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

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

Patterns of Gene Expression in Peripheral Blood Mononuclear Cells and Outcomes from Patients with Sepsis Secondary to Community Acquired Pneumonia

Mechanisms governing the inflammatory response during sepsis have been shown to be complex, involving cross-talk between diverse signaling pathways. Current knowledge regarding the mechanisms underlying sepsis provides an incomplete picture of the syndrome, justifying additional efforts to understand this condition. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. in this study, we investigate whole-genome expression profiles in mononuclear cells from survivors (n = 5) and non-survivors (n = 5) of sepsis. To circumvent the heterogeneity of septic patients, only patients admitted with sepsis caused by community-acquired pneumonia were included. Blood samples were collected at the time of sepsis diagnosis and seven days later to evaluate the role of biological processes or genes possibly involved in patient recovery. Principal Components Analysis (PCA) profiling discriminated between patients with early sepsis and healthy individuals. Genes with differential expression were grouped according to Gene Ontology, and most genes related to immune defense were up-regulated in septic patients. Additionally, PCA in the early stage was able to distinguish survivors from non-survivors. Differences in oxidative phosphorylation seem to be associated with clinical outcome because significant differences in the expression profile of genes related to mitochondrial electron transport chain (ETC) I-V were observed between survivors and non-survivors at the time of patient enrollment. Global gene expression profiles after seven days of sepsis progression seem to reproduce, to a certain extent, patterns collected at the time of diagnosis. Gene expression profiles comparing admission and follow-up samples differed between survivors and non-survivors, with decreased expression of genes related to immune functions in non-survivors. in conclusion, genes related to host defense and inflammatory response ontology were up-regulated during sepsis, consistent with the need for a host response to infection, and the sustainability of their expression in follow-up samples was associated with outcomes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Albert Einstein Research and Education Institute - Hospital Israelita Albert EinsteinHosp Israelita Albert Einstein, Inst Israelita Ensino & Pesquisa, Ctr Expt Res, São Paulo, BrazilHosp Israelita Albert Einstein, Intens Care Unit, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, EPM, Hosp São Paulo, Div Infect Dis, São Paulo, BrazilHosp Sirio Libanes, Intens Care Unit, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, EPM, Dept Gynecol, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, Intens Care Unit, Hosp São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, EPM, Hosp São Paulo, Div Infect Dis, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, EPM, Dept Gynecol, São Paulo, BrazilUniversidade Federal de São Paulo Unifesp, Intens Care Unit, Hosp São Paulo, São Paulo, BrazilFAPESP: FAPESP 2006/58744-1Web of Scienc

Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

This study was designed to assess the effect of GB virus (GBV)-C on the immune response to human immunodeficiency virus (HIV) in chronically HIV-infected and HIV- hepatitis C virus (HCV)-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ) and interleukin (IL)-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8%) presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.Universidade Federal de São Paulo (UNIFESP) Laboratório de Virologia e Imunologia Disciplina de InfectologiaFleury Medicina DiagnósticaUNIFESP, Laboratório de Virologia e Imunologia Disciplina de InfectologiaSciEL