RESUMO
We previously reported that myeloperoxidase-deficient (MPO-/-) mice develop more severe neutrophil-rich lung inflammation than wild-type mice following intranasal Zymosan administration. Interestingly, we found that these mutant mice with severe lung inflammation also displayed pronounced neutrophilia and anemia, characterized by increased granulopoiesis and decreased erythropoiesis in the bone marrow, compared to wild-type mice. This condition was associated with higher concentrations of granulocyte-colony stimulating factor (G-CSF) in both the lungs and serum, a factor known to enhance granulopoiesis. Neutrophils accumulating in the lungs of MPO-/- mice produced greater amounts of G-CSF than those in wild-type mice, indicating that they are a significant source of G-CSF. In vitro experiments using signal transduction inhibitors and Western blot analysis revealed that MPO-/- neutrophils express higher levels of G-CSF mRNA in response to Zymosan, attributed to the upregulation of the IκB kinase/nuclear factor (NF)-κB pathway and the extracellular-signal-regulated kinase/NF-κB pathway. These findings highlight MPO as a critical regulator of granulopoiesis and erythropoiesis in inflamed tissues.
RESUMO
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated in vitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding in vivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 in vitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. In vivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Modelos Animais de Doenças , Armadilhas Extracelulares , Camundongos Knockout , Fator 2 Relacionado a NF-E2 , Neutrófilos , Peroxidase , Espécies Reativas de Oxigênio , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Neutrófilos/imunologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peroxidase/metabolismo , Peroxidase/genética , Camundongos , Humanos , Estresse Oxidativo/imunologia , Camundongos Endogâmicos C57BL , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/etiologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Masculino , Rim/patologia , Rim/imunologia , Transdução de Sinais/imunologiaRESUMO
OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Anticorpos Anticitoplasma de Neutrófilos , Peptidil-Prolil Isomerase F , Animais , Camundongos , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Inflamação , Necrose , Neutrófilos/metabolismo , RNARESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency wherein phagocytes are unable to produce reactive oxygen species (ROS) owing to a defect in the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) complex. Patients with CGD experience bacterial and fungal infections and excessive inflammatory disorders. Bone marrow transplantation and gene therapy are theoretically curative; however, residual pathogenic components cause inflammation and/or organic damage in patients. Moreover, antibiotic treatments may not help in preventing excessive inflammation due to the residual presence of fungal cell wall ß-glucan. Thus, better treatment strategies against CGD are urgently required. Polyethylene glycol-conjugated recombinant porcine D-amino acid oxidase (PEG-pDAO) supplies ROS to defective NADPH oxidase in neutrophils of patients with CGD, following which the neutrophils regain bactericidal activity in vitro. In this study, we employed an in vivo nonviable Candida albicans (nCA)-induced lung inflammation model of gp91-phox knockout CGD mice and supplied novel PEG conjugates of Fusarium spp. D-amino acid oxidase (PEG-fDAO), as it exhibits higher enzyme activity than PEG-pDAO. The body weight, lung weight, and lung pathology were evaluated using three experimental strategies with the in vivo lung inflammation model to test the efficacy of the ROS-generating enzyme replacement therapy with PEG-fDAO. The lung weight and pathological findings suggest the condition was ameliorated by administration PEG-fDAO, followed by intraperitoneal injection of D-phenylalanine or D-proline. Although a more precise protocol is essential, these data reveal the targeted delivery of PEG-fDAO to the nCA-induced inflammation site and show that PEG-fDAO can be used to treat inflammation in CGD in vivo.
Assuntos
Doença Granulomatosa Crônica , Pneumonia , Aminoácidos , Animais , Modelos Animais de Doenças , Doença Granulomatosa Crônica/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Camundongos , Camundongos Knockout , NADPH Oxidases/genética , Neutrófilos , Polietilenoglicóis/farmacologia , Espécies Reativas de Oxigênio , SuínosRESUMO
Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin DNA and contents of granules, such as myeloperoxidase (MPO) and neutrophil elastase (NE). NETs are usually released from neutrophils undergoing NETosis, a neutrophil-specific cell death mode characterized by the collapse and disappearance of cell membranes and nuclear envelopes. It is well known that production of reactive oxygen species (ROS) triggers NETosis and NET formation. However, details of intracellular signaling downstream of ROS production during NETosis and NET formation remains uncertain. Here, we demonstrated that the peroxidation of phospholipids plays a critical role in NETosis and NET formation induced by phorbol 12-myristate13-acetate (PMA) or immune complex in vitro and by lipopolysaccharide (LPS) in vivo. This phospholipid peroxidation is mediated by the enzymatic activity of MPO. On the other hand, NE, which was previously reported to be released from granules to cytosol by MPO during NET formation, is not required for either the peroxidation of phospholipids or the execution of NETosis, but contributes to chromatin decondensation and nuclear swelling independently of MPO-mediated oxidized phospholipids. Analysis of isolated nuclei clearly demonstrated that oxidized phospholipids and NE differently yet synergistically execute chromatin decondensation and nuclear swelling, and the subsequent release of nuclear contents. These findings indicate the dual roles of MPO in NETosis and NET formation, and provide new insight into the molecular mechanism of these phenomena.
RESUMO
Patients with chronic granulomatous disease (CGD) who have mutated phagocyte NADPH oxidase are susceptible to infections due to reduced reactive oxygen species production and exhibit autoimmune and inflammatory diseases in the absence of evident infection. Neutrophils and macrophages have been extensively studied since phagocyte NADPH oxidase is mainly found only in them, while the impact of its deficiency on lymphocyte cellularity is less well characterized. We showed herein a zymosan-induced systemic inflammation model that CGD mice deficient in the phagocyte NADPH oxidase gp91phox subunit (NOX2) exhibited more severe thymic atrophy associated with peripheral blood and splenic lymphopenia and reduced lymphopoiesis in the bone marrow in comparison with the wild-type mice. Conversely, the zymosan-exposed CGD mice suffered from more remarkable neutrophilic lung inflammation, circulating and splenic neutrophilia, and enhanced granulopoiesis compared with those in zymosan-exposed wild-type mice. Overall, this study provided evidence that NOX2 deficiency exhibits severe thymic atrophy and lymphopenia concomitant with enhanced neutrophilic inflammation in a zymosan-induced systemic inflammation model.
Assuntos
Linfopenia/metabolismo , Linfopoese/fisiologia , NADPH Oxidase 2/deficiência , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Timo/metabolismo , Zimosan/toxicidade , Animais , Atrofia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doença Granulomatosa Crônica/induzido quimicamente , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/patologia , Timo/efeitos dos fármacos , Timo/patologiaRESUMO
Myeloperoxidase (MPO) is a heme-containing peroxidase expressed mainly in neutrophils and to a lesser degree in monocytes. In the presence of hydrogen peroxide and halides, MPO catalyzes the formation of reactive oxygen intermediates, including hypochlorous acid (HOCl). The MPO/HOCl system plays an important role in microbial killing by neutrophils. In addition, MPO has been demonstrated to be a local mediator of tissue damage and the resulting inflammation in various inflammatory diseases. These findings have implicated MPO as an important therapeutic target in the treatment of inflammatory conditions. In contrast to its injurious effects at sites of inflammation, recent studies using animal models of various inflammatory diseases have demonstrated that MPO deficiency results in the exaggeration of inflammatory response, and that it affects neutrophil functions including cytokine production. Given these diverse effects, a growing interest has emerged in the role of this well-studied enzyme in health and disease.
Assuntos
Inflamação/enzimologia , Neutrófilos/imunologia , Peroxidase/metabolismo , Animais , Humanos , Camundongos , Camundongos Knockout , Peroxidase/genéticaRESUMO
OBJECTIVES: Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. METHODS: To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. RESULTS: Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. CONCLUSIONS: Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Proteína C-Reativa/imunologia , Imunoglobulina G/sangue , Erros Inatos do Metabolismo/imunologia , Proteínas do Tecido Nervoso/imunologia , Peroxidase/imunologia , Animais , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Proteína C-Reativa/administração & dosagem , Proteína C-Reativa/metabolismo , DNA/imunologia , DNA/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Peroxidase/administração & dosagem , Peroxidase/deficiência , Peroxidase/genéticaRESUMO
Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1ß and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1ß mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1ß production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.
Assuntos
Inflamação/enzimologia , NADPH Oxidases/deficiência , Fagócitos/enzimologia , Pneumonia/enzimologia , Animais , Candida albicans/patogenicidade , Quimiocinas/metabolismo , Doença Granulomatosa Crônica , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Myeloperoxidase (MPO), a major component of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Phagocytosis is a critical event induced by neutrophils for host defense and inflammation. Interestingly, we found that MPO-deficient (MPO-/-) neutrophils engulfed larger amounts of zymosan than wild-type neutrophils. Blocking of the CD11b subunit of complement receptor 3 (CR3) as well as inhibition of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) dramatically reduced zymosan phagocytosis. In contrast, blocking of dectin-1, toll-like receptor 2 (TLR2), or spleen tyrosine kinase (Syk) had no significant effects on phagocytosis. Western blotting analysis showed that inhibition of FAK decreased the phosphorylation of ERK1/2, indicating that ERK1/2 is a downstream regulator of FAK in neutrophils. Importantly, we found that cell surface expression of CD11b and phosphorylation of ERK1/2 was significantly higher in zymosan-stimulated MPO-/- neutrophils than in zymosan-stimulated wild-type neutrophils. Pretreatment with the MPO inhibitor 4-aminobenzoic acid hydrazide dramatically enhanced both zymosan phagocytosis and the surface expression of CD11b in wild-type neutrophils, but not in MPO-/- neutrophils. Collectively, these results strongly suggest that up-regulation of the CD11b/FAK/ERK signaling pathway due to absence of MPO enhances the zymosan phagocytic activity of mouse neutrophils.
Assuntos
Antígenos CD11/metabolismo , Erros Inatos do Metabolismo/metabolismo , Fagocitose/imunologia , Zimosan/metabolismo , Animais , Camundongos , Espécies Reativas de Oxigênio , Regulação para CimaRESUMO
Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Canais Iônicos/metabolismo , Neutrófilos/metabolismo , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Membrana Celular/metabolismo , Exocitose , Feminino , Imunoglobulina G/imunologia , Canais Iônicos/genética , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Knockout , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Peroxidase/metabolismo , Ligação Proteica , Transporte Proteico , Vesículas Secretórias/metabolismoRESUMO
OBJECTIVE: We have previously reported that myeloperoxidase-deficient (MPO(-/-)) neutrophils produce greater amounts of macrophage inflammatory protein-2 (MIP-2) upon in vitro stimulation with zymosan than wild-type neutrophils. This study aimed to examine the effect of MPO deficiency on the expression of other cytokines and chemokines. METHODS: Wild-type and MPO(-/-) neutrophils isolated from peritoneal cavity were stimulated with zymosan in vitro. Secretion of MIP-1α, MIP-1ß, interleukin (IL)-1α, IL-1ß, and tumor necrosis factor (TNF)-α by neutrophils was quantified by ELISA. mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) in neutrophils was analyzed by western blot. For in vivo studies, mice were inoculated with zymosan intranasally, and the levels of these cytokines and chemokines were measured in the lungs. RESULTS: The MPO(-/-) neutrophils stimulated by zymosan expressed and secreted significantly higher levels of MIP-1α, MIP-1ß, IL-1α, IL-1ß, and TNF-α than the stimulated wild-type cells. Expression of all of these inflammatory mediators was blocked by pre-treatment with BAY11-7082, U0126, and SB203580, which are inhibitors of nuclear factor (NF)-κB, ERK1/2, and p38 MAPK, respectively. Enhanced expression of these inflammatory mediators is associated with elevated activation of ERK1/2 in stimulated MPO(-/-) neutrophils. In vivo, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-1α, MIP-1ß, IL-1α, IL-1ß, and TNF-α relative to the responses seen in wild-type mice within 24 h of zymosan administration. CONCLUSION: MPO deficiency upregulates the expression of several proinflammatory cytokines and chemokines in mouse neutrophils.
Assuntos
Citocinas/metabolismo , Pulmão/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Peroxidase/genética , Zimosan/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Citocinas/genética , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: This study aimed to evaluate the effect of myeloperoxidase (MPO) deficiency on lung inflammation induced by nonviable Candida albicans (nCA). METHODS: Mice were inoculated intranasally with nCA, and accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid was analyzed by flow cytometry. The levels of macrophage inflammatory protein 2 (MIP-2), keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß in the lung were measured by ELISA. Production of MIP-2 and KC from neutrophils and macrophages was quantified in vitro. MIP-2 mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the extent of phosphorylation of ERK1/2 and Syk in the neutrophils was analyzed by Western blotting. RESULTS: The MPO(-/-) mice that received nCA showed more severe pneumonia than wild-type mice. Within 12 h of nCA administration, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-2 and KC relative to the responses seen in wild-type mice. Neutralization of MIP-2 and KC in vivo significantly reduced neutrophil infiltration. In vitro, production of MIP-2, but not that of KC, was enhanced in the nCA-stimulated neutrophils from MPO(-/-) mice, concomitant with up-regulation of Syk and ERK1/2. At 1 and 3 days after nCA administration, MPO(-/-) mice had significantly higher lung concentrations of TNF-α and IL-1ß than wild-type mice. CONCLUSION: Pulmonary administration of nCA produced an altered inflammatory response in MPO(-/-) mice relative to wild-type mice. Enhanced MIP-2 production by MPO(-/-) neutrophils may at least partly contribute to exacerbated inflammation in mutant mice.
Assuntos
Candida albicans/imunologia , Erros Inatos do Metabolismo/imunologia , Pneumonia/imunologia , Animais , Células da Medula Óssea/citologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/genética , Citocinas/imunologia , Fêmur/citologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Pneumonia/patologiaRESUMO
Neutrophil accumulation is a critical event in the pathogenesis of inflammation. The generation of hypochlorous acid by myeloperoxidase (MPO) in neutrophils is crucial to the host defense response. MPO-deficient (MPO-KO) mice showed severely reduced cytotoxicity to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and other microorganisms, demonstrating that an MPO-dependent oxidative system is important for in vivo host defense against fungi. On the other hand, impaired reactive oxygen species (ROS) production by neutrophils has previously been shown to cause an abnormal inflammatory response. In the present study, we have found that MPO-KO mice exhibit more severe pulmonary inflammation than wild-type mice when challenged with an intranasal administration of zymosan. In addition to measuring the kinetics of neutrophil accumulation, we also measured the production of macrophage inflammatory protein-2 (MIP-2) in the lung, and we correlate the degree of neutrophil accumulation with the production of this mediator. Our results demonstrate that MPO regulates the production of MIP-2, which may modulate neutrophil accumulation during lung inflammation.
Assuntos
Inflamação/imunologia , Pneumopatias Fúngicas/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Quimiocina CXCL2/metabolismo , Quimiocina CXCL2/fisiologia , Suscetibilidade a Doenças/imunologia , Humanos , Ácido Hipocloroso/metabolismo , Pulmão/metabolismo , Erros Inatos do Metabolismo/imunologia , NADPH Oxidases/fisiologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Peroxidase/fisiologia , Fagócitos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Zimosan/farmacologiaRESUMO
STUDY DESIGN: An animal study using myeloperoxidase-knockout (MPO-KO) mice to examine the in vivo role of myeloperoxidase (MPO) in spinal cord injury (SCI). OBJECTIVE: To clarify the influence of MPO on inflammatory cell infiltration, tissue damage, and functional recovery after SCI. SUMMARY OF BACKGROUND DATA: MPO is considered to be important in spreading tissue damage after SCI because it generates strong neurotoxic oxidant hypochlorous acid (HOCl). However, the direct involvement of MPO in the pathophysiology of SCI remains to be elucidated. METHODS: To compare the inflammatory reaction, tissue damage, and neurological recovery after SCI, a moderate contusion injury was created at the ninth thoracic level in MPO-KO mice and wild-type mice. A HOCl-specific probe solution was injected into the lesion epicenter to assess the spatiotemporal production of MPO-derived HOCl. Inflammatory reactions were quantified by flow cytometry and quantitative real-time polymerase chain reaction, and tissue damage was evaluated by an immunohistochemical analysis. The motor function recovery was assessed by the open-field locomotor score. RESULTS: Prominent production of HOCl was observed during the hyperacute phase of SCI at the lesion site in the wild-type mice; however, little expression was observed in the MPO-KO mice. In this phase, the number of infiltrated neutrophils was significantly reduced in the MPO-KO mice compared with the wild-type mice. In addition, significant differences were observed in the expression levels of proinflammatory cytokines and apoptosis-related genes between 2 groups. In the histological sections, fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells and more spared myelin were observed at the lesion site in MPO-KO mice. Consistent with these results, better functional recovery was observed in the MPO-KO mice than in the wild-type mice after SCI. CONCLUSION: These results clearly indicated that MPO exacerbated secondary injury and impaired the functional recovery not only by generating strong oxidant HOCl, but also by enhancing neutrophil infiltration after SCI.
Assuntos
Infiltração de Neutrófilos , Estresse Oxidativo , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismos da Medula Espinal/enzimologia , Medula Espinal/enzimologia , Animais , Apoptose/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Ácido Hipocloroso/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Peroxidase/genética , Reação em Cadeia da Polimerase em Tempo Real , Recuperação de Função Fisiológica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/imunologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/patologia , Peroxidase/metabolismo , Síndrome do Desconforto Respiratório/patologia , Animais , Citocinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Peroxidase/deficiência , Pneumonia Viral/patologiaRESUMO
OBJECTIVE AND DESIGN: This study examines the role of myeloperoxidase (MPO), a major constituent of neutrophils that generates hypochlorous acid, in neutrophil recruitment into the zymosan-exposed lung of mice. METHODS: Mice were inoculated intranasally with zymosan. The accumulation of neutrophils and other inflammatory cells within the lung was analyzed by flow cytometry. Macrophage inflammatory protein 2 (MIP-2) expression in the lung was quantified, and the contribution of this chemokine to neutrophil accumulation was examined by intranasal administration of MIP-2 antibody. The cellular sources of MIP-2 were identified, and the production of this chemokine from macrophages and neutrophils was quantified in vitro. RESULTS: Zymosan exposure led to greater neutrophil infiltration into the lungs of MPO(-/-) mice relative to wild-type mice. This was associated with higher MIP-2 levels in the mutant mice. Neutralization of MIP-2 in vivo significantly reduced neutrophil infiltration. Neutrophils from MPO(-/-) mice produced more MIP-2, and the production was reduced when MPO was added exogenously. CONCLUSIONS: MPO deficiency results in severe lung inflammation in mice exposed to zymosan. Relatively high MIP-2 levels likely contribute to the strong inflammatory response in these animals.
Assuntos
Quimiocina CXCL2/imunologia , Neutrófilos/imunologia , Peroxidase/imunologia , Pneumonia/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/deficiência , Peroxidase/genética , Pneumonia/induzido quimicamente , Pneumonia/patologia , ZimosanRESUMO
Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.
Assuntos
Quimiocina CCL5/metabolismo , Células Epiteliais/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Peroxidase/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL5/administração & dosagem , Quimiocina CCL5/genética , Quimiocinas/efeitos dos fármacos , Quimiocinas/genética , Quimiocinas/fisiologia , Citocinas/efeitos dos fármacos , Citocinas/genética , Citocinas/fisiologia , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/farmacologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Ativação Linfocitária , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/virologia , Peroxidase/administração & dosagem , RNA Interferente Pequeno , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: Glomerular neutrophil infiltration has been thought to be a key pathological event in the development of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis involving glomerulonephritis. Accordingly, we sought to explore the molecules responsible for glomerular neutrophil accumulation. METHODS: Glomerular neutrophil infiltration and renal chemokine expression in mice treated with anti-MPO IgG were evaluated. Chemokine expression in vitro induced by anti-MPO IgG was measured in the primary mouse glomerular endothelial cells (mGEC). The target molecule reacted with anti-MPO IgG on the mGEC was determined by peptide mass fingerprint analysis. RESULTS: A significant glomerular neutrophil infiltration was observed in the mice administered with anti-MPO IgG. The expressions of CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), were significantly increased in the renal cortex, indicating that these chemokines contribute to the neutrophil infiltration. Based on the previous findings of upregulation of adhesion molecule expression in mGEC treated with anti-MPO IgG, we examined whether mGEC secrete these chemokines in response to anti-MPO IgG. Indeed, anti-MPO IgG induced secretion of KC and MIP-2, leading to neutrophil chemotaxis in vitro. Furthermore, complete depletion of MPO in mGEC and serum using MPO-deficient mice showed an upregulation of intercellular adhesion molecule-1, indicating cross-reactive molecule(s) were existing on mGEC. We identified the molecule as moesin by a proteomic approach. CONCLUSIONS: The endothelial CXC chemokines, KC and MIP-2, contribute to infiltration of neutrophils in MPO-ANCA-associated vasculitis involving glomerulonephritis. The activation of glomerular endothelial cells by anti-MPO IgG appeared to directly involve a signaling through moesin.