RESUMO
Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.
Assuntos
Acetofenonas , Injúria Renal Aguda , Traumatismo por Reperfusão , Camundongos , Animais , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Camundongos Endogâmicos C57BL , Rim/metabolismo , Traumatismo por Reperfusão/genética , Xantina Desidrogenase/metabolismo , RNA MensageiroRESUMO
Antibodies to neutrophil and monocyte myeloperoxidase and proteinase 3 are a feature of anti-neutrophil cytoplasmic antibody vasculitis, a disease with significant morbidity for which new treatments are needed. Mice with a myeloid-specific deletion of the anti-apoptotic protein Mcl1 have reduced numbers of circulating neutrophils. Here, we assessed if myeloid-specific Mcl1 was required in murine anti-myeloperoxidase vasculitis and whether inhibition of myeloperoxidase was protective. In a murine model of anti-neutrophil cytoplasmic antibody vasculitis, induced by anti-myeloperoxidase antibody, mice with a myeloid-specific deletion of Mcl1 were protected from disease. They had fewer crescents, neutrophils, and macrophages in the glomeruli, lower serum creatinine levels and reduced albuminuria compared with controls. At baseline and day six after disease induction they had fewer circulating neutrophils than controls. At day six there were also fewer circulating monocytes. Myeloperoxidase inhibition with AZD5904 had no effect on histological or biochemical parameters of disease, and there was also no reduction in albuminuria at day one, two, five or seven after disease induction. These findings persisted when disease was induced without granulocyte-colony stimulating factor, which increases disease severity. A second myeloperoxidase inhibitor, AZM198, also showed no evidence of an effect, although both AZD5904 and AZM198 inhibited human neutrophil extracellular trap formation in vitro. Thus, our results show that while myeloid-specific Mcl1 is required in this model of anti-myeloperoxidase vasculitis, myeloperoxidase inhibition is not protective.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Glomerulonefrite , Vasculite , Camundongos , Humanos , Animais , Anticorpos Anticitoplasma de Neutrófilos , Proteínas Reguladoras de Apoptose/metabolismo , Albuminúria/prevenção & controle , Albuminúria/metabolismo , Vasculite/prevenção & controle , Neutrófilos , Peroxidase , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismoRESUMO
Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro-generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo-differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor-derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Proteínas de Homeodomínio/metabolismo , Inflamação/imunologia , Células Progenitoras Mieloides/imunologia , Neutrófilos/imunologia , Animais , Diferenciação Celular , Linhagem Celular Transformada , Quimiotaxia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Transgenes/genéticaRESUMO
A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.
Assuntos
Membrana Celular/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Pseudópodes/metabolismo , Quinase Syk/metabolismo , Animais , Células Cultivadas , Humanos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Camundongos , Camundongos Knockout , Fagocitose , Transdução de SinaisRESUMO
A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.
RESUMO
Capsaicin-sensitive peptidergic sensory nerves play complex, mainly protective regulatory roles in the inflammatory cascade of the joints via neuropeptide mediators, but the mechanisms of the hyperacute arthritis phase has not been investigated. Therefore, we studied the involvement of these afferents in the early, "black box" period of a rheumatoid arthritis (RA) mouse model. Capsaicin-sensitive fibres were defunctionalized by pretreatment with the ultrapotent capsaicin analog resiniferatoxin and arthritis was induced by K/BxN arthritogenic serum. Disease severity was assessed by clinical scoring, reactive oxygen species (ROS) burst by chemiluminescent, vascular permeability by fluorescent in vivo imaging. Contrast-enhanced magnetic resonance imaging was used to correlate the functional and morphological changes. After sensory desensitization, both early phase ROS-burst and vascular leakage were significantly enhanced, which was later followed by the increased clinical severity scores. Furthermore, the early vascular leakage and ROS-burst were found to be good predictors of later arthritis severity. We conclude that the anti-inflammatory role of peptidergic afferents depends on their activity in the hyperacute phase, characterized by decreased cellular and vascular inflammatory components presumably via anti-inflammatory neuropeptide release. Therefore, these fibres might serve as important gatekeepers in RA.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Capsaicina/farmacologia , Inflamação Neurogênica/prevenção & controle , Neuropeptídeos/farmacologia , Fármacos do Sistema Sensorial/farmacologia , Animais , Artrite Experimental/etiologia , Artrite Experimental/patologia , Artrite Reumatoide/etiologia , Artrite Reumatoide/patologia , Diterpenos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismoRESUMO
V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator of T cells. We assessed VISTA deficient mice in the murine nephrotoxic nephritis models of acute and chronic immune-complex mediated glomerulonephritis. We show that VISTA deficiency protects from crescentic glomerulonephritis, with no effect on the nephritogenic adaptive immune response. The early neutrophil influx was unaffected but proteinuria was reduced suggesting a reduction in neutrophil activation. In vivo, there was reduced neutrophil degranulation in VISTA deficienct mice and, in vitro, VISTA-deficient neutrophils had an impaired response to immune complexes but not to fMLP or PMA. Mice with a genetic deficiency of neutrophils due to myeloid-specific deletion of myeloid cell leukemia 1 (Mcl-1) were also protected from crescentic glomerulonephritis, indicating an essential role for neutrophils. Therefore, VISTA deficiency inhibits neutrophil activation by immune complexes and neutrophil-dependent crescentic glomerulonephritis. This suggests that VISTA is a therapeutic target for inflammatory disease. However, this would need to be balanced against a potential enhancing effect on autoimmunity.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Glomerulonefrite/imunologia , Glomérulos Renais/patologia , Proteínas de Membrana/deficiência , Neutrófilos/imunologia , Animais , Modelos Animais de Doenças , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Glomerulonefrite/sangue , Glomerulonefrite/patologia , Humanos , Glomérulos Renais/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Ativação de Neutrófilo , Neutrófilos/metabolismoRESUMO
Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.
Assuntos
Neutrófilos , Quinases da Família src , Animais , Membrana Basal , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas , Quinases da Família src/genéticaRESUMO
Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2 Cre/Cre Mcl1 flox/flox (Mcl1 ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1 ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1 ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1 ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1 ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1 flox/flox (Mcl1 ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1 ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.
Assuntos
Artrite/genética , Candida albicans/fisiologia , Candidíase/genética , Epidermólise Bolhosa Adquirida/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neutropenia/genética , Neutrófilos/fisiologia , Infecções Estafilocócicas/genética , Staphylococcus aureus/fisiologia , Animais , Modelos Animais de Doenças , Fertilidade/genética , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genéticaRESUMO
Neutrophils have traditionally been thought to play only a peripheral role in the genesis of many autoimmune and inflammatory diseases. However, recent studies in a variety of animal models suggest that these cells are central to the initiation and propagation of autoimmunity. The use of mouse models, which allow either deletion of neutrophils or the targeting of specific neutrophil functions, has revealed the many complex ways these cells contribute to autoimmune/inflammatory processes. This includes generation of self antigens through the process of NETosis, regulation of T-cell and dendritic cell activation, production of cytokines such as BAFF that stimulate self-reactive B-cells, as well as indirect effects on epithelial cell stability. In comparing the many different autoimmune models in which neutrophils have been examined, a number of common underlying themes emerge - such as a role for neutrophils in stimulating vascular permeability in arthritis, encephalitis and colitis. The use of animal models has also stimulated the development of new therapeutics that target neutrophil functions, such as NETosis, that may prove beneficial in human disease. This review will summarize neutrophil contributions in a number of murine autoimmune/inflammatory disease models.
Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade , Modelos Animais de Doenças , Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/patologia , Comunicação Celular/imunologia , Citocinas/metabolismo , Suscetibilidade a Doenças , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Humanos , Imunomodulação , Mediadores da Inflamação , Neutrófilos/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Neutrophils play a critical role in antimicrobial host defense, but their improper activation also contributes to inflammation-induced tissue damage. Therefore, understanding neutrophil biology is important for the understanding, diagnosis, and therapy of both infectious and inflammatory diseases. Neutrophils express a large number of cell-surface receptors that sense extracellular cues and trigger various functional responses through complex intracellular signaling pathways. During the last several years, we and others have shown that tyrosine kinases play a critical role in those processes. In particular, Src-family and Syk tyrosine kinases couple Fc-receptors and adhesion receptors (integrins and selectins) to various neutrophil effector functions. This pathway shows surprising similarity to lymphocyte antigen receptor signaling and involves various other enzymes (e.g. PLCγ2), exchange factors (e.g. Vav-family members) and adapter proteins (such as ITAM-containing adapters, SLP-76, and CARD9). Those mediators trigger various antimicrobial functions and play a critical role in coordinating the inflammatory response through the release of inflammatory mediators, such as chemokines and LTB4 . Interestingly, however, tyrosine kinases have a limited direct role in the migration of neutrophils to the site of inflammation. Here, we review the role of tyrosine kinase signaling pathways in neutrophils and how those pathways contribute to neutrophil activation in health and disease.
Assuntos
Neutrófilos/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Quimiocinas/metabolismo , Humanos , Imunidade Inata , Integrinas/metabolismo , Ativação de Neutrófilo , Receptores de Antígenos/metabolismo , Receptores Fc/metabolismo , Selectinas/metabolismoRESUMO
As the first line of innate immune defense, neutrophils need to mount a rapid and robust antimicrobial response. Recent studies implicate various positive feedback amplification processes in achieving that goal. Feedback amplification ensures effective migration of neutrophils in shallow chemotactic gradients, multiple waves of neutrophil recruitment to the site of inflammation, and the augmentation of various effector functions of the cells. We review here such positive feedback loops including intracellular and autocrine processes, paracrine effects mediated by lipid (LTB4), chemokine, and cytokine mediators, and bidirectional interactions with the complement system and with other immune and non-immune cells. These amplification mechanisms are not only involved in antimicrobial immunity but also contribute to neutrophil-mediated tissue damage under pathological conditions.
Assuntos
Comunicação Celular , Retroalimentação Fisiológica , Imunidade Inata , Infecções/imunologia , Inflamação/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Animais , Quimiotaxia , Proteínas do Sistema Complemento/metabolismo , Humanos , Leucotrieno B4/metabolismoRESUMO
BACKGROUND: The circadian time-measuring system enables the organism to anticipate and effectively respond to regular daily changes in the environment and is therefore a crucial factor of adaptation. A large body of epidemiological data underlines the circadian characteristics of human immune functions. Circadian control of neutrophil responsiveness contributes to daily changes in the pathology of both acute and chronic inflammation and may therefore time-dependently influence the outcome of therapeutic approaches. AIM: This review summarizes recent data on the role of the circadian clock in the control of immune responses, particularly of those linked to neutrophil activity, and possible mechanisms of the regulation. DISCUSSION: In the first section of this review we present the recent model of the mammalian molecular clock by introducing the main transcription-translation feedback loops and discussing the pace-setting role of post-translational modifications. The next sections summarize clinical, epidemiological and experimental data regarding the daily control of immune responses and studies analysing expression of clock components in various leukocytes and particularly, in human peripheral neutrophils. As the latter data indicate that expression of components of the cell-autonomous clock is relatively low in neutrophils, in the last section we review recent findings suggesting a role for systemic and local factors in the regulation of rhythmic neutrophil responses.
Assuntos
Relógios Circadianos/imunologia , Ritmo Circadiano/imunologia , Imunidade Celular/fisiologia , Neutrófilos/fisiologia , Citocinas/imunologia , Humanos , Leucócitos/imunologia , Neutrófilos/imunologiaRESUMO
Semicarbazide-sensitive amine oxidase (SSAO) produces tissue irritants by deamination of primary amines, which activate transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly on nociceptors. Since there are no data about its functions in pain, we studied the effects and mechanisms of action of our novel SSAO inhibitor and dual TRPA1/TRPV1 antagonist multi-target drug SZV 1287 in different pain models. Acute chemonociception was induced by TRPV1 and TRPA1 activation (resiniferatoxin and formalin, respectively), chronic arthritis by K/BxN serum transfer, traumatic mononeuropathy by sciatic nerve ligation. SZV 1287 (20â¯mg/kg i.p.) was investigated in C57BL/6J wildtype (WT), TRPA1- (TRPA1-/-) and TRPV1-deficient (TRPV1-/-) mice. Paw mechanonociception was measured by aesthesiometry, thermonociception by hot plate, nocifensive behavior by licking duration, volume by plethysmometry, myeloperoxidase activity by luminescence and plasma extravasation by fluorescence imaging, glia activation in pain-related brain regions by immunohistochemistry. SZV 1287 significantly inhibited both TRPA1 and TRPV1 activation-induced acute chemonociception and hyperalgesia. In K/BxN arthritis, daily SZV 1287 injections significantly decreased hyperalgesia, L4-L6 spinal dorsal horn microgliosis, edema and myeloperoxidase activity. SZV 1287-evoked antihyperalgesic and anti-edema effects were absent in TRPV1-/-, and remarkably reduced in TRPA1-/- mice. In contrast, myeloperoxidase-inhibitory effect was absent in TRPA1-/-, but not in TRPV1-/- animals. Acute SZV 1287 administration resulted in approximately 50% significant reduction of neuropathic hyperalgesia 7â¯days after nerve ligation, which was not observed in either TRPA1-/- or TRPV1-/- mice. SZV 1287 inhibits chronic inflammatory and neuropathic pain via TRPV1 and TRPA1/TRPV1 activation, respectively, highlighting its drug developmental potential.
Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Analgésicos/uso terapêutico , Dor Crônica/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Oxazóis/uso terapêutico , Oximas/uso terapêutico , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Analgésicos/farmacologia , Animais , Dor Crônica/genética , Dor Crônica/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Masculino , Camundongos Endogâmicos C57BL , Neuralgia/tratamento farmacológico , Neuralgia/genética , Neuralgia/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genéticaRESUMO
OBJECTIVE: Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis. METHODS: Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14+ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted. RESULTS: EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14+ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect. CONCLUSIONS: Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.
Assuntos
Artrite Psoriásica/patologia , Artrite Reumatoide/patologia , Vesículas Extracelulares/patologia , Osteoclastos/patologia , Adulto , Idoso , Artrite Psoriásica/sangue , Artrite Reumatoide/sangue , Diferenciação Celular , Linhagem Celular , Exossomos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoclastos/citologia , Osteogênese , Ligante RANK/análise , Receptor Ativador de Fator Nuclear kappa-B/análiseRESUMO
Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.
Assuntos
Hidroliases/metabolismo , Macrófagos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Succinatos/farmacologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Feminino , Glicólise , Hidroliases/genética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Malonatos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Translocases Mitocondriais de ADP e ATP/metabolismo , Fosforilação Oxidativa , Rotenona/farmacologia , Succinato-CoA Ligases/metabolismoRESUMO
BACKGROUND/AIMS: Evidence suggests that tyrosine-kinase inhibitors may attenuate lung inflammation and fibrosis in experimental acute respiratory distress syndrome (ARDS). We hypothesized that dasatinib, a tyrosine-kinase inhibitor, might act differently depending on the ARDS etiology and the dose. METHODS: C57/BL6 mice were divided to be pre-treated with dasatinib (1mg/kg or 10mg/kg) or vehicle (1% dimethyl-sulfoxide) by oral gavage. Thirty-minutes after pre-treatment, mice were subdivided into control (C) or ARDS groups. ARDS animals received Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). A new dose of dasatinib or vehicle was administered at 6 and 24h. RESULTS: Forty-eight hours after ARDS induction, dasatinib 1mg/kg yielded: improved lung morphofunction and reduced cells expressing toll-like receptor (TLR)-4 in lung, independent of ARDS etiology; reduced neutrophil and levels of interleukin (IL)-6, IL-10 and transforming growth factor (TGF)-ß in ARDSp. The higher dose of dasatinib caused no changes in lung mechanics, diffuse alveolar damage, neutrophil, or cells expressing TLR4, but increased IL-6, vascular endothelial growth factor (VEGF), and cells expressing Fas receptor in lung in ARDSp. In ARDSexp, it improved lung morphofunction, increased VEGF, and reduced cells expressing TLR4. Conclusion: Dasatinib may have therapeutic potential in ARDS independent of etiology, but careful dose monitoring is required.
Assuntos
Dasatinibe/uso terapêutico , Pulmão/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/etiologia , Animais , Dasatinibe/administração & dosagem , Interleucina-10/análise , Interleucina-6/análise , Pulmão/patologia , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/administração & dosagem , Síndrome do Desconforto Respiratório/patologia , Receptor 4 Toll-Like/análise , Fator de Crescimento Transformador beta/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
OBJECTIVE: The K/BxN serum-transfer arthritis is a widely-used translational mouse model of rheumatoid arthritis, in which the immunological components have thoroughly been investigated. In contrast, little is known about the role of sensory neural factors and the complexity of neuro-immune interactions. Therefore, we analyzed the involvement of capsaicin-sensitive peptidergic sensory nerves in autoantibody-induced arthritis with integrative methodology. METHODS: Arthritogenic K/BxN or control serum was injected to non-pretreated mice or resiniferatoxin (RTX)-pretreated animals where capsaicin-sensitive nerves were inactivated. Edema, touch sensitivity, noxious heat threshold, joint function, body weight and clinical arthritis severity scores were determined repeatedly throughout two weeks. Micro-CT and in vivo optical imaging to determine matrix-metalloproteinase (MMP) and neutrophil-derived myeloperoxidase (MPO) activities, semiquantitative histopathological scoring and radioimmunoassay to measure somatostatin in the joint homogenates were also performed. RESULTS: In RTX-pretreated mice, the autoantibody-induced joint swelling, arthritis severity score, MMP and MPO activities, as well as histopathological alterations were significantly greater compared to non-pretreated animals. Self-control quantification of the bone mass revealed decreased values in intact female mice, but significantly greater arthritis-induced pathological bone formation after RTX-pretreatment. In contrast, mechanical hyperalgesia from day 10 was smaller after inactivating capsaicin-sensitive afferents. Although thermal hyperalgesia did not develop, noxious heat threshold was significantly higher following RTX pretreatment. Somatostatin-like immunoreactivity elevated in the tibiotarsal joints in non-pretreated, which was significantly less in RTX-pretreated mice. CONCLUSIONS: Although capsaicin-sensitive sensory nerves mediate mechanical hyperalgesia in the later phase of autoantibody-induced chronic arthritis, they play important anti-inflammatory roles at least partially through somatostatin release.
Assuntos
Artrite Experimental , Artrite Reumatoide , Diterpenos/farmacologia , Hiperalgesia , Nociceptores/efeitos dos fármacos , Animais , Capsaicina/farmacologia , Modelos Animais de Doenças , Edema , Membro Posterior , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/fisiologia , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Peroxidase/metabolismo , Espécies Reativas de Oxigênio , Fármacos do Sistema Sensorial/farmacologia , Somatostatina/metabolismo , Canais de Cátion TRPV/agonistas , Tarso Animal/diagnóstico por imagem , Tarso Animal/metabolismo , Tarso Animal/patologia , Microtomografia por Raio-XRESUMO
ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased ß2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of ß2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.