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1.
Proc Natl Acad Sci U S A ; 121(10): e2309957121, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38422022

RESUMO

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.


Assuntos
Linfoma de Células B , Proteínas Repressoras , Animais , Camundongos , Hipóxia/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Microambiente Tumoral
2.
Nat Immunol ; 15(4): 343-53, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24531343

RESUMO

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Caspase 1/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Imunidade/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Transgenes/genética , Proteínas Virais/genética , Homólogo LST8 da Proteína Associada a mTOR
3.
Development ; 148(3)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462113

RESUMO

Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1+ myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.


Assuntos
Coração/crescimento & desenvolvimento , Vasos Linfáticos , Macrófagos/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/genética , Adesão Celular , Linhagem Celular , Células Endoteliais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Humanos , Inflamação , Linfangiogênese , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Saco Vitelino
4.
Nat Immunol ; 13(4): 379-86, 2012 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-22388040

RESUMO

Tolerance to endotoxins that is triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism with which to dampen inflammatory cytokines. The receptor-interacting protein RIP140 interacts with the transcription factor NF-κB to regulate the expression of genes encoding proinflammatory cytokines. Here we found lipopolysaccharide stimulation of kinase Syk-mediated tyrosine phosphorylation of RIP140 and interaction of the NF-κB subunit RelA with RIP140. These events resulted in more recruitment of the E3 ligase SCF to tyrosine-phosphorylated RIP140, which degraded RIP140 to inactivate genes encoding inflammatory cytokines. Macrophages expressing nondegradable RIP140 were resistant to the establishment of endotoxin tolerance for specific 'tolerizable' genes. Our results identify RelA as an adaptor with which SCF fine tunes NF-κB target genes by targeting the coactivator RIP140 and show an unexpected role for RIP140 degradation in resolving inflammation and endotoxin tolerance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endotoxinas/imunologia , Tolerância Imunológica/imunologia , Inflamação/imunologia , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Immunoblotting , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/imunologia , Proteínas Nucleares/imunologia , Proteína 1 de Interação com Receptor Nuclear , Proteínas Ligases SKP Culina F-Box/imunologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
5.
Immunity ; 43(2): 264-76, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26275995

RESUMO

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.


Assuntos
Síndromes Periódicas Associadas à Criopirina/imunologia , Células Dendríticas/imunologia , Inflamassomos/metabolismo , Macrófagos Peritoneais/imunologia , Monócitos/imunologia , Peritonite/imunologia , Ribonucleoproteínas/metabolismo , Animais , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica/genética , Homeostase , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/induzido quimicamente , Multimerização Proteica/genética , RNA Interferente Pequeno/genética , Ribonucleoproteínas/genética
6.
FASEB J ; 33(5): 6154-6167, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30799631

RESUMO

Cannabinoid receptor (CB)2 is an immune cell-localized GPCR that has been hypothesized to regulate the magnitude of inflammatory responses. However, there is currently no consensus as to the mechanism by which CB2 mediates its anti-inflammatory effects in vivo. To address this question, we employed a murine dorsal air pouch model with wild-type and CB2-/- 8-12-wk-old female and male C57BL/6 mice and found that acute neutrophil and lymphocyte antigen 6 complex, locus Chi monocyte recruitment in response to Zymosan was significantly enhanced in CB2-/- mice. Additionally, levels of matrix metalloproteinase 9 and the chemokines C-C motif chemokine ligand (CCL)2, CCL4, and C-X-C motif chemokine ligand 10 in CB2-/- pouch exudates were elevated at earlier time points. Importantly, using mixed bone marrow chimeras, we revealed that the proinflammatory phenotype in CB2-/- mice is neutrophil-intrinsic rather than stromal cell-dependent. Indeed, neutrophils isolated from CB2-/- mice exhibited an enhanced migration-related transcriptional profile and increased adhesive phenotype, and treatment of human neutrophils with a CB2 agonist blocked their endothelial transmigration. Overall, we have demonstrated that CB2 plays a nonredundant role during acute neutrophil mobilization to sites of inflammation and, as such, it could represent a therapeutic target for the development of novel anti-inflammatory compounds to treat inflammatory human diseases.-Kapellos, T. S., Taylor, L., Feuerborn, A., Valaris, S., Hussain, M. T., Rainger, G. E., Greaves, D. R., Iqbal, A. J. Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment.


Assuntos
Movimento Celular , Neutrófilos/imunologia , Receptor CB2 de Canabinoide/deficiência , Transcriptoma , Animais , Adesão Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Feminino , Humanos , Imunidade Inata , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Receptor CB2 de Canabinoide/genética
7.
Am J Respir Crit Care Med ; 200(1): 84-97, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649895

RESUMO

Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.


Assuntos
Apoptose/fisiologia , Macrófagos Alveolares/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fagocitose/genética , Fagossomos/fisiologia , Pneumonia Bacteriana , Animais , Apoptose/efeitos dos fármacos , Bactérias , Compostos de Bifenilo/farmacologia , Caspases/metabolismo , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Haemophilus influenzae , Humanos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Óxido Nítrico/metabolismo , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus , Streptococcus pneumoniae , Sulfonamidas/farmacologia
8.
Proc Biol Sci ; 286(1904): 20190730, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31161905

RESUMO

In both cells and animals, cannibalism can transfer harmful substances from the consumed to the consumer. Macrophages are immune cells that consume their own dead via a process called cannibalistic efferocytosis. Macrophages that contain harmful substances are found at sites of chronic inflammation, yet the role of cannibalism in this context remains unexplored. Here we take mathematical and experimental approaches to study the relationship between cannibalistic efferocytosis and substance accumulation in macrophages. Through mathematical modelling, we deduce that substances which transfer between individuals through cannibalism will concentrate inside the population via a coalescence process. This prediction was confirmed for macrophage populations inside a closed system. We used image analysis of whole slide photomicrographs to measure both latex microbead and neutral lipid accumulation inside murine bone marrow-derived macrophages (104-[Formula: see text]) following their stimulation into an inflammatory state ex vivo. While the total number of phagocytosed beads remained constant, cell death reduced cell numbers and efferocytosis concentrated the beads among the surviving macrophages. As lipids are also conserved during efferocytosis, these cells accumulated lipid derived from the membranes of dead and consumed macrophages (becoming macrophage foam cells). Consequently, enhanced macrophage cell death increased the rate and extent of foam cell formation. Our results demonstrate that cannibalistic efferocytosis perpetuates exogenous (e.g. beads) and endogenous (e.g. lipids) substance accumulation inside macrophage populations. As such, cannibalism has similar detrimental consequences in both cells and animals.


Assuntos
Macrófagos/fisiologia , Fagocitose , Animais , Morte Celular , Células Cultivadas , Células Espumosas/citologia , Células Espumosas/metabolismo , Células Espumosas/fisiologia , Metabolismo dos Lipídeos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL
9.
Arterioscler Thromb Vasc Biol ; 37(2): 258-263, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27908893

RESUMO

OBJECTIVE: To create a model of atherosclerosis using green fluorescent protein (GFP)-targeted monocytes/macrophages, allowing analysis of both endogenous GFP+ and adoptively transferred GFP+ myeloid cells in arterial inflammation. APPROACH AND RESULTS: hCD68GFP reporter mice were crossed with ApoE-/- mice. Expression of GFP was localized to macrophages in atherosclerotic plaques and in angiotensin II-induced aortic aneurysms and correlated with galectin 3 and mCD68 expression. Flow cytometry confirmed GFP+ expression in CD11b+/CD64+, CD11c+/MHC-IIHI, and CD11b+/F4/80+ myeloid cells. Adoptive transfer of GFP+ monocytes demonstrated monocyte recruitment to both adventitia and atherosclerotic plaque, throughout the aortic root, within 72 hours. We demonstrated the biological utility of hCD68GFP monocytes by comparing the recruitment of wild-type and CCR2-/- monocytes to sites of inflammation. CONCLUSIONS: hCD68GFP/ApoE-/- mice provide a new approach to study macrophage accumulation in atherosclerotic plaque progression and to identify cells recruited from adoptively transferred monocytes.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aorta/metabolismo , Doenças da Aorta/metabolismo , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Placa Aterosclerótica , Transferência Adotiva , Angiotensina II , Animais , Antígenos CD/genética , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Aorta/patologia , Aneurisma Aórtico/induzido quimicamente , Aneurisma Aórtico/genética , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Rastreamento de Células/métodos , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Galectina 3/metabolismo , Predisposição Genética para Doença , Proteínas de Fluorescência Verde/genética , Macrófagos/patologia , Macrófagos/transplante , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/patologia , Monócitos/transplante , Fenótipo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais
10.
J Immunol ; 196(9): 3910-9, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27016601

RESUMO

CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of α4ß7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood-derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and α4ß7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and α4ß7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Imunoglobulinas/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucoproteínas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas CC/metabolismo , Quimiotaxia/efeitos dos fármacos , Humanos , Doenças Inflamatórias Intestinais/imunologia , Receptores CCR/metabolismo , Linfócitos T/fisiologia , Tretinoína/metabolismo
11.
Am J Respir Crit Care Med ; 196(7): 845-855, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28557543

RESUMO

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. OBJECTIVES: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. METHODS: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage-specific expression of human induced myeloid leukemia cell differentiation protein Mcl-1 (CD68.hMcl-1) was used to model the molecular aspects of COPD. MEASUREMENTS AND MAIN RESULTS: COPD AMs had elevated levels of Mcl-1, an antiapoptotic B-cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages and human monocyte-derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected bone marrow-derived macrophages but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2. CONCLUSIONS: Up-regulation of Mcl-1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.


Assuntos
Anti-Infecciosos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Western Blotting , Lavagem Broncoalveolar , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia
12.
Mediators Inflamm ; 2017: 4315412, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28852269

RESUMO

The endocannabinoid system consists of endogenous lipid mediators and cannabinoid receptors (CB) 1 and 2. It has previously been demonstrated that activation of the leukocyte-expressed CB2 has anti-inflammatory effects in vivo. Here, we report its role under baseline conditions and in a model of low-dose endotoxemia by comparing CB2 knockout to littermate control mice. CB2-deficient mice displayed significantly more neutrophils and fewer monocytes in the bone marrow under steady state. In initial validation experiments, administration of 1 mg/kg LPS to male C57BL/6J mice was shown to transiently upregulate systemic proinflammatory mediators (peaked at 2 hours) and mobilise bone marrow neutrophils and monocytes into circulation. In CB2 knockout mice, the level of the metalloproteinase MMP-9 was significantly elevated by 2 hours and we also observed augmented recruitment of neutrophils to the spleen in addition to increased levels of Ccl2, Ccl3, Cxcl10, and Il6. Collectively, our data show that the absence of CB2 receptor increases the levels of innate immune cell populations in the bone marrow under steady state. Furthermore, during an acute systemic inflammatory insult, we observe a highly reproducible and site-specific increase in neutrophil recruitment and proinflammatory chemokine expression in the spleen of CB2 knockout mice.


Assuntos
Endotoxemia/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Quimiocinas/metabolismo , Modelos Animais de Doenças , Endotoxemia/genética , Citometria de Fluxo , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/genética , Cavidade Peritoneal , Reação em Cadeia da Polimerase , Receptor CB2 de Canabinoide/genética
13.
Glia ; 64(1): 105-21, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26337870

RESUMO

Multiple sclerosis (MS) frequently starts near the lateral ventricles, which are lined by subventricular zone (SVZ) progenitor cells that can migrate to lesions and contribute to repair. Because MS-induced inflammation may decrease SVZ proliferation and thus limit repair, we studied the role of galectin-3 (Gal-3), a proinflammatory protein. Gal-3 expression was increased in periventricular regions of human MS in post-mortem brain samples and was also upregulated in periventricular regions in a murine MS model, Theiler's murine encephalomyelitis virus (TMEV) infection. Whereas TMEV increased SVZ chemokine (CCL2, CCL5, CCL, and CXCL10) expression in wild type (WT) mice, this was inhibited in Gal-3(-/-) mice. Though numerous CD45+ immune cells entered the SVZ of WT mice after TMEV infection, their numbers were significantly diminished in Gal-3(-/-) mice. TMEV also reduced neuroblast and proliferative SVZ cell numbers in WT mice but this was restored in Gal-3(-/-) mice and was correlated with increased numbers of doublecortin+ neuroblasts in the corpus callosum. In summary, our data showed that loss of Gal-3 blocked chemokine increases after TMEV, reduced immune cell migration into the SVZ, reestablished SVZ proliferation and increased the number of progenitors in the corpus callosum. These results suggest Gal-3 plays a central role in modulating the SVZ neurogenic niche's response to this model of MS.


Assuntos
Encéfalo/metabolismo , Galectina 3/metabolismo , Esclerose Múltipla/metabolismo , Doença Autoimune do Sistema Nervoso Experimental/metabolismo , Neurogênese , Nicho de Células-Tronco/fisiologia , Adolescente , Adulto , Idoso , Animais , Encéfalo/imunologia , Encéfalo/patologia , Movimento Celular , Criança , Feminino , Galectina 3/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Doença Autoimune do Sistema Nervoso Experimental/imunologia , Doença Autoimune do Sistema Nervoso Experimental/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Poliomielite/metabolismo , Poliomielite/patologia , Theilovirus , Adulto Jovem
14.
Blood ; 124(15): e33-44, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25030063

RESUMO

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Diferenciação Celular , Proteínas de Fluorescência Verde/metabolismo , Macrófagos Peritoneais/citologia , Monócitos/citologia , Regiões Promotoras Genéticas/genética , Transferência Adotiva , Animais , Medula Óssea/metabolismo , Receptor 1 de Quimiocina CX3C , Doença Crônica , Desenvolvimento Embrionário , Citometria de Fluxo , Imunofluorescência , Genes Reporter , Humanos , Inflamação/patologia , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Mycobacterium/patologia , Mycobacterium bovis/fisiologia , Fenótipo , Receptores de Quimiocinas/metabolismo , Baço/metabolismo
15.
J Immunol ; 193(10): 4833-44, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25305319

RESUMO

The signal regulatory protein (SIRP) locus encodes a family of paired receptors that mediate both activating and inhibitory signals and is associated with type 1 diabetes (T1D) risk. The NOD mouse model recapitulates multiple features of human T1D and enables mechanistic analysis of the impact of genetic variations on disease. In this study, we identify Sirpa encoding an inhibitory receptor on myeloid cells as a gene in the insulin-dependent diabetes locus 13.2 (Idd13.2) that drives islet inflammation and T1D. Compared to T1D-resistant strains, the NOD variant of SIRPα displayed greater binding to its ligand CD47, as well as enhanced T cell proliferation and diabetogenic potency. Myeloid cell-restricted expression of a Sirpa transgene accelerated disease in a dose-dependent manner and displayed genetic and functional interaction with the Idd5 locus to potentiate insulitis progression. Our study demonstrates that variations in both SIRPα sequence and expression level modulate T1D immunopathogenesis. Thus, we identify Sirpa as a T1D risk gene and provide insight into the complex mechanisms by which disease-associated variants act in concert to drive defined stages in disease progression.


Assuntos
Antígeno CD47/genética , Diabetes Mellitus Tipo 1/imunologia , Imunidade Inata , Polimorfismo Genético/imunologia , Receptores Imunológicos/genética , Animais , Autoimunidade , Antígeno CD47/imunologia , Proliferação de Células , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Loci Gênicos , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos NOD , Células Mieloides/imunologia , Células Mieloides/patologia , Ligação Proteica , Receptores Imunológicos/imunologia , Fatores de Risco , Transdução de Sinais , Transgenes
16.
Pharmacol Rev ; 65(1): 47-89, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23300131

RESUMO

Chemokines are a family of low molecular weight proteins with an essential role in leukocyte trafficking during both homeostasis and inflammation. The CC class of chemokines consists of at least 28 members (CCL1-28) that signal through 10 known chemokine receptors (CCR1-10). CC chemokine receptors are expressed predominantly by T cells and monocyte-macrophages, cell types associated predominantly with chronic inflammation occurring over weeks or years. Chronic inflammatory diseases including rheumatoid arthritis, atherosclerosis, and metabolic syndrome are characterized by continued leukocyte infiltration into the inflammatory site, driven in large part by excessive chemokine production. Over years or decades, persistent inflammation may lead to loss of tissue architecture and function, causing severe disability or, in the case of atherosclerosis, fatal outcomes such as myocardial infarction or stroke. Despite the existence of several clinical strategies for targeting chronic inflammation, these diseases remain significant causes of morbidity and mortality globally, with a concomitant economic impact. Thus, the development of novel therapeutic agents for the treatment of chronic inflammatory disease continues to be a priority. In this review we introduce CC chemokine receptors as critical mediators of chronic inflammatory responses and explore their potential role as pharmacological targets. We discuss functions of individual CC chemokine receptors based on in vitro pharmacological data as well as transgenic animal studies. Focusing on three key forms of chronic inflammation--rheumatoid arthritis, atherosclerosis, and metabolic syndrome--we describe the pathologic function of CC chemokine receptors and their possible relevance as therapeutic targets.


Assuntos
Receptores CCR/imunologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Quimiocinas/imunologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/imunologia
17.
Blood ; 121(9): e57-69, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23293084

RESUMO

The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4­activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma.


Assuntos
Interleucina-4/farmacologia , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Transcriptoma , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteoma/análise , Proteoma/efeitos dos fármacos , Especificidade da Espécie
18.
Blood ; 121(8): 1436-45, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23264599

RESUMO

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α-dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hematopoese Extramedular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Policitemia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/fisiologia , Células Cultivadas , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Queratinócitos/citologia , Rim/citologia , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/metabolismo , Policitemia/metabolismo , Policitemia/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Índice de Gravidade de Doença , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia
19.
Arterioscler Thromb Vasc Biol ; 34(12): 2554-62, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25359863

RESUMO

OBJECTIVE: The CX3C chemokine fractalkine (CX3CL1) has a critical role in the development of atherogenesis because apolipoprotein-E-deficient mice lacking CX3CL1 or its receptor CX3CR1 develop smaller plaques and polymorphisms in CX3CR1 are associated with altered risk of cardiovascular disease. CX3CR1 is found on numerous cell types involved in atherogenesis but seems to have a key role in monocyte function. We aimed to elucidate the role of CX3CL1 in human monocyte survival and determine the mechanism by which CX3CL1 spares monocytes from apoptosis. APPROACH AND RESULTS: Primary human monocytes were prepared from healthy donors and subjected to serum-starvation to induce spontaneous apoptosis. The addition of CX3CL1, but not other chemokines tested, promoted monocyte survival in a dose-dependent manner with full-length CX3CL1 (including the mucin stalk) having a more potent antiapoptotic effect than chemokine-domain CX3CL1. The prosurvival effect of CX3CL1 was evident in both monocyte subsets although nonclassical monocytes were more prone to spontaneous apoptosis. In addition, we found that the effect of CX3CL1 was independent of CX3CR1 genotype. Serum-starvation increased the level of intracellular reactive oxygen species, and this was reduced by the addition of CX3CL1. Inhibition of oxidative stress with an antioxidant prevented monocyte apoptosis, indicating that this is the dominant mechanism of cell death targeted by CX3CL1. CONCLUSIONS: CX3CL1 has a substantial and highly reproducible antiapoptotic effect on human monocytes, via a mechanism involving a reduction in oxidative stress. This suggests that CX3CL1 is likely to play a key role in human atherogenesis and may provide a novel therapeutic target in cardiovascular disease.


Assuntos
Sobrevivência Celular/fisiologia , Quimiocina CX3CL1/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Animais , Aterosclerose/etiologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Receptor 1 de Quimiocina CX3C , Quimiocina CCL2/metabolismo , Quimiocina CX3CL1/química , Quimiotaxia de Leucócito , Humanos , Interleucina-8/metabolismo , Camundongos , Monócitos/classificação , Estresse Oxidativo , Estrutura Terciária de Proteína , Receptores de Quimiocinas/metabolismo
20.
Bioorg Med Chem ; 23(1): 241-63, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25487422

RESUMO

The cannabinoid receptor 2 (CB2R) has been linked with the regulation of inflammation, and selective receptor activation has been proposed as a target for the treatment of a range of inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB2R agonists with appropriate physicochemical and ADME properties for future evaluation in vivo, we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation studies led to the identification of a new class of selective CB2R agonists. Several examples showed high levels of activity (EC50<200 nM) and binding affinity (Ki<200 nM) for the CB2R, and no detectable activity at the CB1R. The most promising example, DIAS2, also showed favourable in vitro metabolic stability and absorption properties along with a clean selectivity profile when evaluated against a panel of GPCRs and kinases.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Receptor CB2 de Canabinoide/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Agonistas de Receptores de Canabinoides/química , Avaliação Pré-Clínica de Medicamentos/métodos , Cinética , Ligantes , Modelos Moleculares , Receptor CB2 de Canabinoide/química
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