RESUMO
Mesenchymal stromal cells (MSCs) display heterogeneity in origin and functional role in tissue homeostasis. Subsets of MSCs derived from the neural crest express nestin and serve as niches in bone marrow, but the possibility of coaxing MSCs into nestin-expresing cells for enhanced supportive activity is unclear. In this study, as an approach to the chemical coaxing of MSC functions, we screened libraries of clinically approved chemicals to identify compounds capable of inducing nestin expression in MSCs. Out of 2000 clinical compounds, we chose vorinostat as a candidate to coax the MSCs into neural crest-like fates. When treated with vorinostat, MSCs exhibited a significant increase in the expression of genes involved in the pluripotency and epithelial-mesenchymal transition (EMT), as well as nestin and CD146, the markers for pericytes. In addition, these nestin-induced MSCs exhibited enhanced differentiation towards neuronal cells with the upregulation of neurogenic markers, including SRY-box transcription factor 2 (Sox2), SRY-box transcription factor 10 (Sox10) and microtubule associated protein 2 (Map2) in addition to nestin. Moreover, the coaxed MSCs exhibited enhanced supporting activity for hematopoietic progenitors without supporting leukemia cells. These results demonstrate the feasibility of the drug repositioning of MSCs to induce neural crest-like properties through the chemical coaxing of cell fates.
Assuntos
Diferenciação Celular , Reposicionamento de Medicamentos , Células-Tronco Mesenquimais , Nestina , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Nestina/metabolismo , Nestina/genética , Humanos , Diferenciação Celular/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Cultivadas , Crista Neural/citologia , Crista Neural/metabolismo , Crista Neural/efeitos dos fármacosRESUMO
Immune status including the immune cells and cytokine profiles has been implicated in the development of endometriosis. In this study, we analyzed Th17 cells and IL-17A in peritoneal fluid (PF) and endometrial tissues of patients with (n=10) and without (n=26) endometriosis. Our study has shown increased Th17 cell population and IL-17A level in PF with endometriosis patients. To determine the roles of IL-17A and Th17 cells in the development of endometriosis, the effect of IL-17A, major cytokine of Th17, on endometrial cells isolated from endometriotic tissues was examined. Recombinant IL-17A promoted survival of endometrial cells accompanied by increased expression of anti-apoptotic genes, including Bcl-2 and MCL1, and the activation of ERK1/2 signaling. In addition, treatment of IL-17A to endometrial cells inhibited NK cell mediated cytotoxicity and induced HLA-G expression on endometrial cells. IL-17A also promoted migration of endometrial cells. Our data suggest that Th17 cells and IL-17A play critical roles in the development of endometriosis by promoting endometrial cell survival and conferring a resistance to NK cell cytotoxicity through the activation of ERK1/2 signaling. Targeting IL-17A has potential as a new strategy for the treatment of endometriosis.
RESUMO
The low therapeutic efficacy of current cancer immunotherapy is related to nonimmunogenic and immunosuppressive tumor microenvironments (TMEs). To overcome these limitations, both the immune priming of antitumoral lymphocytes and the reprogramming of immunosuppressive factors in TMEs are essential. Here, we suggest a nanoemulsion (NE)-based immunotherapeutic platform that can not only modulate tumor-induced suppression but also induce an effective cell-mediated immune response for T cell proliferation. Multifunctional NEs can be fabricated by integrating the efficacy of NEs as delivery systems and the multifaceted immunomodulation characteristics (i.e., immunostimulation and reprogramming of immunosuppression) of small molecule-based Toll-like receptor 7/8 agonists. Local in situ vaccination of melanoma and cervical tumor models with tumor antigens (protein and peptide) adjuvanted with NE loaded with TLR7/8 agonists [NE (TLR7/8a)] induced the recruitment and activation of innate immune cells, infiltration of lymphocytes, and polarization of tumor-associated M2 macrophages, which resulted in inhibition of tumor growth and prolonged survival in both primary and rechallenged tumor models. Antibody-depletion experiments also suggested that macrophages, type I IFN (IFN-α and IFN-ß), CD8+ T cells, and NK1.1+ cells contributed to the antitumor effect of NE (TLR7/8a). The combination of antitumoral lymphocytes and reprogramming of immunosuppressive TMEs induced by NE (TLR7/8a) treatment evoked a synergistic antitumor immune response with immune checkpoint blockade therapy (anti-PD-1 and anti-PD-L1).
Assuntos
Vacinas Anticâncer , Imunoterapia/métodos , Glicoproteínas de Membrana/agonistas , Nanoestruturas/química , Receptor 7 Toll-Like/agonistas , Microambiente Tumoral/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Emulsões/química , Emulsões/farmacologia , Feminino , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Receptor 8 Toll-Like/agonistasRESUMO
Natural killer (NK) cells play important roles in immune surveillance. However, the tumor microenvironment suppresses NK cell function and allows cancer cells to evade immune detection. In this study, we investigated whether the thyroid cancer cell microenvironment has this effect on NK cells. We found that prostaglandin (PG) E2 produced by thyroid cancer cells suppressed the cytolytic activity of NK cells by inhibiting the expression of the natural cytotoxicity receptors NKp44 and NKp30 and the death receptor tumor necrosis factor-related apoptosis-inducing ligand. PGE2 and cyclooxygenase-2 were highly expressed in thyroid cancer cells; moreover, anaplastic thyroid cancer cells released higher amounts of PGE2 than the papillary subtype, which was associated with suppression of NK cell-inducing nuclear factor-κB and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways via PGE2 receptor (EP) 2 and EP4 expressed on the NK cell surface. In addition, PGE2 inhibited the functional maturation of NK cells and reduced their cytotoxicity against target cells. These results indicate that PGE2 promotes thyroid cancer progression by inhibiting NK cell maturation and cytotoxicity. Thus, therapeutic strategies that target PGE2 in thyroid cancer could potentiate the immune response and improve patient prognosis.
Assuntos
Diferenciação Celular/imunologia , Citotoxicidade Imunológica , Dinoprostona/biossíntese , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/metabolismo , Evasão Tumoral/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Células Matadoras Naturais/citologia , NF-kappa B/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Microambiente TumoralRESUMO
The present study was performed to know the effects of taurine on the lipid level of plasma and liver, lipid peroxidation and antioxidative enzyme activities of liver tissue in ovariectomized (OVX) rats fed cholesterol. Twenty-four female SD rats (200 ± 5 g) were grouped; sham and ovariectomy groups, which were each randomly subgrouped; fed control and control supplemented with taurine (20 g/kg diet). The serum total cholesterol, TG (triglyceride), LDL-cholesterol, athrogenic index, and HDL-cholesterol of taurine diet group were not statistically different. Also the levels of liver total cholesterol, triglyceride were not considerably different in different diets. The lipid peroxidation of malondialdehyde concentration was considerably lower in taurine-feeding group than control-feeding group in ovariectomy group. The superoxide dismutase activity in liver tissue was significantly higher in rats fed taurine than in rats fed control diet in OVX rats. GSH-Px (glutathione peroxidase) activity was statistically greater at the rats fed taurine diets compared to rats fed control diet in ovariectomy group. Activity of catalase was higher in taurine group than in control group in ovariectomy group, but it was not significantly different. In conclusion, taurine supplementation was beneficial on antioxidative enzyme activities of liver tissue in ovariectomized rats fed cholesterol.
Assuntos
Antioxidantes/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Taurina/farmacologia , Animais , Feminino , Lipídeos/sangue , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
STUDY QUESTION: Is the decreased natural killer (NK) cell cytolytic activity in the peritoneal fluid (PF) of endometriosis patients due to primary cytokine activity? SUMMARY ANSWER: An increased level of interleukin-6 (IL-6) in the PF of patients with endometriosis suppresses NK cell cytolytic activity by down-regulating cytolytic granule components, such as granzyme B and perforin, through the modulation of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) expression. WHAT IS ALREADY KNOWN: Endometriosis is known to be related to a defect in NK cell cytolytic activity. Additionally, the levels of inflammatory cytokines are elevated in the PF of women with endometriosis. STUDY DESIGN, SIZE, DURATION: The effects of PF on the differentiation and functional activity of NK cells were investigated in patients with or without endometriosis, and cytokines that reduce NK cell cytolytic activity in endometriosis patients were examined. The study included women who underwent laparoscopic examination for the diagnosis of endometriosis from August 2012 to July 2013 (33 women with, and 15 women without, endometriosis). PARTICIPANTS/MATERIALS, SETTING, METHODS: Women of reproductive age (20-40 years old) who underwent laparoscopic examination for endometriosis were included. Cytokines present in the PF were identified by enzyme-linked immunosorbent assay. The cytolytic activity of NK cells in the PF was also analyzed using a calcein-acetoxy methyl ester (AM) release assay. MAIN RESULTS AND THE ROLE OF CHANCE: PF from patients with endometriosis suppressed the differentiation and cytotoxicity of NK cells compared with PF from controls (P < 0.05). Increased levels of IL-6 were also found in the PF of patients with endometriosis (P < 0.01), and IL-6 levels were negatively correlated with the cytolytic activity of NK cells (rs = -0.558, P = 0.03). Furthermore, IL-6 reduced the cytolytic activity of NK cells, concomitantly with the down-regulation of granzyme B and perforin (P < 0.05), by modulating SHP-2. Importantly, the addition of anti-IL-6 to the PF of endometriosis patients restored the activity of NK cells (P < 0.01), suggesting that IL-6 plays a crucial role in the reduction of NK cell activity in the PF of patients with endometriosis. LIMITATIONS, REASONS FOR CAUTION: PF contains various inflammatory cytokines in addition to IL-6 and so it is possible that other cytokines may affect the differentiation and activity of NK cells. WIDER IMPLICATIONS OF THE FINDINGS: Our results imply that the suppression of IL-6 using an anti-IL-6 antibody or soluble IL-6 receptor could rescue the impairment of NK cell activity in patients with endometriosis. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the KRIBB Creative Research Program (KCS3051312); the STP project (DTM0111221) of the Ministry of Knowledge & Economy and the Basic Science Research Program (RBM0271312) of the National Research Foundation of Korea (NRF) from the Ministry of Education, Science & Technology. There are no conflicts of interest.
Assuntos
Líquido Ascítico/imunologia , Endometriose/imunologia , Interleucina-6/fisiologia , Células Matadoras Naturais/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Adulto , Líquido Ascítico/citologia , Líquido Ascítico/metabolismo , Diferenciação Celular , Endometriose/metabolismo , Endometriose/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) that belong to the transforming growth factor-ß (TGF-ß) superfamily cytokines, play crucial roles in hematopoiesis. However, roles of Smad6 in hematopoiesis remained unknown in contrast to the other inhibitory Smad (I-Smad), Smad7. Here we show that Smad6 inhibits erythropoiesis in human CD34(+) cord blood hematopoietic stem cells (HSCs). Smad6 was specifically expressed in CD34(+) cord blood HSCs, which was correlated with the expression of BMP2/4/6/7 and BMP type I receptor (BMPRI). BMP-specific receptor-regulated Smads (R-Smads), Smad1 and Smad5 in cooperation with Smad4 induced transcription of the Smad6 gene. Instead of affecting cell cycle, apoptosis, self-renewal, and stemness of CD34(+) cells, Smad6 knockdown enhanced, whereas Smad6 overexpression suppressed erythropoiesis in stem cell culture and colony formation assay. Consistently, Smad6 suppressed the expression of the genes essential for erythropoiesis, such as Kruppel-like factor 1 (erythroid) (KLF1/EKLF) and GATA binding protein 2 (GATA-2). Promoter analyses showed that Smad6 repressed Smad5/4-induced transcription of the Klf1 gene. Thus, our data suggest that Smad6 indirectly maintains stemness by preventing spontaneous erythropoiesis in HSCs.
Assuntos
Eritropoese/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Proteína Smad6/metabolismo , Antígenos CD34/análise , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 7/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Células Cultivadas , Sangue Fetal/citologia , Fator de Transcrição GATA2/genética , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Regiões Promotoras Genéticas , Proteína Smad6/genética , Transcrição GênicaRESUMO
With contrasting observations on the effects of beta-catenin on hematopoietic stem cells (HSCs), the precise role of Wnt/beta-catenin signals on HSC regulation remains unclear. Here, we show a distinct mode of Wnt/beta-catenin signal that can regulate HSCs in a stroma-dependent manner. Stabilization of beta-catenin in the bone marrow stromal cells promoted maintenance and self-renewal of HSCs in a contact-dependent manner, whereas direct stabilization in hematopoietic cells caused loss of HSCs. Interestingly, canonical Wnt receptors and beta-catenin accumulation were predominantly enriched in the stromal rather than the hematopoietic compartment of bone marrows. Moreover, the active form of beta-catenin accumulated selectively in the trabecular endosteum in "Wnt 3a-stimulated" or "irradiation-stressed," but not in "steady-state" marrows. Notably, notch ligands were induced in Wnt/beta-catenin activated bone marrow stroma and downstream notch signal activation was seen in the HSCs in contact with the activated stroma. Taken together, Wnt/beta-catenin activated stroma and their cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Células Estromais/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proliferação de Células , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Receptor Cross-Talk/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the factors regulating the process remain poorly understood. Here, we show that Interleukin-10 (IL-10), a pleiotropic immune modulating cytokine, can also play a role in regulating HSC self-renewal. First, a quantitative decrease of primitive hematopoietic cell populations, but not more matured cells, was observed in the bone marrows of IL-10 disrupted mice as determined by long-term in vitro cultures or in vivo competitive repopulation assays. In contrast, normal HSCs from 5-fluorouracil treated marrows cultured on the IL-10 secreting stroma displayed an enhanced repopulating activity compared with cells grown on control stroma, with ninefold higher numbers of donor-derived HSCs in the reconstituted recipient marrows. Moreover, limiting dilution transplantation assay demonstrated that exogenous addition of IL-10 in the stroma-free cultures of purified Lin- Sca-1+ c-kit+ cells caused three- to fourfold higher frequencies of HSCs in the 5-day short-term culture without indirect inhibitory effect of IL-10 on tumor necrosis factor-alpha or interferon-gamma secretion. Interestingly, primitive hematopoietic cells, including Lin- Sca-1+ c-kit+ or side population cells, expressed the surface receptor for IL-10, and microenvironmental production of IL-10 was sharply increased in the osteoblasts lining the trabecular regions of the radiation-stressed marrow but not in the steady-state marrows. These results show that IL-10 may be a ligand that can stimulate self-renewal of HSCs to promote their regeneration in addition to being a ligand for immune regulation. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interleucina-10/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-10/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the signaling pathways that regulate this process remain poorly understood. Here we show that transduction of adult mouse bone marrow cells with a constitutively activated form of Stat3 (Stat3-C) increased their regenerative activity in lethally irradiated recipients. Conversely, transduction of these cells with a dominant-negative form of Stat3 suppressed their regenerative activity. Serial transplantation and clonal tracking of the HSC progeny regenerated in vivo from STAT3-C-transduced HSCs demonstrated that the major effect of forced expression of STAT3-C was to enhance HSC self-renewal during the initial phase of hematopoietic recovery. This acquired potential for enhanced self-renewal divisions then became latent, but was reactivated when the cells were transferred to new irradiated recipients. Increased levels of activated STAT3 were also found to be associated with greater preservation of primitive hematopoietic cells in short-term cultures. These results indicate a novel biphasic regulation of HSC self-renewal in vivo in which activated STAT3 promotes HSC self-renewal under stimulated, but not homeostatic, conditions. STAT3 may thus be an important regulator of hematopoietic regeneration and a novel target for HSC engineering.