RESUMO
The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.
RESUMO
A proposal for the use of porcine pancreatic elastase (PPE) to develop a mouse model of pulmonary emphysema raised concerns about introducing contaminating porcine viruses into our barrier facility. Porcine Circovirus (PCV) is a known contaminant of vaccines and cell cultures that have been exposed to porcine-derived reagents. Endemic infection of PCV3 in laboratory mice has been reported, and some evidence supports natural PCV infection in wild mice. PPE samples from 2 different vendors tested positive for DNA from both PCV2 and 3. To allow model development with these reagents to proceed, we developed a protocol that would meet scientific objectives, minimize exposure of mice, and provide information on the potential for the virus to spread. Five d after BALB/c mice received intralaryngeal administration of PPE, lungs were harvested and analyzed for evidence of disease. Tissues from other major organs were submitted to test for disseminated PCV2 and 3 DNA. Similarly, tissues (including lungs) from direct contact nude sentinel mice were analyzed for the presence of the virus. To evaluate the possibility of endemic PCV2/3 infection, we also surveyed non-porcine reagent exposed mice on other studies. PCV2 and 3 was not detected in any of the tissues submitted. Although this study provided no evidence of infection and transmission of PCV2/3 from the contaminated PPE sample over the 5 d study, further work is needed to understand the risks and impact of introducing PCV contaminated cells or reagents into barrier maintained rodent colonies.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Ambiente Controlado , Indicadores e Reagentes , Animais , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Circovirus/imunologia , Abrigo para Animais/normas , Ciência dos Animais de Laboratório , Camundongos , Camundongos Endogâmicos BALB C , Infecções Respiratórias/veterinária , Infecções Respiratórias/virologia , Medição de Risco , Doenças dos Roedores/virologiaRESUMO
Innate lymphoid cells (ILCs) are critical mediators of mucosal immunity, and group 1 ILCs (ILC1 cells) and group 3 ILCs (ILC3 cells) have been shown to be functionally plastic. Here we found that group 2 ILCs (ILC2 cells) also exhibited phenotypic plasticity in response to infectious or noxious agents, characterized by substantially lower expression of the transcription factor GATA-3 and a concomitant switch to being ILC1 cells that produced interferon-γ (IFN-γ). Interleukin 12 (IL-12) and IL-18 regulated this conversion, and during viral infection, ILC2 cells clustered within inflamed areas and acquired an ILC1-like phenotype. Mechanistically, these ILC1 cells augmented virus-induced inflammation in a manner dependent on the transcription factor T-bet. Notably, IL-12 converted human ILC2 cells into ILC1 cells, and the frequency of ILC1 cells in patients with chronic obstructive pulmonary disease (COPD) correlated with disease severity and susceptibility to exacerbations. Thus, functional plasticity of ILC2 cells exacerbates anti-viral immunity, which may have adverse consequences in respiratory diseases such as COPD.
Assuntos
Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Vírus da Influenza A/imunologia , Pulmão/imunologia , Linfócitos/imunologia , Infecções por Orthomyxoviridae/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Células Th1/imunologia , Células Th2/imunologia , Idoso , Animais , Diferenciação Celular , Plasticidade Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Fumar/efeitos adversos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease and is presumed to be central to the altered responsiveness to recurrent infection in these patients. We examined the effects of smoke priming underlying the exacerbated response to viral infection in mice. Lack of interleukin-33 (IL-33) signaling conferred complete protection during exacerbation and prevented enhanced inflammation and exaggerated weight loss. Mechanistically, smoke was required to upregulate epithelial-derived IL-33 and simultaneously alter the distribution of the IL-33 receptor ST2. Specifically, smoke decreased ST2 expression on group 2 innate lymphoid cells (ILC2s) while elevating ST2 expression on macrophages and natural killer (NK) cells, thus altering IL-33 responsiveness within the lung. Consequently, upon infection and release, increased local IL-33 significantly amplified type I proinflammatory responses via synergistic modulation of macrophage and NK cell function. Therefore, in COPD, smoke alters the lung microenvironment to facilitate an alternative IL-33-dependent exaggerated proinflammatory response to infection, exacerbating disease.
Assuntos
Imunidade Inata/efeitos dos fármacos , Interleucinas/imunologia , Infecções por Orthomyxoviridae/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptores de Interleucina/imunologia , Fumaça/efeitos adversos , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Vírus da Influenza A/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/deficiência , Interleucinas/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos Transgênicos , Infecções por Orthomyxoviridae/etiologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais , Nicotiana/química , Redução de PesoRESUMO
Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.
Assuntos
Modelos Animais de Doenças , Hipersensibilidade/patologia , Inflamação/patologia , Interleucina-17/fisiologia , Pneumopatias/patologia , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sinciciais Respiratórios/patogenicidade , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Feminino , Citometria de Fluxo , Granzimas/genética , Granzimas/metabolismo , Humanos , Hipersensibilidade/metabolismo , Hipersensibilidade/virologia , Inflamação/metabolismo , Inflamação/virologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-23/metabolismo , Pneumopatias/metabolismo , Pneumopatias/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Muco/metabolismo , RNA Mensageiro/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Linfócitos T Citotóxicos/virologia , Carga Viral , Proteínas Virais/metabolismoRESUMO
A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5-10 min, and saline-like HR measures restored within 20-40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.
Assuntos
Hidrolases de Éster Carboxílico/genética , Sistema Cardiovascular/efeitos dos fármacos , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Mutação/genética , Proteínas Recombinantes/genética , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Hidrolases de Éster Carboxílico/metabolismo , Relação Dose-Resposta a Droga , Eletrocardiografia/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas/métodos , Macaca mulatta , Masculino , Atividade Motora/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
Activation of the canonical Notch pathways has been implicated in Th cell differentiation, but the role of specific Notch ligands in Th2-mediated allergic airway responses has not been completely elucidated. In this study, we show that delta-like ligand 4 (Dll4) was upregulated on dendritic cells in response to cockroach allergen. Blocking Dll4 in vivo during either the primary or secondary response enhanced allergen-induced pathogenic consequences including airway hyperresponsiveness and mucus production via increased Th2 cytokines. In vitro assays demonstrated that Dll4 regulates IL-2 in T cells from established Th2 responses as well as during primary stimulation. Notably, Dll4 blockade during the primary, but not the secondary, response increased IL-2 levels in lung and lymph node of allergic mice. The in vivo neutralization of Dll4 was associated with increased expansion and decreased apoptosis during the primary allergen sensitization. Moreover, Dll4-mediated Notch activation of T cells during primary stimulation in vitro increased apoptosis during the contraction/resting phase of the response, which could be rescued by exogenous IL-2. Consistent with the role for Dll4-mediated IL-2 regulation in overall T cell function, the frequency of IL-4-producing cells was also significantly altered by Dll4 both in vivo and in vitro. These data demonstrate a regulatory role of Dll4 both in initial Th2 differentiation and in Th2 cytokine production in established allergic responses.
Assuntos
Interleucina-2/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Hipersensibilidade Respiratória/imunologia , Células Th2/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Diferenciação Celular/imunologia , Separação Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Interleucina-2/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th2/citologia , Células Th2/metabolismoRESUMO
In the present studies local neutralization of allergen-induced stem cell factor (SCF) leads to decreased production of Th2 cytokines, a reduction in inflammation, allergen-specific serum IgE/IgG1, and attenuation of severe asthma-like responses. The local blockade of pulmonary SCF also resulted in a significant reduction of IL-17E (IL-25). Sorted cell populations from the lung indicated that IL-25 was produced from c-kit(+) cells, whereas Th2 cytokine production was primarily from c-kit(-) cell populations. SCF stimulated c-kit(+) eosinophils produced IL-25, whereas bone marrow-derived mast cells did not. Using 4get mice that contain a IL-4-IRES-eGFP that when transcribed coexpress GFP and IL-4, our studies identified cells that comprised a CD11b(+), GR1(+), Ly6C(+/-), c-kit(-), CD4(-), CD11c(-), MHC class II(low) cell population as a source of IL-4 in the lung after chronic allergen challenge. In the bone marrow a similar cell was identified with approximately a third of the IL-4(+) cells also expressing c-kit(+). The pulmonary and bone marrow IL-4(+) cell populations were significantly reduced upon local pulmonary anti-SCF treatment. Subsequently, when IL-25R was examined during the chronic allergen responses the expression was found on the IL-4(+) myeloid cell population that expressed CD11b(+)GR1(+). Interestingly, the IL-25R(+) cells in the bone marrow were also all CD11b(+)GR1(+), similar to the lung cells, but they were also all c-kit(+), potentially suggesting a maturation of the bone marrow cell once it enters the lung and/or is stimulated by SCF. Overall, these studies suggest a complex relationship between SCF, bone marrow-derived IL-25-responsive myeloid cells, Th2 cytokines, and chronic allergic disease.
Assuntos
Citocinas/biossíntese , Interleucinas/biossíntese , Pulmão/imunologia , Células Mieloides/imunologia , Receptores de Interleucina-17/biossíntese , Hipersensibilidade Respiratória/imunologia , Fator de Células-Tronco/fisiologia , Células Th2/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Antígeno CD11b/biossíntese , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Doença Crônica , Citocinas/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interleucina-17/biossíntese , Interleucina-4/biossíntese , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Receptores de Quimiocinas/biossíntese , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Fator de Células-Tronco/antagonistas & inibidores , Células Th2/metabolismo , Células Th2/patologiaRESUMO
BACKGROUND: A bacterial cocaine esterase (CocE) produces robust protection and reversal of cocaine toxicity. The aim of this study was to investigate how effectiveness of CocE was changed following its repeated administration together with cocaine. METHODS: Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality in mice. Immunologic responses of CocE were determined using ELISA. In the protection experiment, i.v. CocE 0.3mg was given 1min before a lethal dose of i.p. cocaine 180mg/kg. In the rescue experiment, i.v. CocE 0.3mg was given 1min after the occurrence of convulsions elicited by i.p. cocaine 100mg/kg. In both treatment paradigms, four trials were conducted in the same animals with a 2-week interval. RESULTS: CocE retained its effectiveness to protect or rescue mice during the first two trials and these mice did not show an immune response. In contrast, CocE's effectiveness was gradually reduced in the last two trials, accompanied by 10- and 100-fold increases in anti-CocE antibody titers. Nevertheless, effectiveness of CocE could be partially recovered by increasing the dose of CocE. In addition, escalating the dose of CocE from the minimum effective dose for repeated administration could also retain CocE's effectiveness longer and slow the production of anti-CocE antibodies. CONCLUSIONS: These results indicate that CocE is a weak antigen and it can maintain its protective and rescuing ability initially against cocaine-induced toxicity. Decreased effectiveness of CocE following repeated use can be partially improved by adjusting the dose and frequency of CocE treatment.
Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Cocaína/administração & dosagem , Cocaína/metabolismo , Animais , Anticorpos/sangue , Especificidade de Anticorpos , Hidrolases de Éster Carboxílico/uso terapêutico , Cocaína/imunologia , Cocaína/toxicidade , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Camundongos , Postura , Convulsões/induzido quimicamente , Convulsões/prevenção & controleRESUMO
BACKGROUND: The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease. METHODOLOGY/PRINCIPAL FINDINGS: In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell -/- mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells. CONCLUSIONS/SIGNIFICANCE: Collectively, these results provide evidence for antigen presentation as a novel mechanism by which B cells contribute to chronic allergic disease. These findings give new insight into the mechanisms by which B cells promote asthma and other chronic diseases.
Assuntos
Antígenos/metabolismo , Linfócitos B/imunologia , Hipersensibilidade/imunologia , Pneumopatias/imunologia , Células Th2/metabolismo , Animais , Apresentação de Antígeno , Antígenos/química , Linfócitos B/metabolismo , Baratas/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
Prostaglandin D(2), the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD(2) and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD(2)-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation.
Assuntos
Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/prevenção & controle , Regulação para Baixo/genética , Pneumonia/complicações , Pneumonia/genética , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Alérgenos/imunologia , Animais , Anticorpos/sangue , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/imunologia , Baratas , Citocinas/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/sangue , Pneumonia/imunologiaRESUMO
In the present studies the role of stem cell factor (SCF) in mediating eosinophil and fibroblast activation during their interaction was investigated. SCF was significantly higher in fibroblasts grown from lungs of chronic allergen-challenged mice compared to fibroblasts grown from normal mice. When eosinophils were layered onto fibroblasts from allergic mice, a significant increase in SCF was detected compared to fibroblasts from nonallergic mice. The interaction of fibroblasts with eosinophils also increased the production of asthma-associated chemokines, CCL5 and CCL6, was dependent on cell-to-cell interaction, and was observed only with fibroblasts derived from lungs of chronic allergen-challenged mice and not from those derived from unchallenged normal mice. Chemokine production was significantly decreased when anti-SCF antibodies were added during eosinophil-fibroblast interaction. The interaction of fibroblasts from chronic allergen-challenged mice with eosinophils also increased alpha-smooth muscle cell actin and procollagen I expression as well as induced transforming growth factor-beta. The changes in myofibroblast activation were dependent on SCF-mediated pathways because anti-SCF antibody treatment reduced the expression of all three of these latter fibrosis-associated markers. Thus, our data suggest that SCF mediates an important activation pathway for fibroblasts during chronic allergic responses on interaction with recruited eosinophils and suggest a potential mechanism of airway remodeling during chronic disease.
Assuntos
Alérgenos/metabolismo , Fibroblastos/metabolismo , Fator de Células-Tronco/metabolismo , Animais , Antígenos/química , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Técnicas de Cocultura , Eosinófilos/metabolismo , Feminino , Fibroblastos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Fenótipo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: While glucocorticoids are currently the most effective therapy for asthma, associated side effects limit enthusiasm for their use. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators include the synthetic thiazolidinediones (TZDs) which exhibit anti-inflammatory effects that suggest usefulness in diseases such as asthma. How the ability of TZDs to modulate the asthmatic response compares to that of glucocorticoids remains unclear, however, because these two nuclear receptor agonists have never been studied concurrently. Additionally, effects of PPAR-gamma agonists have never been examined in a model involving an allergen commonly associated with human asthma. METHODS: We compared the effectiveness of the PPAR-gamma agonist pioglitazone (PIO) to the established effectiveness of a glucocorticoid receptor agonist, dexamethasone (DEX), in a murine model of asthma induced by cockroach allergen (CRA). After sensitization to CRA and airway localization by intranasal instillation of the allergen, Balb/c mice were challenged twice at 48-h intervals with intratracheal CRA. Either PIO (25 mg/kg/d), DEX (1 mg/kg/d), or vehicle was administered throughout the period of airway CRA exposure. RESULTS: PIO and DEX demonstrated similar abilities to reduce airway hyperresponsiveness, pulmonary recruitment of inflammatory cells, serum IgE, and lung levels of IL-4, IL-5, TNF-alpha, TGF-beta, RANTES, eotaxin, MIP3-alpha, Gob-5, and Muc5-ac. Likewise, intratracheal administration of an adenovirus containing a constitutively active PPAR-gamma expression construct blocked CRA induction of Gob-5 and Muc5-ac. CONCLUSION: Given the potent effectiveness shown by PIO, we conclude that PPAR-gamma agonists deserve investigation as potential therapies for human asthma.
Assuntos
Alérgenos/toxicidade , Asma/tratamento farmacológico , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Tiazolidinedionas/uso terapêutico , Animais , Antígenos de Plantas , Asma/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos BALB C , PioglitazonaRESUMO
Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs' therapeutic potential as new agonists for the resolution of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Lipoxinas/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Éteres Fenílicos/farmacologia , Hipersensibilidade Respiratória/prevenção & controle , Acetatos/farmacologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antiasmáticos/farmacologia , Antígenos de Plantas , Ciclopropanos , Modelos Animais de Doenças , Antagonistas de Leucotrienos/farmacologia , Lipoxinas/química , Lipoxinas/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Quinolinas/farmacologia , Receptores de Leucotrienos/fisiologia , Hipersensibilidade Respiratória/imunologia , Relação Estrutura-Atividade , SulfetosRESUMO
Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.
Assuntos
Células Dendríticas/imunologia , Pulmão/imunologia , Fator 88 de Diferenciação Mieloide/deficiência , Pneumonia Viral/imunologia , Infecções por Respirovirus/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células da Medula Óssea , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação ao Cálcio , Células Dendríticas/patologia , Eosinófilos/imunologia , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-12/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Fator 88 de Diferenciação Mieloide/genética , Pneumonia Viral/patologia , Infecções por Respirovirus/patologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Cocaine esterase (CocE) is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The aim of this study was to investigate the in vivo potency of CocE in blocking cocaine-induced toxicity in the mouse and to assess CocE's potential immunogenicity. Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality. Intravenous administration of CocE (0.1-1 mg) 1 min before cocaine administration produced dose-dependent rightward shifts of the dose-response curve for cocaine toxicity. More important, i.v. CocE (0.1-1 mg), given 1 min after the occurrence of cocaine-induced convulsions, shortened the recovery time after the convulsions and saved the mice from subsequent death. Effects of repeated exposures to CocE were evaluated by measuring anti-CocE antibody titers and the protective effects of i.v. CocE (0.32 mg) against toxicity elicited by i.p. cocaine (320 mg/kg) (i.e., 0-17% occurrence of convulsions and lethality). CocE retained its potency against cocaine toxicity in mice after a single prior CocE exposure (0.1-1 mg), and these mice did not show an immune response. CocE retained similar effectiveness in mice after three prior CocE exposures (0.1-1 mg/week for 3 weeks), although these mice displayed 10-fold higher antibody titers. CocE partially lost effectiveness (i.e., 33-50% occurrence of convulsions and lethality) in mice with four prior exposures to CocE (0.1-1 mg/2 week for four times), and these mice displayed approximately 100-fold higher antibody titers. These results suggest that CocE produces robust protection and reversal of cocaine toxicity, indicating CocE's therapeutic potential for acute cocaine toxicity. Repeated CocE exposures may increase its immunogenicity and partially reduce its protective ability.
Assuntos
Hidrolases de Éster Carboxílico/imunologia , Hidrolases de Éster Carboxílico/farmacologia , Cocaína/toxicidade , Animais , Anticorpos/sangue , Relação Dose-Resposta a Droga , Masculino , Camundongos , Convulsões/induzido quimicamente , Fatores de TempoRESUMO
In this study we performed comparisons of pulmonary responses between two different respiratory syncytial virus (RSV) antigenic subgroup A strains, A2 and Line 19. Line 19 strain induced significant dose-responsive airway hyperreactivity (AHR) in BALB/c mice at days 6 and 9 after infection, whereas the A2 strain induced no AHR at any dose. Histological examination indicated that A2 induced no goblet cell hyper/metaplasia, whereas the Line 19 induced goblet cell expansion and significant increases in gob5 and MUC5AC mRNA and protein levels in vivo. When examining cytokine responses, A2 strain induced significant interleukin (IL)-10 expression, whereas Line 19 strain induced significant IL-13 expression. When IL-13-/- mice were infected with Line 19 RSV, the AHR responses were abrogated along with gob5 gene expression. There was little difference in viral titer throughout the infection between the line 19- and A2-infected mice. However, the A2 strain grew to significantly higher titers than the Line 19 strain in HEp-2 cells in vitro. Thus, RSV Line 19-induced airway dysfunction does not correlate with viral load in vivo. These data demonstrate that different RSV strains of the same antigenic subgroup can elicit differential immune responses that impact the phenotypic expression of RSV-induced illness.
Assuntos
Hiper-Reatividade Brônquica/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Animais , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/virologia , Linhagem Celular , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica/imunologia , Células Caliciformes/imunologia , Células Caliciformes/patologia , Células Caliciformes/virologia , Hiperplasia/imunologia , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Hiperplasia/virologia , Interleucina-13/deficiência , Interleucina-13/imunologia , Camundongos , Camundongos Knockout , Mucina-5AC , Mucinas/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Especificidade da EspécieRESUMO
The progression and severity of chronic asthma likely depends upon the intensity of the damage and remodeling of the tissue. We have developed a chronic model of allergic asthma using multiple cockroach allergen challenges. Using this clinically relevant allergen we have established significant peribronchial fibrosis and mucus overproduction. These remodeling events are accompanied by intense peribronchial inflammation, including lymphocytes and eosinophils. A cytokine that has been identified as having a prominent role in short-term allergic events, stem cell factor (SCF), appears to have a significant role in this late-stage process. Using our polyclonal antibody specific for SCF administered into the airways of mice during the final allergen challenges, we find a significant effect on the chronic peribronchial allergen-induced fibrotic remodeling. This was characterized by reduced inflammation, especially eosinophils, as well as reduced hydroxyproline levels in anti-SCF compared to control antibody-treated animals. In addition, when we examined chemokines associated with the chronic disease and neutralized SCF in vivo we observed a corresponding decrease in CCL6 and CCL17. Using an inhibitor, imatinib mesylate, that blocks SCF/c-kit-associated RTK, we find similar results as with anti-SCF for attenuating AHR and fibrotic changes, suggesting that a potential clinical treatment for chronic asthma already exists related to this pathway. These results further support the potential use of SCF/c-kit inhibition for targeting chronic severe asthmatic responses.
Assuntos
Alérgenos/farmacologia , Brônquios/patologia , Fator de Células-Tronco/antagonistas & inibidores , Administração Intranasal , Alérgenos/imunologia , Animais , Asma/imunologia , Asma/metabolismo , Asma/patologia , Asma/fisiopatologia , Asma/terapia , Benzamidas , Brônquios/imunologia , Brônquios/metabolismo , Hiper-Reatividade Brônquica , Quimiocina CCL17 , Quimiocinas CC/análise , Doença Crônica , Baratas , Eosinófilos/patologia , Fibrose/patologia , Hidroxiprolina/análise , Mesilato de Imatinib , Pulmão/química , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/uso terapêuticoRESUMO
The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3-/- mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3-/- mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3-/- mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3-/- mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3-/- mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.