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

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

Proteomic study revealed cellular assembly and lipid metabolism dysregulation in sepsis secondary to community-acquired pneumonia

Sepsis is a life-threatening disorder characterized by organ dysfunction and a major cause of mortality worldwide. The major challenge in studying sepsis is its diversity in such factors as age, source of infection and etiology. Recently, genomic and proteomic approaches have improved our understanding of its complex pathogenesis. In the present study, we use quantitative proteomics to evaluate the host proteome response in septic patients secondary to community-acquired pneumonia (CAP). Samples obtained at admission and after 7 days of follow-up were analyzed according to the outcomes of septic patients. The patients' proteome profiles were compared with age-and gender-matched healthy volunteers. Bioinformatic analyses of differentially expressed proteins showed alteration in the cytoskeleton, cellular assembly, movement, lipid metabolism and immune responses in septic patients. Actin and gelsolin changes were assessed in mononuclear cells using immunofluorescence, and a higher expression of gelsolin and depletion of actin were observed in survivor patients. Regarding lipid metabolism, changes in cholesterol, HDL and apolipoproteins were confirmed using enzymatic colorimetric methods in plasma. Transcriptomic studies revealed a massive change in gene expression in sepsis. Our proteomic results stressed important changes in cellular structure and metabolism, which are possible targets for future interventions of sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPqFAPESPUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilHosp Israelita Albert Einstein, Intens Care Unit, BR-05652900 Sao Paulo, BrazilHosp Sirio Libanes, Intens Care Unit, BR-01409001 Sao Paulo, BrazilUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilFAPESP: 2011/20401-4FAPESP: 2013/15636-8CNPq: 305685/2011-2Web of Scienc

CD163 and CD206 expression does not correlate with tolerance and cytokine production in LPS-tolerant human monocytes

BackgroundLipopolysaccharide (LPS)-tolerant monocytes produce small amounts of inflammatory cytokines, which is one of the characteristics of the alternative activated macrophages (AAM). These cells exhibited an increased expression of CD206 and CD163. Given the functional similarities of AAMs with the modulation of monocytes' functions observed during sepsis and LPS-tolerance, we evaluated whether the inhibition of inflammatory cytokine production by LPS-tolerant monocytes is associated with the phenotype of cells expressing CD206 and CD163. MethodsWe investigated whether tolerant human monocytes would modulate their expression of CD206 and CD163, markers of alternative activation, and whether the level of their expression would be related to cytokines detection. Tolerance to LPS was induced in peripheral blood mononuclear cell by pre-incubating the cells with increasing concentrations of LPS. The expression of CD206 and CD163 and intracellular TNF- and IL-6 was determined 24 h after LPS challenge by flow cytometry. ResultsNo differences in CD163 expression were observed between tolerant and non-tolerant cells, while the expression of CD206, which was decreased following LPS stimulation in non-tolerized cells, was further reduced in tolerant cells. Decreased production of inflammatory cytokines was observed in the tolerized cells, regardless of the expression of CD163 and CD206, with the exception of IL-6 in CD206+ monocytes, which was similarly expressed in both tolerized and non-tolerized cells. ConclusionsThe effect of LPS in the expression of CD163 and CD206 on monocytes is not reverted in LPS tolerant cells, and the inhibition of inflammatory cytokines in tolerant cells is not related with modulation of these receptors. (c) 2016 International Clinical Cytometry SocietyFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Fed Sao Paulo, Div Infect Dis, Dept Med, Escola Paulista Med,Hosp Sao Paulo, Sao Paulo, BrazilUniv Fed Sao Paulo, Div Infect Dis, Dept Med, Escola Paulista Med,Hosp Sao Paulo, Sao Paulo, BrazilFAPESP: 2011/20401-4Web of Scienc

Peripheral Blood Mononuclear Cell Activation Induced by Leptospira interrogans Glycolipoprotein

Leptospira interrogans glycolipoprotein (GLP) has been implicated in pathological and functional derangement seen in leptospirosis. The goal of this study was to evaluate GLP's ability to induce cellular activation, as assessed by cytokine production and expression of surface activation markers. GLP extracted from either pathogenic L. interrogans serovar Copenhageni or nonpathogenic Leptospira biflexa serovar Patoc (GLPp) was used to stimulate peripheral blood mononuclear cell cultures from healthy donors. Supernatant cytokine levels were measured by enzyme-linked immunosorbent assay. Expression of CD69 and HLA-DR on lymphocytes and monocytes, as well as lipopolysaccharide (LPS) binding, were measured by flow cytometry. At 6 h of incubation, GLP induced a significant rise in tumor necrosis factor alpha levels, which dropped progressively until 72 h of incubation. Interleukin-10 peak levels were obtained at between 24 and 48 h, with sustained levels until 72 h of incubation. The response magnitude was proportional to the GLP dose. CD69 expression on T lymphocytes and monocytes increased significantly, as did HLA-DR expression on monocytes. GLPp induced no CD69 or HLA-DR expression. GLP did not block biotinylated LPS binding to monocytes, suggesting that different pathways are used to induce cell activation. In conclusion, GLP induces cellular activation and may play a major role in the pathogenesis of leptospirosis

Differential expression of toll-like receptor signaling cascades in LPS-tolerant human peripheral blood mononuclear cells

Pre-exposure to low doses of LPS induces resistance to a lethal challenge, a phenomenon known as endotoxin tolerance. in this study, tolerance was induced in human PBMC by culturing cells with 1 ng/mL LPS for 48 h. Cells were subsequently challenged with 100 ng/mL LPS for 2, 6 and 24 h, and the expression of 84 genes encoding proteins involved in the TLR signaling pathway was evaluated at each time point by PCR array. LPS pretreatment did not modulate the expression of TLR4 and CD14 on the surface of monocytes. A gene was defined as tolerized when LPS pretreatment reversed the effect of LPS challenge on the expression of the gene or as non-tolerized when LPS pretreatment did not reverse the effects of LPS challenge. We observed impaired signal transduction through the NF-kappa B, JNK, ERK and TRIF pathways, whereas expression of p38 pathway-related genes was preserved in LPS-tolerant cells. These results show a distinct regulation of the TLR pathway cascades during tolerance; this may account for the differential gene expression of some inflammatory mediators, such as up-regulation of IL-10 and COX2 as well as down-regulation of INF-alpha and IL-12. Depending on the effect of LPS-induced gene up-regulation or down-regulation, tolerance, as a reversion of such LPS effects, may result in repression or induction of gene expression. (C) 2010 Elsevier GmbH. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Div Infect Dis, Escola Paulista Med, Dept Med, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Div Infect Dis, Escola Paulista Med, Dept Med, BR-04039032 São Paulo, BrazilFAPESP: 2006/58744-1FAPESP: 2007/56960-7Web of Scienc

Percentages of T helper (CD3+CD8−) producing IFN-γ (Th1 - A/D) and IL-17 (Th17 - B/E), and percentages of Treg (CD3+CD4+CD127-Foxp3+ - C) lymphocytes in healthy volunteers and septic patients with paired samples (D0 and D7). n = number of individuals in each group.

<p><i>P</i> values are shown. The Mann-Whitney test was used to compare healthy volunteer and patient samples (D0 and D7), and the Wilcoxon Signed-Rank Test was used to compare D0- and D7-related samples.</p

Absolute counts of CD4+ T lymphocytes and subpopulations (CD3+CD8−) producing IFN-γ and IL-17 after PMA/Io stimulation, and T helper (CD3+CD4+) expressing CD127-Foxp3+ in healthy volunteers (n = 30) and septic patients (D0 samples, n = 46).

<p>Values represent the median and 25–75% quartile. The Mann-Whitney test was applied.</p

Percentages of T helper (CD3+CD8−) producing IFN-γ (Th1 - A/D) and IL-17 (Th17 - B/E), and percentages of Treg (CD3+CD4+CD127Foxp3+ - C) lymphocytes in non-survivors (n = 14) and survivors (n = 17) at admission (D0) and after 7 days (D7) in patients with follow-up samples. *<i>P</i><0.05 (Wilcoxon Signed-Rank Test).

<p>Percentages of T helper (CD3+CD8−) producing IFN-γ (Th1 - A/D) and IL-17 (Th17 - B/E), and percentages of Treg (CD3+CD4+CD127Foxp3+ - C) lymphocytes in non-survivors (n = 14) and survivors (n = 17) at admission (D0) and after 7 days (D7) in patients with follow-up samples. *<i>P</i><0.05 (Wilcoxon Signed-Rank Test).</p

Strategy for the analysis of T regulatory lymphocytes.

<p>Dot plots shown are representative of one healthy volunteer and one septic patient. Fifty thousand events were acquired with CD3+ gating and forward scatter versus side scatter parameters. For the analyses, the first gate was determined based on low SSC and high CD45 APC-Cy7, and the second gate was based on CD4+CD3+ cells. The cells with the phenotype CD45+CD3+CD4+CD127-FOXP3+ were considered to be Treg lymphocytes. Quadrants or gates were established based on the isotype controls.</p