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BACKGROUND: There are several studies that indicate that cancer development may be conditioned by the activation of some biological systems that involve the interaction of different biomolecules, such as adenosine and vascular endothelial growth factor. These biomolecules have been targeted of some drugs for treat of cancer; however, there is little information on the interaction of purine derivatives with adenosine and vascular endothelial growth factor receptor (VEGF-R1). OBJECTIVE: The aim of this research was to determine the possible interaction of purine (1: ) and their derivatives (2-31: ) with A1, A2-adenosine receptors, and VEGF-R1. METHODS: Theoretical interaction of purine and their derivatives with A1, A2-adenosine receptors and VEGF-R1 was carried out using the 5uen, 5mzj and 3hng proteins as theoretical tools. Besides, adenosine, cgs-15943, rolofylline, cvt-124, wrc-0571, luf-5834, cvt-6883, AZD-4635, cabozantinib, pazopanib, regorafenib, and sorafenib drugs were used as controls. RESULTS: The results showed differences in the number of aminoacid residues involved in the interaction of purine and their derivatives with 5uen, 5mzj and 3hng proteins compared with the controls. Besides, the inhibition constants (Ki) values for purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: were lower compared with the controls CONCLUSIONS: Theoretical data suggest that purine and their derivatives 5: , 9: , 10: , 14: , 15: , 16: , and 20: could produce changes in cancer cell growth through inhibition of A1, A2-adenosine receptors and VEGFR-1 inhibition. These data indicate that these purine derivatives could be a therapeutic alternative to treat some types of cancer.
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Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming, and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue-specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6 deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, miR-223, and miR-203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.
Assuntos
Fator de Transcrição GATA6 , Macrófagos Peritoneais , MicroRNAs , Transcriptoma , Animais , Camundongos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Especificidade de Órgãos , Proteínas Proto-Oncogênicas , Transativadores/genética , Transativadores/metabolismoRESUMO
Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Metabolismo dos Lipídeos/genética , Mitocôndrias/efeitos dos fármacos , Oxilipinas/metabolismo , Peritonite/genética , Sepse/genética , Acil-CoA Desidrogenase de Cadeia Longa/sangue , Acil-CoA Desidrogenase de Cadeia Longa/genética , Animais , Carnitina O-Palmitoiltransferase/sangue , Carnitina O-Palmitoiltransferase/genética , Coenzima A Ligases/sangue , Coenzima A Ligases/genética , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Interferon gama/farmacologia , Lipidômica/métodos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/sangue , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Oxirredução , Peritonite/sangue , Peritonite/induzido quimicamente , Peritonite/patologia , Células RAW 264.7 , Sepse/sangue , Sepse/patologiaRESUMO
Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.
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Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Tromboplastina/metabolismo , Coagulação Sanguínea , Plaquetas/metabolismo , Linhagem Celular , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Tromboplastina/genéticaRESUMO
The alarm cytokine interleukin-1ß (IL-1ß) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1ß production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1ß; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1ß from its pro-form. However, despite the important role of IL-1ß in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1ß processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1ß processing, providing a previously unrecognized control of IL-1ß in tissue-resident macrophages.
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Epoprostenol/imunologia , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Macrófagos Peritoneais/imunologia , Animais , Epoprostenol/genética , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-1beta/genética , Macrófagos Peritoneais/patologia , Camundongos , Camundongos TransgênicosRESUMO
A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.
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Proteína ADAM17/química , Proteína ADAM17/metabolismo , Técnicas de Cultura de Células/métodos , Células Dendríticas/enzimologia , Macrófagos/enzimologia , Animais , Linhagem Celular , Proteínas de Homeodomínio , CamundongosRESUMO
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
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Fatores de Coagulação Sanguínea/metabolismo , Hemorragia/enzimologia , Hemorragia/metabolismo , Fosfolipídeos/metabolismo , Trombina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Coagulação Sanguínea , Fatores de Coagulação Sanguínea/genética , Plaquetas , Ponte Cardiopulmonar/efeitos adversos , Proteínas de Transporte , Cisteína Endopeptidases , Fator IX/genética , Fator VIII/genética , Fator VIIa/metabolismo , Fator X/genética , Hemofilia A , Hemorragia/prevenção & controle , Hemostasia , Humanos , Ácidos Hidroxieicosatetraenoicos , Lipoproteínas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas de Neoplasias , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismoRESUMO
The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen-presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte-derived macrophages we noted differential responses to the immune-modulatory cytokine IL-10. In contrast to the suppressive actions of IL-10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL-10 may thus have limited impact on development of potentially tissue-damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune-mediated membrane dysfunction, treatment failure, and increased morbidity.
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Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/patologia , Biomarcadores , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Imunomodulação , Imunofenotipagem , Inflamação/patologia , Interleucina-10/genética , Macrófagos/patologia , Camundongos , Camundongos Knockout , Peritonite/imunologia , Peritonite/metabolismo , Peritonite/patologia , Fenótipo , Receptores CCR2/metabolismoRESUMO
INTRODUCTION: Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. METHODS: Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. RESULTS: In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1ß. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. CONCLUSIONS: These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.
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Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Inflamassomos/imunologia , Interleucina-10/imunologia , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Proteínas de Transporte/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteoclastos/citologia , Osteoclastos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Tissue-resident macrophages are heterogeneous as a consequence of anatomical niche-specific functions. Many populations self-renew independently of bone marrow in the adult, but the molecular mechanisms of this are poorly understood. We determined a transcriptional profile for the major self-renewing population of peritoneal macrophages in mice. These cells specifically expressed the transcription factor Gata6. Selective deficiency of Gata6 in myeloid cells caused substantial alterations in the transcriptome of peritoneal macrophages. Gata6 deficiency also resulted in dysregulated peritoneal macrophage proliferative renewal during homeostasis and in response to inflammation, which was associated with delays in the resolution of inflammation. Our investigations reveal that the tissue macrophage phenotype is under discrete tissue-selective transcriptional control and that this is fundamentally linked to the regulation of their proliferation renewal.
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Proliferação de Células , Fator de Transcrição GATA6/fisiologia , Macrófagos Peritoneais/imunologia , Animais , Fator de Transcrição GATA6/genética , Inflamação/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , FenótipoRESUMO
The general paradigm is that monocytes are recruited to sites of inflammation and terminally differentiate into macrophages. There has been no demonstration of proliferation of peripherally-derived inflammatory macrophages under physiological conditions. Here we show that proliferation of both bone marrow-derived inflammatory and tissue-resident macrophage lineage branches is a key feature of the inflammatory process with major implications for the mechanisms underlying recovery from inflammation. Both macrophage lineage branches are dependent on M-CSF during inflammation, and thus the potential for therapeutic interventions is marked. Furthermore, these observations are independent of Th2 immunity. These studies indicate that the proliferation of distinct macrophage populations provides a general mechanism for macrophage expansion at key stages during inflammation, and separate control mechanisms are implicated.
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Células da Medula Óssea/patologia , Linhagem da Célula , Inflamação/patologia , Macrófagos/patologia , Animais , Antígenos Ly/metabolismo , Proliferação de Células , Feminino , Inflamação/imunologia , Interleucina-4/metabolismo , Cinética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/imunologia , Peritonite/patologia , Receptores de Interleucina-4/metabolismo , ZimosanRESUMO
We have re-investigated the role of the complement system and the non-opsonic pattern recognition receptors dectin-1 and dectin-2 in the recognition of fungal particles by inflammatory neutrophils, monocytes and macrophages. We have used in vivo and ex vivo models to study the recognition and response of these cells: i) We confirm previous observations regarding the importance of complement to neutrophil but not monocytic responses; ii) We show that dectin-1 is important for driving inflammatory cell recruitment to fungal stimuli and that it biases the immediate inflammatory response to one that favors neutrophil over monocyte recruitment; iii) We show that dectin-2 contributes to the physical recognition of fungal particles by inflammatory monocytes/macrophages, but is also expressed on neutrophils, where we show it has the potential to contribute to cellular activation; iv) Additionally, we show that serum-opsonization has the potential to interfere with non-opsonic recognition of fungal particles by dectin-1 and dectin-2, presumably through masking of ligands. Collectively these roles are consistent with previously described roles of dectin-1 and dectin-2 in driving inflammatory and adaptive immune responses and complement in containing fungal burdens. This study emphasizes the importance of heterogeneity of receptor expression across myeloid cell subsets in protective immune responses.
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Regulação da Expressão Gênica , Inflamação/metabolismo , Lectinas Tipo C/biossíntese , Monócitos/microbiologia , Micoses/microbiologia , Neutrófilos/microbiologia , Animais , Proteínas do Sistema Complemento , Citometria de Fluxo/métodos , Sistema Imunitário , Ligantes , Camundongos , Micoses/patologia , Células NIH 3T3 , Neutrófilos/metabolismo , Proteínas Opsonizantes/metabolismo , FenótipoRESUMO
Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G proteincoupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectinlike receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domaincontaining inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.
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Doenças Autoimunes/imunologia , Complemento C5a/imunologia , Imunoglobulina G/imunologia , Lectinas Tipo C/metabolismo , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo , Análise de Variância , Animais , Anticorpos Monoclonais , Western Blotting , Cálcio/metabolismo , Adesão Celular/imunologia , Complemento C5a/administração & dosagem , Feminino , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor da Anafilatoxina C5a , Receptores de IgG/genética , Receptores de IgG/imunologia , Ressonância de Plasmônio de Superfície , Quinase SykRESUMO
In this Viewpoint, we concentrate on the aspects of macrophage biology that we believe are fundamental for an appropriate contextual understanding of macrophage function during acute inflammation. These are the different origins of macrophage populations (and the implications of this for the renewal of these populations in the adult); and the impact of specific homeostatic or disease-associated microenvironments upon cellular heterogeneity, activation and effector functions.
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Macrófagos/imunologia , Doença Aguda , Adulto , Animais , Diferenciação Celular/imunologia , Homeostase/imunologia , Humanos , Inflamação , Ativação de Macrófagos , Regeneração/imunologiaRESUMO
Macrophage (MØ) biology is routinely modelled in the peritoneal cavity, a vascular tissue readily infiltrated by leukocytes during inflammation. After several decades of study, no consensus has emerged regarding the importance of in situ proliferation versus peripheral monocyte recruitment for the maintenance of tissue resident MØs. By applying specific measures of mitosis, we have monitored tissue MØ proliferation during newborn development, adulthood and acute resolving inflammation in young adult mice. Despite the vascular nature of the tissue and ease of peripheral leukocyte entry, tissue MØs in the newborn increase in number by local proliferation. On the contrary, in the adult, tissue MØ proliferation is considerably reduced and most likely provides homeostatic control of cell numbers. Importantly, during an acute inflammatory response, when substantial numbers of inflammatory MØs are recruited from the circulation, tissue-resident MØs survive and then undergo a transient and intense proliferative burst in situ to repopulate the tissue. Our data indicate that local proliferation is a general mechanism for the self-sufficient renewal of tissue MØs during development and acute inflammation and not one restricted to non-vascular tissues, which has implications for the therapeutic modulation of MØ activity during the resolution of inflammation.
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Proliferação de Células , Homeostase/imunologia , Macrófagos/imunologia , Peritonite/imunologia , Doença Aguda , Animais , Ciclo Celular/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , DNA/imunologia , DNA/metabolismo , Feminino , Citometria de Fluxo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Cavidade Peritoneal/patologia , Peritonite/metabolismo , Peritonite/patologia , Fatores de TempoRESUMO
Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.
Assuntos
Alternativas ao Uso de Animais , Engenharia Genética/métodos , Neutrófilos/citologia , Neutrófilos/fisiologia , Animais , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Transdução Genética/métodos , LevedurasRESUMO
The mannose receptor (MR) is an endocytic type I membrane molecule with a broad ligand specificity that is involved in both hemostasis and pathogen recognition. Membrane-anchored MR is cleaved by a metalloproteinase into functional soluble MR (sMR) composed of the extracellular domains of intact MR. Although sMR production was initially considered a constitutive process, enhanced MR shedding has been observed in response to the fungal pathogen Pneumocystis carinii. In this work, we have investigated the mechanism mediating enhanced MR shedding in response to fungi. We show that other fungal species, including Candida albicans and Aspergillus fumigatus, together with zymosan, a preparation of the cell wall of Saccharomyces cerevisiae, mimic the effect of P. carinii on sMR production and that this effect takes place mainly through ß-glucan recognition. Additionally, we demonstrate that MR cleavage in response to C. albicans and bioactive particulate ß-glucan requires expression of dectin-1. Our data, obtained using specific inhibitors, are consistent with the canonical Syk-mediated pathway triggered by dectin-1 being mainly responsible for inducing MR shedding, with Raf-1 being partially involved. As in the case of steady-state conditions, MR shedding in response to C. albicans and ß-glucan particles requires metalloprotease activity. The induction of MR shedding by dectin-1 has clear implications for the role of MR in fungal recognition, as sMR was previously shown to retain the ability to bind fungal pathogens and can interact with numerous host molecules, including lysosomal hydrolases. Thus, MR cleavage could also impact on the magnitude of inflammation during fungal infection.
Assuntos
Fungos/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos Peritoneais/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Micoses/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Fungos/imunologia , Lectinas Tipo C/imunologia , Macrófagos Peritoneais/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Micoses/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores de Superfície Celular/imunologiaRESUMO
We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Proteínas de Homeodomínio/genética , Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/citologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Superfície/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Lectinas Tipo C , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Células Precursoras de Monócitos e Macrófagos/efeitos dos fármacos , Células Precursoras de Monócitos e Macrófagos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico/metabolismo , Ovalbumina/imunologia , Transdução Genética , Zimosan/farmacologia , beta-Glucanas/farmacologiaRESUMO
Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A(2) (cPLA(2)α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the ß-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88(-/-) but not Dectin-1(-/-) macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA(2)α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA(2)α phosphorylation, and COX2 expression in response to particulate ß-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA(2)α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both ß-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA(2)α and eicosanoid production.