RESUMO
Influenza infection predisposes patients to secondary bacterial pneumonia that contributes significantly to morbidity and mortality. Although this association is well documented, the mechanisms that govern this synergism are poorly understood. A window of hyporesponsiveness following influenza infection has been associated with a substantial increase in local and systemic IFN-γ concentrations. Recent data suggest that the oxazolidinone antibiotic linezolid decreases IFN-γ and TNF-α production in vitro from stimulated PBMCs. We therefore sought to determine whether linezolid would reverse immune hyporesponsiveness after influenza infection in mice through its effects on IFN-γ. In vivo dose-response studies demonstrated that oral linezolid administration sufficiently decreased bronchoalveolar lavage fluid levels of IFN-γ at day 7 postinfluenza infection in a dose-dependent manner. The drug also decreased morbidity as measured by weight loss compared with vehicle-treated controls. When mice were challenged intranasally with Streptococcus pneumoniae 7 d postinfection with influenza, linezolid pretreatment led to decreased IFN-γ and TNF-α production, decreased weight loss, and lower bacterial burdens at 24 h postbacterial infection in comparison with vehicle-treated controls. To determine whether these effects were due to suppression of IFN-γ, linezolid-treated animals were given intranasal instillations of rIFN-γ before challenge with S. pneumoniae. This partially reversed the protective effects observed in the linezolid-treated mice, suggesting that the modulatory effects of linezolid are mediated partially by its ability to blunt IFN-γ production. These results suggest that IFN-γ, and potentially TNF-α, may be useful drug targets for prophylaxis against secondary bacterial pneumonia following influenza infection.
Assuntos
Acetamidas/uso terapêutico , Antibacterianos/uso terapêutico , Coinfecção/prevenção & controle , Interferon gama/fisiologia , Infecções por Orthomyxoviridae/complicações , Oxazolidinonas/uso terapêutico , Pneumonia Pneumocócica/prevenção & controle , Acetamidas/administração & dosagem , Acetamidas/farmacologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/química , Contagem de Linfócito CD4 , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/fisiologia , Interferon gama/análise , Interferon gama/biossíntese , Interferon gama/genética , Interferon gama/uso terapêutico , Linezolida , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Oxazolidinonas/administração & dosagem , Oxazolidinonas/farmacologia , Pneumonia Pneumocócica/etiologia , Proteínas Recombinantes/uso terapêutico , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVES: Chronic azithromycin therapy has been associated with improved clinical outcomes in patients with cystic fibrosis (CF) who are chronically infected with Pseudomonas aeruginosa. We have previously demonstrated that azithromycin polarizes macrophages towards an alternatively activated phenotype, thereby blunting inflammation associated with infection. Because this phenotype is pro-fibrotic, it is important to evaluate azithromycin's consequential effects upon fibroblast function and extracellular matrix (ECM) protein production. METHODS: We co-cultured macrophages and fibroblasts together and stimulated them by adding P. aeruginosa or lipopolysaccharide to assess the ability of azithromycin to alter the macrophage phenotype, along with the impact exerted upon the production of fibronectin and other effectors that govern tissue remodelling, including transforming growth factor ß (TGFß), matrix metalloproteinase-9 (MMP-9) and arginase. We supported these studies by evaluating the impact of azithromycin treatment on these proteins in a mouse model of P. aeruginosa infection. RESULTS: Azithromycin increased arginase expression in vitro, as well as the activation of latent TGFß, consistent with polarization to the alternative macrophage phenotype. While the drug increased fibronectin concentrations after stimulation in vitro, secretion of the ECM-degrading enzyme MMP-9 was also increased. Neutralization of active TGFß resulted in the ablation of azithromycin's ability to increase fibronectin concentrations, but did not alter its ability to increase MMP-9 expression. In P. aeruginosa-infected mice, azithromycin significantly decreased MMP-9 and fibronectin concentrations in the alveolar space compared with non-treated, infected controls. CONCLUSIONS: Our results suggest that azithromycin's effect on MMP-9 is regulated independently of TGFß activity. Additionally, the beneficial effects of azithromycin may be partially due to effects on homeostasis in which ECM-degrading mediators like MMP-9 are up-regulated early after infection. This may impact the damaging effects of inflammation that lead to fibrosis in this patient population.
Assuntos
Azitromicina/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fatores Imunológicos/metabolismo , Macrófagos/metabolismo , Pseudomonas aeruginosa/imunologia , Animais , Linhagem Celular , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , CamundongosRESUMO
Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. This study tested the capacity of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation. Both CB2-selective agonists and Δ(9)-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2-selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3(+) cells to these compounds completely inhibited their action in a reconstituted MLR. Further, the CB2-selective agonists completely inhibited proliferation of purified T-cells activated by anti-CD3 and anti-CD28 antibodies. T-cell function was decreased by the CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly decreased in the cannabinoid treated cells. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients.