PD-L1 is required for human endometrial regenerative cells-associated attenuation of experimental colitis in mice.

Endometrial regenerative cells (ERCs) are easily isolated from menstrual blood, and can be cultured in large amounts. Although, ERCs can ameliorate DSS-induced colitis in mice, the molecular mechanism underlying ERCs-mediated immunosuppression is unclear. This study was aimed to assess the function of PD-L1 expressed on ERCs in colitis attenuation. ERCs with and without anti-PD-L1 mAb-pretreatment were administered to mice by injection at 2, 5 and 8 days after colitis induction by DSS treatment. Blood, spleen and colon samples were obtained 15 days post-DSS-induction. Then, clinicopathological alterations, cytokine levels, immune cell types and cell tracking were assessed. ERCs or ERCs preincubated with anti-PD-L1 antibody were co-cultured with splenocytes, whose phenotypes was analyzed by flow cytometry. We found that PD-L1 on ERCs was upregulated by IFN-γ stimulation. The transplanted PKH26-labeled ERCs were engrafted to the lung, liver, spleen and injured colon. Interestingly, ERC-based therapy markedly attenuated mouse colitis, but blockade of PD-L1 on ERCs with a specific monoclonal antibody conferred severe colitis to the animals. These effects of PD-L1 inhibition on colitis were associated with reduced amounts of pro-inflammatory cytokines and infiltrated immune cells, including CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD11c+MHC-II+ Dendritic cells and F4/80+ macrophages, both in vivo and in vitro, as well as with elevated levels of anti-inflammatory cytokines and regulatory immune cells, including CD4+CD25+Foxp3+ Tregs and F4/80+CD206+ macrophages. These findings demonstrated that ERCs-based treatment promotes immune tolerance in mouse colitis, in association with PD-L1, thus indicating that PD-L1 modulates immunosuppression by ERCs.

Positive expression of basic transcription factor 3 predicts poor survival of colorectal cancer patients: possible mechanisms involved.

Basic transcription factor 3 (BTF3) is associated with the development of several cancers. The aim of our study was to elucidate the role of BTF3 in colorectal cancer (CRC) tissues. CRC tissues or their paired adjacent noncancerous (ANCT) tissues were obtained from 90 patients who underwent operations in our hospital from November 2011 to December 2016, and then we implemented a gene microarray assay for detecting significant changes in gene expression and confirmed expression in tissues using immunohistochemistry and real-time PCR. We transfected or injected the silencing BTF3 (BTF3-siRNA) plasmid into cells and nude mice, and measured the tumorigenicity of CRC cells with flow cytometry and studied the expression level of BTF3 downstream genes (MAD2L2, MCM3 and PLK1) in CRC cells. BTF3 expression level was not only significantly higher in CRC tissue than in ANCT tissue (2.61 ± 0.07 vs 1.90 ± 0.03, P < 0.001) but BTF3-siRNA decreased tumor formation in a nude mice model. Furthermore, based on the data of gene microarray analysis, MAD2L2, MCM3 and PLK1 were detected as the downstream target genes of BTF3 and their expressions were positive related with BTF3 expression. Also, through transfecting BTF3-siRNA into HCT116 cells, we found that BTF3-siRNA could decrease cell viability and induced cell apoptosis and blocking the cell cycle. In conclusion, BTF3 is positively related to CRC and BTF3-siRNA attenuated the tumorigenicity of colorectal cancer cells via MAD2L2, MCM3 and PLK1 activity reduction.

Human Endometrial Regenerative Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis in Mice.

Endometrial regenerative cells (ERCs) have been recently evaluated as an attractive novel type of stem cell therapy. Previous studies have demonstrated that most ERCs accumulated in the lung after injection and are successfully used to treat diseases such as cardiac fibrosis. However, relevant studies of ERCs in idiopathic pulmonary fibrosis (IPF) have not been reported. The present study was designed to examine the effects of ERCs on bleomycin-induced pulmonary fibrosis. All IPF models in C57BL/6 mice were induced by administrating 5 mg/kg bleomycin in PBS intratracheally. ERCs were isolated from healthy female menstrual blood and were injected (1 million/mouse, i.v.) 24 hours after induction. Wet/dry weight ratio assay, hydroxyproline content, pathological and immunohistological changes, MDA content, T-SOD activity, cytokine profiles, and RT-qPCR analysis were assessed 2 weeks after disease induction. The results showed that ERC treatment significantly decreased the wet/dry ratio and reduced collagen deposition. Histological analyses, Masson staining, and hydroxyproline content analysis indicated that ERCs could reduce collagen fiber production. Immunohistochemical staining revealed lower expression of TGF-ß after ERC treatment. Furthermore, mice treated with ERCs had lower levels of IL-1ß and TNF-α, but a higher level of IL-10 in both the lung and serum. Gene expression analysis demonstrated that ERCs potently suppressed the proapoptotic gene Bax, while increasing the antiapoptotic gene Bcl-2 and antifibrosis genes HGF and MMP-9. Our results indicate that human ERCs protected the lung from pulmonary fibrosis in mice through immunosuppressive and antifibrosis effects. Moreover, these findings formed a foundation for the further use of ERCs in clinical treatment.

Treatment of experimental colitis by endometrial regenerative cells through regulation of B lymphocytes in mice.

BACKGROUND: Endometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cell derived from menstrual 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 drinking water for 7 days to BALB/c mice. In the treated group, mice were injected intravenously with 1 × 106 ERCs on days 2, 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 in the colon was examined by immunohistochemistry staining. Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA), Western blot, or polymerase chain reaction (PCR) analysis. Adoptive transfer of regulatory B cells (Bregs) into colitis mice was performed. RESULTS: Here, we demonstrated that ERC treatment prolonged the survival of colitis mice and attenuated disease activity with fewer pathological changes in colon tissue. ERCs decreased the proportion of immature plasma cells in the spleen and IgG deposition in the colon. On the other hand, ERCs increased the production of Bregs and the interleukin (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 regulating the B-lymphocyte responses.

Skin Allografting Activates Anti-tumor Immunity and Suppresses Growth of Colon Cancer in Mice.

INTRODUCTION: The tumor cells could escape from the immune elimination through the immunoediting mechanisms including the generation of immunosuppressive or immunoregulative cells. By contrast, allograft transplantation could activate the immune system and induce a strong allogenic response. The aim of this study was to investigate the efficacy of allogenic skin transplantation in the inhibition of tumor growth through the activation of allogenic immune response. METHODS: Full-thickness skin transplantation was performed from C57BL/6 (H-2b) donors to BALB/c (H-2d) recipients that were receiving subcutaneous injection of isogenic CT26 colon cancer cells (2 × 106 cells) at the same time. The tumor size and pathological changes, cell populations and cytokine profiles were evaluated at day 14 post-transplantation. RESULTS: The results showed that as compared to non-transplant group, the allogenic immune response in the skin-grafting group inhibited the growth of tumors, which was significantly associated with increased numbers of intra-tumor infiltrating lymphocytes, increased populations of CD11c+MHC-classII+CD86+ DCs, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD19+ B cells, as well as decreased percentage of CD4+CD25+Foxp3+ T cells in the spleens. In addition, the levels of serum IgM and IgG, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were significantly higher within the tumor in skin transplant groups than that in non-transplant group. CONCLUSIONS: Allogenic skin transplantation suppresses the tumor growth through activating the allogenic immune response, and it may provide a new immunotherapy option for the clinical refractory tumor treatment.

Oral Escherichia coli expressing IL-35 meliorates experimental colitis in mice.

BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) characterized by chronic inflammation of colon. It is commonly believed that the imbalance of immune system and overwhelming production of cytokines are involved in the pathogenesis of UC. Recent studies demonstrated that interleukin-35 (IL-35), a key player in the regulation of inflammation, has been identified as potential therapeutic target to treat UC. However, conventional intravenous administration is costly and inconvenient. The present study was designed to establish a novel IL-35 delivery system and investigate its therapeutic effects on dextran sulfate sodium (DSS)-induced experimental colitis in mice for the first time. METHODS: An engineered Escherichia coli (E. coli/IL-35) expressing IL-35 was constructed. Adult male BALB/c mice randomly got the oral administration of E. coli/IL-35, empty plasmid-transformed E. coli (E. coli0) or PBS for treatment following ingestion of 3% DSS solution for 5 days. Normal mice were used as control group. Colonic and splenic tissues were collected on day 10 post-DSS-induction. Clinical signs, disease activity index (DAI), pathological and immunohistological changes, cytokine profiles and cell populations were evaluated. RESULTS: Intragastric administration of E. coli/IL-35 effectively protected the colitis mice from DSS assimilation including weight loss and colon shortening. Pathological analysis showed significantly lower DAI score and much less intra-colon infiltration of neutrophils and CD3+ cells in the IL-35 treated group. Moreover, E. coli/IL-35-treated mice demonstrated much less CD4+ IL-17A+ Th17 cells and a higher level of CD4+CD25+Foxp3+ Tregs in spleen and mesenteric lymph nodes, as well as increased colon and serum level of IL-10 and IL-35 and decreased levels of IL-6. CONCLUSIONS: Our study showed that E. coli/IL-35 as a novel oral IL-35 delivery system alleviated inflammatory damage of colonic tissue in the colitic mice. Genetic therapeutic strategies using engineered E. coli encoding immunoregulatory cytokines may provide a potential approach for the treatment of IBD.

Stromal Cell-Derived Factor-1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell-Based Therapy.

Endometrial regenerative cells (ERCs) are mesenchymal-like stromal cells, and their therapeutic potential has been tested in the prevention of renal ischemic reperfusion injury, acute liver injury, ulcerative colitis, and immunosuppression. However, their potential in the induction of transplant tolerance has not been investigated. The present study was undertaken to investigate the efficacy of ERCs in inducing cardiac allograft tolerance and the function of stromal cell-derived factor-1 (SDF-1) in the ERC-mediated immunoregulation. The inhibitory efficacy of human ERCs in the presence or absence of rapamycin was examined in both mouse cardiac allograft models between BALB/c (H-2d ) donors and C57BL/6 (H-2b ) recipients and in vitro cocultured splenocytes. AMD3100 was used to inhibit the function of SDF-1. Intragraft antibody (IgG and IgM) deposition and immune cell (CD4+ and CD8+ ) infiltration were measured by immunohistochemical staining, and splenocyte phenotypes were determined by fluorescence-activated cell sorting analysis. The results showed that ERC-based therapy induced donor-specific allograft tolerance, and functionally inhibiting SDF-1 resulted in severe allograft rejection. The negative effects of inhibiting SDF-1 on allograft survival were correlated with increased levels of intragraft antibodies and infiltrating immune cells, and also with reduced levels of regulatory immune cells including MHC class IIlow CD86low CD40low dendritic cells, CD68+ CD206+ macrophages, CD4+ CD25+ Foxp3+ T cells, and CD1dhigh CD5high CD83low IL-10high B cells both in vivo and in vitro. These data showed that human ERC-based therapy induces cardiac allograft tolerance in mice, which is associated with SDF-1 activity, suggesting that SDF-1 mediates the immunosuppression of ERC-based therapy for the induction of transplant tolerance. Stem Cells Translational Medicine 2017;6:1997-2008.

Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice.

BACKGROUND: The endometrial regenerative cell (ERC) is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4)-induced acute liver injury (ALI). METHODS: An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse) into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction. RESULTS: ERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G) was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA) was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs) was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1ß, IL-6, and TNF-α but higher level of IL-10 in both serum and liver. CONCLUSIONS: Human ERCs protect the liver from acute injury in mice through hepatocyte proliferation promotion, as well as through anti-inflammatory and immunoregulatory effects.