RESUMO
BACKGROUND: The term sepsis is used to designate a systemic condition of infection and inflammation associated with hemodynamic changes that result in organic dysfunction. Gestational sepsis can impair the development of the central nervous system and may promote permanent behavior alterations in the offspring. The aim of our work was to evaluate the effects of maternal sepsis on inflammatory cytokine levels and synaptic proteins in the hippocampus, neocortex, frontal cortex, and cerebellum of neonatal, young, and adult mice. Additionally, we analyzed the motor development, behavioral features, and cognitive impairments in neonatal, young and adult offspring. METHODS: Pregnant mice at the 14th embryonic day (E14) were intratracheally instilled with saline 0.9% solution (control group) or Klebsiella spp. (3 × 108 CFU) (sepsis group) and started on meropenem after 5 h. The offspring was sacrificed at postnatal day (P) 2, P8, P30, and P60 and samples of liver, lung, and brain were collected for TNF-α, IL-1ß, and IL-6 measurements by ELISA. Synaptophysin, PSD95, and ß-tubulin levels were analyzed by Western blot. Motor tests were performed at all analyzed ages and behavioral assessments were performed in offspring at P30 and P60. RESULTS: Gestational sepsis induces a systemic pro-inflammatory response in neonates at P2 and P8 characterized by an increase in cytokine levels. Maternal sepsis induced systemic downregulation of pro-inflammatory cytokines, while in the hippocampus, neocortex, frontal cortex, and cerebellum an inflammatory response was detected. These changes in the brain immunity were accompanied by a reduction of synaptophysin and PSD95 levels in the hippocampus, neocortex, frontal cortex, and cerebellum, in all ages. Behavioral tests demonstrated motor impairment in neonates, and depressive-like behavior, fear-conditioned memory, and learning impairments in animals at P30 and P60, while spatial memory abilities were affected only at P60, indicating that gestational sepsis not only induces an inflammatory response in neonatal mouse brains, but also affects neurodevelopment, and leads to a plethora of behavioral alterations and cognitive impairments in the offspring. CONCLUSION: These data suggest that maternal sepsis may be causatively related to the development of depression, learning, and memory impairments in the litter.
Assuntos
Encéfalo/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Sepse/imunologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Disfunção Cognitiva/etiologia , Feminino , Inflamação , Camundongos , Atividade Motora/fisiologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Sepse/complicações , Sinapses/metabolismoRESUMO
Dengue is characterized as one of the most important arthropod-borne human viral diseases, representing a public health problem. Increased activation of immune cells is involved in the progression of infection to severe forms. Recently, our group demonstrated the contribution of platelet-monocyte interaction to inflammatory responses in dengue, adding to evolving evidence that platelets have inflammatory functions and can regulate different aspects of innate immune responses. Furthermore, stimuli-specific-activated platelets can promote phenotypic changes and metabolic reprogramming in monocytes. Thus, this study aimed to evaluate the roles of dengue virus (DENV)-activated platelets on immunometabolic reprogramming of monocytes in vitro, focusing on lipid droplet (LD) biogenesis. We demonstrated that platelets exposed to DENV in vitro form aggregates with monocytes and signal to LD formation and CXCL8/IL-8, IL-10, CCL2, and PGE2 secretion. Pharmacologic inhibition of LD biogenesis prevents PGE2 secretion, but not CXCL8/IL-8 release, by platelet-monocyte complexes. In exploring the mechanisms involved, we demonstrated that LD formation in monocytes exposed to DENV-activated platelets is partially dependent on platelet-produced MIF. Additionally, LD formation is higher in monocytes, which have platelets adhered on their surface, suggesting that beyond paracrine signaling, platelet adhesion is an important event in platelet-mediated modulation of lipid metabolism in monocytes. Together, our results demonstrate that activated platelets aggregate with monocytes during DENV infection and signal to LD biogenesis and the secretion of inflammatory mediators, which may contribute to dengue immunopathogenesis.
Assuntos
Plaquetas/imunologia , Citocinas/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Gotículas Lipídicas/imunologia , Monócitos/imunologia , Transdução de Sinais/imunologia , Plaquetas/patologia , Dengue/patologia , Feminino , Humanos , Gotículas Lipídicas/patologia , Masculino , Monócitos/patologiaRESUMO
BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Heme Oxigenase-1/farmacologia , Malária/complicações , Proteínas de Membrana/farmacologia , Substâncias Protetoras/farmacologia , Trombocitopenia/tratamento farmacológico , Lesão Pulmonar Aguda/etiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Feminino , Contagem de Leucócitos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei/fisiologia , Trombocitopenia/etiologiaRESUMO
Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.
Assuntos
Antígenos CD11/metabolismo , Cadeias alfa de Integrinas/metabolismo , Malária/fisiopatologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Edema Encefálico/fisiopatologia , Antígenos CD11/fisiologia , Cloroquina/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Cadeias alfa de Integrinas/fisiologia , Integrinas/imunologia , Integrinas/metabolismo , Contagem de Leucócitos , Leucócitos/metabolismo , Leucócitos/fisiologia , Macrófagos/metabolismo , Malária/genética , Malária Cerebral/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmodium berghei/metabolismoRESUMO
HIV-infected subjects under virological control still exhibit a persistent proinflammatory state. Thus, chronic HIV infection changes the host homeostasis towards an adapted immune response that may affect the outcome of coinfections. However, little is known about the impact of HIV infection on inflammatory amplification and clinical presentation in dengue. Platelets have been shown to participate in immune response in dengue and HIV. We hypothesized that altered platelet responses in HIV-infected subjects may contribute to altered inflammatory milieu and disease progression in dengue. We prospectively followed a cohort of 84 DENV-infected patients of whom 29 were coinfected with HIV under virological control. We report that dengue and HIV coinfection progress with reduced inflammation and milder disease progression with lower risk of vascular instability. Even though the degree of thrombocytopenia and platelet activation were similar between dengue-infected and HIV plus dengue-coinfected patients, plasma levels of the platelet-derived chemokines RANTES/CCL5 and PF4/CXCL4 were lower in coinfection. Consistently, platelets from coinfected patients presented defective secretion of the stored-chemokines PF4 and RANTES, but not newly synthesized IL-1ß, when cultured ex vivo. These data indicate that platelets from HIV-infected subjects release lower levels of chemokines during dengue illness, which may contribute to milder clinical presentation during coinfection.
Assuntos
Plaquetas/metabolismo , Coinfecção/imunologia , Vírus da Dengue/genética , Dengue/imunologia , Infecções por HIV/imunologia , HIV-1 , Ativação Plaquetária/imunologia , Adulto , Antirreumáticos/uso terapêutico , Quimiocina CCL5/sangue , Coinfecção/virologia , Dengue/virologia , Feminino , Seguimentos , Genoma Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Inflamação/imunologia , Interleucina-1beta/sangue , Masculino , Pessoa de Meia-Idade , Fator Plaquetário 4/sangue , Estudos ProspectivosRESUMO
Cardiovascular diseases and thrombotic events became major clinical problems in the combined antiretroviral therapy (cART) era. Although the precise mechanisms behind these clinical problems have not been fully elucidated, a persistent pro-inflammatory state plays a central role. As platelets play important roles on both, thrombus formation and inflammatory/immune response, we aimed at investigating platelet function in HIV-infected subjects virologically controlled through cART. We evaluate parameters of activation, mitochondrial function and activation of apoptosis pathways in platelets from 30 HIV-infected individuals under stable cART and 36 healthy volunteers. Despite viral control achieved through cART, HIV-infected individuals exhibited increased platelet activation as indicated by P-selectin expression and platelet spreading when adhered on fibrinogen-coated surfaces. Platelets from HIV-infected subjects also exhibited mitochondrial dysfunction and activation of apoptosis pathways. Finally, thrombin stimuli induced lower levels of P-selectin translocation and RANTES secretion, but not TXA2 synthesis, in platelets from HIV-infected individuals compared to control; and labeling of platelet alpha granules showed reduced granule content in platelets from HIV-infected individuals when compared to healthy subjects. In summary, platelets derived from HIV-infected individuals under stable cART exhibit a phenotype of increased activation, activation of the intrinsic pathway of apoptosis and undermined granule secretion in response to thrombin.
Assuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Doenças Cardiovasculares/sangue , Infecções por HIV/tratamento farmacológico , Inflamação/sangue , Trombose/sangue , Adulto , Apoptose/efeitos dos fármacos , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Quimiocina CCL5/genética , Feminino , HIV/patogenicidade , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , Voluntários Saudáveis , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Monócitos , Selectina-P/genética , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Agregação Plaquetária/efeitos dos fármacos , Trombina/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/virologiaRESUMO
ß2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDß2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDß2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDß2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDß2 in bacterial infection.
Assuntos
Antígenos CD11/metabolismo , Antígenos CD18/metabolismo , Cadeias alfa de Integrinas/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/imunologia , Contagem de Leucócitos , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Piroptose/imunologia , Infecções por Salmonella/microbiologiaRESUMO
Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.
Assuntos
Plaquetas/imunologia , Vírus da Dengue/fisiologia , Dengue/imunologia , Proteoma/imunologia , Adulto , Plaquetas/química , Estudos de Coortes , Dengue/sangue , Dengue/genética , Dengue/virologia , Vírus da Dengue/imunologia , Feminino , Humanos , Masculino , Ativação Plaquetária , Proteoma/genéticaRESUMO
INTRODUCTION: Molecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18F-fluoro-2-deoxyglucose (18F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14C-2DG uptake in activated neutrophils. METHODS: Lung uptake of 18F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24h following the intraperitoneal injection of 10mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. RESULTS: Significant uptake of 18F-FDG occurred by 2h following LPS, and progressively increased to 24h. Lung uptake of 18F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. CONCLUSION: Systemic endotoxin-induced ALI results in very early and progressive uptake of 18F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: 18F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14C-2DG uptake in activated neutrophils. 18F-FDG PET may provide a potential mechanism for early diagnosis and therapeutic assessment of ALI/ARDS.
Assuntos
Lesão Pulmonar Aguda/diagnóstico por imagem , Lesão Pulmonar Aguda/imunologia , Fluordesoxiglucose F18 , Lipopolissacarídeos/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Tomografia por Emissão de Pósitrons , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Fluordesoxiglucose F18/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hexoquinase/metabolismo , Pulmão/diagnóstico por imagem , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Masculino , Neutrófilos/citologia , Neutrófilos/imunologia , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/diagnóstico por imagem , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismoRESUMO
BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a potentially lethal complication of clinical malaria. Acute lung injury in MA-ARDS shares features with ARDS triggered by other causes, including alveolar inflammation and increased alveolar-capillary permeability, leading to leak of protein-rich pulmonary oedema fluid. Mechanisms and physiologic alterations in MA-ARDS can be examined in murine models of this syndrome. Integrin αDß2 is a member of the leukocyte, or ß2 (CD18), sub-family of integrins, and emerging observations indicate that it has important activities in leukocyte adhesion, accumulation and signalling. The goal was to perform analysis of the lungs of mice wild type C57Bl/6 (a D (+/+) ) and Knockout C57Bl/6 (a D (-/-) ) with malaria-associated acute lung injury to better determine the relevancy of the murine models and investigate the mechanism of disease. METHODS: C57BL/6 wild type (a D (+/+) ) and deficient for CD11d sub-unit (a D (-/-) ) mice were monitored after infection with 10(5) Plasmodium berghei ANKA. CD11d subunit expression RNA was measured by real-time polymerase chain reaction, vascular barrier integrity by Evans blue dye (EBD) exclusion and cytokines by ELISA. Protein and leukocytes were measured in bronchoalveolar lavage fluid (BALF) samples. Tissue cellularity was measured by the point-counting technique, F4/80 and VCAM-1 expression by immunohistochemistry. Respiratory function was analysed by non-invasive BUXCO and mechanical ventilation. RESULTS: Alveolar inflammation, vascular and interstitial accumulation of monocytes and macrophages, and disrupted alveolar-capillary barrier function with exudation of protein-rich pulmonary oedema fluid were present in P. berghei-infected wild type mice and were improved in αDß2-deficient animals. Key pro-inflammatory cytokines were also decreased in lung tissue from α D (-/-) mice, providing a mechanistic explanation for reduced alveolar-capillary inflammation and leak. CONCLUSIONS: The results indicate that αDß2 is an important inflammatory effector molecule in P. berghei-induced MA-ARDS, and that leukocyte integrins regulate critical inflammatory and pathophysiologic events in this model of complicated malaria. Genetic deletion of integrin subunit αD in mice, leading to deficiency of integrin αDß2, alters lung inflammation and acute lung injury in a mouse model of MA-ARDS caused by P. berghei.
Assuntos
Antígenos CD11/metabolismo , Cadeias alfa de Integrinas/metabolismo , Malária/complicações , Síndrome do Desconforto Respiratório/patologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/análise , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Azul Evans/metabolismo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Contagem de Leucócitos , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade , Plasmodium berghei/crescimento & desenvolvimento , Proteínas/análise , Reação em Cadeia da Polimerase em Tempo Real , Testes de Função RespiratóriaRESUMO
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis. Strategies to block inflammatory mediators such as PAF have been investigated as adjuvant therapies for sepsis. PAF-AH, the enzyme responsible for PAF degradation, showed positive results in pre-clinical studies and phase II clinical trials, but the results of a phase III study were disappointing. In this study, we investigated the potential protective mechanism of PAF-AH in sepsis using the murine model of cecal ligation and puncture (CLP). Treatment with rPAF-AH increased peritoneal fluid levels of the anti-inflammatory mediators MCP-1/CCL2 after CLP. The numbers of bacteria (CFU) in the peritoneal cavity were decreased in the rPAF-AH-treated group, indicating more efficient bacterial clearance after rPAF-AH treatment. Interestingly, we observed increased levels of nitric oxide (NO) after PAF-AH administration, and rPAF-AH treatment did not decrease CFU numbers either in iNOS-deficient mice or in CCR2-deficient mice. We concluded that administration of exogenous rPAF-AH reduced inflammatory injury, altered cytokine levels and favored bacterial clearance with a clear impact on mortality through modulation of MCP-1/CCL2 and NO levels in a clinically relevant sepsis model.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/administração & dosagem , Sepse/tratamento farmacológico , Sepse/microbiologia , Animais , Quimiocina CCL2/biossíntese , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Cavidade Peritoneal/microbiologia , Proteínas Recombinantes/administração & dosagem , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Salmonella typhimurium , Sepse/metabolismoRESUMO
Bacterial clearance is one of the most important beneficial consequences of the innate immune response. Chemokines are important mediators controlling leukocyte trafficking and activation, whereas reactive oxygen and nitrogen species are effectors in bacterial killing. In the present work, we used in vivo and in vitro models of infections to study the role of monocyte chemoattractant protein 1 (MCP-1)/CCL2 and nitric oxide (NO) in the bacterial clearance in sepsis. Our results show that MCP-1/CCL2 and NO levels are increased in the peritoneal cavity of mice 6 h after sepsis induced by cecal ligation and puncture. Pretreatment with anti-MCP-1/CCL2 monoclonal antibodies increased the number of colony-forming units (CFUs) recovered in the peritoneal lavage fluid. Moreover, CFU counts were increased in the peritoneal fluid of CCR2 mice subjected to cecal ligation and puncture. In vitro stimulation of peritoneal macrophages with recombinant MCP-1/CCL2 reduced CFU counts in the supernatant after challenge with Escherichia coli. Conversely, treatment with anti-MCP-1/CCL2 increased CFU counts under the same experimental condition. Stimulation of cultured macrophages with MCP-1/CCL2 and interferon had a synergistic effect on NO production. Macrophages from CCL2 mice showed a consistent decrease in NO production when compared with wild-type controls after stimulation with LPS + interferon. Finally, we showed incubation of macrophages with E. coli, and the ERK inhibitor U0126 increased CFU numbers and decreased intracellular levels of NO. In conclusion, we demonstrated for the first time that MCP-1/CCL2 has a crucial role in the clearance of bacteria by mechanisms involving increased expression of inducible NO synthase and production of NO by ERK signaling pathways.
Assuntos
Infecções Bacterianas/imunologia , Quimiocina CCL2/imunologia , Óxido Nítrico/imunologia , Sepse/imunologia , Animais , Líquido Ascítico/microbiologia , Infecções Bacterianas/microbiologia , Butadienos/farmacologia , Células Cultivadas , Quimiocina CCL2/deficiência , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Nitrilas/farmacologia , Fagocitose/imunologia , Proteínas Recombinantes/imunologia , Sepse/microbiologiaRESUMO
Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Mediadores da Inflamação/uso terapêutico , Lovastatina/uso terapêutico , Malária Cerebral/tratamento farmacológico , Animais , Encéfalo/imunologia , Antígeno CD11b/efeitos dos fármacos , Antígeno CD11b/genética , Quimiocinas/sangue , Cloroquina/uso terapêutico , Transtornos Cognitivos/complicações , Transtornos Cognitivos/parasitologia , Citocinas/sangue , Edema/tratamento farmacológico , Endotélio/efeitos dos fármacos , Endotélio/imunologia , Endotélio/parasitologia , Heme Oxigenase-1/efeitos dos fármacos , Heme Oxigenase-1/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamação/tratamento farmacológico , Mediadores da Inflamação/farmacologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/parasitologia , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/imunologia , RNA Mensageiro/efeitos dos fármacosRESUMO
Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.
Assuntos
Transtornos Cognitivos/patologia , Transtornos Cognitivos/prevenção & controle , Malária Cerebral/complicações , Malária Cerebral/parasitologia , Plasmodium berghei/efeitos dos fármacos , Plasmodium yoelii/efeitos dos fármacos , Animais , Antimaláricos/uso terapêutico , Antioxidantes/uso terapêutico , Comportamento Animal , Combinação de Medicamentos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Parasitemia/patologia , Parasitemia/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Taxa de SobrevidaRESUMO
Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAF- acetylhydrolases (PAF-AHs) terminate PAF's signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0=0.001+/-0.001 nmol/ml/min/g vs Time 6=0.031+/-0.018 nmol/ml/min/g, p=0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/biossíntese , Pulmão/enzimologia , Síndrome do Desconforto Respiratório/enzimologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Animais , Líquido da Lavagem Broncoalveolar/química , Feminino , Imuno-Histoquímica , Cinética , Pulmão/metabolismo , Ácido Oleico , Síndrome do Desconforto Respiratório/induzido quimicamente , Suínos , Fatores de TempoRESUMO
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis and in those with systemic inflammatory response syndrome. Strategies to block inflammatory mediators, often with complicated outcomes, are currently being investigated as new adjuvant therapies for sepsis. Here, we determined if administration of recombinant platelet-activating factor (rPAF)-acetylhydrolase (rPAF-AH), an enzyme that inactivates PAF and PAF-like lipids, protects mice from inflammatory injury and death after administration of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Administration of rPAF-AH increased plasma PAF-AH activity and reduced mortality in both models. Treatment with rPAF-AH increased peritoneal fluid levels of monocyte chemoattractant protein 1/CCL-2 and decreased interleukin 6 and migration inhibitory factor levels after LPS administration or CLP. Administration of a broad-spectrum antibiotic together with rPAF-AH was more protective than single treatment with either of these agents. The combined treatment was associated with reduced interleukin 6 levels in mice subjected to CLP. We observed acute decreases in plasma PAF-AH activity in mice subjected to CLP or challenged with LPS and in human patients with sepsis. We conclude that alterations in the endogenous PAF-AH contribute to the pathophysiology of sepsis and that administration of exogenous rPAF-AH reduces inflammatory injury and mortality in models relevant to the clinical syndrome. Variations in endogenous PAF-AH activity may potentially account for variable responses to exogenous rPAF-AH in previous clinical trials. Serial measurements of plasma PAF-AH activity in murine models demonstrate dynamic regulation of the endogenous enzyme, potentially explaining the variations in human subjects.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/administração & dosagem , Fator de Ativação de Plaquetas/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Citocinas/sangue , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Síndrome de Resposta Inflamatória Sistêmica/sangueRESUMO
Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/fisiologia , Plaquetas/enzimologia , Inflamação/metabolismo , Fator de Ativação de Plaquetas/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Regulação da Expressão Gênica , Fosfolipases A2 , Fator de Ativação de Plaquetas/química , Polimorfismo GenéticoRESUMO
Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.
Assuntos
Animais , /fisiologia , Plaquetas/enzimologia , Inflamação/metabolismo , Fator de Ativação de Plaquetas/fisiologia , /química , /genética , Regulação da Expressão Gênica , Polimorfismo Genético , Fator de Ativação de Plaquetas/químicaRESUMO
Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.
Assuntos
Corpos de Inclusão/metabolismo , Leucócitos/metabolismo , Lipoproteínas LDL/metabolismo , Peroxissomos/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/fisiologia , Ciclo-Oxigenase 2 , Sinergismo Farmacológico , Feminino , Humanos , Isoenzimas/metabolismo , Leucócitos/fisiologia , Lipoproteínas LDL/administração & dosagem , Lipoproteínas LDL/farmacologia , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/fisiologia , Fosfolipídeos/metabolismo , Fator de Ativação de Plaquetas/administração & dosagem , Fator de Ativação de Plaquetas/metabolismo , Cavidade Pleural/citologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Cavidade Torácica , Fatores de Transcrição/metabolismoRESUMO
Oxidized low-density lipoprotein (LDL) contains inflammatory agents, including oxidatively fragmented phospholipids that activate the platelet-activating factor (PAF) receptor, but in vivo events caused by these pathologically generated agents are not well defined. Injection of PAF-like lipids derived from oxidized LDL, or C(4)-PAF that is a major PAF-like lipid in these particles, into the pleural cavity of mice resulted in rapid monocyte, neutrophil, and eosinophil accumulation. Increased numbers of intracellular lipid bodies in these cells show they were in an inflammatory environment. Leukocyte recruitment was abolished by a PAF receptor antagonist, as expected. PAF-like lipids induced 5-lipoxygenase expression in leukocytes, mRNA expression for monocyte chemoattractant protein-1 (MCP-1) and other chemokines, synthesis of MCP-1, and leukotriene B(4). The 5-lipoxygenase inhibitor zileuton impaired neutrophil influx, while MCP-1 had a more global role, as determined with MCP-1(-/-) mice. The lack of MCP-1 abrogated leukocyte accumulation and lipid body formation both in vivo and in vitro and chemokine transcription in vivo, and reduced in vivo leukotriene B(4) production. Thus, PAF-like phospholipids in oxidized LDL induce an inflammatory infiltrate through the PAF receptor, chemokine transcription, lipid body formation, and 5-lipoxygenase expression in leukocytes. MCP-1 has a key role in this inflammatory response, and 5-lipoxygenase products are essential for neutrophil recruitment into the inflamed pleural cavity.