RESUMO
Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.
Assuntos
Coproporfirinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Coproporfirinas/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Células Sf9 , SpodopteraRESUMO
Parasitic worms are receiving much attention as a potential new therapeutic approach to treating autoimmune and allergic conditions but concerns remain regarding their safety. As an alternative strategy, we have focused on the use of defined parasitic worm products and recently taken this one step further by designing drug-like small molecule analogues of one such product, ES-62, which is anti-inflammatory by virtue of covalently attached phosphorylcholine moieties. Previously, we have shown that ES-62 mimics are efficacious in protecting against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus and skin and lung allergy. Given the potential role of chronic inflammation in fibrosis, in the present study we have focused our attention on lung fibrosis, a debilitating condition for which there is no cure and which in spite of treatment slowly gets worse over time. Two mouse models of fibrosis - bleomycin-induced and LPS-induced - in which roles for inflammation have been implicated were adopted. Four ES-62 analogues were tested - 11a and 12b, previously shown to be active in mouse models of allergic and autoimmune disease and 16b and AIK-29/62 both of which are structurally related to 11a. All four compounds were found to significantly reduce disease development in both fibrosis models, as shown by histopathological analysis of lung tissue, indicating their potential as treatments for this condition.
Assuntos
Anti-Inflamatórios/uso terapêutico , Proteínas de Helminto/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease. METHODS: C57BL/6 wild-type (WT) and SP-D-/- mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-ß were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D. RESULTS: Dp-challenged SP-D-/- mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-ß1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-ß1 and IL-13 positive eosinophils in SP-D-/- mice. Purified eosinophils stimulated with Dp produced TGF-ß1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D-/- mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D-/- mice and neutralization of TGF-ß1 reduced sub-epithelial fibrosis in Dp-challenged SP-D-/- mice. CONCLUSION: These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-ß.
Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Fibrose Pulmonar/prevenção & controle , Proteína D Associada a Surfactante Pulmonar/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Antígenos de Dermatophagoides , Proteínas de Artrópodes , Asma/imunologia , Asma/patologia , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/patologia , Genótipo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Pneumonia/imunologia , Pneumonia/patologia , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/metabolismo , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Proteína D Associada a Surfactante Pulmonar/deficiência , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/farmacologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidoresRESUMO
TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue-specific induction of Tregs in the periphery remains unclear. We observed that surfactant protein A (SP-A)-deficient mice have impaired expression of Foxp3 and fewer CD25(+)Foxp3(+) Tregs after ex vivo stimulation and after stimulation with LPS in vivo. The addition of exogenous SP-A completely reversed this phenotype. Although SP-A is known to inhibit T cell proliferation under certain activation conditions, both IL-2 levels as well as active TGF-ß levels increase on extended culture with exogenous SP-A, providing a key mechanism for the maintenance and induction of Tregs. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3(+) Tregs in responder T cell populations in a TGF-ß-dependent manner. In mice treated with LPS in vivo, Tregs increased â¼160% in wild-type mice compared with only a 50% increase in LPS-treated SP-A(-/-) mice 8 d after exposure. Taken together, these findings support the hypothesis that SP-A affects T cell immune function by the induction of Tregs during activation.
Assuntos
Ativação Linfocitária/fisiologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteína A Associada a Surfactante Pulmonar/biossíntese , Proteína A Associada a Surfactante Pulmonar/genética , Linfócitos T Reguladores/metabolismoRESUMO
Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A-deficient mice on a C57BL/6 background (H2b; SP-A(-/-)) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A-deficient recipient mice that have undergone an allogeneic BMT [SP-A(-/-)alloBMT] or SP-A-sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A-deficient recipient mice that have undergone a syngeneic BMT or SP-A-sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A(-/-) alloBMT or SP-A-sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A(-/-)alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A(-/-)alloBMT mice also had increased colon expression of IL-1ß, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.
Assuntos
Gastroenteropatias/imunologia , Gastroenteropatias/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/metabolismo , Proteína A Associada a Surfactante Pulmonar/fisiologia , Animais , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Gastroenteropatias/genética , Doença Enxerto-Hospedeiro/genética , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína A Associada a Surfactante Pulmonar/deficiência , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.
Assuntos
Eosinófilos/metabolismo , Regulação da Expressão Gênica , Hipersensibilidade/imunologia , Pulmão/imunologia , Mycoplasma pneumoniae/metabolismo , Pneumonia por Mycoplasma/metabolismo , Pneumonia por Mycoplasma/microbiologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Albuminas/metabolismo , Animais , Cálcio/metabolismo , Eosinófilos/imunologia , Citometria de Fluxo , Humanos , Hipersensibilidade/metabolismo , Inflamação , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos BiológicosRESUMO
Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Cálcio/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Mitógenos/farmacologia , Proteína A Associada a Surfactante Pulmonar/deficiência , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
RATIONALE: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known. OBJECTIVES: The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM). METHODS: Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps. MEASUREMENTS AND MAIN RESULTS: BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-ß1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-ß1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-ß1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice. CONCLUSIONS: These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.
Assuntos
Bleomicina/toxicidade , Fibrose Pulmonar Idiopática/fisiopatologia , Proteína D Associada a Surfactante Pulmonar/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/fisiopatologia , Remodelação das Vias Aéreas/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Citocinas/fisiologia , Modelos Animais de Doenças , Fibrose Pulmonar Idiopática/induzido quimicamente , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiopatologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína A Associada a Surfactante Pulmonar/análise , Proteína A Associada a Surfactante Pulmonar/fisiologia , Proteína D Associada a Surfactante Pulmonar/análise , RatosRESUMO
Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.
Assuntos
Doenças Renais Policísticas/patologia , Fator de Transcrição STAT6/antagonistas & inibidores , Animais , Linhagem Celular , Crotonatos/uso terapêutico , Modelos Animais de Doenças , Cães , Hidroxibutiratos , Camundongos , Nitrilas , Doenças Renais Policísticas/tratamento farmacológico , Toluidinas/uso terapêuticoRESUMO
Mutations in polycystin-1 (PC1) lead to autosomal-dominant polycystic kidney disease (ADPKD), a leading cause of renal failure for which no treatment is available. PC1 is an integral membrane protein, which has been implicated in the regulation of multiple signaling pathways including the JAK/STAT pathway. Here we show that membrane-anchored PC1 activates STAT3 in a JAK2-dependent manner, leading to tyrosine phosphorylation and transcriptional activity. The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation. Tail-cleavage abolishes the ability of PC1 to directly activate STAT3 but the cleaved PC1 tail now coactivates STAT3 in a mechanism requiring STAT phosphorylation by cytokines or growth factors. This leads to an exaggerated cytokine response. Hence, PC1 can regulate STAT activity by a dual mechanism. In ADPKD kidneys PC1 tail fragments are overexpressed, including a unique â¼15-kDa fragment (P15). STAT3 is strongly activated in cyst-lining epithelial cells in human ADPKD, and orthologous and nonorthologous polycystic mouse models. STAT3 is also activated in developing, postnatal kidneys but inactivated in adult kidneys. These results indicate that STAT3 signaling is regulated by PC1 and is a driving factor for renal epithelial proliferation during normal renal development and during cyst growth.
Assuntos
Fatores de Transcrição STAT/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Animais , Morte Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Cães , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Mutação , Fosforilação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição STAT/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/química , TransfecçãoRESUMO
Although many studies have shown that pulmonary surfactant protein (SP)-A functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4(+) T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an OVA model of allergic hypersensitivity, SP-A(-/-) mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared with wild-type counterparts. Although both OVA-exposed groups had similar proportions of CD86(+) DCs and Foxp3(+) T regulatory cells, the SP-A(-/-) mice had elevated proportions of CD4(+) activated and effector memory T cells in their lungs compared with wild-type mice. Ex vivo recall stimulation of CD4(+) T cell pools demonstrated that cells from the SP-A(-/-) OVA mice had the greatest proliferative and IL-4-producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that CD4(+) activated and effector memory T cells expanded to the greatest extent in the lungs of SP-A(-/-) OVA mice. Taken together, our data suggested that SP-A influences the prevalence, types, and functions of CD4(+) T cells in the lungs during allergic inflammation and that SP deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.
Assuntos
Alérgenos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Memória Imunológica , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Proteína A Associada a Surfactante Pulmonar/deficiência , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Memória Imunológica/genética , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Pulmão/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/fisiologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/prevenção & controle , Índice de Gravidade de Doença , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologiaRESUMO
Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.
Assuntos
Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/fisiopatologia , Óxido Nítrico/fisiologia , Proteína A Associada a Surfactante Pulmonar/deficiência , Animais , Imunidade Inata , Pulmão/efeitos dos fármacos , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/fisiologia , Proteína D Associada a Surfactante Pulmonar/metabolismoRESUMO
The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H(2)O(2). In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca(2+) concentrations [Ca(2+)](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNFα and IFNγ by CD4(+) T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca(2+) ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling.
Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Cálcio/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácidos Indolacéticos/farmacologia , Ativação Linfocitária , Ácidos Naftalenoacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Ácido 2,4-Diclorofenoxiacético/química , Animais , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Cálcio/imunologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Curcumina/farmacologia , Feminino , Peróxido de Hidrogênio/imunologia , Ácidos Indolacéticos/química , Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ionomicina/imunologia , Ionomicina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Naftalenoacéticos/química , Reguladores de Crescimento de Plantas/química , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Acetato de Tetradecanoilforbol/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RATIONALE: Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury. OBJECTIVES: Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment. METHODS: Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed. MEASUREMENTS AND MAIN RESULTS: On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo. CONCLUSIONS: These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Bleomicina , Líquido da Lavagem Broncoalveolar , Técnicas de Cultura de Células/métodos , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Edema , Ensaio de Imunoadsorção Enzimática/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell-T cell interactions are not well known. The consequences of blocking CTLA-4-CD80/CD86 interactions on purified mouse CD4(+) T cells were studied in the context of the strength of signal (SOS). CD4(+) T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca(2+) ionophore, Ionomycin (I), or a sarcoplasmic Ca(2+) ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca(2+)](i)) were greatly reduced upon CTLA-4-CD80/CD86 blockade. Further experiments demonstrated that CTLA-4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)-CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4-CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H(2)O(2) was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca(2+)](i) and ROS play important roles in the modulation of T-cell responses by CTLA-4-CD80/CD86 interactions.
Assuntos
Antígenos CD/metabolismo , Cálcio/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Complexo CD3/imunologia , Antígeno CTLA-4 , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Proliferação de Células , Sobrevivência Celular/imunologia , Células Cultivadas , Feminino , Ionomicina/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia , Acetato de Tetradecanoilforbol/imunologia , Tapsigargina/farmacologiaRESUMO
Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a well-studied T cell costimulatory receptor that is known to inhibit T cell activation. In this study, the relationship between strength of the first signal and costimulatory interactions on primary mouse CD4(+) T cells was investigated. CTLA4-CD80/CD86 interactions differentially modulate T cell cycling based on the mode of CD3 signal: Activation with plate-bound (pb) anti-CD3 generates a strong signal compared with a weak signal with soluble (sol) anti-CD3, resulting in approximately sevenfold higher amounts of interleukin (IL)-2 and an increase in cell cycling. Activation of T cells with sol anti-CD3 (weak signal) together with CTLA4-CD80/CD86 blockade lowers IL-2 production and cell cycling, demonstrating an enhancing role for these interactions. Conversely, blockade of CTLA4-CD80/CD86 interactions on T cells activated with pb anti-CD3 (strong signal) increases proliferation, which is consistent with CTLA4 as a negative regulator. Also, coculture of T cells with Chinese hamster ovary cells expressing CD80 or CD86 demonstrates that the strength of the primary signal plays an important role. It is important that modulation of IL-2 amounts leads to distinct alterations in the functional effects of CTLA4-CD80/CD86 interactions. On increasing IL-2 amounts, activation of T cells stimulated with sol anti-CD3 (weak signal) and CTLA4-CD80/CD86 blockade is greater compared with control. Concurrently, neutralization of IL-2 greatly reduces activation of T cells stimulated with pb anti-CD3 (strong signal) and CTLA4-CD80/CD86 blockade compared with control. These results underscore the importance of strength of first signal, CTLA4-CD80/CD86 interactions, and IL-2 amounts in modulating primary CD4(+) T cell responses.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígeno CTLA-4 , Ciclo Celular/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
Primary cilia are implicated in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD), which results from defects in polycystin-1 (PC1), but the function of PC1 remains poorly understood. Here, we show that PC1 undergoes proteolytic cleavage that results in nuclear translocation of its cytoplasmic tail. The PC1 tail interacts with the transcription factor STAT6 and the coactivator P100, and it stimulates STAT6-dependent gene expression. Under normal conditions, STAT6 localizes to primary cilia of renal epithelial cells. Cessation of apical fluid flow results in nuclear translocation of STAT6. Cyst-lining cells in ADPKD exhibit elevated levels of nuclear STAT6, P100, and the PC1 tail. Exogenous expression of the human PC1 tail results in renal cyst formation in zebrafish embryos. These results identify a novel mechanism of cilia function in the transduction of a mechanical signal to changes of gene expression involving PC1 and show that this pathway is inappropriately activated in ADPKD.
Assuntos
Cílios/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas Nucleares/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas/fisiologia , Fator de Transcrição STAT6/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting/métodos , Western Blotting/métodos , Linhagem Celular , Cílios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Embrião não Mamífero , Endonucleases , Ativação Enzimática/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Imunofluorescência/métodos , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação/métodos , Interleucina-4/farmacologia , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Luciferases/metabolismo , Modelos Biológicos , Biologia Molecular/métodos , Mutagênese/fisiologia , Rim Policístico Autossômico Dominante/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Canais de Cátion TRPP , Transativadores/fisiologia , Transfecção/métodos , Translocação Genética , Peixe-ZebraRESUMO
The mechanisms by which concanavalin A (Con A), a lectin, activates T cells are poorly studied. A low dose of Con A is stimulatory for T cells, whereas a high dose of Con A results in suppression of proliferation and enhanced T cell death. The expression and functional roles of costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA4), and their ligands, CD80 and CD86, on primary mouse CD4(+) T cells after activation with different doses of Con A were studied. CTLA4-CD80/CD86 interactions in this T:T cell activation model demonstrate distinct outcomes depending on the dose of Con A. CTLA4-CD80/CD86 interactions inhibit CD4(+) T cell cycling and survival after activation with a suppressive dose of Con A by increasing oxidative stress and decreasing levels of BclX(L). The enhanced CD4(+) T cell death with a suppressive dose of Con A is dependent on excess H(2)O(2) and nitric oxide but is independent of Fas and caspase activity. It is surprising that the increased proliferation of CD4(+) T cells with a suppressive dose of Con A on blocking CTLA4-CD80/CD86 interactions is largely interleukin (IL)-2-independent but is cyclosporine A-sensitive. On activation with a stimulatory dose of Con A, CTLA4-CD80/CD86 interactions enhance T cell activation and survival by reducing the production of reactive oxygen species, increasing IL-2 and BclX(L) levels. Here IL-10 but not transforming growth factor-beta plays a functional role. In summary, CTLA4-CD80/CD86 interactions on T cells integrate signal strength, based on the dose of Con A, to enhance or inhibit primary mouse CD4(+) T cell cycling and survival.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígeno B7-1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Concanavalina A/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Animais , Antígenos CD/efeitos dos fármacos , Antígenos de Diferenciação/efeitos dos fármacos , Antígeno B7-2 , Antígenos CD28/efeitos dos fármacos , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígeno CTLA-4 , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/metabolismo , Fatores Imunológicos/imunologia , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Proteína bcl-XRESUMO
Cell surface interactions between the T cell costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA4), with their cognate ligands, CD80 and CD86, on antigen-presenting cells play an important role in T cell activation. Although CD80 and CD86 are induced on T cells after activation, not much is known about their role in modulating T cell function. We show that CD80, CD86, and CTLA4 are induced on purified CD4(+) T cells after in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and they play an essential role for proliferation and survival. Blockade of CTLA4-CD80/CD86 interactions greatly reduces PMA and ionomycin-mediated mouse CD4(+) T cell activation. The three key features of this inhibition of activation are: First, late events in T cell activation (after 18 h) are affected; second, these cells do not undergo anergy; and third, CD4(+)CD25(+) regulatory T cells are not responsible. Activation of T cells with PMA and ionomycin together with CTLA4-CD80/CD86 blockade results in decreased induction of CD25 and Bcl-X(L), reduced interleukin (IL)-2, and enhanced transforming growth factor-beta (TGF-beta) production. Furthermore, extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis in a large proportion of cells. This inhibition of T cell proliferation can be rescued completely with anti-CD28 or IL-2 and partially with TGF-beta antagonists. This study reveals a functional role for CD80, CD86, and CTLA4 on CD4(+) T lymphocytes and sheds light on the mechanisms by which these molecules enhance activation and survival with PMA and ionomycin.