RESUMO
Migratory macrophages play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. Whole-transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in the expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60-86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix-degrading capacity. IL-4 further increased motility and matrix-degrading capacity in mature macrophages, with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of the core adhesion proteins paxillin and leupaxin decreased macrophage spreading and the number of adhesions, with distinct effects on adhesion and their distribution, and on matrix degradation. Together, the datasets provide an important resource to increase our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages.
Assuntos
Fator Estimulador de Colônias de Macrófagos , Macrófagos , Animais , Adesão Celular/genética , Movimento Celular/genética , Expressão Gênica , Fator Estimulador de Colônias de Macrófagos/genética , CamundongosRESUMO
The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.
Assuntos
Células da Medula Óssea/fisiologia , Macrófagos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Anticorpos Bloqueadores/metabolismo , Diferenciação Celular , Movimento Celular/genética , Células Cultivadas , Quimiocina CCL2/metabolismo , Feminino , Lipopolissacarídeos/imunologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Quimera por Radiação , Análise Serial de Tecidos , Raios Ultravioleta/efeitos adversosRESUMO
A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice.
Assuntos
Células da Medula Óssea/citologia , Movimento Celular/efeitos da radiação , Células Dendríticas/citologia , Glicólise/fisiologia , Pele/efeitos da radiação , Raios Ultravioleta , Animais , Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Glucose/metabolismo , Ácido Láctico/metabolismo , Camundongos , Pele/metabolismoRESUMO
Colony-stimulating factor-1 (CSF-1), also known as macrophage-colony stimulating factor (M-CSF), is the primary growth factor regulating survival, proliferation and differentiation of macrophages. It is also a potent chemokine for macrophages and monocytes. Signaling via the CSF-1 receptor (CSF-1R) is necessary for the production of almost all tissue resident macrophage populations and these macrophages participate, via trophic mechanisms, in the normal development and homeostasis of tissues and organs in which they reside, including the mammary gland. The drawback of this close interaction between macrophages and parenchymal cells is that dysregulation of macrophage trophic functions assists in the development and progression of many cancers, including breast cancer. Furthermore, tumour cells secrete CSF-1 to attract more macrophages to the tumour microenvironment where CSF-1R signaling frequently drives the behaviour of these tumour-associated macrophages (TAMs) to promote tumour progression and metastasis. Evidence is mounting that treated tumours secrete more CSF-1 and the increased recruitment of TAMs limits treatment efficacy. Thus, therapeutic targeting of the CSF-1R to inhibit TAM function is likely to enhance tumour response and improve patient outcomes in the treatment of cancer, including breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias Mamárias Animais/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Progressão da Doença , Feminino , Humanos , Macrófagos/metabolismoRESUMO
Colony-stimulating factor-1 (CSF-1)-stimulated CSF-1 receptor (CSF-1R) tyrosine phosphorylation initiates survival, proliferation, and differentiation signaling pathways in macrophages. Either activation loop Y807F or juxtamembrane domain (JMD) Y559F mutations severely compromise CSF-1-regulated proliferation and differentiation. YEF, a CSF-1R in which all eight tyrosines phosphorylated in the activated receptor were mutated to phenylalanine, lacks in vitro kinase activity and in vivo CSF-1-regulated tyrosine phosphorylation. The addition of Tyr-807 alone to the YEF backbone (Y807AB) led to CSF-1-independent but receptor kinase-dependent proliferation, without detectable activation loop Tyr-807 phosphorylation. The addition of Tyr-559 alone (Y559AB) supported a low level of CSF-1-independent proliferation that was slightly enhanced by CSF-1, indicating that Tyr-559 has a positive Tyr-807-independent effect. Consistent with the postulated autoinhibitory role of the JMD Tyr-559 and its relief by ligand-induced Tyr-559 phosphorylation, the addition of Tyr-559 to the Y807AB background suppressed proliferation in the absence of CSF-1, but restored most of the CSF-1-stimulated proliferation. Full restoration of kinase activation and proliferation required the additional add back of JMD Tyr-544. Inhibitor experiments indicate that the constitutive proliferation of Y807AB macrophages is mediated by the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways, whereas proliferation of WT and Y559,807AB macrophages is, in addition, contributed to by Src family kinase (SFK)-dependent pathways. Thus Tyr-807 confers sufficient kinase activity for strong CSF-1-independent proliferation, whereas Tyr-559 maintains the receptor in an inactive state. Tyr-559 phosphorylation releases this restraint and may also contribute to the CSF-1-regulated proliferative response by activating Src family kinase.
Assuntos
Regulação da Expressão Gênica , Macrófagos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Tirosina/química , Animais , Proliferação de Células , Sobrevivência Celular , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. Mâ»/â».Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of Mâ»/â».Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P3 production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Adesão Celular , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Invasividade Neoplásica , Paxilina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Ratos , Transdução de SinaisRESUMO
Colony stimulating factor-1 (CSF-1, also known as macrophage-colony stimulating factor, M-CSF) has long been known as the primary growth factor regulating survival, proliferation and differentiation of macrophages and other mononuclear phagocytic (MNP) lineage cells. CSF-1 was subsequently identified as a monocyte/macrophage chemokine, a capacity now recognized to be integral to many of the deleterious as well as positive roles of macrophages in development, homeostasis and disease. The pleiotrophic actions of CSF-1 are all transduced by its high affinity receptor, the CSF-1R, a receptor tyrosine kinase (RTK) and the cellular homologue of the v-fms oncoprotein. While the CSF-1R is the sole receptor for CSF-1, an alternative functional ligand for the receptor, interleukin-34 (IL-34), was recently identified. CSF-1-induced CSF-1R activation triggers autophosphorylation of several intracellular tyrosine residues, leading to initiation of an array of phosphotyrosine-based signaling cascades that mediate the wide variety of cellular responses to CSF-1. Dissecting the contributions of the different phosphorylated tyrosine motifs of the receptor to downstream signaling events in macrophages is not only important for our understanding of CSF-1R function, but also for the development of inhibitors to treat diseases where infiltrating macrophages contribute to their progression. This review will outline our current understanding of the CSF-1/CSF-1R signaling axis and describe how a novel macrophage cell line system, which allows examination of CSF-1R signaling in a mature macrophage context, is helping us to tease apart the diverse signaling pathways initiated by CSF-1R activation.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Doenças Autoimunes/patologia , Diferenciação Celular , Humanos , Inflamação/patologia , Macrófagos/citologia , Neoplasias/patologiaRESUMO
Increased cancer incidence occurs with the emergence of immunosenescence, highlighting the indispensability of the immune system in preventing cancer and its dysregulation with aging. Tumor-associated macrophages (TAMs) are often present in high numbers and are associated with poor clinical outcomes in solid cancers, including mesothelioma. Monocytes and macrophages from the bone marrow and spleen can respond to tumor-derived factors, such as CSF-1, and initiation of the CSF-1R signaling cascade results in their proliferation, differentiation, and migration to the tumor. Age-related changes occur in monocytes and macrophages in terms of numbers and function, which in turn can impact tumor initiation and progression. Whether this is due to changes in CSF-1R expression with aging is currently unknown and was investigated in this study. We examined monocytes and macrophages in the bone marrow and spleen during healthy aging in young (3-4 months) and elderly (20-24 months) female C57BL/6J mice. Additionally, changes to these tissues and in TAMs were examined during AE17 mesothelioma tumor growth. Healthy aging resulted in an expansion of Ly6Chigh monocytes and macrophages in the bone marrow and spleen. CSF-1R expression levels were reduced in elderly splenic macrophages only, suggesting differences in CSF-1R signaling between both cell type and tissue site. In tumor-bearing mice, Ly6Chigh monocytes increased with tumor growth in the spleen in the elderly and increased intracellular CSF-1R expression occurred in bone marrow Ly6Chigh monocytes in elderly mice bearing large tumors. Age-related changes to bone marrow and splenic Ly6Chigh monocytes were reflected in the tumor, where we observed increased Ly6Chigh TAMs earlier and expansion of Ly6Clow TAMs later during AE17 tumor growth in the elderly compared to young mice. F4/80high TAMs increased with tumor growth in both young and elderly mice and were the largest subset of TAMs in the tumor. Together, this suggests there may be a faster transition of Ly6Chigh towards F4/80high TAMs with aging. Amongst TAM subsets, expression of CSF-1R was lowest in F4/80high TAMs, however Ly6Clow TAMs had higher intracellular CSF-1R expression. This suggests downstream CSF-1R signaling may vary between macrophage subsets, which can have implications towards CSF-1R blockade therapies targeting macrophages in cancer.
RESUMO
Cardiovascular disease remains the leading cause of death and disability for patients across the world. Our understanding of atherosclerosis as a primary cholesterol issue has diversified, with a significant dysregulated inflammatory component that largely remains untreated and continues to drive persistent cardiovascular risk. Macrophages are central to atherosclerotic inflammation, and they exist along a functional spectrum between pro-inflammatory and anti-inflammatory extremes. Recent clinical trials have demonstrated a reduction in major cardiovascular events with some, but not all, anti-inflammatory therapies. The recent addition of colchicine to societal guidelines for the prevention of recurrent cardiovascular events in high-risk patients with chronic coronary syndromes highlights the real-world utility of this class of therapies. A highly targeted approach to modification of interleukin-1-dependent pathways shows promise with several novel agents in development, although excessive immunosuppression and resulting serious infection have proven a barrier to implementation into clinical practice. Current risk stratification tools to identify high-risk patients for secondary prevention are either inadequately robust or prohibitively expensive and invasive. A non-invasive and relatively inexpensive method to identify patients who will benefit most from novel anti-inflammatory therapies is required, a role likely to be fulfilled by functional imaging methods. This review article outlines our current understanding of the inflammatory biology of atherosclerosis, upcoming therapies and recent landmark clinical trials, imaging modalities (both invasive and non-invasive) and the current landscape surrounding functional imaging including through targeted nuclear and nanobody tracer development and their application.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Doença da Artéria Coronariana , Humanos , Inflamação/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Anti-Inflamatórios/uso terapêutico , Macrófagos/metabolismo , Doenças Cardiovasculares/diagnóstico por imagem , Doença da Artéria Coronariana/induzido quimicamenteRESUMO
Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.
RESUMO
Earlier work has shown that the transcription factor C/EBPalpha induced a transdifferentiation of committed lymphoid precursors into macrophages in a process requiring endogenous PU.1. Here we have examined the effects of PU.1 and C/EBPalpha on fibroblasts, a cell type distantly related to blood cells and akin to myoblasts, adipocytes, osteoblasts, and chondroblasts. The combination of the two factors, as well as PU.1 and C/EBPbeta, induced the up-regulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells. They also up-regulated these markers in mouse embryo- and adult skin-derived fibroblasts. Based on cell morphology, activation of macrophage-associated genes, and extinction of fibroblast-associated genes, cell lines containing an attenuated form of PU.1 and C/EBPalpha acquired a macrophage-like phenotype. The lines also display macrophage functions: They phagocytose small particles and bacteria, mount a partial inflammatory response, and exhibit strict CSF-1 dependence for growth. The myeloid conversion is primarily induced by PU.1, with C/EBPalpha acting as a modulator of macrophage-specific gene expression. Our data suggest that it might become possible to induce the transdifferentiation of skin-derived fibroblasts into cell types desirable for tissue regeneration.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Transdiferenciação Celular , Fibroblastos/citologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Transdiferenciação Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células NIH 3T3 , Fagocitose/genética , Proteínas Proto-Oncogênicas/genética , Retroviridae/genética , Transativadores/genética , Transfecção , Regulação para CimaRESUMO
The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body's inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed.
Assuntos
Mesotelioma Maligno , Mesotelioma , Células Epiteliais , Humanos , Imunidade , Imunoterapia , Mesotelioma/patologia , Mesotelioma/terapiaRESUMO
Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow-derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.
Assuntos
Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Feminino , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Taxa de SobrevidaRESUMO
Most tissue macrophages and osteoclasts are regulated by colony-stimulating factor-1 (CSF-1, also known as macrophage CSF). The effects of CSF-1 are mediated by the CSF-1 receptor tyrosine kinase (CSF-1R), through autophosphorylation of CSF-1R and the subsequent phosphorylation of downstream molecules. Triggering this phosphorylation cascade increases gene transcription and protein translation, and induces cytoskeletal remodeling by several signaling pathways, leading to the survival, proliferation and differentiation of target cells. CSF-1-regulated tissue macrophages are important for innate immunity and for tissue development and function. Because CSF-1 regulates the survival, proliferation and chemotaxis of macrophages and supports their activation, this factor is involved in the pathogenesis of several diseases.
Assuntos
Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Animais , Humanos , Ativação de Macrófagos , FosforilaçãoRESUMO
CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Macrófagos/citologia , Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Animais , Western Blotting , Células da Medula Óssea/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Imunoglobulina G/imunologia , Imunoprecipitação , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação/genética , Fragmentos de Peptídeos/imunologia , Fenótipo , Fosforilação , Coelhos , Retroviridae/genética , Transdução de Sinais , Tirosina/genética , Tirosina/metabolismoRESUMO
Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1-induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1-induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/fisiologia , Pseudópodes/fisiologia , Actinas/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Animais , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas do Citoesqueleto/farmacologia , Relação Dose-Resposta a Droga , Imunofluorescência , Corantes Fluorescentes , Regulação da Expressão Gênica , Imuno-Histoquímica , Cinética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia de Vídeo , Faloidina/metabolismo , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Retroviridae/genética , Rodaminas , Frações Subcelulares/metabolismoRESUMO
Most cancers emerge in the elderly, including lung cancer and mesothelioma, yet the elderly remain an underrepresented population in pre-clinical cancer studies and clinical trials. The immune system plays a critical role in the effectiveness of many anti-cancer therapies in young hosts via tumor-specific T cells. However, immunosuppressive macrophages can constitute up to 50% of the tumor burden and impair anti-tumor T cell activity. Altered macrophage phenotype and function during aging may further impact anti-tumor T cell responses. Yet, the impact of macrophages on anti-tumor T cell responses and immunotherapy in the elderly is unknown. Therefore, we examined macrophages and their interaction with T cells in young (3 months) and elderly (20-24 months) AE17 mesothelioma-bearing female C57BL/6J mice during tumor growth. Mesothelioma tumors grew faster in elderly compared with young mice, and this corresponded with an increase in tumor-associated macrophages. During healthy aging, macrophages increase in bone marrow and spleens suggesting that these sites have an increased potential to supply cancer-promoting macrophages. Interestingly, in tumor-bearing mice, bone marrow macrophages increased proliferation whilst splenic macrophages had reduced proliferation in elderly compared with young mice, and macrophage depletion using the F4/80 antibody slowed tumor growth in young and elderly mice. We also examined responses to treatment with intra-tumoral IL-2/anti-CD40 antibody immunotherapy and found it was less effective in elderly (38% tumor regression) compared to young mice (90% regression). Tumor-bearing elderly mice decreased in vivo anti-tumor cytotoxic T cell activity in tumor draining lymph nodes and spleens. Depletion of macrophages using F4/80 antibody in elderly, but not young mice, improved IL-2/anti-CD40 immunotherapy up to 78% tumor regression. Macrophage depletion also increased in vivo anti-tumor T cell activity in elderly, but not young mice. All the tumor-bearing elderly (but not young) mice had decreased body weight (i.e., exhibited cachexia), which was greatly exacerbated by immunotherapy; whereas macrophage depletion prevented this immunotherapy-induced cachexia. These studies strongly indicate that age-related changes in macrophages play a key role in driving cancer cachexia in the elderly, particularly during immunotherapy, and sabotage elderly anti-tumor immune responses.
RESUMO
The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, thereby promoting cell-cycle progression. Cyclin D1 is overexpressed in hematopoetic and epithelial malignancies correlating with poor prognosis and metastasis in several cancer types. Because tumor-associated macrophages have been shown to enhance malignant progression and metastasis, and cyclin D1-deficient mice are resistant to oncogene-induced malignancies, we investigated the function of cyclin D1-/- bone marrow-derived macrophages. Cyclin D1 deficiency increased focal complex formation at the site of substratum contact, and enhanced macrophage adhesion, yielding a flattened, circular morphology with reduced membrane ruffles. Migration in response to wounding, cytokine-mediated chemotaxis, and transendothelial cell migration of cyclin D1-/- bone marrow-derived macrophages were all substantially reduced. Thus, apart from proliferative and possible motility defects in the tumor cells themselves, the reduced motility and invasiveness of cyclin D1-/- tumor-associated macrophages may contribute to the tumor resistance of these mice.
Assuntos
Movimento Celular/fisiologia , Ciclina D1/metabolismo , Macrófagos/metabolismo , Animais , Adesão Celular/fisiologia , Quimiotaxia/fisiologia , Ciclina D1/deficiência , Fator Estimulador de Colônias de Macrófagos/metabolismo , CamundongosRESUMO
Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.
Assuntos
Neoplasias da Mama/patologia , Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/patologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Fator de Crescimento Epidérmico/biossíntese , Receptores ErbB/antagonistas & inibidores , Retroalimentação Fisiológica , Humanos , Fator Estimulador de Colônias de Macrófagos/biossíntese , Macrófagos/metabolismo , Camundongos , Invasividade Neoplásica , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidoresRESUMO
Macrophages interact with cells in every organ to facilitate tissue development, function and repair. However, the close interaction between macrophages and parenchymal cells can be subverted in disease, particularly cancer. Motility is an essential capacity for macrophages to be able to carry out their various roles. In cancers, the macrophage's interstitial migratory ability is frequently co-opted by tumor cells to enable escape from the primary tumor and metastatic spread. Macrophage accumulation within and movement through a tumor is often stimulated by tumor cell production of the mononuclear phagocytic growth factor, colony-stimulating factor-1 (CSF-1). CSF-1 also regulates macrophage survival, proliferation and differentiation, and its many effects are transduced by its receptor, the CSF-1R, via phosphotyrosine motif-activated signals. Mutational analysis of CSF-1R signaling indicates that the major mediators of CSF-1-induced motility are phosphatidyl-inositol-3 kinase (PI3K) and one or more Src family kinase (SFK), which activate signals to adhesion, actin polymerization, polarization and, ultimately, migration and invasion in macrophages. The macrophage transcriptome, including that of the motility machinery, is very complex and highly responsive to the environment, with selective expression of proteins and splice variants rarely found in other cell types. Thus, their unique motility machinery can be specifically targeted to block macrophage migration, and thereby, inhibit tumor invasion and metastasis.