RESUMO
BACKGROUND: Injury to the lung is a common, clinically serious inflammatory disease. However, its pathogenesis remains unclear, and the existing treatments, including cytokine therapy, stem cell therapy, and hormone therapy, are not completely effective in treating this disease. Dimethyl itaconate (DMI) is a surfactant with important anti-inflammatory effects. OBJECTIVE: The present study used alveolar type II (AT II) and bronchial epithelial cells as models to determine the role of DMI in lung injury. MATERIAL AND METHODS: First, the effects of DMI were established on the survival, inflammatory release, and apoptosis in lipopolysaccharide (LPS)-induced AT II and bronchial epithelial cells. The association between DMI and Sirtuin1 (SIRT1) was assessed using molecular docking. Next, by constructing interference plasmids to inhibit surfactant protein (SP)-A and SP-D expressions, the effect of DMI was observed on inflammatory release and apoptosis. RESULTS: The results revealed that DMI increased the survival rate and expression levels of SP-A, SP-D, and SIRT1, and inhibited inflammatory factors as well as apoptosis in LPS-induced cells. Furthermore, DMI could bind to SIRT1 to regulate SP-A and SP-D expressions. After SP-A and SP-D expressions were inhibited, the inhibitory effect of DMI was reversed on inflammatory release and apoptosis. CONCLUSION: The findings of the present study revealed that DMI inhibited LPS-induced inflammatory release and apoptosis in cells by targeting SIRT1 and then activating SP-A and SP-D. This novel insight into the pharmacological mechanism of DMI lays the foundation for its later use for alleviating lung injury.
Assuntos
Lesão Pulmonar , Surfactantes Pulmonares , Humanos , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/farmacologia , Sirtuína 1/metabolismo , Sirtuína 1/farmacologia , Lesão Pulmonar/metabolismo , Simulação de Acoplamento Molecular , Células Epiteliais/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Apoptose , Tensoativos/metabolismo , Tensoativos/farmacologiaRESUMO
Background Injury to the lung is a common, clinically serious inflammatory disease. However, its pathogenesis remains unclear, and the existing treatments, including cytokine therapy, stem cell therapy, and hormone therapy, are not completely effective in treating this disease. Dimethyl itaconate (DMI) is a surfactant with important anti-inflammatory effects. Objective The present study used alveolar type II (AT II) and bronchial epithelial cells as models to determine the role of DMI in lung injury. Material and Methods First, the effects of DMI were established on the survival, inflammatory release, and apoptosis in lipopolysaccharide (LPS)-induced AT II and bronchial epithelial cells. The association between DMI and Sirtuin1 (SIRT1) was assessed using molecular docking. Next, by constructing interference plasmids to inhibit surfactant protein (SP)-A and SP-D expressions, the effect of DMI was observed on inflammatory release and apoptosis. Results The results revealed that DMI increased the survival rate and expression levels of SP-A, SP-D, and SIRT1, and inhibited inflammatory factors as well as apoptosis in LPS-induced cells. Furthermore, DMI could bind to SIRT1 to regulate SP-A and SP-D expressions. After SP-A and SP-D expressions were inhibited, the inhibitory effect of DMI was reversed on inflammatory release and apoptosis. Conclusion The findings of the present study revealed that DMI inhibited LPS-induced inflammatory release and apoptosis in cells by targeting SIRT1 and then activating SP-A and SP-D. This novel insight into the pharmacological mechanism of DMI lays the foundation for its later use for alleviating lung injury (AU)
Assuntos
Humanos , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacologia , Células Epiteliais/metabolismo , Apoptose , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Lesão Pulmonar/metabolismo , Simulação de Acoplamento Molecular , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/farmacocinética , Sirtuína 1/metabolismo , Sirtuína 1/farmacologiaRESUMO
Objective: To study the mechanism of chronic obstructive pulmonary disease (COPD) in diagnosing alveolar factors and analyze the effect of miR-149-3p on alveolar inflammatory factors and the expression of surfactant protein D (SP-D) and SP-A on the lung surface mediated by Wnt pathway. Methods: Patients with stable COPD were taken as the research subjects, and healthy volunteers as the control group. Cardiac color Doppler ultrasound was adopted to measure the ventricular structure of patients. The ultrasound simulation method was introduced in the ultrasound imaging. The ultrasound image was processed based on the intelligent ultrasound simulation algorithm. The changes in the structure of the left and right ventricles were analyzed and compared in the two groups. The expression changes of miR-149-3p, Wnt1, ß-catenin, RhoA, and Wnt5a in lung tissues of mice in three groups were detected, as well as the content of tumor necrosis factor- (TNF-) α, IL-1ß, interleukin (IL-6), nuclear factor kB (NF-kB), and other inflammatory factors in bronchoalveolar tissues of mice in three groups. Results: The position where the attenuation ratio was less than 0.92 in the experiment under the ultrasonic simulation algorithm had a gray value of 50. Compared with the control group, the right ventricular mass index of patients with stable COPD was statistically considerable (P < 0.05). In patients with stable COPD, the overall right ventricular longitudinal strain, right ventricular diastolic longitudinal strain rate (RV DLSR), right ventricular diastolic circumferential strain rate, and right ventricular longitudinal displacement were significantly impaired (P < 0.05). The content of miR-149-3p in the lung tissue of the model group was dramatically inferior to that of the control group and the interference group (P < 0.05). The contents of Wnt1, ß-catenin, RhoA, and Wnt5a in the lung tissue of the model group were dramatically superior to those of the control group (P < 0.05). In addition, the expressions of TNF-α, IL-1ß, IL-6, and NF-kB in the alveolar lavage fluid of the model group were statistically different from those of control group (P < 0.05). The expression levels of SP-D and surfactant protein A (SP-A) in the COPD group were also statistically different from those of control group (P < 0.05). Conclusion: miR-149-3p regulated the expression of Wnt1, ß-catenin, RhoA, and Wnt5a, which also affected the signal transmission of the Wnt pathway, causing changes in the expression of alveolar inflammatory factors. Eventually, it affected the development of COPD.
Assuntos
MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Animais , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Pulmão , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , NF-kappa B/metabolismo , NF-kappa B/farmacologia , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/farmacologia , Tensoativos/metabolismo , Tensoativos/farmacologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo , beta Catenina/farmacologiaRESUMO
As key components of innate immunity, lung antimicrobial proteins play a critical role in warding off invading respiratory pathogens. Lung surfactant protein A (SP-A) exerts synergistic antimicrobial activity with the N-terminal segment of the SP-B proprotein (SP-BN) against Klebsiella pneumoniae K2 in vivo. However, the factors that govern SP-A/SP-BN antimicrobial activity are still unclear. The aim of this study was to identify the mechanisms by which SP-A and SP-BN act synergistically against K. pneumoniae, which is resistant to either protein alone. The effect of these proteins on K. pneumoniae was studied by membrane permeabilization and depolarization assays and transmission electron microscopy. Their effects on model membranes of the outer and inner bacterial membranes were analyzed by differential scanning calorimetry and membrane leakage assays. Our results indicate that the SP-A/SP-BN complex alters the ultrastructure of K. pneumoniae by binding to lipopolysaccharide molecules present in the outer membrane, forming packing defects in the membrane that may favor the translocation of both proteins to the periplasmic space. The SP-A/SP-BN complex depolarized and permeabilized the inner membrane, perhaps through the induction of toroidal pores. We conclude that the synergistic antimicrobial activity of SP-A/SP-BN is based on the capability of this complex, but not either protein alone, to alter the integrity of bacterial membranes.
Assuntos
Antibacterianos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Pulmão/metabolismo , Surfactantes Pulmonares/farmacologia , Antibacterianos/metabolismo , Líquido da Lavagem Broncoalveolar/química , Sinergismo Farmacológico , Humanos , Imunidade Inata/fisiologia , Infecções por Klebsiella/patologia , Infecções por Klebsiella/prevenção & controle , Klebsiella pneumoniae/imunologia , Pulmão/química , Pulmão/imunologia , Pulmão/microbiologia , Testes de Sensibilidade Microbiana , Proteína A Associada a Surfactante Pulmonar/isolamento & purificação , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Surfactantes Pulmonares/isolamento & purificação , Surfactantes Pulmonares/metabolismo , Infecções Respiratórias/patologia , Infecções Respiratórias/prevenção & controleRESUMO
Glucocorticoid (GC) hormone has been commonly used to treat systemic inflammation and immune disorders. However, the side effects associated with long-term use of high-dose GC hormone limit its clinical application seriously. GC hormone that can specifically target the lung might decrease the effective dosage and thus reduce GC-associated side effects. In this study, we successfully prepared human lung-targeting liposomal methylprednisolone crosslinked with nanobody (MPS-NSSLs-SPANb). Our findings indicate that MPS-NSSLs-SPANb may reduce the effective therapeutic dosage of MPS, achieve better efficacy, and reduce GC-associated side effects. In addition, MPS-NSSLs-SPANb showed higher efficacy and lower toxicity than conventional MPS.
Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Metilprednisolona/administração & dosagem , Metilprednisolona/farmacologia , Proteína A Associada a Surfactante Pulmonar/administração & dosagem , Proteína A Associada a Surfactante Pulmonar/farmacologia , Animais , Química Farmacêutica , Portadores de Fármacos/química , Ensaio de Imunoadsorção Enzimática , Humanos , Lipossomos/química , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/farmacologiaRESUMO
Asthma is an allergic inflammatory lung disease affecting nearly 300 million people worldwide. To better understand asthma, new regulators must be identified. We conducted a study to investigate the effect and mechanisms of action of surfactant protein A (SPA) in OVA-induced asthmatic mice. Treatment with SPA delayed the onset of asthma, decreased its severity, as well as notably suppressed pro-inflammatory cytokine production. Furthermore, SPA-treated mice possessed more leukocytes; more CD4+ T cells infiltrated the spleen in the SPA-treated mice than in the control mice, and there were decreased percentages of Th1 and Th17 cells in vivo. In addition, expression levels of the T-bet (Th1 transcription factor) and RORγt (Th17 transcription factor) genes were significantly downregulated by SPA treatment. Moreover, SPA reduced the production and mRNA expression of pro-inflammatory cytokine mRNAs in activated T cells in vivo. Mechanistically, SPA could inhibit STAT1/4 and STAT3 phosphorylation, resulting in the differentiation of Th1 and suppression of Th17 cells, respectively. In the presence of CD3/CD28 expression, STAT1/4 and STAT3 were activated but suppressed by SPA, which was responsible for the augmentation of Th1 and Th17 differentiation. This result showed that SPA can effectively modulate the JAK/STAT pathway by suppressing Th1 and Th17 differentiation, thus preventing asthma. The present study reveals the novel immunomodulatory activity of SPA and highlights the importance of further investigating the effects of SPA on asthma.
Assuntos
Asma/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Asma/induzido quimicamente , Asma/fisiopatologia , Feminino , Janus Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/efeitos adversos , Fatores de Transcrição STAT/metabolismo , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
BACKGROUND: A surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model. METHODS: The outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625-10 µg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 µg LPS/g body weight and treated with 50 µg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test. RESULTS: The results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation. CONCLUSIONS: These findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 µg SPA4 peptide, comparable to 2.5 µg/g body weight, alleviates LPS-induced lung inflammation.
Assuntos
Fragmentos de Peptídeos/farmacologia , Pneumonia/imunologia , Proteína A Associada a Surfactante Pulmonar/farmacologia , Receptor 4 Toll-Like/metabolismo , Animais , Feminino , Imunoglobulina G/sangue , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/sangue , Receptor 4 Toll-Like/imunologiaRESUMO
Surfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in SP-A2, corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.
Assuntos
Asma/complicações , Eosinofilia/fisiopatologia , Proteína A Associada a Surfactante Pulmonar/fisiologia , Animais , Apoptose/efeitos dos fármacos , Quimiocina CCL11/análise , Variação Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/farmacologiaAssuntos
Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Citocinas/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiologia , Medicina de Precisão/métodos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína A Associada a Surfactante Pulmonar/fisiologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/fisiologiaRESUMO
Lung surfactant, besides alveolar stability, also provides defence against pathogens by surfactant proteins (SP), SP-A and SP-D. The hydrophobic proteins SP-B and SP-C enhance surface activity. An unusual and paradoxical effect of bovine LS and synthetic model LS with SP-B/-C was bactericidal to Staphylococcus aureus and Escherichia coli. Bacterial proliferation were investigated with bovine lung surfactant extract (BLES), dipalmitoylphosphatdylcholine, palmitooleylglycerol, in combination with SP-B/-C using standard microbiological colony forming unit (CFU) counts and structural imaging. BLES and other surfactant-SP-B/-C mixtures inhibit bacterial growth in the concentration range of 0 -7.5 mg/mL, at > 10 mg/mL paradoxical growth of both the bacterial species suggest antibiotic resistance. The lipid only LS have no effect on bacterial proliferation. Smaller peptide mimics of SP-B or SP-B1-25, were less efficient than SP-Cff. Ultra structural studies of the bacterial CFU using electron and atomic force microscopy suggest some membrane damage of S. aereus at inhibitory concentration of BLES, and some structural alteration of E. coli at dividing zones, suggesting utilization and incorporation of surfactant lipid species by both bacteria. The results depicted from in vitro studies are also in agreement with protein-protein interactions obtained from PatchDock, FireDock and ClasPro algorithm. The MD-simulation decipher a small range fluctuation of gyration radius of the LS proteins and their peptide mimics. The studies have alarming implications in the use of high dosages (100 mg/mL/kg body weight) of exogenous surfactant for treatment of respiratory distress syndrome, genetic knock-out abnormalities associated with these proteins, and the novel roles played by SP-B/C as bactericidal agents.
Assuntos
Antibacterianos , Surfactantes Pulmonares/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Interações Hidrofóbicas e Hidrofílicas , Lipossomos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína B Associada a Surfactante Pulmonar/farmacologia , Proteína C Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Nanobody (Nb) is a promising vector for targeted drug delivery. This study aims to identify an Nb that can specifically target the lung by binding human pulmonary surfactant protein A (SP-A). Human lung frozen tissue sections were used for 3 rounds of biospanning of our previously constructed Nb library for rat SP-A to establish a sub-library of Nb, which specifically bound human lung tissues. Phage-ELISA was performed to screen the sub-library to identify Nb4, which specifically bound human SP-A. The binding affinity Kd of Nb4 to recombinant human SP-A was 7.48 × 10-7 M. Nb4 (19 kDa) was stable at 30 °C-37 °C and pH 7.0-7.6 and specifically bound the SP-A in human lung tissue homogenates, human lung A549 cells, and human lung tissues, whereas didn't react with human liver L-02 cells, kidney 293T cells, and human tissues from organs other than the lung. Nb4 accumulated in the lung of nude mice 5 minutes after a tail vein injection of Nb4 and was excreted 3 hours. Short-term exposure (one month) to Nb4 didn't cause apparent liver and kidney toxicity in rats, whereas 3-month exposure resulted in mild liver and kidney injuries. Nb4 may be a promising vector to specifically deliver drugs to the lung.
Assuntos
Sistemas de Liberação de Medicamentos , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Linhagem Celular , Feminino , Humanos , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Proteínas Recombinantes , Distribuição TecidualRESUMO
Human ß-defensin 3 (hBD3) is known to be involved in mast cell activation. However, molecular mechanisms underlying the regulation of hBD3-induced mast cell activation have been poorly understood. We previously reported that SP-A and SP-A-derived peptide 01 (SAP01) regulate the function of hBD3. In this study, we focused on the effects of SP-A and SAP01 on the activation of mast cells induced by hBD3. SAP01 directly bound to hBD3. Mast cell-mediated vascular permeability and edema in hBD3 administered rat ears were decreased when injected with SP-A or SAP01. Compatible with the results in rat ear model, both SP-A and SAP01 inhibited hBD3-induced chemotaxis of mast cells in vitro. Direct interaction between SP-A or SAP01 and hBD3 seemed to be responsible for the inhibitory effects on chemotaxis. Furthermore, SAP01 attenuated hBD3-induced accumulation of mast cells and eosinophils in tracheas of the OVA-sensitized inflammatory model. SP-A might contribute to the regulation of inflammatory responses mediated by mast cells during infection.
Assuntos
Quimiotaxia/efeitos dos fármacos , Inflamação/imunologia , Mastócitos/imunologia , Proteína A Associada a Surfactante Pulmonar/imunologia , beta-Defensinas/imunologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Edema/tratamento farmacológico , Edema/imunologia , Humanos , Inflamação/tratamento farmacológico , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/farmacologia , Ratos Sprague-DawleyRESUMO
BACKGROUND: Reactive aldehydes such as acetaldehyde and malondialdehyde generated as a result of alcohol metabolism and cigarette smoke exposure lead to the formation of malondialdehyde-acetaldehyde-adducted proteins (MAA adducts). These aldehydes can adduct to different proteins such as bovine serum albumin and surfactant protein A or surfactant protein D (SPD). Macrophages play an important role in innate immunity, but the effect of MAA adducts on macrophage function has not yet been examined. Because macrophage scavenger receptor A (SRA; CD204) mediates the uptake of modified proteins, we hypothesized that the effects of MAA-modified proteins on macrophage function are primarily mediated through SRA. METHODS: We tested this hypothesis by exposing SPD-MAA to macrophages and measuring functions. SPD-MAA treatment significantly stimulated pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) release in the macrophage cell line, RAW 264.7. RESULTS: A significant reduction in phagocytosis of zymosan particles was also observed. SPD-MAA stimulated a significant dose-dependent increase in TNF-α and interleukin (IL)-6 release from peritoneal macrophages (PMs) of wild-type (WT) mice. But significantly less TNF-α and IL-6 were released from PMs of SRA-/- mice. We observed a significant reduction in phagocytosis of zymosan particles in PMs from WT mice treated with SPD-MAA. No further SPD-MAA-induced reduction was seen in PMs from SRA-/- mice. SPD-MAA treatment significantly increased SRA mRNA expression, but had no effect on surface receptor protein expression. Protein kinase C alpha inhibitor and NF-κB inhibitor significantly reduced pro-inflammatory cytokine release in response to SPD-MAA. CONCLUSIONS: In conclusion, our data demonstrate that SRA is important for MAA-adducted protein-mediated effect on macrophage functions.
Assuntos
Acetaldeído/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Malondialdeído/química , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Receptores Depuradores Classe A/genética , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/química , Ratos , Receptores Depuradores Classe A/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.
Assuntos
Complexo 2 de Proteínas Adaptadoras/imunologia , Cadeias Pesadas de Clatrina/imunologia , Proteína A Associada a Surfactante Pulmonar/farmacologia , Animais , Caseína Quinase II/metabolismo , Células Cultivadas , Endocitose/efeitos dos fármacos , Humanos , Separação Imunomagnética , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules that belong to the C-type lectin family. In lungs, they play an important role in the clearance of pathogens and control of inflammation. SP-A and SP-D are also expressed in the female reproductive tract where they play an important role in pregnancy and parturition. However, the role of SP-A and SP-D expressed at the feto-maternal interface (decidua) remains unclear. Here, we have examined the expression of SP-A and SP-D in the murine decidua at 17.5 (pre-parturition) and 19.5dpc (near parturition) and their effect on lipopolysaccharide (LPS)-treated decidual macrophages. SP-A and SP-D were localized to stromal cells in the murine decidua at 17.5 and 19.5dpc in addition to cells lining the maternal spiral artery. Purified pre-parturition decidual cells were challenged with LPS with and without SP-A or SP-D, and expression of F4/80 and TNF-α were measured by flow cytometry. On their own, SP-A or SP-D did not affect the percentage of F4/80 positive cells while they suppressed the percentage of TNF-α positive cells. However, simultaneous addition of SP-A or SP-D, together with LPS, reduced TNF-α secreting F4/80 positive cells. It is likely that exogenous administration of SP-A and SP-D in decidua can potentially control infection and inflammation mediators during spontaneous term labor and infection-induced preterm labor. Thus, the presence of SP-A and SP-D in the murine decidua is likely to play a protective role against intrauterine infection during pregnancy.
Assuntos
Decídua/imunologia , Macrófagos/imunologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Decídua/citologia , Decídua/efeitos dos fármacos , Feminino , Feto , Regulação da Expressão Gênica , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Cultura Primária de Células , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/farmacologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Pulmonary surfactant proteins SP-A and SP-D are pattern recognition innate immune molecules. However, there is extrapulmonary existence, especially in the amniotic fluid and at the feto-maternal interface. There is sufficient evidence to suggest that SP-A and SP-D are involved in the initiation of labour. This is of great importance given that preterm birth is associated with increased mortality and morbidity. In this study, we investigated the effects of recombinant forms of SP-A and SP-D (rhSP-A and rhSP-D, the comprising of trimeric lectin domain) on contractile events in vitro, using a human myometrial cell line (ULTR) as an experimental model. Treatment with rhSP-A or rhSP-D increased the cell velocity, distance travelled and displacement by ULTR cells. rhSP-A and rhSP-D also affected the contractile response of ULTRs when grown on collagen matrices showing reduced surface area. We investigated this effect further by measuring contractility-associated protein (CAP) genes. Treatment with rhSP-A and rhSP-D induced expression of oxytocin receptor (OXTR) and connexin 43 (CX43). In addition, rhSP-A and rhSP-D were able to induce secretion of GROα and IL-8. rhSP-D also induced the expression of IL-6 and IL-6 Ra. We provide evidence that SP-A and SP-D play a key role in modulating events prior to labour by reconditioning the human myometrium and in inducing CAP genes and pro-inflammatory cytokines thus shifting the uterus from a quiescent state to a contractile one.
Assuntos
Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Contração Uterina/efeitos dos fármacos , Líquido Amniótico/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Conexina 43/genética , Citocinas/metabolismo , Feminino , Humanos , Miométrio/citologia , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Parto , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Ocitocina/genética , Proteínas Recombinantes/farmacologia , Contração Uterina/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 µM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.
Assuntos
Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Concentração de Íons de Hidrogênio , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/metabolismo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína B Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína B Associada a Surfactante Pulmonar/farmacologia , Proteínas Associadas a Surfactantes Pulmonares/genética , Proteínas Associadas a Surfactantes Pulmonares/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.
Assuntos
Aminas/química , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Proteína A Associada a Surfactante Pulmonar/farmacologia , Administração por Inalação , Aminas/toxicidade , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/administração & dosagem , Nanopartículas/química , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Poliestirenos/química , Poliestirenos/toxicidade , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismoRESUMO
The ability of pulmonary surfactant to reduce surface tension at the alveolar surface is impaired in various lung diseases. Recent animal studies indicate that elevated levels of cholesterol within surfactant may contribute to its inhibition. It was hypothesized that elevated cholesterol levels within surfactant inhibit human surfactant biophysical function and that these effects can be reversed by surfactant protein A (SP-A). The initial experiment examined the function of surfactant from mechanically ventilated trauma patients in the presence and absence of a cholesterol sequestering agent, methyl-ß-cyclodextrin. The results demonstrated improved surface activity when cholesterol was sequestered in vitro using a captive bubble surfactometer (CBS). These results were explored further by reconstitution of surfactant with various concentrations of cholesterol with and without SP-A, and testing of the functionality of these samples in vitro with the CBS and in vivo using surfactant depleted rats. Overall, the results consistently demonstrated that surfactant function was inhibited by levels of cholesterol of 10% (w/w phospholipid) but this inhibition was mitigated by the presence of SP-A. It is concluded that cholesterol-induced surfactant inhibition can actively contribute to physiological impairment of the lungs in mechanically ventilated patients and that SP-A levels may be important to maintain surfactant function in the presence of high cholesterol within surfactant.
Assuntos
Colesterol/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Respiração Artificial/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Colesterol/farmacologia , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/metabolismo , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Microscopia de Força Atômica , Pessoa de Meia-Idade , Oxigênio/sangue , Fosfolipídeos/metabolismo , Fosfolipídeos/farmacologia , Pressão , Proteína A Associada a Surfactante Pulmonar/farmacologia , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacologia , Ratos , Tensão Superficial/efeitos dos fármacos , Adulto Jovem , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Surfactant protein A (SP-A) is involved in lung innate immunity. Humans have two SP-A genes, SFTPA1 and SFTPA2, each with several variants. We examined the in vivo effects of treatment with specific SP-A variants on the alveolar macrophage (AM) proteome from SP-A knockout (KO) mice. KO mice received either SP-A1, SP-A2, or both. AM were collected and their proteomes examined with 2D-DIGE. We identified 90 proteins and categorized them as related to actin/cytoskeleton, oxidative stress, protease balance/chaperones, regulation of inflammation, and regulatory/developmental processes. SP-A1 and SP-A2 had different effects on the AM proteome and these effects differed between sexes. In males more changes occurred in the oxidative stress, protease/chaperones, and inflammation groups with SP-A2 treatment than with SP-A1. In females most SP-A1-induced changes were in the actin/cytoskeletal and oxidative stress groups. We conclude that after acute SP-A1 and SP-A2 treatment, sex-specific differences were observed in the AM proteomes from KO mice, and that these sex differences differ in response to SP-A1 and SP-A2. Females are more responsive to SP-A1, whereas the gene-specific differences in males were minimal. These observations not only demonstrate the therapeutic potential of exogenous SP-A, but also illustrate sex- and gene-specific differences in the response to it. BIOLOGICAL SIGNIFICANCE: This study shows that changes occur in the alveolar macrophage proteome in response to a single in vivo treatment with exogenous SP-A1 and/or SP-A2. We demonstrate that SP-A1 and SP-A2 have different effects on the AM proteome and that sex differences exist in the response to each SP-A1 and SP-A2 gene product. This study illustrates the potential of exogenous SP-A1 and SP-A2 treatment for the manipulation of macrophage function and indicates that the specific SP-A variant used for treatment may vary with sex and with the cellular functions being modified. The observed changes may contribute to sex differences in the incidence of some lung diseases.