RESUMO
Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential.
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Vacinas Anticâncer , Camundongos , Humanos , Animais , Idoso , Poli I-C , Fosfatidilserinas , Cisplatino , Antígenos de Neoplasias , EritrócitosRESUMO
OBJECTIVES: The presence of proinflammatory low-density granulocytes (LDG) has been demonstrated in autoimmune and infectious diseases. Recently, regulatory neutrophilic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) were identified in systemic lupus erythematosus (SLE). Because LDG and PMN-MDSC share a similar phenotype with contrasting functional effects, we explored these cells in a cohort of patients with SLE. METHODS: LDG and normal-density granulocytes (NDG) were isolated from fresh blood of healthy donors (HD) and patients with SLE. Associations between LDG and clinical manifestations were analysed. Multicolor flow cytometry and confocal imaging were performed to immunophenotype the cells. The ability of LDG and NDG to suppress T cell function and induce cytokine production was quantified. RESULTS: LDG prevalence was elevated in SLE versus HD, associated with the interferon (IFN) 21-gene signature and disease activity. Also, the LDG-to-lymphocyte ratio associated better with SLE disease activity index than neutrophil-to-lymphocyte ratio. SLE LDG exhibited significantly heightened surface expression of various activation markers and also of lectin-like oxidised low-density lipoprotein receptor-1, previously described to be associated with PMN-MDSC. Supernatants from SLE LDG did not restrict HD CD4+ T cell proliferation in an arginase-dependent manner, suggesting LDG are not immunosuppressive. SLE LDG supernatants induced proinflammatory cytokine production (IFN gamma, tumour necrosis factor alpha and lymphotoxin alpha) from CD4+ T cells. CONCLUSIONS: Based on our results, SLE LDG display an activated phenotype, exert proinflammatory effects on T cells and do not exhibit MDSC function. These results support the concept that LDG represent a distinct proinflammatory subset in SLE with pathogenic potential, at least in part, through their ability to activate type 1 helper responses.
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Linfócitos T CD4-Positivos/imunologia , Granulócitos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/imunologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Proliferação de Células , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Lúpus Eritematoso Sistêmico/sangue , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
The role of the microbiota in many diseases including cancer has gained increasing attention. Paired with this is our expanding appreciation for the heterogeneity of the neutrophil compartment regarding surface marker expression and functionality. In this review, we will discuss the influence of the microbiota on granulopoiesis and consequent activity of neutrophils in cancer. As evidence for this microbiota-neutrophil-cancer axis builds, it exposes new therapeutic targets to improve a cancer patient's outcome.
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Microbiota/imunologia , Neoplasias/imunologia , Neoplasias/microbiologia , Neutrófilos/imunologia , Adulto , Envelhecimento/imunologia , Animais , Antígenos de Diferenciação/metabolismo , Biomarcadores , Dieta , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Camundongos , Mielopoese , Proteínas de Neoplasias/fisiologia , Neoplasias/terapia , Neutrófilos/classificação , Ratos , Células Estromais/fisiologia , Microambiente TumoralRESUMO
OBJECTIVE: To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease. METHODS: We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples. RESULTS: We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients' blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects. CONCLUSION: Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants.
Assuntos
Alelos , Complemento C1r/deficiência , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Complemento C1r/genética , Consanguinidade , Exoma , Feminino , Genótipo , Humanos , Interferon Tipo I/sangue , Leucócitos Mononucleares/citologia , Lúpus Eritematoso Sistêmico/sangue , Masculino , Neutrófilos/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Índice de Gravidade de Doença , TurquiaRESUMO
Commensal microorganisms (the microbiota) live on all the surface barriers of our body and are particularly abundant and diverse in the distal gut. The microbiota and its larger host represent a metaorganism in which the cross talk between microbes and host cells is necessary for health, survival, and regulation of physiological functions locally, at the barrier level, and systemically. The ancestral molecular and cellular mechanisms stemming from the earliest interactions between prokaryotes and eukaryotes have evolved to mediate microbe-dependent host physiology and tissue homeostasis, including innate and adaptive resistance to infections and tissue repair. Mostly because of its effects on metabolism, cellular proliferation, inflammation, and immunity, the microbiota regulates cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. Here, we review the mechanisms underlying the interaction of the microbiota with cancer and the evidence suggesting that the microbiota could be targeted to improve therapy while attenuating adverse reactions.
Assuntos
Imunidade Inata , Imunoterapia/métodos , Mucosa Intestinal/imunologia , Microbiota/imunologia , Neoplasias/imunologia , Imunidade Adaptativa , Animais , Antineoplásicos/uso terapêutico , Carcinogênese , Humanos , Inflamação , Neoplasias/microbiologia , Neoplasias/terapia , CicatrizaçãoRESUMO
OBJECTIVE: Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized. METHODS: MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice. RESULTS: Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies. CONCLUSION: Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage.
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Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologia , Animais , Feminino , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Doenças Vasculares/imunologiaRESUMO
OBJECTIVES: Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation. METHODS: Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA. RESULTS: Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation. CONCLUSIONS: Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk.
Assuntos
Fator 3 Ativador da Transcrição/biossíntese , Citocinas/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacologia , Lúpus Eritematoso Sistêmico/sangue , RNA Mensageiro/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Fator 3 Ativador da Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Amidas/farmacologia , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos , Camundongos , NF-kappa B/metabolismo , Oxirredução , Peptídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe E/genética , Receptores Depuradores Classe E/metabolismo , Esfingomielinas/farmacologia , Baço/citologia , Receptores Toll-Like/metabolismo , Transcrição Gênica/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have an increased risk of cardiovascular disease (CVD) and impaired endothelial repair. Although vitamin D deficiency is associated with increased CVD risk in the general population, a causal relationship has not been demonstrated. We aimed to determine whether vitamin D deficiency directly modulates endothelial dysfunction and immune responses in a murine model of SLE. METHODS: Vitamin D deficiency was induced in lupus-prone MRL/lpr mice by dietary restriction for 6 weeks. Endothelium-dependent vasorelaxation was quantified using aortic ring myography, and endothelial repair mechanisms were assessed by evaluating the phenotype and function of bone marrow endothelial progenitor cells (EPCs) and with the use of an in vivo Matrigel plug model. Lupus disease activity was determined by evaluating expression of interferon-stimulated genes (ISGs) in splenic tissue, positivity for serum autoantibodies, and renal histology. To validate the findings, expression of ISGs was also measured in whole blood from vitamin D-deficient and vitamin D-sufficient patients with SLE. RESULTS: Vitamin D deficiency resulted in impaired endothelium-dependent vasorelaxation and decreases in neoangiogenesis without a change in the total number of EPCs. There were no differences in anti-double-stranded DNA titers, proteinuria, or glomerulonephritis (activity or chronicity) between vitamin D-deficient or sufficient mice. Vitamin D deficiency was associated with a trend toward increased ISG expression both in mice and in patients with SLE. CONCLUSION: These findings indicate that vitamin D deficiency is associated with hampered vascular repair and reduced endothelial function, and may modulate type I interferon responses.
Assuntos
Endotélio Vascular/fisiopatologia , Interferon Tipo I/genética , Lúpus Eritematoso Sistêmico/fisiopatologia , Deficiência de Vitamina D/fisiopatologia , Animais , Aorta , Autoanticorpos/imunologia , Estudos de Casos e Controles , Diferenciação Celular , Cromatografia Líquida , Modelos Animais de Doenças , Células Progenitoras Endoteliais , Expressão Gênica , Humanos , Rim/patologia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos MRL lpr , Miografia , Hormônio Paratireóideo/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Vasodilatação , Vitamina D/análogos & derivados , Vitamina D/sangue , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/genéticaRESUMO
Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.
Assuntos
DNA Mitocondrial/metabolismo , Armadilhas Extracelulares/imunologia , Doença Granulomatosa Crônica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Mitocôndrias/metabolismo , Neutrófilos/imunologia , Adulto , Animais , Complexo Antígeno-Anticorpo , Armadilhas Extracelulares/metabolismo , Feminino , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Interferon Tipo I/imunologia , Células Jurkat , Rim/imunologia , Rim/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos , Microscopia de Fluorescência , NADPH Oxidases/genética , Oxirredução , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , RibonucleoproteínasRESUMO
RATIONALE: Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E2 (PGE2), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens. OBJECTIVES: To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE2 signaling. METHODS: Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE2 signaling modifiers. MEASUREMENTS AND MAIN RESULTS: NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3-208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE2 treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A-dependent manner. CONCLUSIONS: Our results suggest blockade of the PGE2-EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.
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Dinoprostona/imunologia , Armadilhas Extracelulares/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Adulto , Idoso , Animais , Dinoprostona/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Adulto JovemRESUMO
PURPOSE OF REVIEW: Recent discoveries implicate neutrophils as important regulators of innate and adaptive immunity and in the development of organ damage in systemic autoimmune diseases, including systemic lupus erythematosus (SLE). RECENT FINDINGS: Various putative SLE biomarkers are neutrophil-related, including neutrophil granular proteins and histones undergoing post-translational modifications during neutrophil extracellular trap (NET) formation. In the bone marrow, lupus neutrophils can drive B and T cell abnormalities, at least in part, by their enhanced production of type-I interferons, tumor necrosis factor-alpha (TNFα) and the B-cell stimulating factors B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Lupus neutrophils and, in particular, lupus low-density granulocytes (a distinct pathogenic subset) display epigenetic modifications and genomic alterations that may be relevant to their deleterious roles in SLE. Proteins and enzymes externalized by lupus NETs can affect vascular health by inducing endothelial apoptosis and oxidizing lipoproteins. Hampering NET formation through peptidylarginine deiminase inhibitors abrogates lupus phenotype and atherosclerosis in murine studies. SUMMARY: Recent discoveries support the notion that neutrophils, low-density granulocytes and aberrant NET formation and clearance play important roles in lupus pathogenesis. Future studies should focus on how to selectively target these immunostimulatory pathways in this disease.
Assuntos
Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/imunologia , Imunidade Adaptativa , Armadilhas Extracelulares/imunologia , Humanos , Imunidade InataRESUMO
OBJECTIVE: SLE is an autoimmune disease characterized by autoantibody generation, organ damage and an increased risk of cardiovascular disease. Generally considered an anti-inflammatory cytokine, IL-10 is increased in SLE and correlates with poor cardiovascular outcomes in the general population. The aim of this study was to explore the putative role of IL-10 in modulating endothelial function in SLE by examining the effects of this cytokine on endothelial progenitor cell/circulating angiogenic cell (EPC/CAC) differentiation. METHODS: Human and murine control and lupus EPCs/CACs were differentiated into mature endothelial cells (ECs) in the presence or absence of graded concentrations of recombinant IL-10 with or without recombinant IFN-α or a neutralizing antibody to IL-10. IL-10-deficient mice were examined to assess the role of this cytokine in type I IFN-mediated inhibition of EC differentiation and neo-angiogenesis using an in vivo Matrigel plug assay. Serum IL-10 concentrations were measured via ELISA. RESULTS: IL-10 hampers EC differentiation in a dose-dependent manner. In murine EPC cultures, IL-10 is required to observe the inhibitory effects of type I IFNs on EPC function and neo-angiogenesis. In human SLE EPC/CAC cultures, neutralization of IL-10 significantly improved the differentiation of EPCs, and IL-10 enhanced type I IFN-mediated EPC/CAC dysfunction. The presence of IL-10 in serum inversely correlated with EPC/CAC function in SLE but not in control cells. CONCLUSION: IL-10 interferes with endothelial differentiation and may enhance the effects of type I IFN on vascular repair in SLE. IL-10 may be a relevant target for improving cardiovascular risk in SLE.
Assuntos
Diferenciação Celular/fisiologia , Células Progenitoras Endoteliais/metabolismo , Interferon-alfa/metabolismo , Interleucina-10/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Adulto , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. METHODS: NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice. RESULTS: Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease. CONCLUSIONS: PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.
Assuntos
Amidinas/farmacologia , Armadilhas Extracelulares/metabolismo , Hidrolases/antagonistas & inibidores , Nefropatias/prevenção & controle , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Doenças Vasculares/prevenção & controle , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Nefropatias/etiologia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Desiminases de Arginina em Proteínas , Doenças Vasculares/etiologiaRESUMO
OBJECTIVE: Oxidative stress and oxidized high-density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC). METHODS: Plasma MPO levels and CEC activity were examined in controls and lupus patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from reactive nitrogen species) were quantified in human HDL. Multivariable linear models were used to estimate and test differences between groups. HDL was exposed to NETs from control and lupus neutrophils in the presence or absence of MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was quantified after NET inhibition in vivo. RESULTS: SLE patients displayed higher MPO levels and diminished CEC compared to controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than control HDL, with site-specific oxidation signatures on apolipoprotein A-I. Experiments with human and murine NETs confirmed that chlorination was mediated by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation. CQ inhibited NET formation in vitro. CONCLUSION: Active NOS, NOX, and MPO within NETs significantly modify HDL, rendering the lipoprotein proatherogenic. Since NET formation is enhanced in SLE, these findings support a novel role for NET-derived lipoprotein oxidation in SLE-associated CVD and identify additional proatherogenic roles of neutrophils and putative protective roles of antimalarials in autoimmunity.
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Lipoproteínas HDL/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Neutrófilos/enzimologia , Estresse Oxidativo/fisiologia , Adulto , Animais , Doenças Cardiovasculares/enzimologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , NADPH Oxidases/sangue , Óxido Nítrico Sintase/sangue , Oxirredução , Peroxidase/sangueRESUMO
Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1ß and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Proteínas de Transporte/fisiologia , Inflamassomos/fisiologia , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Adulto , Animais , Comunicação Autócrina , Proteínas de Transporte/genética , Caspase 1/fisiologia , Ativação Enzimática , Feminino , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Transporte de Íons , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neutrófilos/metabolismo , Potássio/metabolismo , Receptores Purinérgicos P2X7/deficiência , Receptores Purinérgicos P2X7/metabolismo , Organismos Livres de Patógenos Específicos , CatelicidinasRESUMO
OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. METHODS: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. RESULTS: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. CONCLUSION: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.