RESUMO
Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.
Assuntos
Apoptose/fisiologia , Macrófagos Alveolares/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fagocitose/genética , Fagossomos/fisiologia , Pneumonia Bacteriana , Animais , Apoptose/efeitos dos fármacos , Bactérias , Compostos de Bifenilo/farmacologia , Caspases/metabolismo , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Haemophilus influenzae , Humanos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Óxido Nítrico/metabolismo , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus , Streptococcus pneumoniae , Sulfonamidas/farmacologiaRESUMO
RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. OBJECTIVES: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. METHODS: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage-specific expression of human induced myeloid leukemia cell differentiation protein Mcl-1 (CD68.hMcl-1) was used to model the molecular aspects of COPD. MEASUREMENTS AND MAIN RESULTS: COPD AMs had elevated levels of Mcl-1, an antiapoptotic B-cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages and human monocyte-derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected bone marrow-derived macrophages but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2. CONCLUSIONS: Up-regulation of Mcl-1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.
Assuntos
Anti-Infecciosos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Western Blotting , Lavagem Broncoalveolar , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.
Assuntos
Apoptose , Proteína Ligante Fas/metabolismo , Monócitos/imunologia , Infecções Pneumocócicas/imunologia , Linfócitos T/fisiologia , Animais , Bacteriemia , Proteínas de Bactérias , Complexo CD3/biossíntese , Células Cultivadas , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Receptores de Lipopolissacarídeos/biossíntese , Pulmão/microbiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/microbiologia , Necrose , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade , Estreptolisinas , Linfócitos T/imunologia , Linfócitos T/microbiologiaRESUMO
A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.
Assuntos
Asma/imunologia , Infecções por Haemophilus/imunologia , Haemophilus influenzae/patogenicidade , Interleucina-17/imunologia , Neutrófilos/imunologia , Animais , Asma/microbiologia , Asma/patologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Imunidade Celular , Inflamação/patologia , Interferon gama/imunologia , Interleucina-13/imunologia , Interleucina-17/metabolismo , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Fenótipo , Linfócitos T Reguladores/imunologia , Células Th17/citologia , Células Th17/imunologiaRESUMO
Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1-regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1alpha upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.
Assuntos
Infecções por Escherichia coli/imunologia , Imunidade Inata , Infecções por Klebsiella/imunologia , Infecções Meningocócicas/imunologia , Monócitos/imunologia , Monócitos/patologia , Infecções por Neisseriaceae/imunologia , Morte Celular/imunologia , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , DNA/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/patologia , Monócitos/metabolismo , Monócitos/microbiologia , Infecções por Neisseriaceae/microbiologia , Infecções por Neisseriaceae/patologiaRESUMO
Neutrophilic asthma is a prevalent, yet recently described phenotype of asthma. It is characterized by neutrophilic rather than eosinophilic airway inflammation and airways hyperresponsiveness (AHR) and may have an infectious origin. Chlamydial respiratory infections are associated with asthma, but how these Th1-inducing bacteria influence Th2-mediated asthma remains unknown. The effects of chlamydial infection on the development of asthma were investigated using a BALB/c mouse model of OVA-induced allergic airways disease (AAD). The effects of current and resolved Chlamydia muridarum infection during OVA sensitization on AAD were assessed and compared with uninfected and nonsensitized controls. Current, but not resolved, infection attenuated hallmark features of AAD: pulmonary eosinophil influx, T cell production of IL-5, mucus-secreting cell hyperplasia, and AHR. Current infection also induced robust OVA-driven neutrophilic inflammation and IFN-gamma release from T cells. The phenotype of suppressed but persistent Th2 responses in association with enhanced neutrophilia is reminiscent of neutrophilic asthma. This phenotype was also characterized by increased pulmonary IL-12 and IL-17 expression and activation of APCs, as well as by reduced thymus- and activation-regulated chemokine. Inhibition of pulmonary neutrophil influx during infection blocked OVA-induced neutrophilic inflammation and T cell IFN-gamma production and reversed the suppressive effects on mucus-secreting cell hyperplasia and AHR during AAD. These changes correlated with decreased IL-12 and IL-17 expression, increased thymus- and activation-regulated chemokine and altered APC activation. Blocking IFN-gamma and IL-17 during OVA challenge had no effect. Thus, active chlamydial respiratory infection during sensitization enhances subsequent neutrophilic inflammation and Th1/Th17 responses during allergen exposure and may have a role in the pathogenesis of neutrophilic asthma.
Assuntos
Alérgenos/administração & dosagem , Alérgenos/imunologia , Asma/imunologia , Asma/patologia , Infecções por Chlamydia/imunologia , Chlamydia muridarum/imunologia , Infiltração de Neutrófilos/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Asma/microbiologia , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Neutrófilos/patologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Hipersensibilidade Respiratória/microbiologia , Hipersensibilidade Respiratória/patologia , Células Th1/imunologia , Células Th1/microbiologia , Células Th1/patologia , Células Th2/imunologia , Células Th2/microbiologia , Células Th2/patologiaRESUMO
Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD(3)) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD(3) and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.
Assuntos
Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Western Blotting , Linhagem Celular , Citocinas/metabolismo , Humanos , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Monócitos/citologia , Óxido Nítrico/biossíntese , FagocitoseRESUMO
Streptococcus pneumoniae (the pneumococcus) is a major global cause of human disease. Since the publication of the entire sequence of TIGR4 in 2001, our understanding of this human pathogen has increased significantly. Genetic studies, and the use of mutant strains have refined our understanding of the pathogenic mechanisms of classic pneumococcal virulence factors, including the polysaccharide capsule, pneumolysin and surface-expressed proteins. Genetic screens are identifying novel virulence factors. Characterization of pili and bacteriocins, as well as genes associated with competence, metabolism and resistance to oxidative stress has provided new insights into the genetic diversity of the pneumococcus. Further appreciation of the molecular basis of pneumococcal pathogenesis will lead to more effective strategies for the prevention and management of pneumococcal disease.
Assuntos
Pneumonia Pneumocócica/microbiologia , Streptococcus pneumoniae/metabolismo , Fatores de Virulência/metabolismo , Humanos , Modelos Biológicos , Mutação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/genéticaRESUMO
RATIONALE: Chlamydial lung infection has been associated with asthma in children and adults. However, how chlamydial infection influences the development of immune responses that promote asthma remains unknown. OBJECTIVES: To determine the effect of chlamydial infection at various ages on the development of allergic airway disease (AAD). METHODS: Mouse models of chlamydial lung infection and ovalbumin-induced AAD were established in neonatal and adult BALB/c mice. Neonatal or adult mice were given a chlamydial infection and 6 weeks later were sensitized and subsequently challenged with ovalbumin. Features of AAD and inflammation were compared between uninfected or unsensitized controls. MEASUREMENTS AND MAIN RESULTS: Mild Chlamydia-induced lung disease was observed 10-15 days after infection, as evidenced by increased bacterial numbers and histopathology in the lung and a reduction in weight gain. After 6 weeks, infection and histopathology had resolved and the rate of weight gain had recovered. Neonatal but not adult infection resulted in significant decreases in interleukin-5 production from helper T cells and by the numbers of eosinophils recruited to the lung in response to ovalbumin exposure. Remarkably, the effects of early-life infection were associated with the generation of both type 1 and 2 ovalbumin-specific helper T-cell cytokine and antibody responses. Furthermore, although neonatal infection significantly attenuated eosinophilia, the generation of the mixed T-cell response exacerbated other hallmark features of asthma: mucus hypersecretion and airway hyperresponsiveness. Moreover, infection prolonged the expression of AAD and these effects were restricted to early-life infection. CONCLUSIONS: Early-life chlamydial infection induces a mixed type 1 and 2 T-cell response to antigen, which differentially affects the development of key features of AAD in the adult.
Assuntos
Asma/imunologia , Infecções por Chlamydia/imunologia , Imunidade Celular/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Asma/etiologia , Asma/patologia , Chlamydia/isolamento & purificação , Infecções por Chlamydia/patologia , Contagem de Colônia Microbiana , Citocinas/imunologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , PrognósticoRESUMO
Asthma is a common inflammatory disease of the airways. Current therapies alleviate symptoms but do not treat the disease. We aim to develop effective immunomodulatory therapies (IMTs) for asthma that target the underlying causes of disease based on Streptococcus pneumoniae (Spn). The effect of Spn IMT on the development of asthma [allergic airways disease (AAD)] was determined in mice. Killed Spn was administered before, during or after ovalbumin sensitization, and the subsequent development of AAD was assessed. IMT attenuated T cell cytokine production, goblet cell hyperplasia, airways hyperresponsiveness (AHR), and eosinophil numbers in the blood, bronchoalveolar lavage fluid and peribronchial tissue. This indicates the potential of Spn as an IMT for asthma.