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1.
PLoS One ; 15(2): e0228484, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32017809

RESUMO

Rhesus macaque (RM) rhadinovirus (RRV) is a simian gamma-2 herpesvirus closely related to human Kaposi's sarcoma-associated herpesvirus (KSHV). RRV is associated with the development of diseases in simian immunodeficiency virus (SIV) co-infected RM that resemble KSHV-associated pathologies observed in HIV-infected humans, including B cell lymphoproliferative disorders (LPD) and lymphoma. Importantly, how de novo KSHV infection affects the expression of host genes in humans, and how these alterations in gene expression affect viral replication, latency, and disease is unknown. The utility of the RRV/RM infection model provides a novel approach to address these questions in vivo, and utilizing the RRV bacterial artificial chromosome (BAC) system, the effects of specific viral genes on host gene expression patterns can also be explored. To gain insight into the effects of RRV infection on global host gene expression patterns in vivo, and to simultaneously assess the contributions of the immune inhibitory viral CD200 (vCD200) molecule to host gene regulation, RNA-seq was performed on pre- and post-infection lymph node (LN) biopsy samples from RM infected with either BAC-derived WT (n = 4) or vCD200 mutant RRV (n = 4). A variety of genes were identified as being altered in LN tissue samples due to RRV infection, including cancer-associated genes activation-induced cytidine deaminase (AICDA), glypican-1 (GPC1), CX3C chemokine receptor 1 (CX3CR1), and Ras dexamethasone-induced 1 (RasD1). Further analyses also indicate that GPC1 may be associated with lymphomagenesis. Finally, comparison of infection groups identified the differential expression of host gene thioredoxin interacting protein (TXNIP), suggesting a possible mechanism by which vCD200 negatively affects RRV viral loads in vivo.


Assuntos
Perfilação da Expressão Gênica/veterinária , Infecções por Herpesviridae/veterinária , Rhadinovirus/patogenicidade , Infecções Tumorais por Vírus/veterinária , Animais , Receptor 1 de Quimiocina CX3C/genética , Transformação Celular Neoplásica/genética , Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Glipicanas/genética , Infecções por Herpesviridae/genética , Tecido Linfoide/metabolismo , Macaca mulatta , Análise de Sequência de RNA/veterinária , Infecções Tumorais por Vírus/genética , Latência Viral , Replicação Viral , Proteínas ras/genética
2.
Proc Natl Acad Sci U S A ; 117(2): 1129-1138, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879345

RESUMO

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Glioma/tratamento farmacológico , Células Mieloides/metabolismo , Receptores CCR2/efeitos dos fármacos , Receptores CCR2/metabolismo , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CCL2 , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioma/patologia , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/metabolismo , Receptor de Morte Celular Programada 1 , Receptores CCR2/genética , Análise de Sobrevida , Microambiente Tumoral/efeitos dos fármacos
3.
Immunity ; 51(6): 1043-1058.e4, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31810882

RESUMO

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.


Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Biomarcadores/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Feminino , Granzimas/genética , Granzimas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/biossíntese , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/genética
4.
Nat Neurosci ; 22(11): 1771-1781, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636449

RESUMO

Microglia dynamically survey the brain parenchyma. Microglial processes interact with neuronal elements; however, what role neuronal network activity plays in regulating microglial dynamics is not entirely clear. Most studies of microglial dynamics use either slice preparations or in vivo imaging in anesthetized mice. Here we demonstrate that microglia in awake mice have a relatively reduced process area and surveillance territory and that reduced neuronal activity under general anesthesia increases microglial process velocity, extension and territory surveillance. Similarly, reductions in local neuronal activity through sensory deprivation or optogenetic inhibition increase microglial process surveillance. Using pharmacological and chemogenetic approaches, we demonstrate that reduced norepinephrine signaling is necessary for these increases in microglial process surveillance. These findings indicate that under basal physiological conditions, noradrenergic tone in awake mice suppresses microglial process surveillance. Our results emphasize the importance of awake imaging for studying microglia-neuron interactions and demonstrate how neuronal activity influences microglial process dynamics.


Assuntos
Microglia/fisiologia , Neurônios/fisiologia , Norepinefrina/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C/genética , Clozapina/análogos & derivados , Clozapina/farmacologia , Isoflurano/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microinjeções , Muscimol/farmacologia , Norepinefrina/farmacologia , Optogenética , Propanolaminas/farmacologia , Propranolol/farmacologia , Receptores Purinérgicos P2Y12/genética , Privação Sensorial/fisiologia , Córtex Somatossensorial/efeitos dos fármacos , Tetrodotoxina/farmacologia , Vigília
5.
Nat Neurosci ; 22(11): 1782-1792, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636451

RESUMO

Microglia are the brain's resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice as compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacologic stimulation of ß2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of ß2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.


Assuntos
Microglia/fisiologia , Plasticidade Neuronal/fisiologia , Norepinefrina/fisiologia , Córtex Visual/fisiologia , Animais , Benzilaminas/farmacologia , Receptor 1 de Quimiocina CX3C/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Ritmo Circadiano/fisiologia , Clembuterol/farmacologia , Dexmedetomidina/farmacologia , Dominância Ocular , Feminino , Fentanila/farmacologia , Locus Cerúleo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Nadolol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Norepinefrina/metabolismo , Propanolaminas/farmacologia , Restrição Física/fisiologia , Terbutalina/farmacologia , Vigília , Ferimentos e Lesões/fisiopatologia
6.
Nat Commun ; 10(1): 3931, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477722

RESUMO

Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1, HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56bright and CD56dim NK cells. Finally, a donor with GATA2T354M mutation exhibits reduced percentage of CD56bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.


Assuntos
Medula Óssea/metabolismo , Células Matadoras Naturais/metabolismo , Análise de Célula Única/métodos , Transcriptoma/genética , Células da Medula Óssea/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Heterogeneidade Genética , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo
7.
Int J Mol Med ; 44(3): 939-948, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31524247

RESUMO

Chemokine C­X3­C motif ligand 1 (CX3CL1) and its sole receptor, CX3CR1, are known to be involved in neuronal damage/death following brain ischemia. In the present study, time­dependent expression changes of CX3CL1 and CX3CR1 proteins were investigated in the hippocampal CA1 field following 5 min of transient global cerebral ischemia (tgCI) in gerbils. To induce tgCI in gerbils, bilateral common carotid arteries were occluded for 5 min using aneurysm clips. Expression changes of CX3CL1 and CX3CR1 proteins were assessed at 1, 2 and 5 days after tgCI using western blotting and immunohistochemistry. CX3CL1 immunoreactivity was strong in the CA1 pyramidal cells of animals in the sham operation group. Weak CX3CL1 immunoreactivity was detected at 6 h after tgCI, recovered at 1 day after tgCI and disappeared from 5 days after tgCI. CX3CR1 immunoreactivity was very weak in CA1 pyramidal cells of the sham animals. CX3CR1 immunoreactivity in CA1 pyramidal cells was significantly increased at 1 days after tgCI and gradually decreased thereafter. On the other hand, CX3CR1 immunoreactivity was significantly increased in microglia from 5 days after tgCI. These results showed that CX3CL1 and CX3CR1 protein expression levels in pyramidal cells and microglia in the hippocampal CA1 field following tgCI were changed, indicating that tgCI­induced expression changes of CX3CL1 and CX3CR1 proteins might be closely associated with tgCI­induced delayed neuronal death and microglial activation.


Assuntos
Isquemia Encefálica/metabolismo , Região CA1 Hipocampal/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Receptor 1 de Quimiocina CX3C/genética , Morte Celular , Quimiocina CX3CL1/genética , Expressão Gênica , Gerbillinae , Imuno-Histoquímica , Masculino , Microglia/metabolismo , Neurônios/metabolismo
8.
Immunohorizons ; 3(7): 262-273, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31356156

RESUMO

Bone marrow (BM)-derived classical monocytes are critical to wound repair, where they differentiate into macrophages and purge foreign materials and dead cells while also laying the framework for tissue repair and regeneration. A subset of this recruited population persists in the wound and acquires alternative activation states to promote cell proliferation and matrix remodeling. In diabetes, this phenotypic switch is impaired and inflammation persists in an elevated state, contributing to delayed wound healing. Long-term tissue-resident macrophages can also play a key role in the resolution of inflammation to varying degrees across different organs. In this study, we investigated different macrophage subpopulations in nondiabetic and diabetic wounds over time using Cx3CR1eGFP transgenic mice and BM transplants. We show Cx3CR1eGFP-hi macrophages in skin wounds are derived from long-term tissue-resident macrophages and predominantly exhibit an alternative activation state, whereas cells expressing low-intermediate Cx3CR1eGFP are derived from the BM, contribute to both early and later stages of wound healing, and show both classical and alternative activation states. Diabetic mice showed significant differences in the dynamics of these subpopulations, which likely contribute to elevated and persisting inflammatory states over time. In particular, failure of Cx3CR1int macrophages to mature into Cx3CR1hi links maturation to resolution of inflammation. Thus strategies to promote macrophage maturation may be effective therapeutic tools in chronic inflammatory environments.


Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Diabetes Mellitus Experimental/metabolismo , Macrófagos/metabolismo , Cicatrização/fisiologia , Animais , Células da Medula Óssea , Transplante de Medula Óssea , Receptor 1 de Quimiocina CX3C/genética , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Inflamação/metabolismo , Macrófagos/classificação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Monócitos/metabolismo , Receptores CCR2/metabolismo , Receptores para Leptina/deficiência
9.
Glia ; 67(8): 1434-1448, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31179602

RESUMO

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study, we utilize the CX3CR1GFP/+ CCR2RFP/+ genetic mouse model, in which CX3CR1+ resident microglia and CCR2+ monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony-stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.


Assuntos
Proliferação de Células , Gliose/fisiopatologia , Microglia/fisiologia , Monócitos/fisiologia , Estado Epiléptico/fisiopatologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular , Modelos Animais de Doenças , Gliose/etiologia , Hipocampo/fisiopatologia , Ácido Caínico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Neurônios/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Estado Epiléptico/complicações , Técnicas de Cultura de Tecidos
10.
Nat Neurosci ; 22(7): 1075-1088, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209379

RESUMO

Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1-/- and Cx3cl1-/- synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.


Assuntos
Proteína ADAM10/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Receptor 1 de Quimiocina CX3C/fisiologia , Quimiocina CX3CL1/fisiologia , Proteínas de Membrana/fisiologia , Microglia/fisiologia , Córtex Sensório-Motor/fisiopatologia , Tato/fisiologia , Vibrissas/lesões , Proteína ADAM10/antagonistas & inibidores , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Contagem de Células , Feminino , Regulação da Expressão Gênica , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas Analíticas Microfluídicas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Córtex Sensório-Motor/metabolismo , Córtex Sensório-Motor/patologia , Transdução de Sinais/fisiologia , Análise de Célula Única , Transcriptoma , Vibrissas/fisiologia
11.
J Ovarian Res ; 12(1): 42, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077234

RESUMO

BACKGROUND: Chemokines are involved in the homing of various cancer cells, including those of ovarian cancer (OvCa), to distant organs. They may also promote or inhibit cancer progression and metastasis. Hypoxia, a common phenomenon in malignant tumors, promotes cell proliferation regulated by HIF-1α. Hypoxia-induced genes are involved in metastasis-associated functions and in the epithelial-to-mesenchymal transition (EMT). RESULTS: Tissue microarrays of human OvCa showed elevated expression of CX3CR1 and HIF-1α compared to normal cells, and their levels were higher in adenocarcinoma stages II and III. To substantiate these observations, we performed studies using OvCa cells. Following exposure to hypoxia, OVCAR-3, SW 626, and TOV-112D cells showed high expression of CX3CR1, a transmembrane protein involved in the adhesion and migration of leukocytes, causing an increased chemotactic response to CX3CL1, the ligand for CX3CR1. As determined by flow cytometry, immunofluorescence, RT-PCR, and western blots, there were higher expressions of CX3CR1 and HIF-1α in OvCa cell lines exposed to hypoxia. Further, OvCa cells expressing CX3CR1 were sensitive to the CX3CL1 ligand. Chemotaxis based on chemokine receptors was influential in elevating the expression of EMT markers and matrix metalloproteinases, which are involved in the progression and metastasis of cancer cells. CONCLUSIONS: In OvCa cells, CX3CR1 was upregulated in a process involving hypoxia-mediated regulation of HIF-1α. The elevated levels of CX3CR1, which were sensitive to CX3CL1, increased EMT markers that led to the progression and metastasis of OvCa. Thus, CX3CR1 and HIF-1α are suitable targets for treatment of OvCa.


Assuntos
Receptor 1 de Quimiocina CX3C/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptor 1 de Quimiocina CX3C/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quimiocina CX3CL1/biossíntese , Quimiocina CX3CL1/genética , Transição Epitelial-Mesenquimal , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Circ Res ; 124(9): 1323-1336, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30832557

RESUMO

RATIONALE: Targeting inflammation has been shown to provide clinical benefit in the field of cardiovascular diseases. Although manipulating regulatory T-cell function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. IL (interleukin)-35, an immunosuppressive cytokine mainly produced by regulatory T cells, is a novel member of the IL-12 family and is composed of an EBI3 (Epstein-Barr virus-induced gene 3) subunit and a p35 subunit. However, the role of IL-35 in infarct healing remains elusive. OBJECTIVE: This study aimed to determine whether IL-35 signaling is involved in healing and cardiac remodeling after myocardial infarction (MI) and, if so, to elucidate the underlying molecular mechanisms. METHODS AND RESULTS: IL-35 subunits (EBI3 and p35), which are mainly expressed in regulatory T cells, were upregulated in mice after MI. After IL-35 inhibition, mice showed impaired infarct healing and aggravated cardiac remodeling, as demonstrated by a significant increase in mortality because of cardiac rupture, decreased wall thickness, and worse cardiac function compared with wild-type MI mice. IL-35 inhibition also led to decreased expression of α-SMA (α-smooth muscle actin) and collagen I/III in the hearts of mice after MI. Pharmacological inhibition of IL-35 suppressed the accumulation of Ly6Clow and major histocompatibility complex IIlow/C-C motif chemokine receptor type 2- (MHC IIlow CCR2-) macrophages in infarcted hearts. IL-35 activated transcription of CX3CR1 (C-X3-C motif chemokine receptor 1) and TGF (transforming growth factor) ß1 in macrophages by inducing GP130 signaling, via IL12Rß2 and phosphorylation of STAT1 (signal transducer and activator of transcription family) and STAT4 and subsequently promoted Ly6Clow macrophage survival and extracellular matrix deposition. Moreover, compared with control MI mice, IL-35-treated MI mice showed increased expression of α-SMA and collagen within scars, correlating with decreased left ventricular rupture rates. CONCLUSIONS: IL-35 reduces cardiac rupture, improves wound healing, and attenuates cardiac remodeling after MI by promoting reparative CX3CR1+Ly6Clow macrophage survival.


Assuntos
Interleucinas/fisiologia , Macrófagos/fisiologia , Infarto do Miocárdio/fisiopatologia , Cicatrização/fisiologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/farmacologia , Receptor 1 de Quimiocina CX3C/biossíntese , Receptor 1 de Quimiocina CX3C/genética , Sobrevivência Celular , Cicatriz/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Regulação da Expressão Gênica/fisiologia , Ruptura Cardíaca Pós-Infarto/fisiopatologia , Ruptura Cardíaca Pós-Infarto/prevenção & controle , Interleucinas/antagonistas & inibidores , Interleucinas/biossíntese , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Miocárdio/metabolismo , Receptores de Citocinas/antagonistas & inibidores , Receptores de Citocinas/biossíntese , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Regulação para Cima , Remodelação Ventricular/fisiologia
13.
Science ; 363(6432)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872492

RESUMO

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.


Assuntos
Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Animais , Antígenos Ly , Receptor 1 de Quimiocina CX3C/genética , Linhagem da Célula , Derme/imunologia , Modelos Animais de Doenças , Fibrose , Glicoproteínas/análise , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Miocárdio/imunologia , Transportadores de Ânions Orgânicos/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma
14.
J Neuroinflammation ; 16(1): 34, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755236

RESUMO

BACKGROUND: Chronic pain is a major clinical problem with limited treatment options. Previous studies have demonstrated that activation of adenosine monophosphate-activated protein kinase (AMPK) can attenuate neuropathic pain. Inflammation/immune response at the site of complete Freund's adjuvant (CFA) injection is known to be a critical trigger of the pathological changes that produce inflammatory pain. However, whether activation of AMPK produces an analgesic effect through inhibiting the proinflammatory cytokines, including interleukin-1ß (IL-1ß), in inflammatory pain remains unknown. METHODS: Inflammatory pain was induced in mice injected with CFA. The effects of AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside, an AMPK activator), Compound C (an AMPK inhibitor), and IL-1ra (an IL-1 receptor antagonist) were tested at day 4 after CFA injection. Inflammatory pain was assessed with von Frey filaments and hot plate. Immunoblotting, hematoxylin and eosin (H&E) staining, and immunofluorescence were used to assess inflammation-induced biochemical changes. RESULTS: The AMPK activator AICAR produced an analgesic effect and inhibited the level of proinflammatory cytokine IL-1ß in the inflamed skin in mice. Moreover, activation of AMPK suppressed CFA-induced NF-κB p65 translocation from the cytosol to the nucleus in activated macrophages (CD68+ and CX3CR1+) of inflamed skin tissues. Subcutaneous injection of IL-1ra attenuated CFA-induced inflammatory pain. The AMPK inhibitor Compound C and AMPKα shRNA reversed the analgesic effect of AICAR and the effects of AICAR on IL-1ß and NF-κB activation in inflamed skin tissues. CONCLUSIONS: Our study provides new information that AMPK activation produces the analgesic effect by inhibiting NF-κB activation and reducing the expression of IL-1ß in inflammatory pain.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/uso terapêutico , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Dor/metabolismo , Ribonucleotídeos/uso terapêutico , Aminoimidazol Carboxamida/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Adjuvante de Freund/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Inflamação/induzido quimicamente , Inflamação/complicações , Inflamação/tratamento farmacológico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dor/tratamento farmacológico , Dor/etiologia , Limiar da Dor/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
15.
Mol Immunol ; 108: 23-33, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30776726

RESUMO

Membrane expression of fractalkine (CX3CL1)-receptor (CX3CR1) is relevant in monocytes (Mo) because CX3CR1-CX3CL1 interactions might participate on both, homeostatic and pathologic conditions. We have previously demonstrated that CX3CR1 levels are decreased during culture and when Mo are differentiated into dendritic cells, but enhanced when differentiated into macrophages. Regarding soluble factors, lipopolysaccharide (LPS) accelerated the loss of CX3CR1, while interleukin (IL)-10 and Interferon-gamma (IFN-γ) prevented it. However, the comprehensive knowledge about the intracellular pathways that underlay the level of CX3CR1 expression in Mo is still incomplete. In the current work, we studied the effect of anti-inflammatory cytokines (IL-4, IL-13, IL-10), alone or together with IFN- γ on CX3CR1 expression. We found that only IL-10 and IFN-γ separately were able to prevent CX3CR1 down-modulation during culture of human Mo. Besides, Mo incubated with IL-10 plus IFN-γ showed the highest CX3CR1 expression by cell, suggesting cooperation between two different mechanism used by both cytokines. By studying intracellular mechanisms triggered by IL-10 and IFN-γ, we demonstrated that they specifically induced PI3K-dependent serine-phosphorylation of signal transducer and activator of transcription (STAT)3 or STAT1, respectively. Moreover, chemical inhibitors of STAT1 or STAT3 abrogated IFN-γ or IL-10 effects on CX3CR1 expression. Strikingly, only IL-10 increased CX3CR1 mRNA level, as consequence of augmenting mRNA stability. CX3CR1 mRNA increase was PI3K-dependent, supporting the causal link between the action of IL-10 at the CX3CR1 transcript and CX3CR1 protein level on Mo. Thus, both cytokines up-regulate CX3CR1 expression on human Mo by different intracellular mechanisms.


Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Monócitos/metabolismo , Regulação para Cima , Receptor 1 de Quimiocina CX3C/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição STAT/metabolismo , Serina/metabolismo
16.
Brain Struct Funct ; 224(3): 1301-1314, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30706162

RESUMO

Microglia represent resident immune cells of the central nervous system (CNS), which have been shown to be involved in the pathophysiology of practically every neuropathology. As microglia were described to participate in the formation of the astroglial glia limitans around CNS vessels, they are part of the neurovascular unit (NVU). Since the NVU is a highly specialized structure, being functionally and morphologically adapted to differing demands in the arterial, capillary, and venous segments, the present study was aimed to systematically investigate the microglial contribution to the glia limitans along the vascular tree. Thereby, the microglial participation in the glia limitans was demonstrated for arteries, capillaries, and veins by immunoelectron microscopy in wild-type mice. Furthermore, analysis by confocal laser scanning microscopy revealed the highest density of microglial endfeet contacting the glial basement membrane around capillaries, with significantly lower densities around arteries and veins. Importantly, this pattern appeared to be unaltered in the setting of experimental autoimmune encephalomyelitis (EAE) in CX3CR1CreERT2:R26-Tomato reporter mice, although perivascular infiltrates of blood-borne leukocytes predominantly occur at the level of post-capillary venules. However, EAE animals exhibited significantly increased contact sizes of individual microglial endfeet around arteries and veins. Noteworthy, under EAE conditions, the upregulation of MHC-II was not limited to microglia of the glia limitans of veins showing infiltrates of leukocytes, but also appeared at the capillary level. As a microglial contribution to the glia limitans was also observed in human brain tissue, these findings may help characterizing microglial alterations within the NVU in various neuropathologies.


Assuntos
Artérias/patologia , Capilares/patologia , Encefalomielite Autoimune Experimental/patologia , Neuroglia/patologia , Veias/fisiologia , Actinas/metabolismo , Animais , Artérias/metabolismo , Artérias/fisiologia , Artérias/ultraestrutura , Encéfalo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/ultraestrutura , Capilares/metabolismo , Capilares/ultraestrutura , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Veias/metabolismo , Veias/patologia , Veias/ultraestrutura
17.
Nature ; 566(7742): 110-114, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30675063

RESUMO

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.


Assuntos
Bactérias/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Extensões da Superfície Celular/metabolismo , Intestino Delgado/citologia , Intestino Delgado/microbiologia , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Bactérias/imunologia , Receptor 1 de Quimiocina CX3C/deficiência , Receptor 1 de Quimiocina CX3C/genética , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/imunologia , Feminino , Células HEK293 , Humanos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Ácido Láctico/farmacologia , Lactobacillus helveticus/metabolismo , Masculino , Metanol , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ácido Pirúvico/farmacologia , Receptores Acoplados a Proteínas-G/deficiência , Receptores Acoplados a Proteínas-G/genética , Salmonella/imunologia , Salmonella/metabolismo
18.
J Clin Invest ; 129(3): 1345-1358, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30652972

RESUMO

In the stomach, chronic inflammation causes metaplasia and creates a favorable environment for the evolution of gastric cancer. Glucocorticoids are steroid hormones that repress proinflammatory stimuli, but their role in the stomach is unknown. In this study, we show that endogenous glucocorticoids are required to maintain gastric homeostasis. Removal of circulating glucocorticoids in mice by adrenalectomy resulted in the rapid onset of spontaneous gastric inflammation, oxyntic atrophy, and spasmolytic polypeptide-expressing metaplasia (SPEM), a putative precursor of gastric cancer. SPEM and oxyntic atrophy occurred independently of lymphocytes. However, depletion of monocytes and macrophages by clodronate treatment or inhibition of gastric monocyte infiltration using the Cx3cr1 knockout mouse model prevented SPEM development. Our results highlight the requirement for endogenous glucocorticoid signaling within the stomach to prevent spontaneous gastric inflammation and metaplasia, and suggest that glucocorticoid deficiency may lead to gastric cancer development.


Assuntos
Gastrite , Glucocorticoides/metabolismo , Lesões Pré-Cancerosas , Neoplasias Gástricas , Estômago/patologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Gastrite/genética , Gastrite/metabolismo , Gastrite/prevenção & controle , Glucocorticoides/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/prevenção & controle , Metaplasia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/prevenção & controle
19.
Nat Immunol ; 20(2): 163-172, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643263

RESUMO

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.


Assuntos
Fibrose Pulmonar Idiopática/imunologia , Pulmão/patologia , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Animais , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Bleomicina/imunologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Receptor 1 de Quimiocina CX3C/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/citologia , Pulmão/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Regulação para Cima
20.
J Neuroinflammation ; 16(1): 4, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30616691

RESUMO

BACKGROUND: Microglia/macrophages (M/Ms) with multiple functions derived from distinct activation states are key surveillants maintaining brain homeostasis. However, their activation status and role during the brain metastasis of malignant tumors have been poorly characterized. METHODS: Heterozygous CX3CR1-GFP transgenic mice were used to visualize the dynamic changes of M/Ms during the development of experimental brain metastasis through long-term intravital imaging equipped with redesigned bilateral cranial windows. The occurrence of experimental brain metastasis was evaluated after M/Ms were depleted with PLX3397, a CSF-1R inhibitor. The possible mediators of M/Ms in facilitating the brain metastasis were determined using reverse transcription-PCR, immunofluorescence, correlational analysis, and MMP inhibition. RESULTS: Here, we showed that M/Ms were persistently activated and facilitated the formation of melanoma brain metastasis in vivo. We observed that M/Ms gradually and massively accumulated in the metastasis, with a 2.89-fold increase. To precisely depict the dynamic changes in the activation state of M/Ms, we defined the branching parameter to quantify their morphological alterations. The quantitative data showed that the extent of activation of M/Ms in metastatic foci was enhanced, with a 2.27-fold increase from day 1 to day 21. Along with the activation, the M/Ms increased their moving velocity (4.15-fold) and established a rapid, confined, and discontinuous motility behavior. The occurrence of melanoma brain metastasis was significantly hindered under M/M elimination, indicating the key role of M/Ms in the experimental brain metastasis. Interestingly, we found that M/Ms highly expressed matrix metalloproteinase 3 (MMP3), which were strongly correlated with M/M activation and the decrease of tight junction protein zonula occludens-1 (ZO-1). An MMP inhibitor moderately decreased the occurrence of melanoma brain metastasis, suggesting that MMP3 secreted by M/Ms may facilitate melanoma cell growth. CONCLUSIONS: Our results indicated that the activated M/Ms were essential in the development of melanoma brain metastasis, suggesting that M/Ms are a potential therapeutic target for tumor brain metastasis.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Encéfalo/diagnóstico por imagem , Regulação Neoplásica da Expressão Gênica/fisiologia , Macrófagos/patologia , Microglia/patologia , Aminopiridinas/administração & dosagem , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/secundário , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Lateralidade Funcional , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Intravital/métodos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Pirróis/administração & dosagem , Fatores de Tempo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
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