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1.
Sci Rep ; 14(1): 14524, 2024 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-38914598

RESUMO

NLRP3 inflammasome has been implicated in neutrophil polarization and extrusion of neutrophil extracellular traps (NETs) in vitro and facilitates secretion of Il1-beta (IL-1ß). Permanent ligation of the left anterior descending artery was used to induce MI in WT and NLRP3-/- mice as well as in NLRP3-/- recipient mice transfused with either WT or NLRP3-/- neutrophils. NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury and preserved left ventricular (LV) function at 12 h after MI as assessed by echocardiography and triphenyltetrazolium chloride staining of live tissue. Transfusion of WT but not NLRP3-/- neutrophils after MI increased infarct size in NLRP3-/- mice and significantly reduced LV function. The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology and increased tissue levels of IL-1ß and plasma levels of von Willebrand Factor (VWF). Flow cytometry analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI. Our data suggest a key role for neutrophil NLRP3 in the production of IL-1ß and deposition of NETs in cardiac tissue exacerbating injury following MI. We provide evidence for a link between neutrophil NLRP3 and VWF release likely enhancing thromboinflammation in the heart. Neutrophil NLRP3 deficiency conferred similar cardioprotective effects to general NLRP3 deletion in MI rendering anti-neutrophil NLRP3 therapy a promising target for early cardioprotective treatment.


Assuntos
Armadilhas Extracelulares , Interleucina-1beta , Camundongos Knockout , Infarto do Miocárdio , Miocárdio , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neutrófilos , Fator de von Willebrand , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neutrófilos/metabolismo , Interleucina-1beta/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Camundongos , Armadilhas Extracelulares/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Fator de von Willebrand/metabolismo , Fator de von Willebrand/genética , Camundongos Endogâmicos C57BL , Masculino , Inflamassomos/metabolismo , Modelos Animais de Doenças
2.
J Thromb Haemost ; 22(5): 1496-1509, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38325598

RESUMO

BACKGROUND: Obesity is an increasing problem in our current society and is expected to keep rising in incidence. With its multiorigin, complex pathophysiology, it is difficult to treat and easy to acquire unnoticeably. During obesity, it has been established that the body is in a constant state of low-grade inflammation, thereby causing changes in immune cell physiology. OBJECTIVES: Here, we investigated the influence of neutrophils, more specifically as a result of peptidylarginine deiminase 4 (PAD4) activity and the release of neutrophil extracellular traps (NETs), during obesity-induced chronic inflammation. METHODS: Wild-type mice were placed on a high-fat diet (HFD) and investigated over a period of 10 weeks for NET formation and its impact on the heart. Neutrophil-selective PAD4 knockout (Ne-PAD4-/-) mice were studied in parallel. RESULTS: As a result of high fat intake, we observed clear alteration in the priming status of isolated neutrophils toward NET release, including early stages of speck formation and histone citrullination of apoptosis-associated speck-like protein containing a CARD. Ne-PAD4-/- mice deficient in NET formation did not increase bodyweight to the same extent as their littermate controls, with Ne-PAD4-/- mice being leaner after 10 weeks of HFD feeding. Interestingly, obesity progression led to cardiac remodeling and diastolic dysfunction in wild-type mice after 10 weeks, while this remodeling and subsequent decrease in function were absent in Ne-PAD4-/- mice. Surprisingly, HFD did not alter NET content or thrombus formation in the inferior vena cava stenosis model. CONCLUSION: Detrimental physiological effects, the result of obesity progression, can in part be attributed to neutrophil PAD4 and NETs in response to chronic inflammation.


Assuntos
Dieta Hiperlipídica , Armadilhas Extracelulares , Inflamação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos , Obesidade , Proteína-Arginina Desiminase do Tipo 4 , Animais , Neutrófilos/imunologia , Neutrófilos/metabolismo , Obesidade/enzimologia , Armadilhas Extracelulares/metabolismo , Modelos Animais de Doenças , Masculino , Doença Crônica , Camundongos , Citrulinação
3.
Proc Natl Acad Sci U S A ; 120(35): e2303814120, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37603754

RESUMO

Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1ß and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3-/-) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3-/- neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.


Assuntos
Quimiotaxia , Leucotrieno B4 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Inflamassomos , Leucotrieno B4/metabolismo , Neutrófilos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
4.
Life Sci Alliance ; 6(10)2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37500179

RESUMO

Rheumatoid arthritis is a prototypic inflammatory condition with affected patients being at greater risk of incident heart failure (HF). Targeting innate immune cell function in the pathogenesis of HF bears the potential to guide the development of future therapies. A collagen-induced arthritis (CIA) model in DBA/1 J mice was used to generate arthritis. Mice with CIA developed concentric hypertrophic myocardial remodeling, left ventricular (LV) diastolic dysfunction, and HF with elevated plasma B-type natriuretic peptide levels but preserved LV ejection fraction. Key features of HF in CIA were increased infiltration of activated neutrophils, deposition of neutrophil extracellular traps in the myocardium, and increased tissue levels of the proinflammatory cytokine IL-1ß. Specific inhibition of protein arginine deiminase 4 (PAD4) by an orally available inhibitor (JBI-589), administered after the onset of clinical arthritis, prevented HF with reduced neutrophil infiltration. We identify PAD4-mediated neutrophil activation and recruitment as the key thromboinflammatory pathway driving HF development in arthritis. Targeting PAD4 may be a viable therapeutic approach for the prevention of HF secondary to chronic inflammation.


Assuntos
Artrite , Insuficiência Cardíaca , Camundongos , Animais , Proteína-Arginina Desiminase do Tipo 4 , Camundongos Endogâmicos DBA , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/prevenção & controle , Inflamação
5.
Sci Rep ; 13(1): 3189, 2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36823444

RESUMO

Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 - deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.


Assuntos
Artrite Reumatoide , Armadilhas Extracelulares , Proteína-Arginina Desiminase do Tipo 4 , Animais , Humanos , Camundongos , Artrite Reumatoide/metabolismo , Armadilhas Extracelulares/metabolismo , Camundongos Knockout , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores
6.
Sci Rep ; 12(1): 21313, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494392

RESUMO

NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1ß, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3-/- and Nlrp3+/+ mice. Nlrp3-/- mice recruited fewer neutrophils than Nlrp3+/+ into the peritoneum and showed lower IL-1ß in peritoneal lavage fluid. The higher production of IL-1ß in Nlrp3+/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1ß production. The Nlrp3+/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3-/- neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3+/+ venules than in Nlrp3-/-. Nlrp3-/- endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3+/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1ß in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/metabolismo , Células Endoteliais/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Caspase 1/metabolismo , Peritonite/induzido quimicamente , Peritonite/metabolismo , Camundongos Endogâmicos C57BL
7.
Arterioscler Thromb Vasc Biol ; 42(9): 1103-1112, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35861953

RESUMO

The activating interplay of thrombosis and inflammation (thromboinflammation) has been established as a major underlying pathway, driving not only cardiovascular disease but also autoimmune disease and most recently, COVID-19. Throughout the years, innate immune cells have emerged as important modulators of this process. As the most abundant white blood cell in humans, neutrophils are well-positioned to propel thromboinflammation. This includes their ability to trigger an organized cell death pathway with the release of decondensed chromatin structures called neutrophil extracellular traps. Decorated with histones and cytoplasmic and granular proteins, neutrophil extracellular traps exert cytotoxic, immunogenic, and prothrombotic effects accelerating disease progression. Distinct steps leading to extracellular DNA release (NETosis) require the activities of PAD4 (protein arginine deiminase 4) catalyzing citrullination of histones and are supported by neutrophil inflammasome. By linking the immunologic function of neutrophils with the procoagulant and proinflammatory activities of monocytes and platelets, PAD4 activity holds important implications for understanding the processes that fuel thromboinflammation. We will also discuss mechanisms whereby vascular occlusion in thromboinflammation depends on the interaction of neutrophil extracellular traps with ultra-large VWF (von Willebrand Factor) and speculate on the importance of PAD4 in neutrophil inflammasome assembly and neutrophil extracellular traps in thromboinflammatory diseases including atherosclerosis and COVID-19.


Assuntos
Aterosclerose , COVID-19 , Armadilhas Extracelulares , Trombose , Aterosclerose/metabolismo , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/metabolismo , Neutrófilos/metabolismo , Tromboinflamação , Trombose/etiologia , Trombose/metabolismo , Fator de von Willebrand/metabolismo
8.
J Thromb Haemost ; 20(10): 2386-2393, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35875933

RESUMO

BACKGROUND: Patients with rheumatoid arthritis (RA) have frequent thrombotic events with endothelial dysfunction. Von Willebrand factor (VWF) has been shown to bind neutrophil extracellular traps (NETs) and NETs are part of RA etiology. OBJECTIVES: This study aims to elucidate whether this prothrombotic status exacerbates inflammation in arthritis. Here we focus on the involvement of A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving VWF and its effect on NET deposition and RA development. METHODS: We evaluated the influence of the Adamts13 gene and recombinant human ADAMTS-13 (rhADAMTS-13) on arthritis in the mouse models of collagen-induced arthritis (CIA). We also assessed VWF and NETs in synovial tissue. RESULTS: Several Adamts13-/- mice developed arthritis, while Adamts13+/+ siblings did not. Synovial tissue from Adamts13-/- showed accumulation of NETs. Treatment of DBA/1 J mice, an arthritis-susceptible strain, with well-tolerated doses of rhADAMT13 reduced arthritis incidence and alleviated the severity of arthritis. Mice treated with rhADAMT13 presented less serum interleukin 6 and less bone erosion determined by micro-computed tomography. The effects on arthritis severity were observed both when administering rhADAMTS-13 prophylactically and also when given after arthritis has developed. In both conditions, rhADAMTS-13 reduced VWF and NET deposition on proliferated synovial tissue evaluated by immunoblotting. CONCLUSIONS: Our results demonstrate the inhibitory role of Adamts13 in murine arthritis and the effectiveness of rhADAMTS-13 treatment. Additionally, this study suggests that deposition of VWF in the synovium and subsequent pathogenic NET retention promotes arthritis. Treatment with rhADAMTS-13 provides a potential therapeutic approach targeting inflammation and pro-thrombotic state in arthritis.


Assuntos
Proteína ADAMTS13/metabolismo , Artrite , Trombose , Animais , Artrite/metabolismo , Artrite/patologia , Desintegrinas , Humanos , Inflamação , Interleucina-6 , Camundongos , Camundongos Endogâmicos DBA , Microtomografia por Raio-X , Fator de von Willebrand/metabolismo
9.
J Exp Med ; 219(6)2022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35522219

RESUMO

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.


Assuntos
Neoplasias , Neutrófilos , Humanos , Imunidade Inata , Inflamação , Neoplasias/genética , Fenótipo
10.
Arthritis Rheumatol ; 74(7): 1139-1146, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35166055

RESUMO

OBJECTIVE: Genome-wide association studies have connected PADI4, encoding peptidylarginine deiminase 4 (PAD4), with rheumatoid arthritis (RA). PAD4 promotes neutrophil extracellular trap (NET) formation. This study was undertaken to investigate the origin of PAD4 and the importance of NET formation in a C57BL/6 mouse model of arthritis. METHODS: To permit the effective use of C57BL/6 mice in the collagen-induced arthritis (CIA) model, we introduced the administration of granulocyte colony-stimulating factor (G-CSF) for 4 consecutive days in conjunction with the booster immunization on day 21. Mice with global Padi4 deficiency (Padi4-/- ) and mice with hematopoietic lineage-specific Padi4 deficiency (Padi4Vav1Cre/+ ) were evaluated in the model. RESULTS: G-CSF significantly increased the incidence and severity of CIA. G-CSF-treated mice showed elevated citrullinated histone H3 (Cit-H3) levels in plasma, while vehicle-treated mice did not. Immunofluorescence microscopy revealed deposition of Cit-H3 in synovial tissue in G-CSF-treated mice. Padi4-/- mice developed less severe arthritis and had lower levels of serum interleukin-6 and plasma Cit-H3, lower levels of Cit-H4 in synovial tissue, and less bone erosion on micro-computed tomography than Padi4+/+ mice in the G-CSF-modified CIA model. Similarly, Padi4Vav1Cre/+ mice developed less severe arthritis, compared with Padi4fl/fl mice, and presented the same phenotype as Padi4-/- mice. CONCLUSION: We succeeded in developing an arthritis model suitable for use in C57BL/6 mice that is fully compliant with high animal welfare standards. We observed a >90% incidence of arthritis in male mice and detectable NET markers. This model, with some features consistent with human RA, demonstrates that hematopoietic PAD4 is an important contributor to arthritis development and may prove useful in future RA research.


Assuntos
Artrite Experimental , Artrite Reumatoide , Proteína-Arginina Desiminase do Tipo 4 , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/enzimologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/enzimologia , Colágeno , Estudo de Associação Genômica Ampla , Fator Estimulador de Colônias de Granulócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Desiminases de Arginina em Proteínas , Microtomografia por Raio-X
11.
Blood Adv ; 6(7): 2001-2013, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-34991159

RESUMO

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.


Assuntos
COVID-19 , Armadilhas Extracelulares , Animais , Armadilhas Extracelulares/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Neutrófilos/metabolismo , SARS-CoV-2
14.
Cell Chem Biol ; 28(12): 1728-1739.e5, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34352225

RESUMO

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.


Assuntos
Antifibrinolíticos/farmacologia , Antitrombinas/farmacologia , Inativadores de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Trombose Venosa/tratamento farmacológico , Animais , Antifibrinolíticos/química , Antitrombinas/química , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/metabolismo , Inativadores de Plasminogênio/química , Inibidores de Serina Proteinase/química , Trombose Venosa/metabolismo
15.
Front Immunol ; 12: 683803, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122445

RESUMO

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.


Assuntos
Armadilhas Extracelulares/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Neutrófilos/enzimologia , Animais , Caspase 1/farmacologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neutrófilos/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Trombose Venosa/sangue , Trombose Venosa/enzimologia , Trombose Venosa/genética
17.
PLoS One ; 16(5): e0251726, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33999963

RESUMO

Peptidylarginine deiminase 4 (PAD4) catalyzes posttranslational modification of many target proteins through converting protein arginine or mono-methylarginine to citrulline. Neutrophil extracellular trap (NET) formation is the most dramatic manifestation of PAD4-mediated hypercitrullination reaction in neutrophils, which is characterized by the release of nuclear chromatin to form a chromatin network in the extracellular space. Histones H4, one of the major protein components of chromatin, is released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury and can also be released during the process of NET formation, along with its citrullinated form. The present study showed that histone H4 can induce NET formation in a calcium and PAD4 dependent manner. Histone H4 caused permeabilization of the neutrophil membrane and sustained rise in intracellular calcium that is necessary for activation of PAD4. In comparison, citrullinated histone H4 induced less calcium influx compared with its native form, leading to reduced NET formation. These studies suggest that citrullinated histone H4 could serve as a brake in the pathology of NETs, slowing down the vicious circle between histone H4 and NETs.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Citrulinação , Armadilhas Extracelulares/metabolismo , Histonas/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/metabolismo , Animais , Células HL-60 , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout
18.
J Thromb Haemost ; 19(2): 387-399, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33058430

RESUMO

PURPOSE: Platelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis. METHODS: We used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet-depleting antibody (anti-GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined. RESULTS: Mice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers. CONCLUSIONS: This work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.


Assuntos
Veia Cava Inferior , Trombose Venosa , Animais , Plaquetas , Modelos Animais de Doenças , Fibrose , Camundongos , Veia Cava Inferior/diagnóstico por imagem , Veia Cava Inferior/patologia , Trombose Venosa/patologia
20.
Annu Rev Cell Dev Biol ; 36: 191-218, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32663035

RESUMO

Neutrophils are critical to innate immunity, including host defense against bacterial and fungal infections. They achieve their host defense role by phagocytosing pathogens, secreting their granules full of cytotoxic enzymes, or expelling neutrophil extracellular traps (NETs) during the process of NETosis. NETs are weblike DNA structures decorated with histones and antimicrobial proteins released by activated neutrophils. Initially described as a means for neutrophils to neutralize pathogens, NET release also occurs in sterile inflammation, promotes thrombosis, and can mediate tissue damage. To effectively manipulate this double-edged sword to fight a particular disease, researchers must work toward understanding the mechanisms driving NETosis. Such understanding would allow the generation of new drugs to promote or prevent NETosis as needed. While knowledge regarding the (patho)physiological roles of NETosis is accumulating, little is known about the cellular and biophysical bases of this process. In this review, we describe and discuss our current knowledge of the molecular, cellular, and biophysical mechanisms mediating NET release as well as open questions in the field.


Assuntos
Armadilhas Extracelulares/metabolismo , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , DNA/metabolismo , Humanos
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