RESUMO
Macrophages are one of the first innate immune cells to reach the site of infection or injury. Diverse functions from the uptake of pathogen or antigen, its killing, and presentation, the release of pro- or anti-inflammatory cytokines, activation of adaptive immune cells, clearing off tissue debris, tissue repair, and maintenance of tissue homeostasis have been attributed to macrophages. Besides tissue-resident macrophages, the circulating macrophages are recruited to different tissues to get activated. These are highly plastic cells, showing a spectrum of phenotypes depending on the stimulus received from their immediate environment. The macrophage differentiation requires colony-stimulating factor-1 (CSF-1) or macrophage colony-stimulating factor (M-CSF), colony-stimulating factor-2 (CSF-2), or granulocyte-macrophage colony-stimulating factor (GM-CSF) and different stimuli activate them to different phenotypes. The richness of tissue macrophages is precisely controlled via the CSF-1 and CSF-1R axis. In this review, we have given an overview of macrophage origin via hematopoiesis/myelopoiesis, different phenotypes associated with macrophages, their clinical significance, and how they are altered in various diseases. We have specifically focused on the function of CSF-1/CSF-1R signaling in deciding macrophage fate and the outcome of aberrant CSF-1R signaling in relation to macrophage phenotype in different diseases. We further extend the review to briefly discuss the possible strategies to manipulate CSF-1R and its signaling with the recent updates.
Assuntos
Relevância Clínica , Fator Estimulador de Colônias de Macrófagos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos , Transdução de Sinais , CitocinasRESUMO
The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).
Assuntos
Colecalciferol/farmacologia , Células Dendríticas/imunologia , Glicólise/fisiologia , Tolerância Imunológica/genética , Leucócitos Mononucleares/imunologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Monócitos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Células Dendríticas/efeitos dos fármacos , Glucose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Interleucinas/farmacologia , Lactatos/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Chronic pain is a debilitating condition that often emerges as a clinical symptom of inflammatory diseases. It has therefore been widely accepted that the immune system critically contributes to the pathology of chronic pain. Microglia, a type of immune cell in the central nervous system, has attracted researchers' attention because in rodent models of neuropathic pain that develop strong mechanical and thermal hypersensitivity, histologically activated microglia are seen in the dorsal horn of spinal cord. Several kinds of cytokines are generated by damaged peripheral neurons and contribute to microglial activation at the distal site of the injury where damaged neurons send their projections. Microglia are known as key players in the surveillance of the local environment in the central nervous system and have a significant role of circuit remodeling by physical contact to synapses. Key molecules for the pathology of neuropathic pain exist in the activated microglia, but the factors driving pain-inducible microglial activation remain unclear. Therefore, to find the key molecules inducing activation of spinal microglia and to figure out the precise mechanism of how microglia modulate neuronal circuits in the spinal cord to form chronic pain state is a critical step for developing effective treatment of neuropathic pain.
Assuntos
Comunicação Celular , Microglia/fisiologia , Neuralgia/etiologia , Neurônios/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Proliferação de Células , Quimiocinas/fisiologia , Dor Crônica/etiologia , Humanos , Fator Estimulador de Colônias de Macrófagos/fisiologiaRESUMO
BACKGROUND: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II- dendritic-like 'L-DC', and CD11b+CD11c+MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished. RESULTS: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL-10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDC-like cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma. CONCLUSION: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF.
Assuntos
Células Dendríticas/imunologia , Hematopoese , Ativação Linfocitária , Fator Estimulador de Colônias de Macrófagos/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Feminino , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Células Estromais/imunologia , Linfócitos T Reguladores/imunologia , TranscriptomaRESUMO
Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αß T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αß T cells have declined.
Assuntos
Fator Estimulador de Colônias de Macrófagos/fisiologia , Malária/prevenção & controle , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Feminino , Humanos , Ativação Linfocitária , Malária/imunologia , Camundongos , Parasitemia/prevenção & controle , RecidivaRESUMO
Myelopoiesis is necessary for the generation of mature myeloid cells during homeostatic turnover and immunological insults; however, the metabolic requirements for this process remain poorly defined. Here, we demonstrate that myelopoiesis, including monocyte and macrophage differentiation, requires mechanistic target of rapamycin complex 1 (mTORC1) signaling and anabolic metabolism. Loss of mTORC1 impaired myelopoiesis under steady state and dampened innate immune responses against Listeria monocytogenes infection. Stimulation of hematopoietic progenitors with macrophage colony-stimulating factor (M-CSF) resulted in mTORC1-dependent anabolic metabolism, which in turn promoted expression of M-CSF receptor and transcription factors PU.1 and IRF8, thereby constituting a feed-forward loop for myelopoiesis. Mechanistically, mTORC1 engaged glucose metabolism and initiated a transcriptional program involving Myc activation and sterol biosynthesis after M-CSF stimulation. Perturbation of glucose metabolism or disruption of Myc function or sterol biosynthesis impaired myeloid differentiation. Integrative metabolomic and genomic profiling further identified one-carbon metabolism as a central node in mTORC1-dependent myelopoiesis. Therefore, the interplay between mTORC1 signaling and metabolic reprogramming underlies M-CSF-induced myelopoiesis.
Assuntos
Fator Estimulador de Colônias de Macrófagos/fisiologia , Complexos Multiproteicos/fisiologia , Mielopoese/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Transporte/fisiologia , Técnicas de Introdução de Genes , Glucose/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Transdução de Sinais/fisiologiaRESUMO
Objective: To investigate the key cytokine which polarizes M2 macrophages and promotes invasion and metastasis in non-small cell lung cancer (NSCLC). Methods: After co-culture with A549 cells in vitro, the proportion of CD14(+) CD163(+) M2 macrophages in monocytes and macrophage colony stimulating factor (M-CSF) levels in culture supernatant were detected by flow cytometry, ELISA assay and real-time qPCR, respectively. The effects of CD14(+) CD163(+) M2 macrophages on invasion of A549 cells and angiogenesis of HUVEC cells were measured by transwell assay and tubule formation assay, respectively. The clinical and prognostic significance of M-CSF expression in NSCLC was further analyzed. Results: The percentage of CD14(+) CD163(+) M2 macrophages in monocytes and the concentration of M-CSF in the supernatant followed by co-culture was (12.03±0.46)% and (299.80±73.76)pg/ml, respectively, which were significantly higher than those in control group [(2.80±1.04)% and (43.07±11.22)pg/ml, respectively, P< 0.05]. Human recombinant M-CSF promoted M2 polarization of macrophages in vitro. M2 macrophages enhanced the invasion of A549 cells (66 cells/field vs. 26 cells/field) and the angiogenesis of HUVEC cells (22 tubes/field vs. 8 tubes/field). The mRNA expression of M-CSF in stage â -â ¡ patients (16.23±4.83) was significantly lower than that in stage â ¢-â £ (53.84±16.08; P<0.05). M-CSF levels were associated with poorer overall survival and disease-free survival in NSCLC patients (P<0.05). Conclusions: Tumor-derived M-CSF can induce CD14(+) CD163(+) M2 polarization of macrophages, which can further promote the metastasis and angiogenesis of NSCLC. M-CSF could be used as a potential therapeutic target of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Polaridade Celular , Neoplasias Pulmonares/patologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/secundário , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/análise , Intervalo Livre de Doença , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Fator Estimulador de Colônias de Macrófagos/análise , Macrófagos/patologia , Monócitos/patologia , Invasividade Neoplásica , Neovascularização PatológicaRESUMO
Controlled regulation of lineage decisions is imperative for hematopoiesis. Yet, the molecular mechanisms underlying hematopoietic lineage choices are poorly defined. Colony-stimulating factor 1 (CSF-1), the cytokine acting as the principal regulator of monocyte/macrophage (M) development, has been shown to be able to instruct the lineage choice of uncommitted granulocyte M (GM) progenitors toward an M fate. However, the intracellular signaling pathways involved are unknown. CSF-1 activates a multitude of signaling pathways resulting in a pleiotropic cellular response. The precise role of individual pathways within this complex and redundant signaling network is dependent on cellular context, and is not well understood. Here, we address which CSF-1-activated pathways are involved in transmitting the lineage-instructive signal in primary bone marrow-derived GM progenitors. Although its loss is compensated for by alternative signaling activation mechanisms, Src family kinase (SFK) signaling is sufficient to transmit the CSF-1 lineage instructive signal. Moreover, c-Src activity is sufficient to drive M fate, even in nonmyeloid cells.
Assuntos
Linhagem da Célula , Fator Estimulador de Colônias de Macrófagos/fisiologia , Monócitos/citologia , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Células Precursoras de Granulócitos/citologia , Hematopoese , CamundongosRESUMO
Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte-macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Células MCF-7/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células MCF-7/metabolismoRESUMO
M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved.
Assuntos
Linhagem da Célula/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Células Mieloides/imunologia , Animais , HumanosRESUMO
During human pregnancy, immune tolerance of the fetal semiallograft occurs in the presence of abundant maternal leukocytes. At the implantation site, macrophages comprise approximately 20% of the leukocyte population and act as primary mediators of tissue remodeling. Decidual macrophages display a balance between anti-inflammatory and proinflammatory phenotypes. However, a shift to an M1 subtype is reported in preeclampsia. Granulocyte-macrophage colony-stimulating-factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are major differentiating factors that mediate M1 and M2 polarization, respectively. Previously, we observed the following: i) the preeclamptic decidua contains an excess of both macrophages and GM-CSF, ii) the preeclampsia-associated proinflammatory cytokines, IL-1ß and tumor necrosis factor-α, markedly enhance GM-CSF and M-CSF expression in cultured leukocyte-free first-trimester decidual cells (FTDCs), iii) FTDC-secreted GM-CSF polarizes macrophages toward an M1 subtype. The microenvironment is a key determinant of macrophage phenotype. Thus, we examined proinflammatory stimulation of FTDC-secreted M-CSF and its role in macrophage development. Immunofluorescence staining demonstrated elevated M-CSF-positive decidual cell numbers in preeclamptic decidua. In FTDCs, IL-1ß and tumor necrosis factor-α signal through the NF-κB pathway to induce M-CSF production, which does the following: i) enhances differentiation of and elevates CD163 expression in macrophages, ii) increases macrophage phagocytic capacity, and iii) inhibits signal-regulatory protein α expression by macrophages. These findings suggest that FTDC-secreted M-CSF modulates the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to proinflammatory stimuli.
Assuntos
Decídua/imunologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Pré-Eclâmpsia/imunologia , Antígenos de Diferenciação/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Decídua/metabolismo , Feminino , Humanos , Interleucina-1beta/fisiologia , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/metabolismo , NF-kappa B/metabolismo , Fagocitose/imunologia , Gravidez , Primeiro Trimestre da Gravidez , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
PURPOSE: The peripheral giant cell lesion (PGCL) is a reactive process associated with a local irritating factor that shows low recurrence after treatment, especially if the irritating factor is eliminated. In contrast, the central giant cell lesion (CGCL) presents variable clinical behavior ranging from slow and asymptomatic growth without recurrence to rapid, painful, and recurrent growth. The immunoexpression of glucose transporter (GLUT)-1, GLUT-3, and macrophage colony-stimulating factor (M-CSF) was compared in CGCL and PGCL. MATERIALS AND METHODS: Twenty nonaggressive CGCLs, 20 aggressive CGCLs, and 20 PGCLs were selected for analysis of the immunoexpression of GLUT-1, GLUT-3, and M-CSF in multinuclear giant cells (MGCs) and mononuclear cells (MCs). RESULTS: There was a difference in the percentage of immunoreactive cells of GLUT-1 and GLUT-3 in MC components among lesions and in the intensity of GLUT-1 in MCG and MC components, GLUT-3 in MGC components, and M-CSF in MC components. CONCLUSIONS: These results suggest that GLUT-1, GLUT-3, and M-CSF could play a role in the pathogenesis of the lesions studied. The stronger immunostaining of these proteins in MCs shows that these cells have greater metabolic activity and osteoclastogenesis, especially in aggressive CGCL. The MCs showed a stronger relation than the MGCs to the pathogenesis of the studied lesions.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Granuloma de Células Gigantes/metabolismo , Doenças Maxilomandibulares/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Células Gigantes/metabolismo , Transportador de Glucose Tipo 1/fisiologia , Transportador de Glucose Tipo 3/fisiologia , Granuloma de Células Gigantes/patologia , Humanos , Doenças Maxilomandibulares/patologia , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/fisiologiaRESUMO
Patients of Alzheimer's disease (AD) frequently have lower bone mineral density and higher rate of hip fracture. Tg2576, a well characterized AD animal model that ubiquitously express Swedish mutant amyloid precursor protein (APPswe), displays not only AD-relevant neuropathology, but also age-dependent bone deficits. However, the underlying mechanisms remain poorly understood. As APP is implicated as a regulator of iron export, and the metal chelation is considered as a potential therapeutic strategy for AD, we examined iron chelation's effect on the osteoporotic deficit in Tg2576 mice. Remarkably, in vivo treatment with iron chelator, clinoquinol (CQ), increased both trabecular and cortical bone-mass, selectively in Tg2576, but not wild type (WT) mice. Further in vitro studies showed that low concentrations of CQ as well as deferoxamine (DFO), another iron chelator, selectively inhibited osteoclast (OC) differentiation, without an obvious effect on osteoblast (OB) differentiation. Intriguingly, both CQ and DFO's inhibitory effect on OC was more potent in bone marrow macrophages (BMMs) from Tg2576 mice than that of wild type controls. The reduction of intracellular iron levels in BMMs by CQ was also more dramatic in APPswe-expressing BMMs. Taken together, these results demonstrate a potent inhibition on OC formation and activation in APPswe-expressing BMMs by iron chelation, and reveal a potential therapeutic value of CQ in treating AD-associated osteoporotic deficits.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Clioquinol/uso terapêutico , Quelantes de Ferro/uso terapêutico , Osteoclastos/fisiologia , Doença de Alzheimer/patologia , Animais , Reabsorção Óssea , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Clioquinol/farmacologia , Avaliação Pré-Clínica de Medicamentos , Quelantes de Ferro/farmacologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Ligante RANK/fisiologiaRESUMO
The purpose of this study was to evaluate the presence of M2-polarized macrophages and their relationships to angiogenesis in keratocystic odontogenic tumor (KCOT). M2-polarized macrophages were detected in KCOT samples by immunohistochemistry and immunofluorescence. Meanwhile, microvessel density measured with antibody against CD31 was closely correlated with the presence of M2-polarized macrophages. In addition, macrophage colony-stimulating factor (M-CSF) significantly contributed to the activation of M2-polarized macrophages. Moreover, the results of in vitro wound healing, cell migration and tube formation assays further revealed the pro-angiogenic function of M2-polarized macrophage-like cells. This function might be associated with secretion of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß) and matrix metalloprotein-9 (MMP-9). This study demonstrates for the first time that M2-polarized macrophages are prevalent in KCOT, and their presence is dependent on M-CSF expression. More importantly, these tumor-supportive cells can also promote tumor angiogenesis by secreting angiogenic cytokines.
Assuntos
Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Neovascularização Patológica , Tumores Odontogênicos/irrigação sanguínea , Citocinas/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Tumores Odontogênicos/fisiopatologiaRESUMO
Established from the calvaria of newborn macrophage colony-stimulating factor (M-CSF)-deficient mice, OP9 is a stromal cell line that used as a feeder layer to support the in vitro differentiation of pluripotent stem cells into various hematopoietic lineage cells, including granulocytes, erythrocytes, lymphocytes, and megakaryocytes. However, as a primary culture cell line, OP9 can be used as stromal cells for only 1 month. Therefore, to obtain functional OP9 cells, numerous M-CSF-deficient newborn mice must be sacrificed. These limitations in some ways restrict the application of OP9 cells in longterm and largescale experiments. In this study, we used human papillomavirus 16 E6 and E7 genes to generate immortalized OP9 stromal cells, designated I-OP9 cells, and then tested their ability to support the megakaryocytic differentiation of pluripotent stem cells in vitro. I-OP9 cells have similar morphology and properties as do parental OP9 cells, and, as expected, have an extended lifespan and can support megakaryocytic differentiation. Our data suggest that the method used in this study, including establishing I-OP9 cells, enables the possibility to enlarge and lengthen the scale of the experiment and, more critically, provides a humanistic approach for preparing stromal cells that support the hematopoietic differentiation of pluripotent stem cells in vitro.
Assuntos
Diferenciação Celular , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Megacariócitos/citologia , Células-Tronco Embrionárias Murinas/citologia , Células Estromais/citologia , Animais , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Embrião de Mamíferos/metabolismo , Citometria de Fluxo , Imunofluorescência , Hematopoese , Humanos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Masculino , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismoRESUMO
The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid-dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow-derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor-mediated signaling by amino acids. Therefore, the amino acid-laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.
Assuntos
Aminoácidos/metabolismo , Fator Estimulador de Colônias de Macrófagos/fisiologia , Complexos Multiproteicos/fisiologia , Pinocitose , Fator de Crescimento Derivado de Plaquetas/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Transporte Biológico , Endossomos/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Cytokines and metabolic pathway-controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8+CD45RClo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ and CD4+ Tregs. IL-34 was involved in the suppressive function of both CD8+ and CD4+ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8+ and CD4+ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8+ and CD4+ FOXP3+ Tregs, with a superior suppressive potential of antidonor immune responses compared with non-IL-34-expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg-specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.
Assuntos
Interleucinas/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante , Transferência Adotiva , Aloenxertos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dependovirus/genética , Fatores de Transcrição Forkhead/análise , Vetores Genéticos , Células HEK293 , Transplante de Coração , Humanos , Interleucinas/biossíntese , Interleucinas/genética , Interleucinas/farmacologia , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos Lew , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Proteínas Recombinantes de Fusão/genética , Análise Serial de Tecidos , Transdução GenéticaRESUMO
Reactive oxygen species (ROS) play a central role in estrogen deficiency-induced bone loss. We previously identified and characterized a novel member of the Peroxiredoxin (PRX) like 2 family that we called PAMM: Peroxiredoxin Activated in M-CSF stimulated Monocytes, a redox regulatory protein that modulates osteoclast differentiation in vitro. In this study, we report increased PAMM expression in H2O2-treated cells and in bones from ovariectomized (OVX) mice 4 weeks after surgery, models for oxidative stress in vitro and in vivo, respectively. We also detected increased PAMM abundance and phosphorylated Akt in OVX mice treated with estrogen. In addition, Wortmannin, a specific PI3Kinase inhibitor and Rapamycin, an inhibitor of the PI3Kinase/Akt pathway, blocked Akt phosphorylation and stimulation of PAMM expression by M-CSF. These results indicate that M-CSF-induced PAMM expression is mediated by Akt phosphorylation. Our data also suggest that estrogen-induced PAMM expression is mediated by phosphorylation of Akt. These findings point to PAMM as a potential candidate for Akt-mediated protection against oxidative stress.
Assuntos
Estresse Oxidativo , Peroxirredoxinas/metabolismo , Animais , Antioxidantes/metabolismo , Densidade Óssea , Células Cultivadas , Estrogênios/fisiologia , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/fisiologia , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Osteoclastos/metabolismo , Ovariectomia , Oxirredução , Peroxirredoxinas/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação TranscricionalRESUMO
Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucosylceramidase (GCase). Deficiency in GCase leads to characteristic visceral pathology and lethal neurological manifestations in some patients. Investigations into neurogenesis have suggested that neurodegenerative disorders, such as GD, could be overcome or at least ameliorated by the generation of new neurons. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are potential candidates for use in the treatment of neurodegenerative disorders because of their ability to promote neurogenesis. Our objective was to examine the mechanism of neurogenesis by BM-MSCs in GD. We found that neural stem cells (NSCs) derived from a neuronopathic GD model exhibited decreased ability for self-renewal and neuronal differentiation. Co-culture of GBA-deficient NSCs with BM-MSCs resulted in an enhanced capacity for self-renewal, and an increased ability for differentiation into neurons or oligodendrocytes. Enhanced proliferation and neuronal differentiation of GBA-deficient NSCs was associated with elevated release of macrophage colony-stimulating factor (M-CSF) from BM-MSCs. Our findings suggest that soluble M-CSF derived from BM-MSCs can modulate GBA-deficient NSCs, resulting in their improved proliferation and neuronal differentiation.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Fator Estimulador de Colônias de Macrófagos/farmacologia , Células-Tronco Neurais/fisiologia , Animais , Células Cultivadas , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase , Glucosilceramidas/metabolismo , Fator Estimulador de Colônias de Macrófagos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Comunicação ParácrinaRESUMO
Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP(+) osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11-7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.