RESUMO
Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.
Assuntos
Macrófagos , Neoplasias , Sepse , Humanos , Sepse/imunologia , Macrófagos/imunologia , Feminino , Neoplasias/imunologia , Neoplasias/terapia , Masculino , Receptores CXCR6/metabolismo , Animais , Linfócitos T/imunologia , Receptores CCR2/metabolismo , Pessoa de Meia-Idade , Camundongos , Idoso , Quimiocinas/metabolismo , AdultoRESUMO
Azithromycin (AZM) is a 15-membered-ring macrolide that presents a broad-spectrum antimicrobial activity against Gram-positive bacteria and atypical microorganisms but suffers from a poor diffusion across the outer-membrane of Gram-negative bacilli, including Pseudomonas aeruginosa (PA). However, AZM has demonstrated clinical benefits in patients suffering from chronic PA respiratory infections, especially cystic fibrosis patients. Since the rise of multidrug-resistant PA has led to a growing need for new therapeutic options, this macrolide has been proposed as an adjunctive therapy. Clinical trials assessing AZM in PA acute pneumonia are scarce. However, a careful examination of the available literature provides good rationales for its use in that context. In fact, 14- and 15-membered-ring macrolides have demonstrated immunomodulatory and immunosuppressive effects that could be of major interest in the management of acute illness. Furthermore, growing evidence supports a downregulation of PA virulence dependent on direct interaction with the ribosomes, and based on the modulation of several key regulators from the Quorum Sensing network. First highlighted in vitro, these interesting properties of AZM have subsequently been confirmed in the animal models. In this review, we systematically analyzed the literature regarding AZM immunomodulatory and anti-PA effects. In vitro and in vivo studies, as well as clinical trials were reviewed, looking for rationales for AZM use in PA acute pneumonia.
RESUMO
Natural killer (NK) cells play a key role in both antibacterial and antitumor immunity. Pseudomonas aeruginosa infection has already been reported to alter NK cell functions. We studied in vitro the effect of P. aeruginosa on NK cell cytotoxic response (CD107a membrane expression) to a lymphoma cell line. Through positive and negative cell sorting and adoptive transfer, we determined the influence of monocytes, lymphocytes, and regulatory T cells (Treg) on NK cell function during P. aeruginosa infection. We also studied the role of the activating receptor natural killer group 2D (NKG2D) in NK cell response to B221. We determined that P. aeruginosa significantly altered both cytotoxic response to B221 and NKG2D expression on NK cells in a Treg-dependent manner and that the NKG2D receptor was involved in NK cell cytotoxic response to B221. Our results also suggested that during P. aeruginosa infection, monocytes participated in Treg-mediated NK cell alteration. In conclusion, P. aeruginosa infection impairs NK cell cytotoxicity and alters antitumor immunity. These results highlight the strong interaction between bacterial infection and immunity against cancer.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Linfócitos T Reguladores/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Humanos , Leucócitos Mononucleares , Receptores de Lipopolissacarídeos/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Monócitos/imunologia , Infecções por Pseudomonas/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Sepse/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Células Cultivadas , Feminino , Humanos , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Sepse/microbiologia , Staphylococcus aureus , Linfócitos T Reguladores/citologiaRESUMO
Sepsis and trauma cause inflammation and elevated susceptibility to hospital-acquired pneumonia. As phagocytosis by macrophages plays a critical role in the control of bacteria, we investigated the phagocytic activity of macrophages after resolution of inflammation. After resolution of primary pneumonia, murine alveolar macrophages (AMs) exhibited poor phagocytic capacity for several weeks. These paralyzed AMs developed from resident AMs that underwent an epigenetic program of tolerogenic training. Such adaptation was not induced by direct encounter of the pathogen but by secondary immunosuppressive signals established locally upon resolution of primary infection. Signal-regulatory protein α (SIRPα) played a critical role in the establishment of the microenvironment that induced tolerogenic training. In humans with systemic inflammation, AMs and also circulating monocytes still displayed alterations consistent with reprogramming six months after resolution of inflammation. Antibody blockade of SIRPα restored phagocytosis in monocytes of critically ill patients in vitro, which suggests a potential strategy to prevent hospital-acquired pneumonia.
Assuntos
Epigênese Genética , Inflamação/etiologia , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Animais , Biomarcadores , Reprogramação Celular , Citocinas/metabolismo , Humanos , Tolerância Imunológica , Imunofenotipagem , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/imunologia , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Interleukin (IL)-22 is a cytokine involved in tissue protection and repair following lung pathologies. Interferon (IFN)-λ cytokines displayed similar properties during viral infection and a synergy of action between these two players has been documented in the intestine. We hypothesize that during Pseudomonas aeruginosa challenge, IL-22 up-regulates IFN-λ and that IFN-λ exhibits protective functions during Pseudomonas aeruginosa acute pneumonia model in mice. METHODS: Using an in vitro human alveolar epithelial cell line A549, we assessed the ability of IL-22 to enhance IFN-λ expression during infection. IFN-λ protective function was evaluated in an acute mouse pneumonia model. RESULTS: We first demonstrated in murine lungs that only type-II alveolar cells express IL-22 receptor and that IL-22 treatment of A549 cell line up-regulates IFN-λ expression. In a murine acute pneumonia model, IL-22 administration maintained significant IFN-λ levels in the broncho-alveolar fluids whereas IL-22 neutralization abolished IFN-λ up-regulation. In vivo administration of IFN-λ during Pseudomonas aeruginosa pneumonia improves mice outcome by dampening neutrophil recruitment and decreasing epithelium damages. DISCUSSION: We show here that IL-22 regulates IFN-λ levels during Pseudomonas aeruginosa pneumonia.
Assuntos
Interferons/imunologia , Interleucinas/imunologia , Pneumonia/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Células A549 , Células Epiteliais Alveolares/imunologia , Animais , Brônquios/imunologia , Linhagem Celular Tumoral , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/imunologia , Camundongos , Infiltração de Neutrófilos/imunologia , Receptores de Interleucina/imunologia , Regulação para Cima/imunologia , Interleucina 22RESUMO
OBJECTIVES: In patients with spinal cord injury, spinal cord injury-immune depression syndrome induces pneumonia. We aimed to develop a new spinal cord injury-immune depression syndrome mouse model and to test antiprogrammed cell death 1 therapy. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: RjOrl: SWISS and BALB/cJ mice. INTERVENTIONS: Mouse model of spinal cord injury-immune depression syndrome followed by a methicillin-susceptible Staphylococcus aureus pneumonia. Lung injuries were assessed by histologic analysis. Membrane markers and intracytoplasmic cytokines were assessed by flow cytometry. Cytokine production was assessed by quantitative polymerase chain reaction (messenger RNA) and enzyme-linked immunosorbent assay (protein). Animals were treated with blocking antiprogrammed cell death 1 antibodies (intraperitoneal injection). MEASUREMENTS AND MAIN RESULTS: Spinal cord injury mice were more susceptible to methicillin-susceptible S. aureus pneumonia (increased mortality rate). An early inflammatory response was observed in spinal cord injury mice characterized in lungs by a decreased percentage of aerated tissue, an increased production of proinflammatory cytokines (tumor necrosis factor-α). In spleen, an increased expression of major histocompatibility complex class II molecules on dendritic cells, and an increased production of proinflammatory cytokines (interleukin-12, interferon-γ) was observed. Following this pulmonary and systemic inflammation, spinal cord injury-immune depression syndrome was observed in spleens as acknowledged by a decrease of spleen's weight, a lymphopenia, a decrease of major histocompatibility complex class II expression on dendritic cells. An increase of interleukin-10 production and the increase of a cell exhaustion marker expression, programmed cell death 1 receptor on T-cell were also observed. Blockade of programmed cell death 1 molecules, improved survival of spinal cord injury infected mice and enhanced interferon-γ production by natural killer T cells as well as number of viable CD4 T cells. CONCLUSIONS: This model of spinal cord injury in mice mimics a clinical scenario rendering animals prone to a secondary pneumonia. We show for the first time an acute T-cell exhaustion-like phenomenon following an initial inflammatory response. Finally, inhibition of exhaustion pathway should be considered as a new therapeutic option to overcome spinal cord injury-immune depression syndrome and to decrease the rate of nosocomial pneumonia.
Assuntos
Anticorpos/farmacologia , Pneumonia Bacteriana/tratamento farmacológico , Receptor de Morte Celular Programada 1/imunologia , Traumatismos da Medula Espinal/complicações , Staphylococcus aureus/imunologia , Animais , Citocinas/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/microbiologia , Baço/metabolismo , Linfócitos T/imunologiaRESUMO
Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts.
Assuntos
Escherichia coli/patogenicidade , Osteoblastos/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Aderência Bacteriana , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura , Endocitose , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/toxicidade , Perfilação da Expressão Gênica , Proteínas Hemolisinas/toxicidade , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , L-Lactato Desidrogenase/análise , Microscopia Confocal , Ortopedia , Osteoblastos/fisiologia , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVE: Trauma induces a state of immunosuppression, which is responsible for the development of nosocomial infections. Hydrocortisone reduces the rate of pneumonia in patients with trauma. Because alterations of dendritic cells and natural killer cells play a central role in trauma-induced immunosuppression, we investigated whether hydrocortisone modulates the dendritic cell/natural killer cell cross talk in the context of posttraumatic pneumonia. DESIGN: Experimental study. SETTINGS: Research laboratory from an university hospital. SUBJECTS: Bagg Albino/cJ mice (weight, 20-24 g). INTERVENTIONS: First, in an a priori substudy of a multicenter, randomized, double-blind, placebo-controlled trial of hydrocortisone (200 mg/d for 7 d) in patients with severe trauma, we have measured the blood levels of five cytokines (tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-12, interleukin-17) at day 1 and day 8. In a second step, the effects of hydrocortisone on dendritic cell/natural killer cell cross talk were studied in a mouse model of posttraumatic pneumonia. Hydrocortisone (0.6 mg/mice i.p.) was administered immediately after hemorrhage. Twenty-four hours later, the mice were challenged with Staphylococcus aureus (7 × 10 colony-forming units). MEASUREMENTS AND MAIN RESULTS: Using sera collected during a multicenter study in patients with trauma, we found that hydrocortisone decreased the blood level of interleukin-10, a cytokine centrally involved in the regulation of dendritic cell/natural killer cell cluster. In a mouse model of trauma-hemorrhage-induced immunosuppression, splenic natural killer cells induced an interleukin-10-dependent elimination of splenic dendritic cell. Hydrocortisone treatment reduced this suppressive function of natural killer cells and increased survival of mice with posthemorrhage pneumonia. The reduction of the interleukin-10 level in natural killer cells by hydrocortisone was partially dependent on the up-regulation of glucocorticoid-induced tumor necrosis factor receptor-ligand (TNFsf18) on dendritic cell. CONCLUSIONS: These data demonstrate that trauma-induced immunosuppression is characterized by an interleukin-10-dependent elimination of dendritic cell by natural killer cells and that hydrocortisone improves outcome by limiting this immunosuppressive feedback loop.
Assuntos
Anti-Inflamatórios/farmacologia , Hidrocortisona/farmacologia , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Ferimentos e Lesões/fisiopatologia , Adolescente , Adulto , Idoso , Animais , Infecção Hospitalar/prevenção & controle , Citocinas/imunologia , Células Dendríticas/imunologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Pneumonia Bacteriana/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Índices de Gravidade do Trauma , Adulto JovemRESUMO
OBJECTIVES: Pseudomonas aeruginosa infection is a clinically relevant infection involved in pneumonia in ICUs. Understanding the type of immune response initiated by the host during pneumonia would help defining new strategies to interfere with the bacteria pathogenicity. In this setting, the role of natural killer cells remains controversial. We assessed the role of systemic natural killer cells in a Pseudomonas aeruginosa mouse pneumonia model. DESIGN: Experimental study. SETTING: Research laboratory from a university hospital. SUBJECTS: RjOrl:SWISS and BALB/cJ mice (weight, 20-24 g). INTERVENTIONS: Lung injuries were assessed by bacterial load, myeloperoxidase activity, endothelial permeability (pulmonary edema), immune cell infiltrate (histological analysis), proinflammatory cytokine release, and Ly6-G immunohistochemistry. Bacterial loads were assessed in the lungs and spleen. Natural killer cell number and status were assessed in spleen (flow cytometry and quantitative polymerase chain reaction). Depletion of natural killer cells was achieved through an IV anti-asialo-GM1 antibody injection. MEASUREMENTS AND MAIN RESULTS: Pseudomonas aeruginosa tracheal instillation led to an acute pneumonia with a rapid decrease of bacterial load in lungs and with an increase of endothelial permeability, proinflammatory cytokines (tumor necrosis factor-α and interleukin-1ß), and myeloperoxidase activity followed by Ly6-G positive cell infiltrate in lungs. Pseudomonas aeruginosa was detected in the spleen. Membrane markers of activation and maturation (CD69 and KLRG1 molecules) were increased in splenic natural killer cells during Pseudomonas aeruginosa infection. Splenic natural killer cells activated upon Pseudomonas aeruginosa infection produced interferon-γ but not interleukin-10. Ultimately, mice depleted of natural killer cells displayed an increased neutrophil numbers in the lungs and an increased mortality rate without bacterial load modifications in the lungs, indicating that mice depleted of natural killer cells were much more susceptible to infection compared with control animals. CONCLUSIONS: We report for the first time that natural killer cells play a major role in the mice susceptibility toward a Pseudomonas aeruginosa-induced acute pneumonia model.
Assuntos
Suscetibilidade a Doenças/imunologia , Células Matadoras Naturais/imunologia , Pulmão/imunologia , Neutrófilos/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa , Baço/imunologia , Animais , Separação Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interferon gama/análise , Interleucina-10/análise , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos , Peroxidase/análise , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fator de Necrose Tumoral alfa/análiseRESUMO
BACKGROUND: Linezolid is considered as a therapeutic alternative to the use of glycopeptides for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Clinical studies reported a potent survival advantage conferred by the oxazolidinone and called into question the use of glycopeptides as first-line therapy. METHODS: In a mouse model of MRSA-induced pneumonia, quantitative bacteriology, proinflammatory cytokine concentrations in lung, myeloperoxidase activity, Ly6G immunohistochemistry, and endothelial permeability were assessed to compare therapeutic efficacy and immunomodulative properties of linezolid and vancomycin administered subcutaneously every 12 hours. RESULTS: Significant antibacterial activity was achieved after 48 hours of treatment for linezolid and vancomycin. Levels of interleukin 1ß, a major proinflammatory cytokine, and macrophage inflammatory protein 2, a chemokine involved in the recruitment of neutrophils, were decreased by both antimicrobials. Only linezolid was able to dramatically reduce the production of tumor necrosis factor α. Analysis of myeloperoxidase activity and Ly6G immunostaining showed a dramatic decrease of neutrophil infiltration in infected lung tissues for linezolid-treated animals. A time-dependent increase of endothelial permeability was observed for the control and vancomycin regimens. Of interest, in the linezolid group, decreased endothelial permeability was detected 48 hours after infection. CONCLUSIONS: Our results indicate that linezolid could be superior to vancomycin for the management of MRSA pneumonia by attenuating an excessive inflammatory reaction and protecting the lung from pathogen-associated damages.
Assuntos
Acetamidas/administração & dosagem , Antibacterianos/administração & dosagem , Fatores Imunológicos/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Neutrófilos/efeitos dos fármacos , Oxazolidinonas/administração & dosagem , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/patologia , Animais , Antígenos Ly/análise , Carga Bacteriana , Citocinas/análise , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Injeções Subcutâneas , Linezolida , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Neutrófilos/imunologia , Peroxidase/análise , Pneumonia Estafilocócica/imunologia , Vancomicina/administração & dosagemRESUMO
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF) and Nuclear factor kappaB (NF-κB) pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of P. aeruginosa into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG). Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on P. aeruginosa infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with P. aeruginosa significantly reduced mortality. Thus, hypoxia reduces P. aeruginosa internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of P. aeruginosa infection.
Assuntos
Células Epiteliais/imunologia , Pneumonia/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Amidas/farmacologia , Aminoácidos Dicarboxílicos/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Células CACO-2 , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Hep G2 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Pneumonia/microbiologia , Pneumonia/prevenção & controle , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/fisiologia , Piridinas/farmacologia , Interferência de RNA , Análise de Sobrevida , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/imunologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
OBJECTIVES: To assess the activity of ceftolozane, a novel oxyimino-cephalosporin, in comparison with ceftazidime and piperacillin/tazobactam against a multidrug-resistant Pseudomonas aeruginosa strain using a murine model of pneumonia. METHODS: Quantitative bacteriology, survival, histological examination, myeloperoxidase activity, proinflammatory cytokine levels in lungs and endothelial permeability were evaluated to determine the effects of ceftolozane and comparators on P. aeruginosa-induced pneumonia. RESULTS: After 48 h of treatment, ceftolozane reduced the bacterial load by 3-4 log(10) cfu/g of lung. Systemic dissemination of the pulmonary infection and development of lung damage were inhibited in all ß-lactam-treated animals. P. aeruginosa-induced pneumonia led to elevated concentrations of tumour necrosis factor-α, interleukin (IL)-1ß and macrophage inflammatory protein (MIP)-2 in the lungs. While the levels of proinflammatory cytokines decreased following ceftazidime and piperacillin/tazobactam therapy, ceftolozane exhibited increased concentrations of IL-1ß and MIP-2 after 24 h of infection, resulted in significantly increased levels of recruited neutrophils within the infected lung without increasing lung endothelial permeability. CONCLUSIONS: These data strongly support ceftolozane as an effective option for the treatment of severe P. aeruginosa respiratory infections by improving the early pulmonary inflammatory response without impairing 48 h post-infection homeostasis.
Assuntos
Anti-Infecciosos/farmacologia , Cefalosporinas/farmacologia , Modelos Animais de Doenças , Pneumonia Bacteriana/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Doença Aguda , Animais , Anti-Infecciosos/uso terapêutico , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Cefalosporinas/uso terapêutico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Testes de Sensibilidade Microbiana/métodos , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Resultado do TratamentoRESUMO
TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was independent of TNF signaling. Notably, NF-κB signaling through IκB kinase ß protected crypt IECs but did not protect villus IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium and death. Consistent with direct activation of the TLR3-TRIF-caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3, type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that dsRNA activation of the TLR3-TRIF-caspase 8 signaling pathway in IECs has a significant impact on the structure and function of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral pathogens.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Caspase 8/fisiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Poli I-C/toxicidade , Receptor 3 Toll-Like/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Viral/toxicidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/deficiênciaRESUMO
Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and â¼500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-ß production, and investigated the importance of IFN-ß production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-ß through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-ß and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-ß production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , RNA Helicases DEAD-box/fisiologia , Interferon beta/biossíntese , Mucosa Intestinal/imunologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Rotavirus/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Linhagem Celular , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/deficiência , Células HT29 , Humanos , Helicase IFIH1 Induzida por Interferon , Interferon beta/fisiologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/virologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , RNA Helicases/genética , RNA Helicases/fisiologia , RNA Viral/biossíntese , RNA Viral/genética , Receptores de Superfície Celular , Receptores Imunológicos , Elementos de Resposta/imunologia , Rotavirus/genética , Transdução de Sinais/genética , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.
Assuntos
Células Epiteliais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/fisiologia , Antracenos/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Expressão Gênica , Células HT29 , Humanos , Imidazóis/farmacologia , Interleucina-8/metabolismo , Intestinos/patologia , Antígeno 96 de Linfócito/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/fisiologia , TransfecçãoRESUMO
BACKGROUND: Epidemiological studies have revealed a protective effect of NSAIDs, which principally target cyclooxygenase (COX)-1 and COX-2, on the development of colorectal cancer. Increased expression of COX-2 was shown in colorectal adenocarcinoma. However, some effects were shown to be COX-independent. Here, we compared two selective COX-2 inhibitors for their effect on the growth of colorectal tumour cells in vitro. MATERIALS AND METHODS: Fifteen tumour cell lines were characterized for COX-1 and COX-2 expression by Western blot and RT-PCR. The effect of celecoxib and rofecoxib on their growth was assessed by staining of DNA with crystal violet. RESULTS: COX-2 expression varied among cell lines, whereas COX-1 was always expressed. Rofecoxib displayed a limited dose-related effect on cell proliferation, whereas celecoxib strongly inhibited cell growth at high concentrations. Both effects appeared COX-2-independent. CONCLUSION: Rofecoxib, which is devoid of apoptotic effect at high concentration but efficient at pharmacological concentrations, revealed a potential new mechanism of action of NSAIDs towards colorectal cancer.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Lactonas/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Células CACO-2 , Celecoxib , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Células HT29 , Humanos , Proteínas de Membrana , Fenótipo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.