Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Divisão de Moléstias InfecciosasUNIFESP, EPM, Divisão de Moléstias InfecciosasFAPESP: 2006/58744-1SciEL

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Disciplina de InfectologiaMax-Planck-Institute for ImmunobiologyUNIFESP, Disciplina de InfectologiaFAPESP: 2006/58744-1SciEL

Increased Percentages of T Helper Cells Producing IL-17 and Monocytes Expressing Markers of Alternative Activation in Patients with Sepsis

BACKGROUND: A shift from Th1 to Th2 as well as an increase in Treg CD4+T cell subsets has been reported in septic patients (SP). Furthermore, these patients display modulation of monocyte function, with reduced production of pro-inflammatory cytokines upon LPS stimulus, which resembles the phenotype of alternatively activated macrophages. In this study, we evaluated the percentages of T cells differentiated into Th1, Th17 and Treg subsets, as well as the percentage of monocytes expressing markers of alternatively activated monocytes/macrophages (AAM) in SP. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMC) were obtained from 32 healthy volunteers (HV) and from SP at admission (D0, n = 67) and after 7 days of therapy (D7, n = 33). Th1 and Th17 (CD3+CD8-) lymphocytes were identified by the intracellular detection of IFN-γ and IL-17, respectively, spontaneously and after PMA/Io stimulation, and Treg cells were identified by Foxp3+CD127- expression. Monocytes were evaluated for CD206 and CD163 expression. Absolute numbers of CD4+T lymphocytes were measured in whole blood samples by flow cytometry. The Mann-Whitney or Wilcoxon test was applied, as appropriate. The percentage of Th1 cells was lower in SP than in HV at admission after PMA/Io stimulation, whereas the percentage of Th17 cells was higher. In patients' follow-up samples, a higher percentage of Th1 cells and a lower percentage of Th17 cells were observed on D7 compared with the D0 samples. Treg cells remained unchanged. Septic patients showed a markedly increased proportion of monocytes expressing CD163 and CD206. CONCLUSIONS/SIGNIFICANCE: Upon in vitro stimulus, the percentage of T helper lymphocytes producing IL-17 was higher in SP than in HV at admission, and the percentage producing IFN-γ was lower, a pattern that was reversed during follow-up. The increased expression of CD163 and CD206 indicates that monocytes may acquire the AAM phenotype during sepsis

Characterization of an animal model of severe sepsis associated with respiratory dysfunction

PURPOSE: Pathophysiological studies in humans regarding sepsis are difficult to perform due to ethical and methodological concerns. In this context, animal models of sepsis can be useful to better understand this condition and to test therapeutic strategies. The purpose of this study was to characterize a feasible and clinically relevant model of sepsis in pigs that could be useful for testing different therapeutic interventions. METHODS: 5 White Large pigs were anesthetized, arterial and pulmonary catheters were introduced, and sepsis was induced by fecal peritonitis. Several biochemical indicators of organ dysfunction and infectious parameters were measured. The pigs were monitored until death, when fragments of organs were removed for pathology. Three animals without peritonitis served as controls and were sacrificed 24 hours after surgery without developing significant changes in organ function. RESULTS: Septic pigs survived 17 hours on average (range, 16-18 h), and Escherichia coli was recovered from blood cultures. They developed a significant decrease in left ventricular work and a nonsignificant reduction in mixed venous oxygen saturation. Respiratory dysfunction was characterized by a decrease in the PaO2/FiO2 ratio and respiratory compliance. Pathology of the lungs revealed areas of pulmonary collapse, hemorrhage, pulmonary congestion, and discrete neutrophil infiltrate. CONCLUSIONS: Fecal peritonitis in pigs is a clinically relevant model of sepsis associated with acute lung injury without direct pulmonary insult. This model may prove to be useful for studying pathogenic aspects of secondary lung injury as well as for validating ventilatory or pharmacologic interventions.PROPOSTA: Estudos sobre sepse envolvendo sua fisiopatologia são difíceis de serem realizados devido a razões éticas e metodológicas. Neste sentido, modelos animais criam oportunidades de estudos para entender a fisiopatologia e testar estratégias terapêuticas. O objetivo deste estudo foi criar um modelo relevante de choque séptico em porcos para testar e entender diferentes intervenções. MÉTODOS: 5 porcos da raça "White Large" foram anestesiados e monitorizados com uma linha arterial e um cateter de artéria pulmonar. Uma peritonite fecal foi induzida através de laparotomia. Marcadores de disfunções orgânicas e infecciosos foram mensurados. Todos porcos evoluíram até a morte e amostras de órgãos foram coletadas para exame anátomo patológico. Três animais controles com o mesmo preparo cirúrgico e sem peritonite foram sacrificados após 24 horas de evolução, sem desenvolver mudanças significativas nas funções orgânicas. RESULTADOS: Os animais séptico sobreviveram na média 17 horas (16 - 18h), e Escherichia coli foi cultivada nas amostras de sangue. Os animais sépticos evoluíram com redução do trabalho de ventrículo esquerdo. A disfunção respiratória foi caracterizada por uma redução na relação PaO2/FiO2 e na complacência respiratória. A anatomia patológica dos pulmões revelou colapso pulmonar, hemorragia, congestão e infiltrado neutrofílico. CONCLUSÕES: A peritonite fecal em porcos é um modelo de choque séptico clinicamente relevante e associada a uma lesão pulmonar sem um insulto direto. Este é um modelo que pode ser utilizado para estudar aspectos fisiopatológicos das lesões pulmonares secundárias, assim como para estudar intervenções ventilatórias ou farmacológicas

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood

Surfacen (R) is a clinical surfactant preparation of porcine origin. in the present study, we have evaluated the effect of Surfacen (R) in the modulation of oxidative burst in monocytes and neutrophils in human blood and proinflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen (R) added to whole blood monocytes and neutrophils. the time course experiments of pre-incubation with Surfacen (R) showed no significant increase of ROS level at 2 h; however, the ROS level decreased when pre incubated for 4 h and 6 h with Surfacen (R). Pre-incubation of PBMC cells with Surfacen (R) at 0.125 and 0.5 mg/mL showed a dose-dependent suppression of TNF-alpha levels measured after 4 h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24 h. A similar behavior was observed in IL-6 release. in summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen (R) in human monocytes and neutrophils in vitro. (C) 2014 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Ctr Nacl Sanidad Agr CENSA, San Jose de Las Lajas, Mayabeque, CubaUniversidade Federal de São Paulo, Escola Paulista Med, Hosp São Paulo, Div Infect Dis, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Hosp São Paulo, Div Infect Dis, São Paulo, BrazilCAPES: AUX-PE-MESCUBA: 103/10Web of Scienc

Cytokine Kinetics in Febrile Neutropenic Children: Insights on the Usefulness as Sepsis Biomarkers, Influence of Filgrastim, and Behavior of the IL-23/IL-17 Pathway

Background. The study aimed to describe the kinetics of various cytokines from day 1 to day 14 of the onset of fever in neutropenic children and to evaluate their performances as discriminators of sepsis in the first 24 hours of fever, the possible influence of filgrastim, and the functioning of the IL-23/IL-17 axis. Methods. IL-1 beta, TNF-alpha, IL-10, IL-12/23p40, IL-21, IL-6, IL-8, IL-17, G-CSF, and GM-CSF were measured in plasma on days 1, 2, 3, 5, and 14 from the onset of fever in 35 patients. Results. Thirteen patients (37.1%) developed sepsis. In mixed models, IL-6, IL-8, IL-10, and G-CSF showed higher estimated means in septic patients (P < 0 005), and IL-12/23p40 and IL-17 in nonseptic patients (P < 0 05). On day 1, IL-6, IL-8, and IL-10 appeared upregulated in patients who received filgrastim. Only IL-6, IL-8, IL-10, and procalcitonin were useful as discriminators of sepsis. Associating the markers with each other or to a risk assessment model improved performance. Conclusions. Cytokines kinetics showed proinflammatory and anti-inflammatory responses similar to what is described in nonneutropenic patients. IL-8, IL-6, IL-10, and procalcitonin are useful as early biomarkers of sepsis. Filgrastim upregulates expression of these markers, and we observed deficiency in the IL-23-IL-17 axis accompanying sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)GRAACC/Instituto de Oncologia PediatricaSao Paulo Fed Univ UNIFESP, Grp Apoio Adolescente & Crianca Canc GRAACC, IOP, Rua Pedro de Toledo 572, BR-04039001 Sao Paulo, SP, BrazilSao Paulo Fed Univ UNIFESP, Div Infect Dis, Dept Med, Escola Paulista Med, Rua Pedro de Toledo 669,10th Floor, BR-04039001 Sao Paulo, SP, BrazilSao Paulo Fed Univ UNIFESP, Grp Apoio Adolescente & Crianca Canc GRAACC, IOP, Rua Pedro de Toledo 572, BR-04039001 Sao Paulo, SP, BrazilSao Paulo Fed Univ UNIFESP, Div Infect Dis, Dept Med, Escola Paulista Med, Rua Pedro de Toledo 669,10th Floor, BR-04039001 Sao Paulo, SP, BrazilFAPESP: 2011/20401-4Web of Scienc