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1.
Arterioscler Thromb Vasc Biol ; 39(1): 25-36, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30580568

RESUMO

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.


Assuntos
Citometria de Fluxo/métodos , Monócitos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Aterosclerose/etiologia , Movimento Celular , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/etiologia , Humanos , Receptores de Lipopolissacarídeos/análise , Camundongos , Pessoa de Meia-Idade , Monócitos/imunologia , Receptores de IgG/análise
2.
Cell Rep ; 24(9): 2329-2341.e8, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30157427

RESUMO

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.


Assuntos
Medula Óssea/metabolismo , Neutrófilos/metabolismo , Células-Tronco/metabolismo , Animais , Humanos , Camundongos
3.
Arterioscler Thromb Vasc Biol ; 37(11): 2043-2052, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28935758

RESUMO

OBJECTIVE: Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature. APPROACH AND RESULTS: To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein. CONCLUSIONS: Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.


Assuntos
Aterosclerose/metabolismo , Antígenos CD36/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Artéria Femoral/metabolismo , Migração e Rolagem de Leucócitos , Monócitos/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Antígenos CD36/deficiência , Antígenos CD36/genética , Dieta Ocidental , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Vascular/patologia , Artéria Femoral/patologia , Predisposição Genética para Doença , Humanos , Microscopia Intravital , Lipoproteínas LDL/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Fenótipo , Transdução de Sinais , Fatores de Tempo , Quinases da Família src/metabolismo
4.
J Clin Invest ; 126(12): 4603-4615, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27820700

RESUMO

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.


Assuntos
Antígenos CD/imunologia , Movimento Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Células Dendríticas/imunologia , Cadeias alfa de Integrinas/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores de Esteroides/imunologia , Receptores dos Hormônios Tireóideos/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos CD/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Citrobacter rodentium/imunologia , Proteínas de Ligação a DNA/genética , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/imunologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Imidazóis/farmacologia , Cadeias alfa de Integrinas/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptores CCR7/genética , Receptores CCR7/imunologia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linfócitos T/imunologia
5.
Immunity ; 45(5): 975-987, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27814941

RESUMO

Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes, we present a proof-of-principle approach that addresses these limitations. Combining ChIP-seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. Because Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.


Assuntos
Diferenciação Celular/imunologia , Macrófagos/imunologia , Melanoma Experimental/imunologia , Monócitos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Animais , Antígenos Ly/imunologia , Separação Celular , Imunoprecipitação da Cromatina , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Reação em Cadeia da Polimerase
6.
Nat Immunol ; 16(12): 1228-34, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26523867

RESUMO

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.


Assuntos
Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Sistema Nervoso Simpático/imunologia , Animais , Linhagem Celular , Células Cultivadas , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Expressão Gênica/imunologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Norepinefrina/imunologia , Norepinefrina/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sistema Nervoso Simpático/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismo
7.
J Immunol ; 195(8): 3515-9, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26363057

RESUMO

The transcription factor IFN regulatory factor (IRF)4 was shown to play a crucial role in the protective CD8(+) T cell response; however, regulation of IRF4 expression in CD8(+) T cells remains unclear. In this article, we report a critical role for Nr4a1 in regulating the expansion, differentiation, and function of CD8(+) T cells through direct transcriptional repression of Irf4. Without Nr4a1, the regulation of IRF4 is lost, driving an increase in Irf4 expression and, in turn, resulting in a faster rate of CD8 T cell proliferation and expansion. Nr4a1-deficient mice show increases in CD8 T cell effector responses with improved clearance of Listeria monocytogenes. Our data support a novel and critical role for Nr4a1 in the regulation of CD8(+) T cell expansion and effector function through transcriptional repression of Irf4.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Fatores Reguladores de Interferon/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Fatores Reguladores de Interferon/genética , Listeriose/genética , Listeriose/patologia , Camundongos , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética
8.
Sci Rep ; 5: 9059, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25762306

RESUMO

The NR4A nuclear receptor family member Nr4a1 is strongly induced in thymocytes undergoing selection, and has been shown to control the development of Treg cells; however the role of Nr4a1 in CD8(+) T cells remains undefined. Here we report a novel role for Nr4a1 in regulating the development and frequency of CD8(+) T cells through direct transcriptional control of Runx3. We discovered that Nr4a1 recruits the corepressor, CoREST to suppress Runx3 expression in CD8(+) T cells. Loss of Nr4a1 results in increased Runx3 expression in thymocytes which consequently causes a 2-fold increase in the frequency and total number of intrathymic and peripheral CD8(+) T cells. Our findings establish Nr4a1 as a novel and critical player in the regulation of CD8 T cell development through the direct suppression of Runx3.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Transcrição Gênica , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Proteínas Correpressoras , Regulação para Baixo , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Timo/citologia , Timo/imunologia , Quimeras de Transplante
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