RESUMO
The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2â¯h) and slower accumulation of androstenedione and testosterone (24â¯h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.
Assuntos
Androgênios , Desidroepiandrosterona , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Androgênios/metabolismo , Androgênios/biossíntese , Desidroepiandrosterona/metabolismo , Testosterona/metabolismo , Androstenodiona/metabolismo , Rotenona/farmacologia , Glândulas Suprarrenais/metabolismo , Metabolismo Energético , Linhagem Celular , Linhagem Celular Tumoral , Complexo I de Transporte de Elétrons/metabolismoRESUMO
Empagliflozin has been successfully repurposed for treating neutropenia and neutrophil dysfunction in patients with glycogen storage disease type 1b (GSD 1b), however, data in infants are missing. We report on efficacy and safety of empagliflozin in infants with GSD 1b. This is an international retrospective case series on 21 GSD 1b infants treated with empagliflozin (total treatment time 20.6 years). Before starting empagliflozin (at a median age of 8.1 months with a median dose of 0.3 mg/kg/day) 12 patients had clinical signs and symptoms of neutrophil dysfunction. Six of these previously symptomatic patients had no further neutropenia/neutrophil dysfunction-associated findings on empagliflozin. Eight patients had no signs and symptoms of neutropenia/neutrophil dysfunction before start and during empagliflozin treatment. One previously asymptomatic individual with a horseshoe kidney developed a central line infection with pyelonephritis and urosepsis during empagliflozin treatment. Of the 10 patients who were treated with G-CSF before starting empagliflozin, this was stopped in four and decreased in another four. Eleven individuals were never treated with G-CSF. While in 17 patients glucose homeostasis remained stable on empagliflozin, four showed glucose homeostasis instability in the introductory phase. In 17 patients, no other side effects were reported, while genital (n = 2) or oral (n = 1) candidiasis and skin infection (n = 1) were reported in the remaining four. Empagliflozin had a good effect on neutropenia/neutrophil dysfunction-related signs and symptoms and a favourable safety profile in infants with GSD 1b and therefore qualifies for further exploration as first line treatment.
Assuntos
Compostos Benzidrílicos , Glucosídeos , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Neutrófilos , Humanos , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/complicações , Neutropenia/tratamento farmacológico , Masculino , Feminino , Lactente , Compostos Benzidrílicos/uso terapêutico , Compostos Benzidrílicos/administração & dosagem , Estudos Retrospectivos , Neutrófilos/efeitos dos fármacos , Glucosídeos/uso terapêutico , Glucosídeos/farmacologia , Glucosídeos/administração & dosagem , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Resultado do Tratamento , Fator Estimulador de Colônias de Granulócitos/uso terapêuticoRESUMO
Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1ß production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1ß production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions.
Assuntos
Inflamassomos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Inflamassomos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Cálcio/metabolismo , Macrófagos/metabolismo , Imunidade Inata , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismoAssuntos
Asma , Armadilhas Extracelulares , Humanos , Receptores Proteína Tirosina Quinases , NeutrófilosRESUMO
Neutrophils are a specialized subset of white blood cells, which have the ability to store pre-formed mediators in their cytoplasmic granules. Neutrophils are well-known effector cells involved in host protection against pathogens through diverse mechanisms such as phagocytosis, degranulation, extracellular traps, and oxidative burst. In this study, we provide evidence highlighting the significance of the SNARE proteins syntaxin-4 and synaptosomal-associated protein (SNAP) 23 in the release of azurophilic granules, specific granules, and the production of reactive oxygen species in human neutrophils. In contrast, the specific blockade of either syntaxin-4 or SNAP23 did not prevent the release of mitochondrial dsDNA in the process of neutrophil extracellular trap (NET) formation. These findings imply that degranulation and the release of mitochondrial dsDNA involve at least partially distinct molecular pathways in neutrophils.
Assuntos
Armadilhas Extracelulares , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Humanos , DNA Mitocondrial/metabolismo , Exocitose , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismoRESUMO
Neutrophil extracellular traps (NETs) and oxidative stress are considered to be beneficial in the innate immune defense against pathogens. However, defective clearance of NETs in the lung of acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients could lead to severe respiratory syndrome infection, the so-called coronavirus disease 2019 (COVID-19). To elucidate the pathways that are related to NETs within the pathophysiology of COVID-19, we utilized RNA sequencing (RNA-seq) as well as immunofluorescence and immunohistochemistry methods. RNA-seq analysis provided evidence for increased oxidative stress and the activation of viral-related signaling pathways in post-mortem lungs of COVID-19 patients compared to control donors. Moreover, an excess of neutrophil infiltration and NET formation were detected in the patients' lungs, where the extracellular DNA was oxidized and co-localized with neutrophil granule protein myeloperoxidase (MPO). Interestingly, staining of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) depicted high colocalization with NETs and was correlated with the neutrophil infiltration of the lung tissues, suggesting that it could serve as a suitable marker for the identification of NETs and the severity of the disease. Moreover, local inhalation therapy to reduce the excess lipid oxidation and NETs in the lungs of severely infected patients might be useful to ameliorate their clinical conditions.
Assuntos
COVID-19 , Armadilhas Extracelulares , Humanos , COVID-19/metabolismo , Armadilhas Extracelulares/metabolismo , SARS-CoV-2 , Pulmão , Estresse Oxidativo , Neutrófilos/metabolismoRESUMO
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/metabolismoRESUMO
Neutrophils are the most abundant innate immune cells in human blood, emerging as important players in a variety of diseases. Mitochondria are bioenergetic, biosynthetic, and signaling organelles critical for cell fate and function. Mitochondria have been overlooked in neutrophil research owing to the conventional view that neutrophils contain few, if any, competent mitochondria and do not rely on these organelles for adenosine triphosphate production. A growing body of evidence suggests that mitochondria participate in neutrophil biology at many levels, ranging from neutrophil development to chemotaxis, effector function, and cell death. Moreover, mitochondria and mitochondrial components, such as mitochondrial deoxyribonucleic acid, can be released by neutrophils to eliminate infection and/or shape immune response, depending on the specific context. In this review, we provide an update on the functional role of mitochondria in neutrophils, highlight mitochondria as key players in modulating the neutrophil phenotype and function during infection and inflammation, and discuss the possibilities and challenges to exploit the unique aspects of mitochondria in neutrophils for disease treatment.
Assuntos
Mitocôndrias , Neutrófilos , Humanos , Mitocôndrias/metabolismo , Inflamação , Diferenciação Celular , QuimiotaxiaRESUMO
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 , AutofagiaRESUMO
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/metabolismoRESUMO
Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Imunidade Inata , Redes e Vias Metabólicas , FagocitoseRESUMO
Eosinophils are a subset of granulocytes characterized by a high abundance of specific granules in their cytoplasm. To act as effector cells, eosinophils degranulate and form eosinophil extracellular traps (EETs), which contain double-stranded DNA (dsDNA) co-localized with granule proteins. The exact molecular mechanism of EET formation remains unknown. Although the term "EET release" has been used in scientific reports, it is unclear whether EETs are pre-formed in eosinophils and subsequently released. Moreover, although eosinophil degranulation has been extensively studied, a precise time-course of granule protein release has not been reported until now. In this study, we investigated the time-dependent release of eosinophil peroxidase (EPX) and mitochondrial DNA (mtDNA) following activation of both human and mouse eosinophils. Unexpectedly, maximal degranulation was already observed within 1 min with no further change upon complement factor 5 (C5a) stimulation of interleukin-5 (IL-5) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-primed eosinophils. In contrast, bulk mtDNA release in the same eosinophil populations occurred much slower and reached maximal levels between 30 and 60 min. Although no single-cell analyses have been performed, these data suggest that the molecular pathways leading to degranulation and mtDNA release are at least partially different. Moreover, based on these data, it is likely that the association between the mtDNA scaffold and granule proteins in the process of EET formation occurs in the extracellular space.
Assuntos
DNA Mitocondrial/metabolismo , Peroxidase de Eosinófilo/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , CinéticaRESUMO
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/genéticaRESUMO
Cytokine-primed neutrophils can undergo a nonapoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and NADPH oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF-primed human neutrophils. Specifically, we observed that several inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α) were able to block CD44-mediated reactive oxygen species production and cell death, but not FAS receptor-mediated apoptosis. To understand how FAP-α is involved in this nonapoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 MAPK, PI3K, and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL, and p38 MAPK activation but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis underwent a GM-CSF-independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3-MLKL pathway activates NADPH oxidase but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated nonapoptotic neutrophil death, leading to tissue damage.
Assuntos
Artrite Reumatoide/imunologia , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina Endopeptidases/metabolismo , Células Cultivadas , Endopeptidases , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Terapia de Alvo Molecular , NADPH Oxidases/metabolismo , Necroptose , Ativação de Neutrófilo , Neutrófilos/imunologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Endophilin B1, also known as BAX-interacting protein 1 (BIF-1), is part of the endophilin B protein family, and is a multifunctional protein involved in the regulation of apoptosis, autophagy, and mitochondrial morphology. The role of BIF-1 in cancer is controversial since previous reports indicated to both tumor-promoting and tumor-suppressive roles, perhaps depending on the cancer cell type. In the present study, we report that BIF-1 is significantly downregulated in both primary and metastatic melanomas, and that patients with high levels of BIF-1 expression exhibited a better overall survival. Depleting BIF-1 using CRISPR/Cas9 technology in melanoma cells resulted in higher proliferation rates both in vitro and in vivo, a finding that was associated with increased ATP production, metabolic acidification, and mitochondrial respiration. We also observed mitochondrial hyperpolarization, but no increase in the mitochondrial content of BIF-1-knockout melanoma cells. In contrast, such knockout melanoma cells were equally sensitive to anticancer drug- or UV irradiation-induced cell death, and exhibited similar autophagic activities as compared with control cells. Taken together, it appears that downregulation of BIF-1 contributes to tumorigenesis in cutaneous melanoma by upregulating mitochondrial respiration and metabolism, independent of its effect on apoptosis and autophagy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Trifosfato de Adenosina/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Mitocôndrias/metabolismo , Neoplasias Cutâneas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Regulação para Baixo , Glicólise , Humanos , Estimativa de Kaplan-Meier , Melanoma/metabolismo , Melanoma/terapia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Extracellular DNA trap formation is a cellular function of neutrophils, eosinophils, and basophils that facilitates the immobilization and killing of invading microorganisms in the extracellular milieu. To form extracellular traps, granulocytes release a scaffold consisting of mitochondrial DNA in association with granule proteins. As we understand more about the molecular mechanism for the formation of extracellular DNA traps, the in vivo function of this phenomenon under pathological conditions remains an enigma. In this article, we critically review the literature to summarize the evidence for extracellular DNA trap formation under in vivo conditions. Extracellular DNA traps have not only been detected in infectious diseases but also in chronic inflammatory diseases, as well as in cancer. While on the one hand, extracellular DNA traps clearly exhibit an important function in host defense, it appears that they can also contribute to the maintenance of inflammation and metastasis, suggesting that they may represent an interesting drug target for such pathological conditions.
Assuntos
DNA/genética , Armadilhas Extracelulares/metabolismo , Animais , Humanos , CamundongosRESUMO
Activation of neutrophils is an important mechanism in the pathology of granulomatosis with polyangiitis (GPA). In this study, we evaluated whether extracellular vesicles (EVs) circulating in the plasma of GPA patients could contribute to this process. EVs from the plasma of GPA patients in the active stage of the disease (n = 10) and healthy controls (n = 10) were isolated by ultracentrifugation and characterized by flow cytometry (CD63, CD8) and nanoparticle tracking analysis. Targeted oxylipin lipidomics of EVs was performed by HPLC-MS/MS. EV/oxylipin-induced neutrophil extracellular traps (NETs) were analyzed by confocal microscopy, and released double-stranded DNA (dsDNA) was quantified by PicoGreen fluorescent dye. Reactive oxygen species (ROS) production and neutrophils' EV binding/uptake were evaluated by flow cytometry. Brief priming with granulocyte-macrophage colony-stimulating factor was required for EV-mediated ROS production and dsDNA release. It was observed that priming also increased EV binding/uptake by neutrophils only for EVs from GPA patients. EVs from GPA patients had higher concentrations of leukotriene (LT)B4 and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB4 or 5-oxo-ETE produced ROS and released dsDNA in a concentration-dependent manner. These results reveal the potential role of EVs containing oxylipin cargo on ROS production and NET formation by activated neutrophils.
Assuntos
Ácidos Araquidônicos/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Granulomatose com Poliangiite/tratamento farmacológico , Leucotrieno B4/farmacologia , Neutrófilos/efeitos dos fármacos , DNA/análise , DNA/metabolismo , Relação Dose-Resposta a Droga , Vesículas Extracelulares/metabolismo , Granulomatose com Poliangiite/sangue , Granulomatose com Poliangiite/metabolismo , Humanos , Neutrófilos/metabolismo , Oxilipinas/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Encapsulation of porphyrinic photosensitizers (PSs) into polymeric carriers plays an important role in enhancing their efficiency as drugs in photodynamic therapy (PDT). Porphyrin aggregation and low solubility as well as the preservation of the advantageous photophysical properties pose a challenge on the design of efficient PS-carrier systems. Block copolymer micelles (BCMs) and polyvinylpyrrolidone (PVP) are promising drug delivery vehicles for physical entrapment of PSs. BCMs exhibit enhanced dynamics as compared to the less flexible PVP network. In the current work the question is addressed how these different dynamics affect PS encapsulation, release from the carrier, reaction with serum proteins, and cellular uptake. The porphyrinic compounds serine-amide of chlorin e6 (SerCE) and chlorin e4 (CE4) were used as model PSs with different lipophilicity and aggregation properties. 1H NMR and fluorescence spectroscopy were applied to study their interactions with PVP and BCMs consisting of Kolliphor P188 (KP). Both chlorins were well encapsulated by the carriers and had improved photophysical properties. Compared to SerCE, the more lipophilic CE4 exhibited stronger hydrophobic interactions with the BCM core, stabilizing the system and preventing exchange with the surrounding medium as was shown by NMR NOESY and DOSY experiments. PVP and BCMs protected the encapsulated chlorins against interaction with human transferrin (Tf). However, SerCE and CE4 were released from BCMs in favor of binding to human serum albumin (HSA) while PVP prevented interaction with HSA. Fluorescence spectroscopic studies revealed that HSA binds to the surface of PVP forming a protein corona. PVP and BCMs reduced cellular uptake of the chlorins. However, encapsulation into BCMs resulted in more efficient cell internalization for CE4 than for SerCE. HSA significantly lowered both, free and carrier-mediated cell uptake for CE4 and SerCE. In conclusion, PVP appears as the more universal delivery system covering a broad range of host molecules with respect to polarity, whereas BCMs require a higher drug-carrier compatibility. Poorly soluble hydrophobic PSs benefit stronger from BCM-type carriers due to enhanced bioavailability through disaggregation and solubilization allowing for more efficient cell uptake. In addition, increased PS-carrier hydrophobic interactions have a stabilizing effect. For more hydrophilic PSs, the main advantage of polymeric carriers like PVP or poloxamer micelles lies in their protection during the transport through the bloodstream. HSA binding plays an important role for drug release and cell uptake in carrier-mediated delivery to the target tissue.
Assuntos
Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Povidona/química , Células Cultivadas , Clorofilídeos , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Fármacos Fotossensibilizantes/química , Polímeros/química , Porfirinas/química , Serina/química , Albumina Sérica Humana/metabolismo , Solubilidade , Transferrina/metabolismoRESUMO
Microtubules (MTs) are critically involved in the transport of material within cells, but their roles in chemotactic leukocyte motility and effector functions are still obscure. Resting neutrophils contain few MTs assembled in an MT organizing center (MTOC) behind their multilobular nuclei. Using a probe of real-time tubulin polymerization, SiR-tubulin, we found that neutrophils elongated their MTs within minutes in response to signals from the two prototypic chemotactic peptides, CXCL1 and fMLP. Taxol, a beta-tubulin binding and MT stabilizing drug, was found to abolish this CXCL1- and fMLP-stimulated MT polymerization. Nevertheless, taxol treatment as well as disruption of existing and de novo generated MTs did not impair neutrophil protrusion and squeezing through IL-1ß-stimulated endothelial monolayers mediated by endothelial deposited CXCL1 and neutrophil CXCR2. Notably, CXCL1-dependent neutrophil TEM was not associated with neutrophil MT polymerization. Chemokinetic neutrophil motility on immobilized CXCL1 was also not associated with MT polymerization, and taxol treatment did not interfere with this motility. Nevertheless, and consistent with its ability to suppress MT polymerization induced by soluble CXCL1 and fMLP, taxol treatment inhibited neutrophil chemotaxis toward both chemotactic peptides. Taxol treatment also suppressed CXCL1- and fMLP-triggered elastase-dependent neutrophil invasion through collagen I barriers. Collectively, our results highlight de novo chemoattractant-triggered MT polymerization as key for neutrophil chemotaxis and elastase-dependent invasion but not for chemotactic neutrophil crossing of inflamed endothelial barriers.