RESUMO
Interleukin (IL)-22 plays a critical role in regulating the maintenance of the mucosal barrier. As airway epithelial regeneration is abnormal in cystic fibrosis (CF), we investigated IL-22 integrity in CF. We first demonstrated, using Il-22-/- mice, that IL-22 is important to prevent lung damage induced by the CF pathogen Pseudomonas aeruginosa. Next, IL-22 receptor was found normally expressed at the airway epithelial surfaces of CF patients. In wound-healing assays, IL-22-treated CF cultures had higher wound-closure rate than controls, suggesting that IL-22 signaling per se could be functional in a CF context. However, persistence of neutrophil-derived serine-proteases is a major feature of CF airways. Remarkably, IL-22 was found altered in this protease-rich inflammatory microenvironment; the serine protease-3 being the most prone to fully degrade IL-22. Consequently, we suspect an acquired deficiency of the IL-22 pathway in the lungs of CF patients due to IL-22 cleavage by the surrounding neutrophil serine-proteases.
Assuntos
Interleucinas/imunologia , Pulmão/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/imunologia , Adolescente , Adulto , Idoso , Animais , Criança , Fibrose Cística , Feminino , Humanos , Interleucinas/genética , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologia , Interleucina 22RESUMO
Chronic obstructive pulmonary disease (COPD) is punctuated by episodes of infection-driven acute exacerbations. Despite the life-threatening nature of these exacerbations, the underlying mechanisms remain unclear, although a high number of neutrophils in the lungs of COPD patients is known to correlate with poor prognosis. Interleukin (IL)-22 is a cytokine that plays a pivotal role in lung antimicrobial defence and tissue protection. We hypothesised that neutrophils secrete proteases that may have adverse effects in COPD, by altering the IL-22 receptor (IL-22R)-dependent signalling.Using in vitro and in vivo approaches as well as reverse transcriptase quantitative PCR, flow cytometry and/or Western blotting techniques, we first showed that pathogens such as the influenza virus promote IL-22R expression in human bronchial epithelial cells, whereas Pseudomonas aeruginosa, bacterial lipopolysaccharide or cigarette smoke do not. Most importantly, neutrophil proteases cleave IL-22R and impair IL-22-dependent immune signalling and expression of antimicrobial effectors such as ß-defensin-2. This proteolysis resulted in the release of a soluble fragment of IL-22R, which was detectable both in cellular and animal models as well as in sputa from COPD patients with acute exacerbations.Hence, our study reveals an unsuspected regulation by the proteolytic action of neutrophil enzymes of IL-22-dependent lung host response. This process probably enhances pathogen replication, and ultimately COPD exacerbations.
Assuntos
Células Epiteliais/enzimologia , Imunidade Inata/efeitos dos fármacos , Neutrófilos/metabolismo , Doença Pulmonar Obstrutiva Crônica/microbiologia , Receptores de Interleucina/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Humanos , Imunidade Inata/fisiologia , Camundongos , Neutrófilos/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina/imunologia , Estudos de Amostragem , Sensibilidade e Especificidade , Fumar/efeitos adversos , Estatísticas não Paramétricas , beta-Defensinas/farmacologiaRESUMO
RATIONALE: Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES: We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS: We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS: Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS: Poly-L-lysine may be an alternative to dornase-α to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.
Assuntos
Antibacterianos/farmacologia , Fibrose Cística/tratamento farmacológico , DNA/efeitos dos fármacos , Lisina/farmacologia , Peptídeo Hidrolases/efeitos dos fármacos , Escarro/efeitos dos fármacos , Adulto , Idoso , Animais , Catepsina G/efeitos dos fármacos , Catepsina G/metabolismo , Fibrose Cística/metabolismo , DNA/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo/métodos , Humanos , Elastase de Leucócito/efeitos dos fármacos , Elastase de Leucócito/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Escarro/metabolismo , Staphylococcus aureus/efeitos dos fármacosRESUMO
The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.
Assuntos
Modelos Animais de Doenças , Neutrófilos/enzimologia , Pneumonia/enzimologia , Serina Proteases/metabolismo , Animais , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Degranulação Celular , Humanos , Técnicas In Vitro , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Pneumonia/sangue , Pseudomonas aeruginosa/fisiologia , Inibidores de Serina Proteinase/farmacologia , Especificidade da Espécie , Staphylococcus aureus/fisiologia , SuínosRESUMO
Uncontrolled proteolysis by neutrophil serine proteases (NSPs) in lung secretions is a hallmark of cystic fibrosis (CF). We have shown that the active neutrophil elastase, protease 3, and cathepsin G in CF sputum resist inhibition in part by exogenous protease inhibitors. This resistance may be due to their binding to neutrophil extracellular traps (NETs) secreted by the activated neutrophils in CF sputum and to genomic DNA released from senescent and dead neutrophils. Treating CF sputum with DNase dramatically increases its elastase activity, which can then be stoichiometrically inhibited by exogenous elastase inhibitors. However, DNase treatment does not increase the activities of protease 3 and cathepsin G, indicating their different distribution and/or binding in CF sputum. Purified blood neutrophils secrete NETs when stimulated by the opportunistic CF bacteria Pseudomonas aeruginosa and Staphylococcus aureus. The activities of the three proteases were unchanged in these conditions, but subsequent DNase treatment produced a dramatic increase in all three proteolytic activities. Neutrophils activated with a calcium ionophore did not secrete NETs but released huge amounts of active proteases whose activities were not modified by DNase. We conclude that NETs are reservoirs of active proteases that protect them from inhibition and maintain them in a rapidly mobilizable status. Combining the effects of protease inhibitors with that of DNA-degrading agents could counter the deleterious proteolytic effects of NSPs in CF lung secretions.
Assuntos
Fibrose Cística/enzimologia , Fibrose Cística/imunologia , DNA/metabolismo , Neutrófilos/enzimologia , Serina Proteases/metabolismo , Escarro/imunologia , Catepsina G/metabolismo , Fibrose Cística/genética , Desoxirribonucleases/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Elastase Pancreática/metabolismo , Proteólise , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidade , Inibidores de Serina Proteinase/farmacologia , Escarro/efeitos dos fármacos , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
BACKGROUND: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process. METHODS: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated. RESULTS: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed. CONCLUSIONS: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.
Assuntos
Fibrose Cística/sangue , Fibrose Cística/patologia , Neutrófilos/fisiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Ácidos Graxos Insaturados/metabolismo , Feminino , Masculino , Modelos Teóricos , Proteômica , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
Background: The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Objective: Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. Results: We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Limitations: Further studies are now required to confirm the feasibility of the whole process during routine practice. Conclusion: Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.
RESUMO
Pseudomonas aeruginosa is an opportunistic bacteria and a major cause of nosocomial pneumonia. P. aeruginosa has many virulence factors contributing to its ability to colonize the host. LoxA is a lipoxygenase enzyme secreted by P. aeruginosa that oxidizes polyunsaturated fatty acids. Based on previous in vitro biochemical studies, several biological roles of LoxA have been hypothesized, including interference of the host lipid signaling, and modulation of bacterial invasion properties. However, the contribution of LoxA to P. aeruginosa lung pathogenesis per se remained unclear. In this study, we used complementary in vitro and in vivo approaches, clinical strains of P. aeruginosa as well as lipidomics technology to investigate the role of LoxA in lung infection. We found that several P. aeruginosa clinical isolates express LoxA. When secreted in the lungs, LoxA processes a wide range of host polyunsaturated fatty acids, which further results in the production of bioactive lipid mediators (including lipoxin A4). LoxA also inhibits the expression of major chemokines (e.g., MIPs and KC) and the recruitment of key leukocytes. Remarkably, LoxA promotes P. aeruginosa persistence in lungs tissues. Hence, our study suggests that LoxA-dependent interference of the host lipid pathways may contribute to P. aeruginosa lung pathogenesis.
RESUMO
Polymorphonuclear neutrophils (PMNs) can contribute to the regulation of the host immune response by crosstalk with innate and adaptive leukocytes, including NK cells. Mechanisms by which this immunoregulation process occurs remain incompletely understood. Here, we focused on the effect of human neutrophil-derived serine proteases on NKp46, a crucial activating receptor expressed on NK cells. We used flow cytometry, Western blotting, and mass spectrometry (MS) analysis to reveal that cathepsin G [CG; and not elastase or proteinase 3 (PR3)] induces a time- and concentration-dependent, down-regulatory effect on NKp46 expression through a restricted proteolytic mechanism. We also used a functional assay to demonstrate that NKp46 cleavage by CG severely impairs NKp46-mediated responses of NK cells, including IFN-γ production and cell degranulation. Importantly, sputa of cystic fibrosis (CF) patients, which have high concentrations of CG, also alter NKp46 on NK cells. Hence, we have identified a new immunoregulatory mechanism of neutrophils that proteolytically disarms NK cell responses.
Assuntos
Células Matadoras Naturais/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neutrófilos/metabolismo , Catepsina G/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo , Humanos , Células K562 , Receptor 1 Desencadeador da Citotoxicidade Natural/química , Ativação de NeutrófiloRESUMO
The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.
Assuntos
Interleucinas/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/metabolismo , Animais , Infecção Hospitalar , Humanos , Evasão da Resposta Imune , Interleucinas/deficiência , Interleucinas/genética , Interleucinas/imunologia , Pulmão/fisiopatologia , Camundongos , Camundongos Knockout , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/metabolismo , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Proteólise , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Interleucina 22RESUMO
Recent in silico studies suggested that the transcription cofactor LIM-only protein FHL2 is a major transcriptional regulator of mouse natural killer (NK) cells. However, the expression and role of FHL2 in NK cell biology are unknown. Here, we confirm that FHL2 is expressed in both mouse and human NK cells. Using FHL2-/- mice, we found that FHL2 controls NK cell development in the bone marrow and maturation in peripheral organs. To evaluate the importance of FHL2 in NK cell activation, FHL2-/- mice were infected with Streptococcus pneumoniae. FHL2-/- mice are highly susceptible to this infection. The activation of lung NK cells is altered in FHL2-/- mice, leading to decreased IFNγ production and a loss of control of bacterial burden. Collectively, our data reveal that FHL2 is a new transcription cofactor implicated in NK cell development and activation during pulmonary bacterial infection.
RESUMO
The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases.