RESUMO
Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Lipopolissacarídeos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Camundongos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Transporte Biológico , Testes de Sensibilidade Microbiana , Humanos , Microscopia Crioeletrônica , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Modelos Animais de Doenças , Feminino , Transportadores de Cassetes de Ligação de ATPRESUMO
BACKGROUND: Cigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited. METHODS: We used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship. RESULTS: Cigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar-capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3 + cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features. CONCLUSION: This work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.
Assuntos
Fumar Cigarros , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Animais , Fumar Cigarros/efeitos adversos , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , NicotianaRESUMO
The upper respiratory tract is highly exposed to airborne pathogens and serves as an important inductive site for protective antibody responses, including mucosal IgA and systemic IgG. However, it is currently unknown to what extent inhaled environmental toxins, such as a cigarette smoke, affect the ability to induce antibody-mediated immunity at this site. Using a murine model of intranasal lipopolysaccharide and ovalbumin (LPS/OVA) immunization, we show that cigarette smoke exposure compromises the induction of antigen-specific IgA in the upper airways and systemic circulation. Deficits in OVA-IgA were observed in conjunction with a reduced accumulation of OVA-specific IgA antibody-secreting cells (ASCs) in the nasal mucosa, inductive tissues (NALT, cervical lymph nodes, spleen) and the blood. Nasal OVA-IgA from smoke-exposed mice also demonstrated reduced avidity during the acute post-immunization period in association with an enhanced mutational burden in the cognate nasal Igha repertoire. Mechanistically, smoke exposure attenuated the ability of the nasal mucosa to upregulate VCAM-1 and pIgR, suggesting that cigarette smoke may inhibit both nasal ASC homing and IgA transepithelial transport. Overall, these findings demonstrate the immunosuppressive nature of tobacco smoke and illustrate the diversity of mechanisms through which this noxious stimulus can interfere with IgA-mediated immunity in the upper airways.
Assuntos
Formação de Anticorpos/imunologia , Antígenos/imunologia , Imunidade nas Mucosas , Imunoglobulina A Secretora/imunologia , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Fumar Tabaco/efeitos adversos , Animais , Biomarcadores , Quimiocinas CC/metabolismo , Imunização , Imunofenotipagem , Lipopolissacarídeos/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Ovalbumina/imunologia , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Hipermutação Somática de Imunoglobulina , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Competition among microorganisms is a key determinant of successful host colonization and persistence. For Streptococcus pneumoniae, lower than predicted rates of co-colonizing strains suggest a competitive advantage for resident bacteria over newcomers. In light of evolutionary theory, we hypothesized that S. pneumoniae use owner-intruder asymmetries to settle contests, leading to the disproportionate success of the initial resident 'owner', regardless of the genetic identity of the 'intruder'. We investigated the determinants of within-host competitive success utilizing S. pneumoniae colonization of the upper respiratory tract of infant mice. Within 6 h, colonization by the resident inhibited colonization by an isogenic challenger. The competitive advantage of the resident was dependent on quorum sensing via the competence (Com) regulon and downstream choline binding protein D (CbpD) and on the competence-induced bacteriocins A and B (CibAB) implicated in fratricide. CbpD and CibAB are highly conserved across pneumococcal lineages, indicating evolutionary advantages for asymmetric competitive strategies within the species. Mathematical modelling supported a significant role for quorum sensing via the Com regulon in competition, even for strains with different competitive advantages. Our study suggests that asymmetric owner-intruder competitive strategies do not require complex cognition and are used by a major human pathogen to determine 'ownership' of human hosts.
Assuntos
Amidoidrolases/genética , Proteínas de Bactérias/genética , Bacteriocinas/genética , Interações Microbianas/fisiologia , Percepção de Quorum/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiologia , Animais , Bacteriocinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Interações Microbianas/genética , Modelos Teóricos , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologiaRESUMO
Cigarette smoking is the main risk factor for chronic obstructive pulmonary disease, and to date, existing pharmacologic interventions have been ineffective at controlling inflammatory processes associated with the disease. To address this issue, we used the Connectivity Map (cMap) database to identify drug candidates with the potential to attenuate cigarette smoke-induced inflammation. We queried cMap using three independent in-house cohorts of healthy nonsmokers and smokers. Potential drug candidates were validated against four publicly available human datasets, as well as six independent datasets from cigarette smoke-exposed mice. Overall, these analyses yielded two potential drug candidates: kaempferol and bethanechol. Subsequently, the efficacy of each drug was validated in vivo in a model of cigarette smoke-induced inflammation. BALB/c mice were exposed to room air or cigarette smoke and treated with each of the two candidate drugs either prophylactically or therapeutically. We found that kaempferol, but not bethanechol, was able to reduce cigarette smoke-induced neutrophilia, both when administered prophylactically and when administered therapeutically. Mechanistically, kaempferol decreased expression of IL-1α and CXCL5 concentrations in the lung. Our data suggest that cMap analyses may serve as a useful tool to identify novel drug candidates against cigarette smoke-induced inflammation.
Assuntos
Betanecol/farmacologia , Fumar Cigarros/efeitos adversos , Avaliação Pré-Clínica de Medicamentos/métodos , Quempferóis/farmacologia , Pneumonia/induzido quimicamente , Adulto , Idoso , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Pneumonia/tratamento farmacológico , Pneumonia/prevenção & controle , Reprodutibilidade dos TestesRESUMO
Smokers have nasal microbiota dysbiosis, with an increased frequency of colonizing bacterial pathogens. It is possible that cigarette smoke increases pathogen acquisition by perturbing the microbiota and decreasing colonization resistance. However, it is difficult to disentangle microbiota dysbiosis due to cigarette smoke exposure from microbiota changes caused by increased pathogen acquisition in human smokers. Using an experimental mouse model, we investigated the impact of cigarette smoke on the nasal microbiota in the absence and presence of nasal pneumococcal colonization. We observed that cigarette smoke exposure alone did not alter the nasal microbiota composition. The microbiota composition was also unchanged at 12 h following low-dose nasal pneumococcal inoculation, suggesting that the ability of the microbiota to resist initial nasal pneumococcal acquisition was not impaired in smoke-exposed mice. However, nasal microbiota dysbiosis occurred as a consequence of established high-dose nasal pneumococcal colonization at day 3 in smoke-exposed mice. Similar to clinical reports on human smokers, an enrichment of potentially pathogenic bacterial genera such as Fusobacterium, Gemella, and Neisseria was observed. Our findings suggest that cigarette smoke exposure predisposes to pneumococcal colonization independent of changes to the nasal microbiota and that microbiota dysbiosis observed in smokers may occur as a consequence of established pathogen colonization.
Assuntos
Microbiota/efeitos dos fármacos , Nariz/microbiologia , Fumaça/efeitos adversos , Streptococcus pneumoniae/fisiologia , Animais , Modelos Animais de Doenças , Disbiose , Fusobacterium/isolamento & purificação , Gemella/isolamento & purificação , Humanos , Pulmão/microbiologia , Camundongos , Neisseria/isolamento & purificação , Infecções Pneumocócicas/microbiologia , Pneumonia/microbiologia , Produtos do Tabaco/efeitos adversosRESUMO
BACKGROUND: Chronic cigarette smoke exposure is known to activate the adaptive immune system; however, the functional role of these processes is currently unknown. Given the role of oxidized lipids in driving innate inflammatory responses to cigarette smoke, we investigated whether an adaptive immune response against damaged lipids was induced following chronic cigarette smoke exposure. METHODS AND RESULTS: Using a well-established mouse model, we showed that cigarette smoke exposure led to a progressive increase in pulmonary antibodies against oxidized low-density lipoprotein (OxLDL). Functionally, we found that intranasal delivery of an antibody against oxidized phosphatidylcholine (anti-OxPC; clone E06) increased lipid and particle uptake by pulmonary macrophages without exacerbating cigarette smoke-induced neutrophilia. We also found that anti-OxPC treatment increased particle uptake following smoking cessation. Finally, the frequency of pulmonary macrophages with internalized particles was increased after prolonged smoke exposure, at which time lung anti-OxPC responses were highest. CONCLUSIONS: Altogether, this is the first report to demonstrate a non-pathogenic, and possibly protective, function of a newly identified autoantibody induced by chronic cigarette smoke exposure.
Assuntos
Formação de Anticorpos , Lipídeos/imunologia , Pulmão/imunologia , Nicotiana , Fumaça , Imunidade Adaptativa/efeitos dos fármacos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Lipoproteínas LDL/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Fosfatidilcolinas/imunologia , Abandono do Hábito de FumarRESUMO
Streptococcus pneumoniae is a leading cause of invasive bacterial infections, with nasal colonization an important first step in disease. While cigarette smoking is a strong risk factor for invasive pneumococcal disease, the underlying mechanisms remain unknown. This is partly due to a lack of clinically relevant animal models investigating nasal pneumococcal colonization in the context of cigarette smoke exposure. We present a model of nasal pneumococcal colonization in cigarette smoke-exposed mice and document, for the first time, that cigarette smoke predisposes to invasive pneumococcal infection and mortality in an animal model. Cigarette smoke increased the risk of bacteremia and meningitis without prior lung infection. Mechanistically, deficiency in interleukin 1α (IL-1α) or platelet-activating factor receptor (PAFR), an important host receptor thought to bind and facilitate pneumococcal invasiveness, did not rescue cigarette smoke-exposed mice from invasive pneumococcal disease. Importantly, we observed cigarette smoke to attenuate nasal inflammatory mediator expression, particularly that of neutrophil-recruiting chemokines, normally elicited by pneumococcal colonization. Smoking cessation during nasal pneumococcal colonization rescued nasal neutrophil recruitment and prevented invasive disease in mice. We propose that cigarette smoke predisposes to invasive pneumococcal disease by suppressing inflammatory processes of the upper respiratory tract. Given that smoking prevalence remains high worldwide, these findings are relevant to the continued efforts to reduce the invasive pneumococcal disease burden.
Assuntos
Portador Sadio/imunologia , Mucosa Nasal/microbiologia , Infecções Pneumocócicas/imunologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Streptococcus pneumoniae/crescimento & desenvolvimento , Animais , Bacteriemia/microbiologia , Bacteriemia/prevenção & controle , Portador Sadio/prevenção & controle , Modelos Animais de Doenças , Resistência à Doença , Meningite Pneumocócica/microbiologia , Meningite Pneumocócica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Nasal/imunologia , Neutrófilos/imunologia , Infecções Pneumocócicas/prevenção & controle , Streptococcus pneumoniae/imunologiaRESUMO
Emerging evidence suggests that autoimmune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. We observed that BAFF levels were elevated in smokers and mice exposed to cigarette smoke. In mice, BAFF expression was rapidly induced in the lungs following 4 days of cigarette smoke exposure and remained elevated following 8 and 24 weeks of exposure. Alveolar macrophages were the major source of BAFF Blockade of BAFF using a BAFF receptor-Fc (BAFFR-Fc) construct prevented pulmonary ANA and TLT formation when delivered concurrent with cigarette smoke exposure. Under these conditions, no impact on lung inflammation was observed. However, administration of BAFFR-Fc following smoking cessation markedly reduced the number of TLTs and ANA levels and, of note, reduced pulmonary neutrophilia. Altogether, this study shows for the first time a central role of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggests that BAFF blockade following smoking cessation could have beneficial effects on persistent inflammatory processes.In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. Data presented show that BAFF plays a central role in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggest a therapeutic potential for BAFF blockade in limiting autoimmune processes associated with smoking.
Assuntos
Anticorpos Antinucleares/metabolismo , Fator Ativador de Células B/imunologia , Fumar Cigarros/efeitos adversos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Nicotiana/efeitos adversos , Animais , Fator Ativador de Células B/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Feminino , Humanos , Imunoglobulina M/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Abandono do Hábito de Fumar , Baço/efeitos dos fármacos , Baço/metabolismo , Estruturas Linfoides Terciárias/induzido quimicamente , Estruturas Linfoides Terciárias/imunologiaRESUMO
BACKGROUND: Previous studies have suggested that asthmatic patients often have comorbid depression; however, temporal associations remain unclear. OBJECTIVES: To determine whether depression predicts asthma and, conversely, whether asthma predicts depression. METHODS: A literature search was conducted without language restrictions using Pubmed, Embase, Cochrane and PsycINFO for studies published before January, 2015. Papers referenced by the obtained articles were also reviewed. Only comparative prospective studies with reported risk estimates of the association between depression and asthma were included. In order to investigate whether one of these conditions was predictive of the other, studies were excluded if enrolled participants had pre-existing depression or asthma. A random-effects model was used to calculate the pooled risk estimates for two outcomes: depression predicting asthma and asthma predicting depression. RESULTS: Seven citations, derived from 8 cohort studies, met our inclusion criteria. Of these, six studies reported that depression predicted incident adult-onset asthma, including 83684 participants and 2334 incident cases followed for 8 to 20 years. Conversely, two studies reported that asthma predicted incident depression. These studies involved 25566 participants and 2655 incident cases followed for 10 and 20 years, respectively. The pooled adjusted relative risks (RRs) of acquiring asthma associated with baseline depression was 1.43 (95% CI, 1.28-1.61) (P<0.001). The adjusted RRs for acquiring depression associated with baseline asthma was 1.23 (95% CI, 0.72-2.10) (P = 0.45). CONCLUSIONS: Depression was associated with a 43% increased risk of developing adult-onset asthma. However, asthma did not increase the risk of depression based on limited studies. Further prospective studies ascertaining the true association between asthma and subsequent risk of depression are warranted.
Assuntos
Asma/epidemiologia , Depressão/epidemiologia , Transtorno Depressivo/epidemiologia , Asma/complicações , Depressão/complicações , Transtorno Depressivo/complicações , Humanos , Incidência , Estudos Prospectivos , RiscoRESUMO
RATIONALE: Nontypeable Haemophilus influenzae (NTHi) causes acute exacerbation of chronic obstructive pulmonary disease (AECOPD). IL-17A is central for neutrophilic inflammation and has been linked to COPD pathogenesis. OBJECTIVES: We investigated whether IL-17A is elevated in NTHi-associated AECOPD and required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. METHODS: Experimental studies with cigarette smoke and NTHi infection were pursued in gene-targeted mice and using antibody intervention. IL-17A was measured in sputum collected from patients with COPD at baseline, during, and after AECOPD. MEASUREMENTS AND MAIN RESULTS: Exacerbated airway neutrophilia in cigarette smoke-exposed mice infected with NTHi was associated with an induction of IL-17A. In agreement, elevated IL-17A was observed in sputum collected during NTHi-associated AECOPD, compared with samples collected before or after the event. NTHi-exacerbated neutrophilia and induction of neutrophil chemoattractants over the background of cigarette smoke, as observed in wild-type mice, was absent in Il17a(-/-) mice and in mice treated with a neutralizing anti-IL-17A antibody. Further studies revealed that IL-1 receptor (R)1 signaling was required for IL-17A-dependent neutrophilia. Moreover, deficiency or therapeutic neutralization of IL-17A did not increase bacterial burden or delay bacterial clearance. CONCLUSIONS: IL-17A is induced during NTHi-associated AECOPD. Functionally, IL-1R1-dependent IL-17A is required for NTHi-exacerbated pulmonary neutrophilia induced by cigarette smoke. Targeting IL-17A in AECOPD may thus be beneficial to reduce neutrophil recruitment to the airways.
Assuntos
Infecções por Haemophilus/metabolismo , Haemophilus influenzae , Interleucina-17/metabolismo , Neutrófilos/fisiologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Idoso , Animais , Modelos Animais de Doenças , Feminino , Infecções por Haemophilus/complicações , Humanos , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Fumar/efeitos adversosRESUMO
Overwhelming evidence links inflammation to the pathogenesis of smoking-related pulmonary diseases, especially chronic obstructive pulmonary disease (COPD). Despite an increased understanding of the disease pathogenesis, mechanisms initiating smoking-induced inflammatory processes remain incompletely understood. To investigate the mechanisms that initiate and propagate smoke-induced inflammation, we used a well-characterised mouse model of cigarette smoke exposure, mice deficient for interleukin (IL)-1α, IL-1ß and Toll-like receptor 4, and antibodies blocking granulocyte-macrophage colony-stimulating factor (GM-CSF). Studies were also pursued using intranasal delivery of human oxidised low-density lipoprotein (hOxLDL), a source of oxidised lipids, to investigate the inflammatory processes associated with impaired lipid homeostasis. We found that cigarette smoke exposure rapidly led to lipid accumulation in pulmonary macrophages, a defining feature of foam cells, which in turn released high levels of IL-1α. In smoke-exposed IL-1α-deficient mice, phospholipids accumulated in the bronchoalveolar lavage, a phenomenon also observed when blocking GM-CSF. Intranasal administration of hOxLDL led to lipid accumulation in macrophages and initiated an inflammatory process that mirrored the characteristics of cigarette smoke-induced inflammation. These findings identify a link between lipid accumulation in macrophages, inflammation and damaged surfactant, suggesting that the response to damaged pulmonary surfactant is a central mechanism that drives cigarette smoke-induced inflammation. Further investigations are required to explore the role of distorted lipid homeostasis in the pathogenesis of COPD.
Assuntos
Interleucina-1alfa/metabolismo , Lipoproteínas LDL/administração & dosagem , Macrófagos Alveolares/patologia , Pneumonia/metabolismo , Fumar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Homeostase , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Pneumonia/fisiopatologia , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Determining the cellular and molecular phenotypes of inflammation in asthma can identify patient populations that may best benefit from targeted therapies. Although elevated IL-6 and polymorphisms in IL-6 signalling are associated with lung dysfunction in asthma, it remains unknown if elevated IL-6 levels are associated with a specific cellular inflammatory phenotype, and how IL-6 blockade might impact such inflammatory responses. METHODS: Patients undergoing exacerbations of asthma were phenotyped according to their airway inflammatory characteristics (normal cell count, eosinophilic, neutrophilic, mixed granulocytic), sputum cytokine profiles, and lung function. Mice were exposed to the common allergen, house dust-mite (HDM), in the presence or absence of endogenous IL-6. The intensity and nature of lung inflammation, and levels of pro-granulocytic cytokines and chemokines under these conditions were analyzed. RESULTS: Elevated IL-6 was associated with a lower FEV1 in patients with mixed eosinophilic-neutrophilic bronchitis. In mice, allergen exposure increased lung IL-6 and IL-6 was produced by dendritic cells and alveolar macrophages. Loss-of-function of IL-6 signalling (knockout or antibody-mediated neutralization) abrogated elevations of eosinophil and neutrophil recruiting cytokines/chemokines and allergen-induced airway inflammation in mice. CONCLUSIONS: We demonstrate the association of pleiotropic cellular airway inflammation with IL-6 using human and animal data. These data suggest that exacerbations of asthma, particularly those with a combined eosinophilic and neutrophilic bronchitis, may respond to therapies targeting the IL-6 pathway and therefore, provide a rational basis for initiation of clinical trials to evaluate this.
RESUMO
Chronic obstructive pulmonary disease (COPD) is characterised by a state of chronic pulmonary inflammation punctuated by microbial exacerbations. Despite advances in treatment options, COPD remains difficult to manage. In this study, we investigated the potential of peroxisome proliferator-activated receptor (PPAR)γ activation as a new therapy against cigarette smoke-induced inflammation and its associated bacterial exacerbation. C57BL/6 mice were exposed to room air or cigarette smoke for either 4 days or 4 weeks and treated either prophylactically or therapeutically with rosiglitazone. The impact of rosiglitazone on cigarette smoke-induced exacerbated response to the bacterial pathogen nontypeable Haemophilus influenzae (NTHi) was studied using the therapeutic treatment protocol. We found that rosiglitazone was able to reduce cigarette smoke-induced neutrophilia both when administered prophylactically or therapeutically. Therapeutic intervention with rosiglitazone was also effective in preventing cigarette smoke-induced neutrophilia exacerbation following NTHi infection. Moreover, the anti-inflammatory effects of rosiglitazone did not lead to an increase in the pulmonary bacterial burden, unlike dexamethasone. Altogether, our data suggest that pharmacological activation of PPARγ may be an effective therapeutic approach to improve COPD management, as it is able to reduce cigarette smoke-induced inflammation and decrease the magnitude of bacterial exacerbations, without compromising the ability of the immune system to control the infection.
Assuntos
Infecções Bacterianas/fisiopatologia , Regulação Bacteriana da Expressão Gênica , Pulmão/efeitos dos fármacos , PPAR gama/metabolismo , Fumar/efeitos adversos , Corticosteroides/uso terapêutico , Animais , Líquido da Lavagem Broncoalveolar , Dexametasona/farmacologia , Feminino , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/microbiologia , Rosiglitazona , Tiazolidinedionas/farmacologia , Produtos do TabacoRESUMO
Cigarette smoke has a broad impact on the mucosal environment with the ability to alter host defense mechanisms. Within the context of a bacterial infection, this altered host response is often accompanied by exacerbated cellular inflammation, characterized by increased neutrophilia. The current study investigated the mechanisms of neutrophil recruitment in a murine model of cigarette smoke exposure and, subsequently, a model of both cigarette smoke exposure and bacterial infection. We investigated the role of IL-1 signaling in neutrophil recruitment and found that cigarette smoke-induced neutrophilia was dependent on IL-1α produced by alveolar macrophages. In addition to being the crucial source of IL-1α, alveolar macrophages isolated from smoke-exposed mice were primed for excessive IL-1α production in response to bacterial ligands. To test the relevance of exaggerated IL-1α production in neutrophil recruitment, a model of cigarette smoke exposure and nontypeable Haemophilus influenzae infection was developed. Mice exposed to cigarette smoke elaborated an exacerbated CXCR2-dependent neutrophilia in response to nontypeable Haemophilus influenzae. Exacerbated neutrophilia was dependent on IL-1α priming of the pulmonary environment by cigarette smoke as exaggerated neutrophilia was dependent on IL-1 signaling. These data characterize a novel mechanism of cigarette smoke priming the lung mucosa toward greater IL-1-driven neutrophilic responses to bacteria, with a central role for the alveolar macrophage in this process.
Assuntos
Haemophilus influenzae/imunologia , Interleucina-1alfa/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-8B/imunologia , Fumaça/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Quimiocina CXCL1/biossíntese , Quimiocina CXCL5/biossíntese , Quimiocina CXCL5/genética , Quimiocina CXCL5/imunologia , Feminino , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Inflamação/imunologia , Contagem de Leucócitos , Pulmão/patologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Mensageiro/biossíntese , Receptores de Interleucina-8B/biossíntese , Receptores de Interleucina-8B/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Nicotiana/efeitos adversosRESUMO
Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4(+/+) or il4(-/-) eosinophils. Eosinophils controlled CD103(+) dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity.
Assuntos
Imunidade Adaptativa/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Trato Gastrointestinal/citologia , Células Th2/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/metabolismo , Antígeno CD11c/metabolismo , Degranulação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , Células Dendríticas/efeitos dos fármacos , Eosinófilos/ultraestrutura , Eritropoetina/farmacologia , Humanos , Imunização , Cadeias alfa de Integrinas/metabolismo , Interleucina-4/biossíntese , Camundongos , Células Th2/efeitos dos fármacosRESUMO
Formation of pulmonary tertiary immune structures is a characteristic feature of advanced COPD. In the current study, we investigated the mechanisms of tertiary lymphoid tissue (TLT) formation in the lungs of cigarette smoke-exposed mice. We found that cigarette smoke exposure led to TLT formation that persisted following smoking cessation. TLTs consisted predominantly of IgM positive B cells, while plasma cells in close proximity to TLTs expressed IgM, IgG, and IgA. The presence of TLT formation was associated with anti-nuclear autoantibody (ANA) production that also persisted following smoking cessation. ANAs were observed in the lungs, but not the circulation of cigarette smoke-exposed mice. Similarly, we observed ANA in the sputum of COPD patients where levels correlated with disease severity and were refractory to steroid treatment. Both ANA production and TLT formation were dependent on interleukin-1 receptor 1 (IL-1R1) expression. Contrary to TLT and ANA, lung neutrophilia resolved following smoking cessation. These data suggest a differential regulation of innate and B cell-related immune inflammatory processes associated with cigarette smoke exposure. Moreover, our study further emphasizes the importance of interleukin-1 (IL-1) signaling pathways in cigarette smoke-related pulmonary pathogenesis.
Assuntos
Anticorpos Antinucleares/biossíntese , Exposição por Inalação/efeitos adversos , Tecido Linfoide/imunologia , Abandono do Hábito de Fumar , Fumar/efeitos adversos , Fumar/metabolismo , Idoso , Animais , Feminino , Humanos , Tecido Linfoide/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fumar/patologia , Escarro/imunologia , Escarro/metabolismo , Fatores de TempoRESUMO
We have evaluated the use of a panel of six fluorogenic cytochrome P450 (CYP) substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 7-benzyloxy-4-(trifluoromethyl)-coumarin, 7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifluoromethyl)-coumarin, and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male rat liver were characterized, their sensitivities to 15 putative inhibitors were determined and compared to similar experiments using nine different complementary DNA (cDNA)-expressed rat CYPs. Inhibitory profiles of the substrates in microsomes were different from each other, with some overlap, suggesting that each substrate is to some extent biotransformed by a different CYP isoform. Ketoconazole and clotrimazole were nonselective inhibitors, while ticlopidine selectively inhibited biotransformation of AMMC. CYP2A1 did not biotransform any of the substrates, and CYP2E1 was insensitive to all the inhibitors tested. Some inhibitors did not affect the biotransformation of the fluorogenic substrates by cDNA-expressed isoforms as predicted by their effects on conventional substrates, e.g., chlorzoxazone and diethyldithiocarbamate were inactive against CYP2E1, and CYP2C6 was not inhibited by sulfaphenazole. When results in microsomes and cDNA-expressed CYPs were compared, only the majority of the biotransformation of AMMC by microsomes could be assigned with full confidence to a specific CYP isoform, namely CYP2D2. Nevertheless, different inhibitory profiles of the substrates indicate that the panel will be useful for rapid functional quantification of global CYP activity in rats under different experimental conditions. Our results also demonstrate the inappropriateness of extrapolating inhibitory data between conventional and fluorogenic CYP substrates.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Cumarínicos/metabolismo , Corantes Fluorescentes/metabolismo , Microssomos Hepáticos/enzimologia , Quinolinas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Biotransformação/efeitos dos fármacos , Cumarínicos/toxicidade , DNA Complementar/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Quinolinas/toxicidade , RatosRESUMO
Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.