RESUMO
Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils.
Assuntos
Escherichia coli , Neutrófilos , Pectinas , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Escherichia coli/efeitos dos fármacos , Pectinas/farmacologia , Trifosfato de Adenosina/metabolismo , Carbonato de Cálcio/farmacologia , Carbonato de Cálcio/química , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Ativação de Neutrófilo/efeitos dos fármacosRESUMO
Cathepsin C (CatC) is an enzyme which regulates the maturation of neutrophil serine proteases (NSPs) essential for neutrophil activation. Activated neutrophils are key players in the innate immune system, and are also implicated in the etiology of various inflammatory diseases. This study aims to demonstrate a therapeutic potential for CatC inhibitors against disorders in which activated neutrophil-derived neutrophil extracellular traps (NETs) play a significant role. We demonstrate that a CatC inhibitor, MOD06051, dose-dependently suppresses the cellular activity of NSPs, including neutrophil elastase (NE), in vitro. Neutrophils derived from MOD06051-administered rats exhibit significantly lower NE activity and NET-forming ability than controls. Furthermore, MOD06051 dose-dependently ameliorates vasculitis and significantly decreases NETs when administered to a rat model of myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody-associated vasculitis (AAV). These findings suggest that CatC inhibition is a promising strategy to reduce neutrophil activation and improve activated neutrophil-mediated diseases such as MPO-AAV.
Assuntos
Catepsina C , Armadilhas Extracelulares , Elastase de Leucócito , Ativação de Neutrófilo , Neutrófilos , Peroxidase , Catepsina C/metabolismo , Catepsina C/antagonistas & inibidores , Animais , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Humanos , Ratos , Elastase de Leucócito/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Masculino , Peroxidase/metabolismo , Peroxidase/antagonistas & inibidores , Serina Proteases/metabolismo , Ratos Sprague-Dawley , Modelos Animais de Doenças , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologiaRESUMO
The present study focused on the efficacy and role of triptolide (TPL) in relieving symptoms of acute gouty arthritis (AGA) in vivo and in vitro. The effects of TPL in AGA were investigated in monosodium urate (MSU)-treated rat ankles, RAW264.7 macrophages, and neutrophils isolated from mouse peritoneal cavity. Observation of pathological changes in the ankle joint of rats. Enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to detect the expression levels of inflammatory factors and chemokines. The levels of the indicators of macrophage M1/M2 polarization, and the mechanistic targets of Akt and rapamycin complex 2, were determined via western blotting and RT-qPCR. The expression levels of CD86 and CD206 were detected using immunohistochemistry. Neutrophil migration was observed via air pouch experiments in vivo and Transwell cell migration assay in vitro. Myeloperoxidase (MPO) and Neutrophil elastase (NE) release was analyzed by via immunohistochemistry and immunofluorescence. The expression levels of beclin-1, LC3B, Bax, Bcl-2, and cleaved caspase-3 in neutrophils were determined via western blotting and immunofluorescence. Neutrophil apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Our results suggest that TPL inhibited inflammatory cell infiltration in rat ankle joints and inflammatory factor and chemokine secretion in rat serum, regulated macrophage polarization through the PI3K/AKT signaling pathway, suppressed inflammatory factor and chemokine expression in neutrophils, and inhibited neutrophil migration, neutrophil extracellular trap formation, transitional autophagy, and apoptosis. This suggests that TPL can prevent and treat MSU-induced AGA by regulating macrophage polarization through the PI3K/Akt pathway and modulating neutrophil activity.
Assuntos
Artrite Gotosa , Diterpenos , Compostos de Epóxi , Macrófagos , Neutrófilos , Fenantrenos , Ácido Úrico , Animais , Diterpenos/farmacologia , Diterpenos/uso terapêutico , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Ratos , Fenantrenos/farmacologia , Fenantrenos/uso terapêutico , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/metabolismo , Artrite Gotosa/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Compostos de Epóxi/farmacologia , Masculino , Modelos Animais de Doenças , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ativação de Neutrófilo/efeitos dos fármacos , Humanos , Ratos Sprague-Dawley , Movimento Celular/efeitos dos fármacosRESUMO
OBJECTIVES: Neutrophils and neutrophil extracellular traps (NETs) contribute to the vascular complications of multiple diseases, but their role in systemic sclerosis (SSc) is understudied. We sought to test the hypothesis that NETs are implicated in SSc vasculopathy and that treatment with prostacyclin analogs may ameliorate SSc vasculopathy not only through vasodilation but also by inhibiting NET release. METHODS: Blood from 125 patients with SSc (87 diffuse cutaneous SSc and 38 limited cutaneous SSc) was collected at a single academic medical center. Vascular complications such as digital ulcers, pulmonary artery hypertension, and scleroderma renal crisis were recorded. The association between circulating NETs and vascular complications was determined using in vitro and ex vivo assays. The impact of the synthetic prostacyclin analog epoprostenol on NET release was determined. RESULTS: Neutrophil activation and NET release were elevated in patients with SSc-associated vascular complications compared to matched patients without vascular complications. Neutrophil activation and NETs positively correlated with soluble E-selectin and VCAM-1, circulating markers of vascular injury. Treatment of patients with digital ischemia with a synthetic prostacyclin analog boosted neutrophil cyclic AMP, which was associated with the blunting of NET release and reduced NETs in circulation. CONCLUSION: Our study demonstrates an association between NETs and vascular complications in SSc. We also identified the potential for an additional therapeutic benefit of synthetic prostacyclin analogs, namely to reduce neutrophil hyperactivity and NET release in SSc patients.
Assuntos
Epoprostenol , Armadilhas Extracelulares , Escleroderma Sistêmico , Humanos , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Feminino , Masculino , Escleroderma Sistêmico/tratamento farmacológico , Pessoa de Meia-Idade , Epoprostenol/análogos & derivados , Epoprostenol/uso terapêutico , Epoprostenol/farmacologia , Adulto , Idoso , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologiaRESUMO
Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Fagocitose , Humanos , Fagocitose/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Serpinas/metabolismo , Serpinas/genética , Serpinas/imunologia , Serpinas/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Catepsina G/metabolismo , Quimotripsina/metabolismoRESUMO
OBJECTIVES: Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator, FTY720, in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation. METHODS: The effects of FTY720 in EAV were evaluated by quantifying haematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary haemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analysed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis). RESULTS: FTY720 treatment significantly attenuated renal injury and pulmonary haemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferator-activated receptor (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1a (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis. CONCLUSION: FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Ácidos Graxos , Cloridrato de Fingolimode , Neutrófilos , Oxirredução , PPAR alfa , Cloridrato de Fingolimode/farmacologia , PPAR alfa/metabolismo , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/metabolismo , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Ratos , Humanos , Ácidos Graxos/metabolismo , Oxirredução/efeitos dos fármacos , Masculino , Peroxidase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , Ativação de Neutrófilo/efeitos dos fármacos , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologiaRESUMO
Senescent cells have a profound impact on the surrounding microenvironment through the secretion of numerous bioactive molecules and inflammatory factors. The induction of therapy-induced senescence by anticancer drugs is known, but how senescent tumor cells influence the tumor immune landscape, particularly neutrophil activity, is still unclear. In this study, we investigate the induction of cellular senescence in breast cancer cells and the subsequent immunomodulatory effects on neutrophils using the CDK4/6 inhibitor palbociclib, which is approved for the treatment of breast cancer and is under intense investigation for additional malignancies. Our research demonstrates that palbociclib induces a reversible form of senescence endowed with an inflammatory secretome capable of recruiting and activating neutrophils, in part through the action of interleukin-8 and acute-phase serum amyloid A1. The activation of neutrophils is accompanied by the release of neutrophil extracellular trap and the phagocytic removal of senescent tumor cells. These findings may be relevant for the success of cancer therapy as neutrophils, and neutrophil-driven inflammation can differently affect tumor progression. Our results reveal that neutrophils, as already demonstrated for macrophages and natural killer cells, can be recruited and engaged by senescent tumor cells to participate in their clearance. Understanding the interplay between senescent cells and neutrophils may lead to innovative strategies to cope with chronic or tumor-associated inflammation.
Assuntos
Neoplasias da Mama , Senescência Celular , Neutrófilos , Piperazinas , Piridinas , Humanos , Piperazinas/farmacologia , Piridinas/farmacologia , Senescência Celular/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Neutrófilos/metabolismo , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Linhagem Celular Tumoral , Ativação de Neutrófilo/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Neutrophils are heterogeneous and plastic, with the ability to polarize from antitumour to protumour phenotype and modulate tumour microenvironment components. While some advances have been made, the neutrophil-targeting therapy remains underexplored. Activation of formyl peptide receptors (FPRs) by formylated peptides is needed for local control of infection through the recruitment of activated neutrophils while the potential contribution of antitumour activity remains underexplored. Here, we demonstrate that neutrophils can be harnessed to suppress tumour growth through the action of the formyl peptide (FP) on the formyl peptide receptor (FPR). Mechanistically, FP efficiently recruits neutrophils to produce reactive oxygen species production (ROS), resulting in the direct killing of tumours. Antitumour functions disappeared when neutrophils were depleted by anti-Ly6G antibodies. Interestingly, extensive T-cell activation was observed in mouse tumours treated with FP, showing the potential to alter the immune suppressed tumour microenvironment (TME) and further sensitize mice to anti-PD1 therapy. Transcriptomic and flow cytometry analyses revealed the mechanisms of FP-sensitized anti-PD1 therapy, mainly including stimulated neutrophils and an altered immune-suppressed tumour microenvironment. Collectively, these data establish FP as an effective combination partner for sensitizing anti-PD1 therapy by stimulating tumour-infiltrated neutrophils.
Assuntos
Imunoterapia , Camundongos Endogâmicos C57BL , Neutrófilos , Receptores de Formil Peptídeo , Linfócitos T , Microambiente Tumoral , Animais , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Camundongos , Imunoterapia/métodos , Receptores de Formil Peptídeo/metabolismo , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Humanos , Feminino , Ativação de Neutrófilo/efeitos dos fármacos , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ativação Linfocitária/efeitos dos fármacos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologiaRESUMO
The recent global resurgence of severe infections caused by the Group A streptococcus (GAS) pathogen, Streptococcus pyogenes, has focused attention on this microbial pathogen, which produces an array of virulence factors, such as the pore-forming toxin, streptolysin O (SOT). Importantly, the interactions of SOT with human neutrophils (PMN), are not well understood. The current study was designed to investigate the effects of pretreatment of isolated human PMN with purified SOT on several pro-inflammatory activities, including generation of reactive oxygen species (ROS), degranulation (elastase release), influx of extracellular calcium (Ca2+) and release of extracellular DNA (NETosis), using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of PMN to SOT alone caused modest production of ROS and elastase release, while pretreatment with the toxin caused significant augmentation of chemoattractant (fMLP)-activated ROS generation and release of elastase by activated PMN. These effects of treatment of PMN with SOT were associated with both a marked and sustained elevation of cytosolic Ca2+concentrations and significant increases in the concentrations of extracellular DNA, indicative of NETosis. The current study has identified a potential role for SOT in augmenting the Ca2+-dependent pro-inflammatory interactions of PMN, which, if operative in a clinical setting, may contribute to hyper-activation of PMN and GAS-mediated tissue injury.
Assuntos
Armadilhas Extracelulares , Neutrófilos , Streptococcus pyogenes , Estreptolisinas , Humanos , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Inflamação/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/metabolismoRESUMO
Polymorphonuclear cells (PMN) provide a rapid response to infection and tissue damage and stress can modify these critical innate immune defences. The study of adrenergic receptor (AR) expression and function in bovine PMNs is limited but both neutrophils and eosinophils express numerous AR genes but differ significantly in their expression of individual AR genes. A flow cytometric technique was developed to differentiate between bovine neutrophils and eosinophils so both neutrophil and eosinophil responses to adrenergic agonists could be analysed. Neutrophils and eosinophils displayed significantly different changes in CD11b, L-selectin, and CD44 expression when activated by bovine serum opsonized zymosan and recombinant bovine interferon gamma. The responses of activated and resting neutrophils and eosinophils were then compared following stimulation with endogenous adrenergic agonists, epinephrine (E) norepinephrine (NE), and synthetic agonists targeting α1-, α2-, or ß-ARs. Both resting and activated neutrophils and eosinophils displayed differences in iROS, CD44, and L-selectin expression following stimulation with E and NE. Resting neutrophils displayed pro-inflammatory responses to both E and NE, while resting eosinophils displayed a pro-inflammatory response to only NE. No single synthetic adrenergic agonist fully recapitulated responses observed with either E or NE and responses to adrenergic agonists were dose-dependent. In conclusion, bovine eosinophils and neutrophils responded to multiple adrenergic agonists by altering expression of proteins involved in immune surveillance and pro-inflammatory responses. Significant differences in neutrophil and eosinophil responses to adrenergic agonists are consistent with their differences in AR gene expression. This highlights the importance of analysing separately these two PMN subpopulations when investigating the effects of either endogenous or synthetic AR agonists.
Assuntos
Eosinófilos , Epinefrina , Selectina L , Neutrófilos , Norepinefrina , Animais , Bovinos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Norepinefrina/farmacologia , Epinefrina/farmacologia , Agonistas Adrenérgicos/farmacologia , Receptores de Hialuronatos/genética , Citometria de Fluxo , Antígeno CD11b , Ativação de Neutrófilo/efeitos dos fármacos , Receptores AdrenérgicosRESUMO
INTRODUCTION: Overwhelming neutrophil activation and oxidative stress significantly contribute to acute respiratory distress syndrome (ARDS) pathogenesis. However, the potential of repurposing ribociclib, a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor used clinically in cancer treatment, for treating neutrophilic ARDS remains uncertain. This study illustrated the ability and underlying mechanism of ribociclib for treating ARDS and neutrophilic inflammation. METHODS: Primary human neutrophils were used to determine the therapeutic effects of ribociclib on respiratory bursts, chemotactic responses, and inflammatory signaling. In vitro and silico analyses were performed to determine the underlying molecular mechanisms. The potential of ribociclib repurposing was evaluated using an in vivo ARDS model in lipopolysaccharide (LPS)-primed mice. RESULTS: We found that treatment using ribociclib markedly limited overabundant oxidative stress (reactive oxygen species [ROS]) production and chemotactic responses (integrin levels and adhesion) in activated human neutrophils. Ribociclib was also shown to act as a selective inhibitor of phosphodiesterase 4 (PDE4), thereby promoting the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, leading to the inhibition of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) phosphorylation, and calcium influx. Notably, prophylactic administration and post-treatment with ribociclib ameliorated neutrophil infiltration, lung inflammation, accumulation of oxidative stress, pulmonary destruction, and mortality in mice with LPS-induced ARDS. CONCLUSION: We demonstrated for the first time that ribociclib serves as a novel PDE4 inhibitor for treating neutrophilic inflammation and ARDS. The repurposing ribociclib and targeting neutrophilic PDE4 offer a potential off-label alternative for treating lung lesions and other inflammatory conditions.
Assuntos
Aminopiridinas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Inflamação , Lipopolissacarídeos , Neutrófilos , Inibidores da Fosfodiesterase 4 , Purinas , Síndrome do Desconforto Respiratório , Aminopiridinas/farmacologia , Purinas/farmacologia , Animais , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Humanos , Camundongos , Inflamação/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismo , Masculino , AMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação de Neutrófilo/efeitos dos fármacosRESUMO
Factors influencing the activation of neutrophils in SCD and the potential neutrophil-mediated ameliorating effects of therapies in SCD.
Assuntos
Anemia Falciforme , Ativação de Neutrófilo , Neutrófilos , Anemia Falciforme/terapia , Anemia Falciforme/tratamento farmacológico , Humanos , Neutrófilos/patologia , Ativação de Neutrófilo/efeitos dos fármacosRESUMO
AIMS: One of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction. METHODS AND RESULTS: In vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls. CONCLUSIONS: Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.
Assuntos
Anti-Inflamatórios/farmacologia , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ativação de Neutrófilo/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Fibrose , Humanos , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/imunologia , Miocárdio/metabolismo , Miocárdio/patologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Transdução de SinaisRESUMO
Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil-platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.
Assuntos
Vesículas Extracelulares/metabolismo , Neutrófilos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Estreptolisinas/farmacologia , Animais , Proteínas de Bactérias/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Humanos , Camundongos , Ativação de Neutrófilo/efeitos dos fármacosRESUMO
Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.
Assuntos
Compostos de Anilina/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Neutrófilos/efeitos dos fármacos , Ácidos Nipecóticos/farmacologia , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C3/farmacologia , Convertases de Complemento C3-C5/antagonistas & inibidores , Convertases de Complemento C3-C5/metabolismo , Complemento C5a/farmacologia , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/efeitos dos fármacos , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/metabolismoRESUMO
An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 µL in healthy subjects and 700-4000 per 1 µL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 µL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.
Assuntos
Colecalciferol/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Armadilhas Extracelulares/efeitos dos fármacos , Ácidos Graxos Ômega-3/uso terapêutico , Úlcera da Perna/tratamento farmacológico , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Deficiência de Vitamina D/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Colecalciferol/efeitos adversos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Ácidos Docosa-Hexaenoicos/uso terapêutico , Quimioterapia Combinada , Ácido Eicosapentaenoico/uso terapêutico , Armadilhas Extracelulares/metabolismo , Ácidos Graxos Ômega-3/efeitos adversos , Feminino , Humanos , Úlcera da Perna/sangue , Úlcera da Perna/diagnóstico , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Projetos Piloto , Fatores de Tempo , Resultado do Tratamento , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Cicatrização/efeitos dos fármacosRESUMO
Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI (caprikd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3ß, and cofilin; and excessive actin polymerization. caprikd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients.
Assuntos
Quimiotaxia de Leucócito/imunologia , Neutrófilos/imunologia , Proteínas Ativadoras de ras GTPase/imunologia , Proteínas ras/antagonistas & inibidores , Actinas/imunologia , Movimento Celular , Polaridade Celular , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/genética , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Receptores Acoplados a Proteínas G/imunologia , Complexo Shelterina/imunologia , Transdução de Sinais , Proteínas de Ligação a Telômeros/imunologia , Proteínas Ativadoras de ras GTPase/deficiência , Proteínas Ativadoras de ras GTPase/genética , Proteínas ras/imunologiaRESUMO
Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood-brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague-Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1ß, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI-CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1ß were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI-CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.
Assuntos
Ácido Cinurênico/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Sepse/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Ácido Cinurênico/análogos & derivados , Ácido Cinurênico/síntese química , Masculino , Permeabilidade , Ratos , Sepse/tratamento farmacológico , Sepse/etiologia , Sepse/patologiaRESUMO
Hepatic cell death occurs in response to diverse stimuli such as chemical and physical damage. The exposure of intracellular contents such as DNA during necrosis induces a severe inflammatory response that has yet to be fully explored therapeutically. Here, we sought means to neutralize the ability of extracellular DNA to induce deleterious tissue inflammation when drug-induced liver injury had already ensued. DNA exposure and inflammation were investigated in vivo in drug-induced liver injury using intravital microscopy. The necrotic DNA debris was studied in murine livers in vivo and in DNA debris models in vitro by using a positively charged chemokine-derived peptide (MIG30; CXCL9[74-103]). Acetaminophen-induced liver necrosis was associated with massive DNA accumulation, production of CXC chemokines, and neutrophil activation inside the injured tissue. The MIG30 peptide bound the healthy liver vasculature and, to a much greater extent, to DNA-rich necrotic tissue. Moreover, MIG30 bound extracellular DNA directly in vivo in a charge-dependent manner and independently of glycosaminoglycans and chemokines. Post-treatment of mice with MIG30 reduced mortality, liver damage, and inflammation significantly. These effects were not observed with a control peptide that does not bind DNA. Mechanistically, MIG30 inhibited the interaction between DNA and histones, and promoted the dissociation of histones from necrotic debris. MIG30 also inhibited the pro-inflammatory effect of CpG DNA, as measured by a reduction in CXCL8 production, indicating that MIG30 disturbs the ability of DNA to induce hepatic inflammation. Conclusion: The use of DNA-binding peptides reduces necrotic liver injury and inflammation, even at late timepoints.
Assuntos
Anti-Inflamatórios/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Degradação Necrótica do DNA/efeitos dos fármacos , Fígado/patologia , Peptídeos/farmacologia , Acetaminofen/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Quimiocina CXCL9/efeitos dos fármacos , Quimiocinas CXC/efeitos dos fármacos , Modelos Animais de Doenças , Matriz Extracelular/genética , Histonas/efeitos dos fármacos , Humanos , Interleucina-8/efeitos dos fármacos , Fígado/efeitos dos fármacos , Camundongos , Necrose/induzido quimicamente , Necrose/patologia , Ativação de Neutrófilo/efeitos dos fármacos , Eletricidade EstáticaRESUMO
Neutrophil extracellular traps (NETs) are extracellular webs of DNA, histones and granular contents that are released by neutrophils to control infections. However, NETs that is not properly regulated can propagate inflammation and thrombosis. It was recognized that viruses can induce NETs. As a synthetic analog of viral double-stranded (ds) RNA, polyinosinic-polycytidylic acid [poly(I:C)] is known to induce inflammation and thrombosis. However, whether and how poly(I:C) modulates NETs remains unclear. Here, we have demonstrated that poly(I:C) induced extracellular DNA traps in human neutrophils in a dose-dependent manner. Further, poly(I:C) or dsRNA virus elevated the levels of myeloperoxidase-DNA complexes and citrullinated histone H3, which are specific markers of NETs, in both neutrophil supernatants and mouse plasma. Interestingly, a potent peptidylarginine deiminase 4 (PAD4) inhibitor, BB-CL-Amidine (BB-CLA) or PAD4 knockdown effectively prevented poly(I:C)-induced NETs formation and release. In addition, BB-CLA abrogated poly(I:C)-triggered neutrophil activation and infiltration, and vascular permeability in lungs. BB-CLA also attenuated poly(I:C)-induced thrombocytopenia in circulation, fibrin deposition and thrombus formation in tissues. Taken together, these results suggest that viral mimetic poly(I:C) may induce NETs-dependent inflammation and thrombosis through PAD4, and that inhibiting PAD4 may become a good strategy to protect against viral infection-caused inflammation/thrombosis-related pathological conditions of diseases.