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1.
Sci Signal ; 16(769): eabm0517, 2023 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-36693132

RESUMO

Neutrophil extracellular traps (NETs) are DNA scaffolds coated with granule proteins that are released by neutrophils to ensnare and kill bacteria. NET formation occurs in response to many stimuli through independent molecular pathways. Although NET release has been equated to a form of lytic cell death, live neutrophils can rapidly release antimicrobial NETs. Gasdermin D (GSDMD), which causes pyroptotic death in macrophages, is thought to be required for NET formation by neutrophils. Through experiments with known physiological activators of NET formation and ligands that activate canonical and noncanonical inflammasome signaling pathways, we demonstrated that Gsdmd-deficient mouse neutrophils were as competent as wild-type mouse neutrophils in producing NETs. Furthermore, GSDMD was not cleaved in wild-type neutrophils during NET release in response to inflammatory mediators. We found that activation of both canonical and noncanonical inflammasome signaling pathways resulted in GSDMD cleavage in wild-type neutrophils but was not associated with cell death. Moreover, NET formation as a result of either pathway of inflammasome activation did not require GSDMD. Together, these data suggest that NETs can be formed by viable neutrophils after inflammasome activation and that this function does not require GSDMD.


Assuntos
Gasderminas , Piroptose , Camundongos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Morte Celular , Neutrófilos/metabolismo
2.
Front Immunol ; 14: 1331151, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38235134

RESUMO

Introduction: Obesity is a metabolic condition that elevates the risk of all-cause mortality. Brown and beige adipose tissues, known for their thermogenic properties, offer potential therapeutic targets for combating obesity. Recent reports highlight the role of immune cells, including eosinophils, in adipose tissue homeostasis, while the underlying mechanisms are poorly understood. Methods: To study the role of autophagy in eosinophils in this process, we used a genetic mouse model lacking autophagy-associated protein 5 (Atg5), specifically within the eosinophil lineage (Atg5 eoΔ). Results: The absence of Atg5 in eosinophils led to increased body weight, impaired glucose metabolism, and alterations in the cellular architecture of adipose tissue. Our findings indicate that Atg5 modulates the functional activity of eosinophils within adipose tissue rather than their abundance. Moreover, RNA-seq analysis revealed upregulation of arginase 2 (Arg2) in Atg5-knockout eosinophils. Increased Arg2 activity was shown to suppress adipocyte beiging. Furthermore, we observed enrichment of the purine pathway in the absence of Atg5 in eosinophils, leading to a pro-inflammatory shift in macrophages and a further reduction in beiging. Discussion: The data shed light on the importance of autophagy in eosinophils and its impact on adipose tissue homeostasis by suppressing Arg2 expression and limiting inflammation in adipose tissue.


Assuntos
Tecido Adiposo , Eosinófilos , Camundongos , Animais , Tecido Adiposo/metabolismo , Adipócitos/metabolismo , Obesidade , Autofagia
3.
PLoS Biol ; 20(9): e3001794, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36108062

RESUMO

In contrast to molecular changes associated with increased inflammatory responses, little is known about intracellular counter-regulatory mechanisms that control signaling cascades associated with functional responses of neutrophils. Active RHO GTPases are typically considered as effector proteins that elicit cellular responses. Strikingly, we show here that RHOH, although being constitutively GTP-bound, limits neutrophil degranulation and the formation of neutrophil extracellular traps (NETs). Mechanistically, RHOH is induced under inflammatory conditions and binds to non-muscle myosin heavy chain IIA (NMHC IIA) in activated neutrophils in order to inhibit the transport of mitochondria and granules along actin filaments, which is partially reverted upon disruption of the interaction with NMHC IIA by introducing a mutation in RhoH at lysine 34 (RhoHK34A). In parallel, RHOH inhibits actin polymerization presumably by modulating RAC1 activity. In vivo studies using Rhoh-/- mice, demonstrate an increased antibacterial defense capability against Escherichia coli (E. coli). Collectively, our data reveal a previously undefined role of RHOH as a molecular brake for actomyosin-mediated neutrophil effector functions, which represents an intracellular regulatory axis involved in controlling the strength of an antibacterial inflammatory response.


Assuntos
Actomiosina , Neutrófilos , Fatores de Transcrição , Proteínas rho de Ligação ao GTP , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Antibacterianos , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina Trifosfato , Lisina , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Neutrófilos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
4.
Blood ; 137(21): 2958-2969, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33598715

RESUMO

Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses.


Assuntos
Proteína 5 Relacionada à Autofagia/fisiologia , Eosinófilos/fisiologia , Mielopoese/fisiologia , Animais , Proteína 5 Relacionada à Autofagia/biossíntese , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Medula Óssea/patologia , Sistemas CRISPR-Cas , Degranulação Celular , Linhagem Celular Tumoral , Células Cultivadas , Citrobacter rodentium , Ensaio de Unidades Formadoras de Colônias , Infecções por Enterobacteriaceae/imunologia , Eosinófilos/citologia , Eosinófilos/imunologia , Humanos , Síndrome Hipereosinofílica/sangue , Síndrome Hipereosinofílica/patologia , Interleucina-5/genética , Contagem de Leucócitos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética
5.
Sci Adv ; 6(44)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33127675

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked fibrosis and low immunogenicity, features that are linked to treatment resistance and poor clinical outcomes. Therefore, understanding how PDAC regulates the desmoplastic and immune stromal components is of great clinical importance. We found that acyl-CoA synthetase long-chain 3 (ACSL3) is up-regulated in PDAC and correlates with increased fibrosis. Our in vivo results show that Acsl3 knockout hinders PDAC progression, markedly reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells, and increases cytotoxic T cell infiltration. This effect is, at least in part, due to decreased plasminogen activator inhibitor-1 (PAI-1) secretion from tumor cells. Accordingly, PAI-1 expression in PDAC positively correlates with markers of fibrosis and immunosuppression and predicts poor patient survival. We found that PAI-1 pharmacological inhibition strongly enhances chemo- and immunotherapeutic response against PDAC, increasing survival of mice. Thus, our results unveil ACSL3-PAI-1 signaling as a requirement for PDAC progression with druggable attributes.


Assuntos
Carcinoma Ductal Pancreático , Coenzima A Ligases , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Coenzima A Ligases/genética , Fibrose , Camundongos , Neoplasias Pancreáticas/patologia , Inibidor 1 de Ativador de Plasminogênio/genética , Serpina E2
6.
J Cell Biol ; 216(12): 4073-4090, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29150539

RESUMO

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein-deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species-regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.


Assuntos
Citoesqueleto/imunologia , Armadilhas Extracelulares/imunologia , Glutationa/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Actinas/genética , Actinas/imunologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Citoesqueleto/ultraestrutura , DNA Mitocondrial/imunologia , DNA Mitocondrial/metabolismo , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/imunologia , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas de Homeodomínio/imunologia , Humanos , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oxirredução , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
7.
Immunology ; 152(3): 517-525, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28703297

RESUMO

The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Eosinófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Imunidade Inata , Neutrófilos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/imunologia , Células Cultivadas , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/patologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Genótipo , Imunidade Inata/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo
8.
J Leukoc Biol ; 95(2): 293-303, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24163421

RESUMO

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.


Assuntos
Movimento Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Proteínas Quinases Associadas com Morte Celular/metabolismo , Eosinófilos/citologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Animais , Adesão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Humanos , Inflamação/patologia , Células Jurkat , Camundongos , Cadeias Leves de Miosina/metabolismo , Neutrófilos/efeitos dos fármacos , Peritonite/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
9.
Antimicrob Agents Chemother ; 54(3): 1323-6, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20065062

RESUMO

Antibiotic-induced bacteriolysis exacerbates inflammation and brain damage in bacterial meningitis. Here the quality and temporal kinetics of cerebrospinal fluid (CSF) inflammation were assessed in an infant rat pneumococcal meningitis model for the nonbacteriolytic antibiotic daptomycin versus ceftriaxone. Daptomycin led to lower CSF concentrations of interleukin 1beta (IL-1beta), IL-10, IL-18, monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1alpha) (P < 0.05). In experimental pneumococcal meningitis, daptomycin treatment resulted in more rapid bacterial killing, lower CSF inflammation, and less brain damage than ceftriaxone treatment.


Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Líquido Cefalorraquidiano/imunologia , Daptomicina/farmacologia , Inflamação , Meningite Pneumocócica/tratamento farmacológico , Animais , Animais Recém-Nascidos , Antibacterianos/administração & dosagem , Ceftriaxona/administração & dosagem , Líquido Cefalorraquidiano/efeitos dos fármacos , Líquido Cefalorraquidiano/microbiologia , Daptomicina/administração & dosagem , Modelos Animais de Doenças , Humanos , Inflamação/líquido cefalorraquidiano , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Meningite Pneumocócica/líquido cefalorraquidiano , Meningite Pneumocócica/imunologia , Meningite Pneumocócica/microbiologia , Ratos , Ratos Wistar , Resultado do Tratamento
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