RESUMEN
Emergency granulopoiesis is the enhanced and accelerated production of granulocytes that occurs during acute infection. The contribution of hematopoietic stem cells (HSCs) to this process was reported; however, how HSCs participate in emergency granulopoiesis remains elusive. Here, using a mouse model of emergency granulopoiesis we observe transcriptional changes in HSCs as early as 4 h after lipopolysaccharide (LPS) administration. We observe that the HSC identity is changed towards a myeloid-biased HSC and show that CD201 is enriched in lymphoid-biased HSCs. While CD201 expression under steady-state conditions reveals a lymphoid bias, under emergency granulopoiesis loss of CD201 marks the lymphoid-to-myeloid transcriptional switch. Mechanistically, we determine that lymphoid-biased CD201+ HSCs act as a first response during emergency granulopoiesis due to direct sensing of LPS by TLR4 and downstream activation of NF-κΒ signaling. The myeloid-biased CD201- HSC population responds indirectly during an acute infection by sensing G-CSF, increasing STAT3 phosphorylation, and upregulating LAP/LAP* C/EBPß isoforms. In conclusion, HSC subpopulations support early phases of emergency granulopoiesis due to their transcriptional rewiring from a lymphoid-biased to myeloid-biased population and thus establishing alternative paths to supply elevated numbers of granulocytes.
Asunto(s)
Células Madre Hematopoyéticas , Lipopolisacáridos , Lipopolisacáridos/metabolismo , Hematopoyesis , Granulocitos/metabolismoRESUMEN
BACKGROUND: Chemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked. METHODS: We employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1-/-, Cxcr2-/- or Ccr1-/-Cxcr2-/- mice. RESULTS: Myeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1-/-Cxcr2-/- BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2-/- BM significantly suppressed tumor growth and liver metastasis. CONCLUSION: Dual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.
Asunto(s)
Ratones Noqueados , Células Mieloides , Receptores CCR1 , Receptores de Interleucina-8B , Microambiente Tumoral , Animales , Receptores CCR1/metabolismo , Receptores CCR1/genética , Receptores CCR1/antagonistas & inhibidores , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Ratones , Células Mieloides/metabolismo , Células Mieloides/inmunología , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Ratones Endogámicos C57BL , Línea Celular Tumoral , Linfocitos T CD8-positivos/inmunologíaRESUMEN
The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.
Asunto(s)
Células Dendríticas , Monocitos , Animales , Diferenciación Celular , Macrófagos , Ratones , Sistema Mononuclear FagocíticoRESUMEN
Myelopoiesis is a process that produces myeloid cells including granulocytes and mononuclear phagocytes. The differentiation and proliferation of hematopoietic stem and progenitor cells are tightly regulated to meet demands for such myeloid cells both at steady state and under stressed conditions. CCAAT/enhancer-binding protein family transcription factors are involved not only in the appropriate regulation of myelopoiesis but also in dysregulated myelopoiesis. A recent study has revealed that inflammation, in addition to the established concepts or mechanisms of dysregulated myelopoiesis, triggers long-term epigenetic memory in hematopoietic stem/progenitor cells. Further, clonal hematopoiesis develops and impairs host health conditions via inflammatory conditions. Intensive studies covering both the basic and clinical aspects of myelopoiesis are required to establish therapeutic and even prophylactic approaches to different types of human diseases including hematopoietic and nonhematopoietic origins.
Asunto(s)
Células Madre Hematopoyéticas , Mielopoyesis , Humanos , Mielopoyesis/fisiología , Diferenciación Celular , Factores de Transcripción , Células MieloidesRESUMEN
Cancer cachexia is a multifactorial disease that causes continuous skeletal muscle wasting. Thereby, it seems to be a key determinant of cancer-related death. Although anamorelin, a ghrelin receptor agonist, has been approved in Japan for the treatment of cachexia, few medical treatments for cancer cachexia are currently available. Myostatin (MSTN)/growth differentiation factor 8, which belongs to the transforming growth factor-ß family, is a negative regulator of skeletal muscle mass, and inhibition of MSTN signaling is expected to be a therapeutic target for muscle-wasting diseases. Indeed, we have reported that peptide-2, an MSTN-inhibiting peptide from the MSTN prodomain, alleviates muscle wasting due to cancer cachexia. Herein, we evaluated the therapeutic benefit of myostatin inhibitory D-peptide-35 (MID-35), whose stability and activity were more improved than those of peptide-2 in cancer cachexia model mice. The biologic effects of MID-35 were better than those of peptide-2. Intramuscular administration of MID-35 effectively alleviated skeletal muscle atrophy in cachexia model mice, and the combination therapy of MID-35 with anamorelin increased food intake and maximized grip strength, resulting in longer survival. Our results suggest that this combination might be a novel therapeutic tool to suppress muscle wasting in cancer cachexia.
Asunto(s)
Productos Biológicos , Neoplasias , Animales , Productos Biológicos/farmacología , Caquexia/tratamiento farmacológico , Caquexia/etiología , Caquexia/patología , Modelos Animales de Enfermedad , Hidrazinas , Ratones , Músculo Esquelético , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/etiología , Atrofia Muscular/patología , Miostatina , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Oligopéptidos , Péptidos/farmacología , Receptores de Ghrelina/uso terapéutico , Factores de Crecimiento Transformadores/farmacología , Factores de Crecimiento Transformadores/uso terapéuticoRESUMEN
The emergence of tyrosine kinase inhibitors as part of a front-line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR-ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)-resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR-ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb-M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR-ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR-ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb-M' consistently downregulated the expression of BCR-ABL1 in these cells and this drug was highly effective even in an imatinib-resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR-ABL1 in these cells conferred relative resistance to Chb-M'. In addition, in vivo experiments with the Ph+ ALL patient-derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR-ABL1. Chb-M' could be a novel drug for patients with TKI-resistant refractory Ph+ ALL.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Fusión bcr-abl/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Ratones , Mutación , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) is highly expressed in monocytes/macrophages. However, its roles in monopoiesis are largely unknown. Here, we investigated the roles of C/EBPß in monopoiesis. Further subdivision of monocytes revealed that Cebpb messenger RNA was highly upregulated in Ly6C- monocytes in bone marrow. Accordingly, the number of Ly6C- monocytes was significantly reduced in Cebpb-/- mice. Bone marrow chimera experiments and Mx1-Cre-mediated deletion of Cebpb revealed a cell-intrinsic and monocyte-specific requirement for C/EBPß in monopoiesis. In Cebpb-/- mice, turnover of Ly6C- monocytes was highly accelerated and apoptosis of Ly6C- monocytes was increased. Expression of Csf1r, which encodes a receptor for macrophage colony-stimulating factor, was significantly reduced in Ly6C- monocytes of Cebpb-/- mice. C/EBPß bound to positive regulatory elements of Csf1r and promoted its transcription. Collectively, these results indicate that C/EBPß is a critical factor for Ly6C- monocyte survival, at least in part through upregulation of Csf1r.
Asunto(s)
Apoptosis/genética , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Monocitos/fisiología , Animales , Antígenos Ly/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Células COS , Diferenciación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Chlorocebus aethiops , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Monocitos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiologíaRESUMEN
A substantial proportion of patients with acute graft-versus-host disease (aGVHD) respond to cell therapy with culture-expanded human bone marrow mesenchymal stromal/stem cells (BM-MSCs). However, the mechanisms by which these cells can ameliorate aGVHD-associated complications remain to be clarified. We show here that BM-MSC-derived extracellular vesicles (EVs) recapitulated the therapeutic effects of BM-MSCs against aGVHD. Systemic infusion of human BM-MSC-derived EVs prolonged the survival of mice with aGVHD and reduced the pathologic damage in multiple GVHD-targeted organs. In EV-treated GVHD mice, CD4+ and CD8+ T cells were suppressed. Importantly, the ratio of CD62L-CD44+ to CD62L + CD44- T cells was decreased, suggesting that BM-MSC-derived EVs suppressed the functional differentiation of T cells from a naive to an effector phenotype. BM-MSC-derived EVs also preserved CD4 + CD25 + Foxp3+ regulatory T cell populations. In a culture of CD3/CD28-stimulated human peripheral blood mononuclear cells with BM-MSC-derived EVs, CD3+ T cell activation was suppressed. However, these cells were not suppressed in cultures with EVs derived from normal human dermal fibroblasts (NHDFs). NHDF-derived EVs did not ameliorate the clinical or pathological characteristics of aGVHD in mice, suggesting an immunoregulatory function unique to BM-MSC-derived EVs. Microarray analysis of microRNAs in BM-MSC-derived EVs versus NHDF-derived EVs showed upregulation of miR-125a-3p and downregulation of cell proliferative processes, as identified by Gene Ontology enrichment analysis. Collectively, our findings provide the first evidence that amelioration of aGVHD by therapeutic infusion of BM-MSC-derived EVs is associated with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of BM-MSC-derived EVs. Stem Cells 2018;36:434-445.
Asunto(s)
Enfermedad Injerto contra Huésped/metabolismo , Células Madre Mesenquimatosas/metabolismo , Envejecimiento/fisiología , Línea Celular , Proliferación Celular/fisiología , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Trasplante de Células Madre Mesenquimatosas , MicroARNs/metabolismoRESUMEN
The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-ß/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-ß/Smad pathway activation.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/genética , Proteína Smad2/genética , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/genética , Animales , Biomarcadores de Tumor/metabolismo , Hipoxia de la Célula , Línea Celular Transformada , Línea Celular Tumoral , Femenino , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos NOD , Mieloma Múltiple/metabolismo , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Trasplante de Neoplasias , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Células Madre/patología , Análisis de Supervivencia , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Under stress conditions such as infection, inflammation, and hematopoietic recovery following chemotherapy or transplantation, the hematopoietic system is required to meet the increasing demands, especially from myeloid cells. Therefore, an understanding of the molecular mechanism underlying stress hematopoiesis is clinically imperative. We previously showed that C/EBPß, which is a transcription factor required for emergency granulopoiesis, plays a pivotal role at the level of hematopoietic stem/progenitor cells under stress conditions. Upon exposure to stress, the C/EBPß protein is upregulated in the hematopoietic stem cells. A close examination of C/EBPß knockout mice revealed that C/EBPß regulates the proliferation and differentiation of hematopoietic stem cells at the cost of the self-renewing activity. Further elucidation of the functions and regulation of C/EBPß in hematopoietic stem cells will facilitate an understanding of stress hematopoiesis.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular , Proliferación Celular , Granulocitos , Células Madre Hematopoyéticas/citología , Animales , Hematopoyesis , Ratones , Ratones NoqueadosRESUMEN
Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34(+) hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Animales , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Quimiotaxis/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Steady-state hematopoiesis responds to extracellular stimuli to meet changing demands and also to pathologically altered intracellular signaling. Granulocyte production increases following infection or in response to cytokine stimulation, and activation of the CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for such stress-induced granulopoiesis, whereas C/EBPα plays a critical role in maintaining steady-state granulopoiesis. Different roles of these C/EBP transcription factors in different modes of hematopoiesis are evolutionally conserved from zebrafish to humans. In addition to reactions against infections, C/EBPß is responsible for cancer-driven myelopoiesis, which promotes cancer progression, at least in part, by abrogating the immune response in the cancer microenvironment. The BCR-ABL fusion protein activates emergency-specific pathway of granulopoiesis by upregulating C/EBPß. This in turn causes chronic phase chronic myeloid leukemia, which is characterized by myeloid expansion. The C/EBPß transcription factor also plays a role in other hematological malignancies of both myeloid and lymphoid lineage origin. Thus, elucidation of the upstream and downstream networks surrounding C/EBPß will lead to the development of novel therapeutic strategies for diseases mediated by non-steady-state hematopoiesis.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Hematopoyesis , Animales , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Neutropenia/congénito , Neutropenia/genética , Neutropenia/metabolismoRESUMEN
γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. γδT cells expanded ex vivo by nitrogen-containing bisphosphonate (N-BP) treatment can kill tumor cells. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for γδT cells. We have previously observed that as they get closer, migrating γδT cells increase in speed toward target multiple myeloma (MM) cells. In the present study, we investigated the γδT cell chemotactic factors involving using a micro total analysis system-based microfluidic cellular analysis device. The addition of supernatant from RPMI8226 MM cells treated with the N-BP zoledronic acid (ZOL) or the addition of IPP to the device induced chemotaxis of γδT cells and increased the speed of migration compared to controls. Analysis of the ZOL-treated RPMI8226 cell supernatant revealed that it contained IPP secreted in a ZOL-dose-dependent manner. These observations indicate that IPP activates the chemotaxis of γδT cells toward target MM cells treated with ZOL.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Difosfonatos/farmacología , Hemiterpenos/farmacología , Imidazoles/farmacología , Mieloma Múltiple/metabolismo , Compuestos Organofosforados/farmacología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/efectos de los fármacos , Línea Celular Tumoral , Medios de Cultivo Condicionados , Hemiterpenos/metabolismo , Humanos , Mieloma Múltiple/patología , Linfocitos T/inmunología , Ácido ZoledrónicoRESUMEN
The CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb(-/-) mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb(-/-) bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb(-/-) mice. These results indicate that C/EBPß is required for the survival of monocytes in peripheral blood.
Asunto(s)
Apoptosis/fisiología , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Monocitos/citología , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Recuento de Linfocitos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
The transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) regulates the differentiation of a variety of cell types. Here, the role of C/EBPß expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B-cell lymphopoiesis was examined. The size of the precursor B-cell population in bone marrow was reduced in C/EBPß-knockout (KO) mice. When bone marrow cells from C/EBPß-KO mice were transplanted into lethally irradiated wild-type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B-cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPß-KO mice did not support the differentiation of WT c-Kit(+) Sca-1(+) Lineage(-) hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell-derived factor-1 by the cocultured C/EBPß-deficient BMMSCs. The ability of C/EBPß-deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPß-deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPß expressed by BMMSCs plays a crucial role in early B-cell lymphopoiesis.
Asunto(s)
Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Linfopoyesis , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Linfocitos B/patología , Proteína beta Potenciadora de Unión a CCAAT/deficiencia , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Microambiente Celular , Quimiocina CXCL12/biosíntesis , Técnicas de Cocultivo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Leucemia de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis , Células Precursoras de Linfocitos B/citologíaRESUMEN
Parathyroid hormone (PTH) stimulates hematopoiesis in mouse models. The involvement of osteoblasts in this process has been well investigated; however, the effects of PTH on human hematopoiesis and bone marrow mesenchymal stromal cells (BM-MSCs) are unclear. Here, we show that BM-MSCs contribute to the hematopoiesis-stimulating effects of PTH via upregulation of cadherin-11 (CDH11). When culture-expanded human BM-MSCs were stimulated with PTH, their ability to expand cocultured CD34(+) hematopoietic progenitor cells (HPCs) was enhanced. Furthermore, when PTH-treated BM-MSCs were subcutaneously implanted into NOD/SCID mice, the induction of hematopoietic cells was enhanced. Culture-expanded human BM-MSCs expressed CDH11, and the level of CDH11 expression increased following PTH stimulation. Depletion of CDH11 expression in BM-MSCs using small interfering RNA abolished the enhancement of HPC expansion by PTH-treated BM-MSCs. In lethally irradiated mice that underwent BM transplantation, CDH11 expression in BM-MSCs was higher and survival was better in PTH-treated mice than in control mice. The number of hematopoietic cells in BM and the number of red blood cells in peripheral blood were higher in PTH-treated mice than in control mice. Our results demonstrate that PTH stimulates hematopoiesis through promoting the upregulation of CDH11 expression in BM-MSCs, at least in part. PTH treatment may be an effective strategy to enhance the ability of BM-MSCs to support hematopoiesis.
Asunto(s)
Células de la Médula Ósea/metabolismo , Cadherinas/metabolismo , Hematopoyesis/fisiología , Células Madre Mesenquimatosas/metabolismo , Hormona Paratiroidea/metabolismo , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Femenino , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Xenoinjertos , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. The role of MDSCs in autoimmune diseases remains controversial, and little is known about the function of MDSCs in autoimmune arthritis. In this study, we clarify that MDSCs play crucial roles in the regulation of proinflammatory immune response in a collagen-induced arthritis (CIA) mouse model. MDSCs accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited the proliferation of CD4(+) T cells and their differentiation into Th17 cells in vitro. Moreover, MDSCs inhibited the production of IFN-γ, IL-2, TNF-α, and IL-6 by CD4(+) T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced the severity of CIA in vivo, which was accompanied by a decrease in the number of CD4(+) T cells and Th17 cells in the draining lymph nodes. However, depletion of MDSCs abrogated the spontaneous improvement of CIA. In conclusion, MDSCs in CIA suppress the progression of CIA by inhibiting the proinflammatory immune response of CD4(+) T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis.
Asunto(s)
Artritis Experimental/inmunología , Linfocitos T CD4-Positivos/inmunología , Inflamación/inmunología , Células Mieloides/inmunología , Traslado Adoptivo , Animales , Artritis Experimental/terapia , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Proliferación Celular , Colágeno , Interferón gamma/biosíntesis , Interleucina-1/biosíntesis , Interleucina-2/biosíntesis , Interleucina-6/biosíntesis , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos DBA , Células Mieloides/metabolismo , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND & AIMS: Loss of the tumor suppressor SMAD4 correlates with progression of colorectal cancer (CRC). In mice, colon tumors that express CCL9 recruit CCR1(+) myeloid cells, which facilitate tumor invasion and metastasis by secreting matrix metalloproteinase 9. METHODS: We used human CRC cell lines to investigate the ability of SMAD4 to regulate expression of CCL15, a human ortholog of mouse CCL9. We used immunohistochemistry to compare levels of CCL15 and other proteins in 141 samples of human liver metastases. RESULTS: In human CRC cell lines, knockdown of SMAD4 increased CCL15 expression, and overexpression of SMAD4 decreased it. SMAD4 bound directly to the promoter region of the CCL15 gene to negatively regulate its expression; transforming growth factor-ß increased binding of SMAD4 to the CCL15 promoter and transcriptional repression. In livers of nude mice, SMAD4-deficient human CRC cells up-regulated CCL15 to recruit CCR1(+) cells and promote metastasis. In human tumor samples, there was a strong inverse correlation between levels of CCL15 and SMAD4; metastases that expressed CCL15 contained 3-fold more CCR1(+) cells than those without CCL15. Patients with CCL15-expressing metastases had significantly shorter times of disease-free survival than those with CCL15-negative metastases. CCR1(+) cells in the metastases expressed the myeloid cell markers CD11b and myeloperoxidase, and also matrix metalloproteinase 9. CONCLUSIONS: In human CRC cells, loss of SMAD4 leads to up-regulation of CCL15 expression. Human liver metastases that express CCL15 contain higher numbers CCR1(+) cells; patients with these metastases have shorter times of disease-free survival. Reagents designed to block CCL15 recruitment of CCR1(+) cells could prevent metastasis of CRC to liver.
Asunto(s)
Adenocarcinoma/metabolismo , Quimiocinas CC/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Células Mieloides/patología , Receptores CCR1/metabolismo , Proteína Smad4/deficiencia , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígeno CD11b/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Xenoinjertos , Humanos , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células Mieloides/metabolismo , Metástasis de la Neoplasia/fisiopatología , Metástasis de la Neoplasia/prevención & control , Peroxidasa/metabolismo , Estudios Retrospectivos , Proteína Smad4/efectos de los fármacos , Proteína Smad4/genética , Tasa de SupervivenciaRESUMEN
HAX1 was identified as the gene responsible for the autosomal recessive type of severe congenital neutropenia. However, the connection between mutations in the HAX1 gene and defective granulopoiesis in this disease has remained unclear, mainly due to the lack of a useful experimental model for this disease. In this study, we generated induced pluripotent stem cell lines from a patient presenting for severe congenital neutropenia with HAX1 gene deficiency, and analyzed their in vitro neutrophil differentiation potential by using a novel serum- and feeder-free directed differentiation culture system. Cytostaining and flow cytometric analyses of myeloid cells differentiated from patient-derived induced pluripotent stem cells showed arrest at the myeloid progenitor stage and apoptotic predisposition, both of which replicated abnormal granulopoiesis. Moreover, lentiviral transduction of the HAX1 cDNA into patient-derived induced pluripotent stem cells reversed disease-related abnormal granulopoiesis. This in vitro neutrophil differentiation system, which uses patient-derived induced pluripotent stem cells for disease investigation, may serve as a novel experimental model and a platform for high-throughput screening of drugs for various congenital neutrophil disorders in the future.