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1.
Am J Respir Cell Mol Biol ; 71(1): 43-52, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38767348

RESUMO

Mechanical ventilation contributes to the morbidity and mortality of patients in intensive care, likely through the exacerbation and dissemination of inflammation. Despite the proximity of the pleural cavity to the lungs and exposure to physical forces, little attention has been paid to its potential as an inflammatory source during ventilation. Here, we investigate the pleural cavity as a novel site of inflammation during ventilator-induced lung injury. Mice were subjected to low or high tidal volume ventilation strategies for up to 3 hours. Ventilation with a high tidal volume significantly increased cytokine and total protein levels in BAL and pleural lavage fluid. In contrast, acid aspiration, explored as an alternative model of injury, only promoted intraalveolar inflammation, with no effect on the pleural space. Resident pleural macrophages demonstrated enhanced activation after injurious ventilation, including upregulated ICAM-1 and IL-1ß expression, and the release of extracellular vesicles. In vivo ventilation and in vitro stretch of pleural mesothelial cells promoted ATP secretion, whereas purinergic receptor inhibition substantially attenuated extracellular vesicles and cytokine levels in the pleural space. Finally, labeled protein rapidly translocated from the pleural cavity into the circulation during high tidal volume ventilation, to a significantly greater extent than that of protein translocation from the alveolar space. Overall, we conclude that injurious ventilation induces pleural cavity inflammation mediated through purinergic pathway signaling and likely enhances the dissemination of mediators into the vasculature. This previously unidentified consequence of mechanical ventilation potentially implicates the pleural space as a focus of research and novel avenue for intervention in critical care.


Assuntos
Camundongos Endogâmicos C57BL , Cavidade Pleural , Lesão Pulmonar Induzida por Ventilação Mecânica , Animais , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Cavidade Pleural/metabolismo , Cavidade Pleural/patologia , Inflamação/patologia , Inflamação/metabolismo , Camundongos , Respiração Artificial/efeitos adversos , Volume de Ventilação Pulmonar , Macrófagos/metabolismo , Macrófagos/patologia , Trifosfato de Adenosina/metabolismo , Vesículas Extracelulares/metabolismo , Masculino , Citocinas/metabolismo , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Interleucina-1beta/metabolismo
2.
Am J Respir Cell Mol Biol ; 68(2): 140-149, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36150169

RESUMO

Blood-borne myeloid cells, neutrophils and monocytes, play a central role in the development of indirect acute lung injury (ALI) during sepsis and noninfectious systemic inflammatory response syndrome. By contrast, the contribution of circulating myeloid cell-derived extracellular vesicles (EVs) to ALI is unknown, despite acute increases in their numbers during sepsis and systemic inflammatory response syndrome. Here, we investigated the direct role of circulating myeloid-EVs in ALI using a mouse isolated perfused lung system and a human cell coculture model of pulmonary vascular inflammation consisting of lung microvascular endothelial cells and peripheral blood mononuclear cells. Total and immunoaffinity-isolated myeloid (CD11b+) and platelet (CD41+) EVs were prepared from the plasma of intravenous LPS-injected endotoxemic donor mice and transferred directly into recipient lungs. Two-hour perfusion of lungs with unfractionated EVs from a single donor induced pulmonary edema formation and increased perfusate concentrations of RAGE (receptor for advanced glycation end products), consistent with lung injury. These responses were abolished in the lungs of monocyte-depleted mice. The isolated myeloid- but not platelet-EVs produced a similar injury response and the acute intravascular release of proinflammatory cytokines and endothelial injury markers. In the in vitro human coculture model, human myeloid- (CD11b+) but not platelet- (CD61+) EVs isolated from LPS-stimulated whole blood induced acute proinflammatory cytokine production and endothelial activation. These findings implicate circulating myeloid-EVs as acute mediators of pulmonary vascular inflammation and edema, suggesting an alternative therapeutic target for attenuation of indirect ALI.


Assuntos
Lesão Pulmonar Aguda , Vesículas Extracelulares , Pneumonia , Sepse , Humanos , Lipopolissacarídeos/farmacologia , Leucócitos Mononucleares , Células Endoteliais , Pulmão , Lesão Pulmonar Aguda/terapia , Inflamação , Monócitos , Síndrome de Resposta Inflamatória Sistêmica
3.
Am J Respir Crit Care Med ; 204(4): 421-430, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848447

RESUMO

Rationale: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator-induced lung injury. eCypA (extracellular CypA [cyclophilin A]) is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to coronavirus disease (COVID-19). Objectives: To explore the involvement of eCypA in the pathophysiology of ventilator-induced lung injury. Methods: Mice were ventilated with a low or high Vt for up to 3 hours, with or without blockade of eCypA signaling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretching to explore the cellular source of eCypA, and CypA concentrations were measured in BAL fluid from patients with acute respiratory distress syndrome to evaluate the clinical relevance. Measurements and Main Results: High-Vt ventilation in mice provoked a rapid increase in soluble CypA concentration in the alveolar space but not in plasma. In vivo ventilation and in vitro stretching experiments indicated the alveolar epithelium as the likely major source. In vivo blockade of eCypA signaling substantially attenuated physiological dysfunction, macrophage activation, and MMPs (matrix metalloproteinases). Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated concentrations of eCypA within BAL fluid. Conclusions: CypA is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. eCypA represents an exciting novel target for pharmacological intervention.


Assuntos
Anti-Inflamatórios/imunologia , Ciclofilina A/imunologia , Inflamação/imunologia , Respiração Artificial/efeitos adversos , Síndrome do Desconforto Respiratório/imunologia , Mucosa Respiratória/imunologia , Lesão Pulmonar Induzida por Ventilação Mecânica/imunologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Animais , COVID-19/genética , COVID-19/fisiopatologia , Células Cultivadas/efeitos dos fármacos , Ciclofilina A/farmacologia , Humanos , Inflamação/fisiopatologia , Masculino , Camundongos , Modelos Animais , Síndrome do Desconforto Respiratório/fisiopatologia , SARS-CoV-2 , Lesão Pulmonar Induzida por Ventilação Mecânica/genética
4.
Am J Physiol Lung Cell Mol Physiol ; 320(1): L73-L83, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33146567

RESUMO

Despite advances in the pathophysiology of chronic obstructive pulmonary disease (COPD), there is a distinct lack of biochemical markers to aid clinical management. Microvesicles (MVs) have been implicated in the pathophysiology of inflammatory diseases including COPD, but their association to COPD disease severity remains unknown. We analyzed different MV populations in plasma and bronchoalveolar lavage fluid (BALF) taken from 62 patients with mild to very severe COPD (51% male; mean age: 65.9 yr). These patients underwent comprehensive clinical evaluation (symptom scores, lung function, and exercise testing), and the capacity of MVs to be clinical markers of disease severity was assessed. We successfully identified various MV subtype populations within BALF [leukocyte, polymorphonuclear leukocyte (PMN; i.e., neutrophil), monocyte, epithelial, and platelet MVs] and plasma (leukocyte, PMN, monocyte, and endothelial MVs) and compared each MV population to disease severity. BALF neutrophil MVs were the only population to significantly correlate with the clinical evaluation scores including forced expiratory volume in 1 s, modified Medical Research Council dyspnea score, 6-min walk test, hyperinflation, and gas transfer. BALF neutrophil MVs, but not neutrophil cell numbers, also strongly correlated with BODE index. We have undertaken, for the first time, a comprehensive evaluation of MV profiles within BALF/plasma of COPD patients. We demonstrate that BALF levels of neutrophil-derived MVs are unique in correlating with a number of key functional and clinically relevant disease severity indexes. Our results show the potential of BALF neutrophil MVs for a COPD biomarker that tightly links a key pathophysiological mechanism of COPD (intra-alveolar neutrophil activation) with clinical severity/outcome.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Micropartículas Derivadas de Células/patologia , Neutrófilos/patologia , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Índice de Gravidade de Doença , Idoso , Micropartículas Derivadas de Células/metabolismo , Citocinas/metabolismo , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Neutrófilos/metabolismo , Alvéolos Pulmonares/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Testes de Função Respiratória
5.
Biol Reprod ; 102(2): 445-455, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31599921

RESUMO

In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-µg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL.


Assuntos
Pressão Arterial/fisiologia , Lipopolissacarídeos/efeitos adversos , Miométrio/metabolismo , Trabalho de Parto Prematuro/induzido quimicamente , Placenta/metabolismo , Receptores CCR2/metabolismo , Animais , Pressão Arterial/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Inflamação/genética , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Miométrio/efeitos dos fármacos , Trabalho de Parto Prematuro/genética , Trabalho de Parto Prematuro/metabolismo , Parto/efeitos dos fármacos , Parto/genética , Parto/metabolismo , Placenta/efeitos dos fármacos , Gravidez , Receptores CCR2/genética
6.
FASEB J ; 33(5): 6442-6455, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30776316

RESUMO

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1ß but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.


Assuntos
Trifosfato de Adenosina/imunologia , Membrana Celular/imunologia , Vesículas Extracelulares/imunologia , Macrófagos/imunologia , Pneumonia/imunologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Doença Aguda , Trifosfato de Adenosina/genética , Animais , Comunicação Celular/genética , Comunicação Celular/imunologia , Membrana Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Vesículas Extracelulares/genética , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Knockout , Pneumonia/induzido quimicamente , Pneumonia/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética
7.
Thorax ; 74(12): 1120-1129, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31278170

RESUMO

BACKGROUND: Despite advances in understanding the pathophysiology of acute respiratory distress syndrome, effective pharmacological interventions have proven elusive. We believe this is a consequence of existing preclinical models being designed primarily to explore biological pathways, rather than predict treatment effects. Here, we describe a mouse model in which both therapeutic intervention and ventilation were superimposed onto existing injury and explored the impact of ß-agonist treatment, which is effective in simple models but not clinically. METHODS: Mice had lung injury induced by intranasal lipopolysaccharide (LPS), which peaked at 48 hours post-LPS based on clinically relevant parameters including hypoxaemia and impaired mechanics. At this peak of injury, mice were treated intratracheally with either terbutaline or tumour necrosis factor (TNF) receptor 1-targeting domain antibody, and ventilated with moderate tidal volume (20 mL/kg) to induce secondary ventilator-induced lung injury (VILI). RESULTS: Ventilation of LPS-injured mice at 20 mL/kg exacerbated injury compared with low tidal volume (8 mL/kg). While terbutaline attenuated VILI within non-LPS-treated animals, it was ineffective to reduce VILI in pre-injured mice, mimicking its lack of clinical efficacy. In contrast, anti-TNF receptor 1 antibody attenuated secondary VILI within pre-injured lungs, indicating that the model was treatable. CONCLUSIONS: We propose adoption of a practical framework like that described here to reduce the number of ultimately ineffective drugs reaching clinical trials. Novel targets should be evaluated alongside interventions which have been previously tested clinically, using models that recapitulate the (lack of) clinical efficacy. Within such a framework, outperforming a failed pharmacologic should be a prerequisite for drugs entering trials.


Assuntos
Respiração Artificial/efeitos adversos , Síndrome do Desconforto Respiratório/terapia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Anticorpos Neutralizantes/uso terapêutico , Modelos Animais de Doenças , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Respiração Artificial/métodos , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/fisiopatologia , Terbutalina/uso terapêutico , Volume de Ventilação Pulmonar , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia
9.
Thorax ; 73(4): 350-360, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28389600

RESUMO

RATIONALE: Primary graft dysfunction in lung transplant recipients derives from the initial, largely leukocyte-dependent, ischaemia-reperfusion injury. Intravascular lung-marginated monocytes have been shown to play key roles in experimental acute lung injury, but their contribution to lung ischaemia-reperfusion injury post transplantation is unknown. OBJECTIVE: To define the role of donor intravascular monocytes in lung transplant-related acute lung injury and primary graft dysfunction. METHODS: Isolated perfused C57BL/6 murine lungs were subjected to warm ischaemia (2 hours) and reperfusion (2 hours) under normoxic conditions. Monocyte retention, activation phenotype and the effects of their depletion by intravenous clodronate-liposome treatment on lung inflammation and injury were determined. In human donor lung transplant samples, the presence and activation phenotype of monocytic cells (low side scatter, 27E10+, CD14+, HLA-DR+, CCR2+) were evaluated by flow cytometry and compared with post-implantation lung function. RESULTS: In mouse lungs following ischaemia-reperfusion, substantial numbers of lung-marginated monocytes remained within the pulmonary microvasculature, with reduced L-selectin and increased CD86 expression indicating their activation. Monocyte depletion resulted in reductions in lung wet:dry ratios, bronchoalveolar lavage fluid protein, and perfusate levels of RAGE, MIP-2 and KC, while monocyte repletion resulted in a partial restoration of the injury. In human lungs, correlations were observed between pre-implantation donor monocyte numbers/their CD86 and TREM-1 expression and post-implantation lung dysfunction at 48 and 72 hours. CONCLUSIONS: These results indicate that lung-marginated intravascular monocytes are retained as a 'passenger' leukocyte population during lung transplantation, and play a key role in the development of transplant-associated ischaemia-reperfusion injury.


Assuntos
Transplante de Pulmão , Monócitos/metabolismo , Traumatismo por Reperfusão , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/fisiopatologia , Transplante de Pulmão/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Pneumonia/fisiopatologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Doadores de Tecidos
10.
Biol Reprod ; 98(3): 376-395, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29145579

RESUMO

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.


Assuntos
Miométrio/efeitos dos fármacos , Parto/efeitos dos fármacos , Progesterona/farmacologia , Animais , Quimiocinas/metabolismo , Conexina 43/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Mifepristona/farmacologia , Monócitos/metabolismo , Miométrio/imunologia , Miométrio/metabolismo , Neutrófilos/metabolismo , Parto/imunologia , Parto/metabolismo
11.
Biol Reprod ; 97(2): 258-272, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044422

RESUMO

Sepsis is the leading cause of direct maternal mortality, but there are no data directly comparing the response to sepsis in pregnant and nonpregnant (NP) individuals. This study uses a mouse model of sepsis to test the hypothesis that the cardiovascular response to sepsis is more marked during pregnancy. Female CD1 mice had radiotelemetry probes implanted and were time mated. NP and day 16 pregnant CD-1 mice received intraperitoneal lipopolysaccharide (LPS; 10 µg, serotype 0111: B4). In a separate study, tissue and serum (for RNA, protein and flow cytometry studies), aorta and uterine vessels (for wire myography) were collected after LPS or vehicle control administration. Administration of LPS resulted in a greater fall in blood pressure in pregnant mice compared to NP mice. This occurred with similar changes in the circulating levels of cytokines, vasoactive factors, and circulating leukocytes, but with a greater monocyte and lesser neutrophil margination in the lungs of pregnant mice. Baseline markers of cardiac dysfunction and apoptosis as well as cytokine expression were higher in pregnant mice, but the response to LPS was similar in both groups as was the ex vivo assessment of vascular function. In pregnant mice, nonfatal sepsis is associated with a more marked hypotensive response but not a greater immune response. We conclude that endotoxemia induces a more marked hypotensive response in pregnant compared to NP mice. These changes were not associated with a more marked systemic inflammatory response in pregnant mice, although monocyte lung margination was greater. The more marked hypotensive response to LPS may explain the greater vulnerability to some infections exhibited by pregnant women.


Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Animais , Biomarcadores , Pressão Sanguínea , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Inflamação/induzido quimicamente , Inflamação/patologia , Camundongos , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Crit Care Med ; 45(8): e831-e839, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28426531

RESUMO

OBJECTIVE: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury. DESIGN: In vivo study. SETTING: University research laboratory. SUBJECTS: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls. INTERVENTIONS: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes. MEASUREMENTS AND MAIN RESULTS: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells. CONCLUSIONS: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.


Assuntos
Dieta Hiperlipídica , Obesidade/fisiopatologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Animais , Gasometria , Citocinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Obesidade/epidemiologia , Edema Pulmonar/fisiopatologia , Edema Pulmonar/prevenção & controle , Mecânica Respiratória , Volume de Ventilação Pulmonar , Lesão Pulmonar Induzida por Ventilação Mecânica/epidemiologia
13.
Thorax ; 71(11): 1020-1029, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27287089

RESUMO

BACKGROUND: Microvesicles (MVs) are important mediators of intercellular communication, packaging a variety of molecular cargo. They have been implicated in the pathophysiology of various inflammatory diseases; yet, their role in acute lung injury (ALI) remains unknown. OBJECTIVES: We aimed to identify the biological activity and functional role of intra-alveolar MVs in ALI. METHODS: Lipopolysaccharide (LPS) was instilled intratracheally into C57BL/6 mice, and MV populations in bronchoalveolar lavage fluid (BALF) were evaluated. BALF MVs were isolated 1 hour post LPS, assessed for cytokine content and incubated with murine lung epithelial (MLE-12) cells. In separate experiments, primary alveolar macrophage-derived MVs were incubated with MLE-12 cells or instilled intratracheally into mice. RESULTS: Alveolar macrophages and epithelial cells rapidly released MVs into the alveoli following LPS. At 1 hour, the dominant population was alveolar macrophage-derived, and these MVs carried substantive amounts of tumour necrosis factor (TNF) but minimal amounts of IL-1ß/IL-6. Incubation of these mixed MVs with MLE-12 cells induced epithelial intercellular adhesion molecule-1 (ICAM-1) expression and keratinocyte-derived cytokine release compared with MVs from untreated mice (p<0.001). MVs released in vitro from LPS-primed alveolar macrophages caused similar increases in MLE-12 ICAM-1 expression, which was mediated by TNF. When instilled intratracheally into mice, these MVs induced increases in BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05). CONCLUSIONS: We demonstrate, for the first time, the sequential production of MVs from different intra-alveolar precursor cells during the early phase of ALI. Our findings suggest that alveolar macrophage-derived MVs, which carry biologically active TNF, may play an important role in initiating ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/fisiopatologia , Micropartículas Derivadas de Células/metabolismo , Macrófagos Alveolares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL
14.
Biol Reprod ; 95(6): 125, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27760748

RESUMO

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 µg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta.


Assuntos
Inflamação/imunologia , Leucócitos/imunologia , Lipopolissacarídeos/farmacologia , Miométrio/imunologia , Trabalho de Parto Prematuro/imunologia , Útero/efeitos dos fármacos , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , NF-kappa B/metabolismo , Trabalho de Parto Prematuro/induzido quimicamente , Trabalho de Parto Prematuro/metabolismo , Gravidez , Transdução de Sinais/fisiologia , Útero/imunologia , Útero/metabolismo
15.
Am J Physiol Lung Cell Mol Physiol ; 309(7): L639-52, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26254421

RESUMO

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined "interstitial" leukocyte populations during models of inflammatory lung diseases.


Assuntos
Leucócitos , Pulmão , Infiltração de Neutrófilos , Pneumonia Aspirativa , Coloração e Rotulagem/métodos , Animais , Anticorpos/farmacologia , Citometria de Fluxo/métodos , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/metabolismo , Leucócitos/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Pneumonia Aspirativa/metabolismo , Pneumonia Aspirativa/patologia
16.
Am J Physiol Lung Cell Mol Physiol ; 308(9): L912-21, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25770178

RESUMO

Mechanical ventilation, through overdistension of the lung, induces substantial inflammation that is thought to increase mortality among critically ill patients. The mechanotransduction processes involved in converting lung distension into inflammation during this ventilator-induced lung injury (VILI) remain unclear, although many cell types have been shown to be involved in its pathogenesis. This study aimed to identify the profile of in vivo lung cellular activation that occurs during the initiation of VILI. This was achieved using a flow cytometry-based method to quantify the phosphorylation of several markers (p38, ERK1/2, MAPK-activated protein kinase 2, and NF-κB) of inflammatory pathway activation within individual cell types. Anesthetized C57BL/6 mice were ventilated with low (7 ml/kg), intermediate (30 ml/kg), or high (40 ml/kg) tidal volumes for 1, 5, or 15 min followed by immediate fixing and processing of the lungs. Surprisingly, the pulmonary endothelium was the cell type most responsive to in vivo high-tidal-volume ventilation, demonstrating activation within just 1 min, followed by the alveolar epithelium. Alveolar macrophages were the slowest to respond, although they still demonstrated activation within 5 min. This order of activation was specific to VILI, since intratracheal lipopolysaccharide induced a very different pattern. These results suggest that alveolar macrophages may become activated via a secondary mechanism that occurs subsequent to activation of the parenchyma and that the lung cellular activation mechanism may be different between VILI and lipopolysaccharide. Our data also demonstrate that even very short periods of high stretch can promote inflammatory activation, and, importantly, this injury may be immediately manifested within the pulmonary vasculature.


Assuntos
Inflamação/imunologia , Mecanotransdução Celular/imunologia , Alvéolos Pulmonares/imunologia , Respiração Artificial/efeitos adversos , Lesão Pulmonar Induzida por Ventilação Mecânica/imunologia , Animais , Endotélio/citologia , Endotélio/patologia , Ativação Enzimática , Epitélio/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inflamação/patologia , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Alvéolos Pulmonares/patologia , Respiração Artificial/mortalidade , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Volume de Ventilação Pulmonar , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Crit Care Med ; 43(7): 1375-85, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25867908

RESUMO

OBJECTIVES: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α-converting enzyme baseline and inducible activity profiles. DESIGN: Observational clinical study. SETTING: Mixed surgical/medical teaching hospital ICU. PATIENTS: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α-converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients' monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α-converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α-converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α-converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α-converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α-converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide. CONCLUSIONS: Monocyte tumor necrosis factor-α-converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α-converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis.


Assuntos
Proteínas ADAM/fisiologia , Inflamação/sangue , Monócitos/enzimologia , Sepse/sangue , Proteína ADAM17 , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
J Immunol ; 190(8): 4274-82, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23487422

RESUMO

The ability of the alveolar epithelium to prevent and resolve pulmonary edema is a crucial determinant of morbidity and mortality in acute lung injury (ALI). TNF has been implicated in ALI pathogenesis, but the precise mechanisms remain undetermined. We evaluated the role of TNF signaling in pulmonary edema formation in a clinically relevant mouse model of ALI induced by acid aspiration and investigated the effects of TNF p55 receptor deletion, caspase-8 inhibition, and alveolar macrophage depletion on alveolar epithelial function. We found that TNF plays a central role in the development of pulmonary edema in ALI through activation of p55-mediated death signaling, rather than through previously well-characterized p55-mediated proinflammatory signaling. Acid aspiration produced pulmonary edema with significant alveolar epithelial dysfunction, as determined by alveolar fluid clearance (AFC) and intra-alveolar levels of the receptor for advanced glycation end-products. The impairment of AFC was strongly correlated with lung caspase-8 activation, which was localized to type 1 alveolar epithelial cells by flow cytometric analysis. p55-deficient mice displayed markedly attenuated injury, with improved AFC and reduced caspase-8 activity but no differences in downstream cytokine/chemokine production and neutrophil recruitment. Caspase-8 inhibition significantly improved AFC and oxygenation, whereas depletion of alveolar macrophages attenuated epithelial dysfunction with reduced TNF production and caspase-8 activity. These results provide in vivo evidence for a novel role for TNF p55 receptor-mediated caspase-8 signaling, without substantial apoptotic cell death, in triggering alveolar epithelial dysfunction and determining the early pathophysiology of ALI. Blockade of TNF-induced death signaling may provide an effective early-phase strategy for ALI.


Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/toxicidade , Lesão Pulmonar Aguda/metabolismo , Animais , Caspase 8/toxicidade , Morte Celular/imunologia , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/toxicidade , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Edema Pulmonar/imunologia , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/toxicidade , Fator de Necrose Tumoral alfa/metabolismo
19.
Ann Surg ; 259(6): 1235-44, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24263322

RESUMO

OBJECTIVE: Postoperative cognitive decline is emerging as a significant complication of surgery among older adults. Animal models indicate a central role of hippocampal inflammatory responses in the pathophysiology of postoperative cognitive decline. We hypothesized that atorvastatin, shown to exert neuroprotective potential in central nervous system (CNS) disorders, would attenuate neuroinflammation and improve cognitive function in mice after surgery and anesthesia. METHODS: C57BL6 adult mice were pretreated with atorvastatin (250 µg) or vehicle, orally, for 5 days before undergoing unilateral nephrectomy under isoflurane anesthesia. We evaluated behavioral parameters related to cognitive function (fear conditioning and Morris Water Maze) and determined systemic and hippocampal interleukin-1ß levels, postoperatively. Endothelial COX-2 expression, gross NF-κB and microglial (IBA1, CD68) activation, synaptic function (synapsin-1, PSD95, COX-2), heme oxygenase-1, and GSK3ß were also examined. RESULTS: Surgery induced a significant reduction in hippocampal-dependent fear response that was attenuated by treatment with atorvastatin, which also preserved spatial memory on day 7 after surgery. Atorvastatin evoked significant protection from hippocampal interleukin-1ß production, but not systemic interleukin-1ß production, accompanied by a marked reduction in hippocampal endothelial COX-2, NF-κB activation and decreased microglial reactivity. Surgery triggered an acute decline in synapsin-1, paralleled by an increase in postsynaptic COX-2 that was partially attenuated by atorvastatin. Furthermore, phosphorylation and inactivation of neuronal GSK3ß was significantly enhanced after atorvastatin treatment. CONCLUSIONS: These findings indicate that cognitive decline is very likely associated with synaptic pathology after systemic and central inflammation induced by peripheral surgery/isoflurane anesthesia and suggest that the anti-inflammatory and neuroprotective properties of atorvastatin provide a rationale for its use as a therapeutic strategy for postoperative cognitive decline.


Assuntos
Transtornos Cognitivos/tratamento farmacológico , Cognição/efeitos dos fármacos , Ácidos Heptanoicos/administração & dosagem , Memória/efeitos dos fármacos , Nefrectomia/efeitos adversos , Pirróis/administração & dosagem , Recuperação de Função Fisiológica/efeitos dos fármacos , Administração Oral , Animais , Atorvastatina , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias , Resultado do Tratamento
20.
Crit Care Med ; 42(1): e49-57, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23963135

RESUMO

OBJECTIVES: Ventilator-induced lung injury has substantive impact on mortality of patients with acute respiratory distress syndrome. Although low tidal volume ventilation has been shown to reduce mortality, clinical benefits of open-lung strategy are controversial. In this study, we investigated the impact of two distinct forms of ventilator-induced lung injury, i.e., volutrauma and atelectrauma, on the progression of lung injury and inflammation, in particular alveolar and systemic cytokine production. DESIGN: Ex vivo study. SETTING: University research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Isolated, buffer-perfused lungs were allocated to one of three ventilatory protocols for 3 hours: control group received low tidal volume (7 mL/kg) with positive end-expiratory pressure (5 cm H2O) and regular sustained inflation; high-stretch group received high tidal volume (30-32 mL/kg) with positive end-expiratory pressure (3 cm H2O) and sustained inflation; and atelectasis group received the same tidal volume as control but neither positive end-expiratory pressure nor sustained inflation. MEASUREMENTS AND MAIN RESULTS: Both injurious ventilatory protocols developed comparable levels of physiological injury and pulmonary edema, measured by respiratory system mechanics and lavage fluid protein. High-stretch induced marked increases in proinflammatory cytokines in perfusate and lung lavage fluid, compared to control. In contrast, atelectasis had no effect on perfusate cytokines compared to control but did induce some up-regulation of lavage cytokines. Depletion of monocytes marginated within the lung microvasculature, achieved by pretreating mice with i.v. liposome-encapsulated clodronate, significantly attenuated perfusate cytokine levels, especially tumor necrosis factor, in the high-stretch, but not atelectasis group. CONCLUSIONS: Volutrauma (high-stretch), but not atelectrauma (atelectasis), directly activates monocytes within the pulmonary vasculature, leading to cytokine release into systemic circulation. We postulate this as a potential explanation why open-lung strategy has limited mortality benefits in ventilated critically ill patients.


Assuntos
Citocinas/fisiologia , Monócitos/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/imunologia , Animais , Citocinas/biossíntese , Edema/etiologia , Edema/imunologia , Edema/fisiopatologia , Citometria de Fluxo , Pulmão/citologia , Pulmão/imunologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Respiração com Pressão Positiva/efeitos adversos , Respiração com Pressão Positiva/métodos , Volume de Ventilação Pulmonar/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia
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