimmun. 87 38–49. 10.1016/j.jaut.2017.12.004
    1. Wang X., Lazorchak A. S., Song L., Li E., Zhang Z., Jiang B., et al. (2016). Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage. Stem Cells 34 380–391. 10.1002/stem.2242
    1. Wang Z. L., He R. Z., Tu B., He J. S., Cao X., Xia H. S., et al. (2018). Drilling combined with adipose-derived stem cells and bone morphogenetic protein-2 to treat femoral head epiphyseal necrosis in juvenile rabbits. Curr. Med. Sci. 38 277–288. 10.1007/s11596-018-1876-3
    1. Wirtz S., Popp V., Kindermann M., Gerlach K., Weigmann B., Fichtner-Feigl S., et al. (2017). Chemically induced mouse models of acute and chronic intestinal inflammation. Nat. Protoc. 12 1295–1309. 10.1038/nprot.2017.044
    1. Yañez R., Oviedo A., Aldea M., Bueren J. A., Lamana M. L. (2010). Prostaglandin E2 plays a key role in the immunosuppressive properties of adipose and bone marrow tissue-derived mesenchymal stromal cells. Exp. Cell Res. 316 3109–3123. 10.1016/j.yexcr.2010.08.008
    1. Yang R., Liu F., Wang J., Chen X., Xie J., Xiong K. (2019). Epidermal stem cells in wound healing and their clinical applications. Stem Cell Res. Ther. 10:229. 10.1186/s13287-019-1312-z
    1. Yang R., Yang S., Zhao J., Hu X., Chen X., Wang J., et al. (2020). Progress in studies of epidermal stem cells and their application in skin tissue engineering. Stem Cell Res. Ther. 11:303. 10.1186/s13287-020-01796-3
    1. Zhang L., Li Y., Guan C. Y., Tian S., Lv X. D., Li J. H., et al. (2018). Therapeutic effect of human umbilical cord-derived mesenchymal stem cells on injured rat endometrium during its chronic phase. Stem Cell Res. Ther. 9:36. 10.1186/s13287-018-0777-5
    1. Zhang Q., Fu L., Liang Y., Guo Z., Wang L., Ma C., et al. (2018). Exosomes originating from MSCs stimulated with TGF-β and IFN-γ promote Treg differentiation. J. Cell. Physiol. 233 6832–6840. 10.1002/jcp.26436
    1. Zhang Q., Shi S., Liu Y., Uyanne J., Shi Y., Shi S., et al. (2009). Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J. Immunol. 183 7787–7798. 10.4049/jimmunol.0902318
    1. Zhu Y., Xu H., Liu W., Qi W., Yang X., Ye L., et al. (2018). Glasgow prognostic score is a practical predictive index for postoperative intra-abdominal septic complications after bowel resection in Crohn’s disease patients. Int. J. Colorectal. Dis. 33 947–953. 10.1007/s00384-018-3035-5

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren