RESUMO
Eosinophils have been previously shown to be able to regulate early humoral responses during systemic vaccination. Here we investigated the role of eosinophils during pulmonary vaccination, comparing vaccine-induced responses in eosinophil-deficient (ΔdblGATA) and wild-type mice using a Th2 adjuvant. We observed that eosinophils were needed to induce a complete vaccine response, thereby eliciting specific antibody-secreting plasma cells in the regional lymph nodes and antibody secretion in the BAL at the early stage of the immune response. Reintroduction of eosinophils in the lungs of ΔdblGATA mice during the priming stage enhanced both specific IgM and IgG plasma cells but not specific IgA plasma cells. Upon vaccination, eosinophils migrated to the lungs and secreted cytokines involved in B-cell activation, which might promote antibody production. Importantly, however, the absence of eosinophils did not impair late immune responses in a prime/boost protocol because, in that setup, we uncovered a compensating mechanism involving a Th17 pathway. In conclusion, our data demonstrate for the first time a new role for eosinophils during lung mucosal vaccination, whereby they accelerate early immune responses (IgM and IgG) while regulating IgA production at the late stages.
Assuntos
Formação de Anticorpos , Eosinófilos , Camundongos , Animais , Eosinófilos/metabolismo , Pulmão/patologia , Vacinação , Imunoglobulina G , Imunoglobulina M , Imunoglobulina A/metabolismo , Camundongos Endogâmicos BALB C , Imunidade nas MucosasRESUMO
Pseudomonas aeruginosa (P.a) infections are a major public health issue in ventilator-associated pneumoniae, cystic fibrosis, and chronic obstructive pulmonary disease exacerbations. P.a is multidrug resistant, and there is an urgent need to develop new therapeutic approaches. Here, we evaluated the effect of direct pulmonary transplantation of gene-modified (elafin and interleukin [IL]-6) syngeneic macrophages in a mouse model of acute P.a infection. Wild-type (WT) or Elafin-transgenic (eTg) alveolar macrophages (AMs) or bone marrow-derived macrophages (BMDMs) were recovered from bronchoalveolar lavage or generated from WT or eTg mouse bone marrow. Cells were modified with adenovirus IL-6 (Ad-IL-6), characterized in vitro, and transferred by oropharyngeal instillation in the lungs of naive mice. The protective effect was assessed during P.a acute infection (survival studies, mechanistic studies of the inflammatory response). We show that a single bolus of genetically modified syngeneic AMs or BMDMs provided protection in our P.a-induced model. Mechanistically, Elafin-modified AMs had an IL-6-IL-10-IL-4R-IL-22-antimicrobial molecular signature that, in synergy with IL-6, enhanced epithelial cell proliferation and tissue repair in the alveolar unit. We believe that this innovative cell therapy strategy could be of value in acute bacterial infections in the lung.
Assuntos
Infecções por Pseudomonas , Animais , Elafina , Imunoterapia , Interleucina-6/genética , Pulmão/microbiologia , Macrófagos , Macrófagos Alveolares , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/genéticaRESUMO
Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, particularly in hospital patients undergoing ventilation and in individuals with cystic fibrosis. Although we and others have investigated mechanisms used by P.a to subvert innate immunity, relatively less is known about the potential strategies used by this bacterium to fight the adaptive immune system and, in particular, T cells. Here, using RAG KO (devoid of 'classical' αß and γδ TCR T lymphocytes) and double RAG γC KO mice (devoid of T, NK and ILC cells), we demonstrate that the lymphocytic compartment is important to combat P.a (PAO1 strain). Indeed, we show that PAO1 load was increased in double RAG γC KO mice. In addition, we show that PAO1 down-regulates IL-23 and IL-22 protein accumulation in the lungs of infected mice while up-regulating their RNA production, thereby pointing towards a specific post-transcriptional regulatory mechanism not affecting other inflammatory mediators. Finally, we demonstrate that an adenovirus-mediated over-expression of IL-1, IL-23 and IL-7 induced lung neutrophil and lymphocytic influx and rescued mice against P.a-induced lethality in all WT, RAG γC KO and RAG γC KO RAG-deficient mice, suggesting that this regimen might be of value in 'locally immunosuppressed' individuals such as cystic fibrosis patients.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Animais , Fibrose Cística/genética , Fibrose Cística/metabolismo , Interleucina-23/metabolismo , Interleucinas , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Interleucina 22RESUMO
BACKGROUND: Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity. OBJECTIVE: We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo. METHODS: Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models. RESULTS: We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways. CONCLUSIONS: Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.
Assuntos
Proteínas de Bactérias , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/imunologia , Células Epiteliais/fisiologia , Imunidade Inata/fisiologia , Interleucina-6/fisiologia , Metaloendopeptidases , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: The lung epithelium constitutes the first barrier against invading pathogens and also a major surface potentially exposed to nanoparticles. In order to ensure and preserve lung epithelial barrier function, the alveolar compartment possesses local defence mechanisms that are able to control bacterial infection. For instance, alveolar macrophages are professional phagocytic cells that engulf bacteria and environmental contaminants (including nanoparticles) and secrete pro-inflammatory cytokines to effectively eliminate the invading bacteria/contaminants. The consequences of nanoparticle exposure in the context of lung infection have not been studied in detail. Previous reports have shown that sequential lung exposure to nanoparticles and bacteria may impair bacterial clearance resulting in increased lung bacterial loads, associated with a reduction in the phagocytic capacity of alveolar macrophages. RESULTS: Here we have studied the consequences of SiO2 nanoparticle exposure on Pseudomonas aeruginosa clearance, Pseudomonas aeruginosa-induced inflammation and lung injury in a mouse model of acute pneumonia. We observed that pre-exposure to SiO2 nanoparticles increased mice susceptibility to lethal pneumonia but did not modify lung clearance of a bioluminescent Pseudomonas aeruginosa strain. Furthermore, internalisation of SiO2 nanoparticles by primary alveolar macrophages did not reduce the capacity of the cells to clear Pseudomonas aeruginosa. In our murine model, SiO2 nanoparticle pre-exposure preferentially enhanced Pseudomonas aeruginosa-induced lung permeability (the latter assessed by the measurement of alveolar albumin and IgM concentrations) rather than contributing to Pseudomonas aeruginosa-induced lung inflammation (as measured by leukocyte recruitment and cytokine concentration in the alveolar compartment). CONCLUSIONS: We show that pre-exposure to SiO2 nanoparticles increases mice susceptibility to lethal pneumonia but independently of macrophage phagocytic function. The deleterious effects of SiO2 nanoparticle exposure during Pseudomonas aeruginosa-induced pneumonia are related to alterations of the alveolar-capillary barrier rather than to modulation of the inflammatory responses.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Nanopartículas/toxicidade , Pneumonia Bacteriana/induzido quimicamente , Infecções por Pseudomonas/induzido quimicamente , Pseudomonas aeruginosa/patogenicidade , Alvéolos Pulmonares/efeitos dos fármacos , Óxidos de Selênio/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Citocinas/análise , Imunoglobulina M/análise , Exposição por Inalação , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/química , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Alvéolos Pulmonares/irrigação sanguínea , Óxidos de Selênio/química , Propriedades de Superfície , Análise de SobrevidaRESUMO
Silver nanoparticles (AgNPs) are a potential antiviral agent due to their ability to disrupt the viral particle or alter the virus metabolism inside the host cell. In vitro, AgNPs exhibit antiviral activity against the most common human respiratory viruses. However, their capacity to modulate immune responses during respiratory viral infections has yet to be explored. This study demonstrates that administering AgNPs directly into the lungs prior to infection can reduce viral loads and therefore virus-induced cytokines in mice infected with influenza virus or murine pneumonia virus. The prophylactic effect was diminished in mice with depleted lymphoid cells. We showed that AgNPs-treatment resulted in the recruitment and activation of lymphocytes in the lungs, particularly natural killer (NK) cells. Mechanistically, AgNPs enhanced the ability of alveolar macrophages to promote both NK cell migration and IFN-γ production. By contrast, following infection, in mice treated with AgNPs, NK cells exhibited decreased activation, indicating that these nanoparticles can regulate the potentially deleterious activation of these cells. Overall, the data suggest that AgNPs may possess prophylactic antiviral properties by recruiting and controlling the activation of lymphoid cells through interaction with alveolar macrophages.
RESUMO
Introduction: Although many studies have underscored the importance of T cells, phenotypically and functionally, fewer have studied the functions of myeloid cells in COVID disease. In particular, the potential role of myeloid cells such as monocytes and low-density neutrophils (LDNs) in innate responses and particular in the defense against secondary bacterial infections has been much less documented. Methods: Here, we compared, in a longitudinal study, healthy subjects, idiopathic fibrosis patients, COVID patients who were either hospitalized/moderate (M-) or admitted to ICU (COV-ICU) and patients in ICU hospitalized for other reasons (non-COV-ICU). Results: We show that COVID patients have an increased proportion of low-density neutrophils (LDNs), which produce high levels of proteases (particularly, NE, MMP-8 and MMP-9) (unlike non-COV-ICU patients), which are partly responsible for causing type II alveolar cell damage in co-culture experiments. In addition, we showed that M- and ICU-COVID monocytes had reduced responsiveness towards further live Pseudomonas aeruginosa (PAO1 strain) infection, an important pathogen colonizing COVID patients in ICU, as assessed by an impaired secretion of myeloid cytokines (IL-1, TNF, IL-8, ). By contrast, lymphoid cytokines (in particular type 2/type 3) levels remained high, both basally and post PAO1 infection, as reflected by the unimpaired capacity of T cells to proliferate, when stimulated with anti-CD3/CD28 beads. Discussion: Overall, our results demonstrate that COVID circulatory T cells have a biased type 2/3 phenotype, unconducive to proper anti-viral responses and that myeloid cells have a dual deleterious phenotype, through their LDN-mediated damaging effect on alveolar cells and their impaired responsiveness (monocyte-mediated) towards bacterial pathogens such as P. aeruginosa.
Assuntos
COVID-19 , Monócitos , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa , SARS-CoV-2 , Humanos , COVID-19/imunologia , Pseudomonas aeruginosa/imunologia , Monócitos/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Infecções por Pseudomonas/imunologia , Neutrófilos/imunologia , Idoso , Citocinas/metabolismo , Citocinas/imunologia , Adulto , Estudos Longitudinais , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/microbiologiaRESUMO
Epidemiological studies showed a positive association between exposure to PM2.5 and the severity of influenza virus infection. However, the mechanisms by which PM2.5 can disrupt antiviral defence are still unclear. From this perspective, the objective of this study was to evaluate the effects of PM2.5 on antiviral signalling in the respiratory epithelium using the bronchial Calu-3 cell line grown at the air-liquid interface. Pre-exposure to PM2.5 before infection with the influenza virus was investigated, as well as a co-exposure. Although a physical interaction between the virus and the particles seems possible, no effect of PM2.5 on viral replication was observed during co-exposure, although a downregulation of IFN-ß release was associated to PM2.5 exposure. However, pre-exposure slightly increased the viral nucleoprotein production and the pro-inflammatory response. Conversely, the level of the myxovirus resistance protein A (MxA), an interferon-stimulated gene (ISG) induced by IFN-ß, was reduced. Therefore, these results suggest that pre-exposure to PM2.5 could alter the antiviral response of bronchial epithelial cells, increasing their susceptibility to viral infection.
Assuntos
Influenza Humana , Orthomyxoviridae , Viroses , Humanos , Interferons , Influenza Humana/genética , Influenza Humana/metabolismo , Mucosa Respiratória , Antivirais , Epitélio/metabolismo , Material Particulado/toxicidadeRESUMO
One billion people worldwide get flu every year, including patients with non-small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Vírus da Influenza A , Influenza Humana , Neoplasias Pulmonares , Infecções por Orthomyxoviridae , Humanos , Animais , Camundongos , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Microambiente Tumoral , Linfócitos T CD8-Positivos , Pulmão , Infecções por Orthomyxoviridae/patologiaRESUMO
Mucins, the main glycoproteins present within mucus, modulate the rheologic properties of airways and participate in lung defense. They are thought to be able to trap and eliminate microorganisms from the lung. Among the mucins secreted in the lung, MUC5AC is the most prominent factor secreted by surface epithelial cells. Although much is known about the signaling pathways involved in the regulation of MUC5AC by host factors such as cytokines or proteases, less is known about the pathways triggered by microorganisms and, specifically, by influenza A virus (IAV). We therefore set up experiments to dissect the molecular mechanisms responsible for the potential modulation of MUC5AC by IAV. Using epithelial cells, C57/Bl6 mice, and IAV strains, we measured MUC5AC expression at the RNA and protein levels, specificity protein 1 (Sp1) activation, and protease activity. Intermediate molecular partners were confirmed using pharmacological inhibitors, blocking antibodies, and small interfering (si)RNAs. We showed in vitro and in vivo that IAV up-regulates epithelial cell-derived MUC5AC and Muc5ac expression in mice, both at transcriptional (through the induction of Sp1) and translational levels. In addition, we determined that this induction was dependent on a protease-epithelial growth factor receptor-extracellular regulated kinase-Sp1 signaling cascade, involving in particular the human airway trypsin. Our data point to MUC5AC as a potential modulatory mechanism by which the lung epithelia respond to IAV infection, and we dissect, for the first time to the best of our knowledge, the molecular partners involved. Future experiments using MUC5AC-targeted strategies should help further unravel the pathophysiological consequences of IAV-induced MUC5AC expression for lung homeostasis.
Assuntos
Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vírus da Influenza A/metabolismo , Pulmão/metabolismo , Mucina-5AC/biossíntese , Peptídeo Hidrolases/genética , Fator de Transcrição Sp1/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Influenza Humana/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC/genética , Mucina-5AC/metabolismo , Peptídeo Hidrolases/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/genética , Tripsina/genética , Tripsina/metabolismo , Regulação para Cima , Replicação Viral/genéticaRESUMO
Eicosanoids are metabolites of arachidonic acid produced by cyclooxygenases (COXs) or lipoxygenases (LOXs). They mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases. The gel-forming mucin MUC5AC is over-expressed in the airways of patients with these diseases. MUC5AC expression is mediated by an extracellular signal-regulated kinase (ERK)/Sp1 dependent mechanism. Our aim was to study the role of eicosanoids and their signalling pathways in MUC5AC expression. Inhibitors of 12-LOX, but not those of COX, 5-LOX or 15-LOX, reduce MUC5AC expression induced by phorbol myristate acetate (PMA) in the bronchial epithelial cell line NCI-H292. These inhibitors also abrogate the production of whole mucus by cell monolayers. Two forms of 12-LOX (R and S) exist in mammals. Using siRNAs we show that 12R-LOX but not 12S-LOX is involved in MUC5AC expression induced by PMA, lipopolysaccharide or transforming growth factor-α. 12R-LOX also participates in MUC2 and MUC5B expression, although to a lesser extent than for MUC5AC. Contrarily, 12R-LOX silencing does not modify interleukin-8 production. 12-LOX inhibitors reduce ERK activation and Sp1 translocation induced by PMA. Moreover, the 12R-LOX product 12(R)-hydroxyeicosatetraenoic acid, induces MUC5AC expression, ERK activation and Sp1 translocation. 12R-LOX is involved in MUC5AC expression. This occurs via ERK- and Sp1-signalling pathways.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Mucina-5AC/biossíntese , Mucosa Respiratória/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Araquidonato 12-Lipoxigenase/genética , Carcinógenos/farmacologia , Linhagem Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Interleucina-8/biossíntese , Lipopolissacarídeos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mucina-2/biossíntese , Mucina-5B/biossíntese , Muco/metabolismo , Transporte Proteico , Mucosa Respiratória/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
Bacterial LPS triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1R-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as IL-33, a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor because it is not found in ST2 knockout mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR.
Assuntos
Citocinas/biossíntese , Interleucinas/fisiologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Animais , Linhagem Celular , Tolerância Imunológica , Inflamação/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Knockout , Receptores de Interleucina/imunologia , Receptores Toll-Like/metabolismoRESUMO
Influenza virus infection leads to severe and complicated disease, particularly in patients with lung cancer. It alters the tumor microenvironment (TME), which may potentiate lung cancer progression and disrupt responses to antitumoral treatments. Consequently, influenza vaccination and antiviral treatments should be recommended to all patients with lung cancer.
Assuntos
Antivirais/uso terapêutico , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/terapia , Neoplasias Pulmonares/mortalidade , Progressão da Doença , Humanos , Influenza Humana/complicações , Influenza Humana/imunologia , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Oncologia/normas , Guias de Prática Clínica como Assunto , Microambiente Tumoral/imunologia , Vacinação/normasRESUMO
In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.
Assuntos
Membranas Extraembrionárias/efeitos dos fármacos , Miométrio/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Fibras de Estresse/efeitos dos fármacos , Actinas/metabolismo , Western Blotting , Células Cultivadas , Membranas Extraembrionárias/metabolismo , Membranas Extraembrionárias/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Microscopia de Fluorescência , Miométrio/fisiologia , Gravidez , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/genética , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fibras de Estresse/fisiologiaRESUMO
Individuals with impaired immune responses, such as ventilated and cystic fibrosis patients are often infected with Pseudomonas aeruginosa (P.a) bacteria, and a co-infection with the Influenza virus (IAV) is often present. It has been known for many years that infection with IAV predisposes the host to secondary bacterial infections (such as Streptococcus pneumoniae or Staphylococcus aureus), and there is an abundance of mechanistic studies, including those studying the role of desensitization of TLR signaling, type I IFN- mediated impairment of neutrophil chemokines and antimicrobial production, attenuation of IL1ß production etc., showing this. However, little is known about the mechanistic events underlying the potential deleterious synergy between Influenza and P.a co-infections. We demonstrate here in vitro in epithelial cells and in vivo in three independent models (two involving mice given IAV +/- P.a, and one involving mice given IAV +/- IL-1ß) that IAV promotes secondary P.a-mediated lung disease or augmented IL-1ß-mediated inflammation. We show that IAV-P.a-mediated deleterious responses includes increased matrix metalloprotease (MMP) activity, and MMP-9 in particular, and that the use of the MMP inhibitor improves lung resilience. Furthermore, we show that IAV post-transcriptionally inhibits the antimicrobial/anti-protease molecule elafin/trappin-2, which we have shown previously to be anti-inflammatory and to protect the host against maladaptive neutrophilic inflammation in P.a infections. Our work highlights the capacity of IAV to promote further P.a-mediated lung damage, not necessarily through its interference with host resistance to the bacterium, but by down-regulating tissue resilience to lung inflammation instead. Our study therefore suggests that restoring tissue resilience in clinical settings where IAV/P.a co-exists could prove a fruitful strategy.
Assuntos
Coinfecção/imunologia , Elafina/metabolismo , Vírus da Influenza A/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Pseudomonas aeruginosa/imunologia , Animais , Linhagem Celular , Coinfecção/induzido quimicamente , Coinfecção/metabolismo , Fibrose Cística/imunologia , Citocinas/metabolismo , Suscetibilidade a Doenças/metabolismo , Células Epiteliais/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Pneumonia/metabolismo , Infecções Estafilocócicas/imunologiaRESUMO
Most of current influenza virus vaccines fail to develop a strong immunity at lung mucosae (site of viral entry) due to sub-optimal vaccination protocols (e.g. inactivated virus administered by parenteral injections). Mucosal immunity could be improved by using locally-delivered vaccines containing appropriate adjuvants. Here we show, in a mouse model, that inclusion of silver nanoparticles (AgNPs) in virus-inactivated flu vaccine resulted in reduction of viral loads and prevention of excessive lung inflammation following influenza infection. Concomitantly, AgNPs enhanced specific IgA secreting plasma cells and antibodies titers, a hallmark of successful mucosal immunity. Moreover, vaccination in the presence of AgNPs but not with gold nanoparticles, protected mice from lethal flu. Compared with other commercial adjuvants (squalene/oil-based emulsion) or silver salts, AgNPs stimulated stronger antigen specific IgA production with lower toxicity by promoting bronchus-associated lymphoid tissue (BALT) neogenesis, and acted as a bona fide mucosal adjuvant.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunidade nas Mucosas , Imunoglobulina A/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Tecido Linfoide/imunologia , Nanopartículas Metálicas/química , Prata/química , Animais , Brônquios/imunologia , Cães , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/metabolismo , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Inflamação/patologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Tecido Linfoide/efeitos dos fármacos , Células Madin Darby de Rim Canino , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , VacinaçãoRESUMO
Surfactant protein A (SP-A) is known to cause bacterial permeabilization. The aim of this work was to gain insight into the mechanism by which SP-A induces permeabilization of rough lipopolysaccharide (Re-LPS) membranes. In the presence of calcium, large interconnected aggregates of fluorescently labeled TR-SP-A were observed on the surface of Re-LPS films by epifluorescence microscopy. Using Re-LPS monolayer relaxation experiments at constant surface pressure, we demonstrated that SP-A induced Re-LPS molecular loss by promoting the formation of three-dimensional lipid-protein aggregates in Re-LPS membranes. This resulted in decreased van der Waals interactions between Re-LPS acyl chains, as determined by differential scanning calorimetry, which rendered the membrane leaky. We also showed that the coexistence of gel and fluid lipid phases within the Re-LPS membrane conferred susceptibility to SP-A-mediated permeabilization. Taken together, our results seem to indicate that the calcium-dependent permeabilization of Re-LPS membranes by SP-A is related to the extraction of LPS molecules from the membrane due to the formation of calcium-mediated protein aggregates that contain LPS.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Cálcio/farmacologia , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Ligação Proteica/efeitos dos fármacos , Propriedades de Superfície , Água/metabolismoRESUMO
Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.
Assuntos
Desmina/metabolismo , Filamentos Intermediários/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Vimentina/metabolismo , Animais , Cálcio/metabolismo , Extratos Celulares , Células Cultivadas , Desmina/deficiência , Desmina/genética , Desmina/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Concentração Osmolar , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/ultraestrutura , Ratos , Espectrometria de Massas em TandemRESUMO
Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as an alternative to antivirals to treat human infectious diseases, especially influenza virus infections where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of an influenza virus infection of lung epithelial cells, that AgNPs down-regulated influenza induced CCL-5 and -IFN-ß release (two cytokines important in antiviral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the antiviral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent antiviral responses and up-regulation of IL-8-dependent antibacterial responses) may have practical implications for their use in the clinic.
Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/química , Mitocôndrias/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Prata/farmacologia , Tretinoína/farmacologia , Animais , Antivirais/química , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cães , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Mitocôndrias/metabolismo , Prata/química , Tretinoína/químicaRESUMO
According to the WHO, and despite reduction in mortality rates, there were an estimated 438 000 malaria deaths in 2015. Therefore new antimalarials capable of limiting organ damage are still required. We show that systemic and lung adenovirus (Ad)-mediated over-expression of trappin-2 (T-2) an antibacterial molecule with anti-inflammatory activity, increased mice survival following infection with the cerebral malaria-inducing Plasmodium berghei ANKA (PbANKA) strain. Systemically, T-2 reduced PbANKA sequestration in spleen, lung, liver and brain, associated with a decrease in pro-inflammatory cytokines (eg TNF-α in spleen and lung) and an increase in IL-10 production in the lung. Similarly, local lung instillation of Ad-T-2 resulted in a reduced organ parasite sequestration and a shift towards an anti-inflammatory/repair response, potentially implicating monocytes in the protective phenotype. Relatedly, we demonstrated in vitro that human monocytes incubated with Plasmodium falciparum-infected red blood cells (Pf-iRBCs) and IgGs from hyper-immune African human sera produced T-2 and that the latter colocalized with merozoites and inhibited Pf multiplication. This array of data argues for the first time for the potential therapeutic usefulness of this host defense peptide in human malaria patients, with the aim to limit acute lung injury and respiratory distress syndrom often observed during malaria episodes.