Patients and healthy volunteers
Patients admitted to the intensive care units of the Sao Paulo, Albert Einstein, and Sirio-Libanes Hospitals with a clinical diagnosis of sepsis according to the ACCP/SCCM consensus conference [1], from April 2014 to June 2015, were enrolled in the study. The protocol was approved by the ethics committees of the participating hospitals.
Blood samples were obtained from 34 septic patients at admission (D0), and 15 of the patients had a second sample collected after 7 days (D7) of therapy. Samples were also collected from 19 healthy volunteers who were matched according to age and gender.
LPS, gram-negative, and gram-positive bacteria
LPS from Salmonella abortus equi was a generous gift from C. Galanos (Max-Planck Institute of Immunobiology, Germany). Pseudomonas aeruginosa (ATCC27853) and S. aureus (ATCC 25923) were purchased from Oxoid Limited, Basingstoke, Hampshire, UK.
Induction and detection of the production of ROS and NO in monocytes in whole blood
ROS and NO were measured constitutively and after stimulation with LPS and heat-killed S. aureus, and P. aeruginosa for 30 min. Based on the dose-response curves, 100 ng/mL LPS and 2.4 × 108 colonies/mL S. aureus were used for induction of ROS and NO. The concentration of P. aeruginosa was 2.4 × 107 colonies/mL for ROS and 2.4 × 108 colonies/mL for NO. The ROS and NO levels were quantified in monocytes in whole blood by measuring the oxidation of 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma, St. Louis, MO) and 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FMDA; Invitrogen, Carlsbad, CA), respectively, as previously described [11, 23]. Briefly, the tubes from each sample were incubated in the presence of 0.06 mM DCFH-DA or 0.01 mM DAF-FMDA in a 37 °C shaking water bath for 30 min. After incubation, 2 mL of 3 mM EDTA (Sigma) or phosphate-buffered saline (PBS) was added to each tube for ROS and NO determination, respectively, and the mixture was then centrifuged (800g for 5 min at 4 °C). Erythrocytes were lysed in hypotonic saline, and the pellets were incubated with 6 μL of CD14-PerCP clone MΦP9 (BD Bioscience, San Jose, CA, USA) and anti-CD163-PE clone GHI/61 (BD Bioscience) at room temperature for 15 min in the dark. Then, 2 ml of PBS was added to each tube, and the mixture was centrifuged (800g for 5 min at 4 °C). The supernatants were discarded, and the pellets were resuspended in 300 μL of PBS for flow cytometric analysis.
Intracellular detection of cytokines in monocytes in whole blood
Whole blood was diluted 1:2 in RPMI and incubated with LPS and heat-killed bacteria (LPS: 100 ng/mL, P. aeruginosa and S. aureus: 2.4 × 108/mL), or without stimulus in 5-mL propylene tubes at 37 °C in the presence of 5 % CO2. After 30 min, 5 μL (1 mg/mL) of Brefeldin A (Sigma, Saint Louis, MO, USA) was added to the samples, and they were incubated for an additional 4 h. After washing, the red blood cells were ruptured with 2 mL lysis solution (FACS lysing solution, BD Bioscience). After washing with 2 mL PBS, the samples were incubated with the fluorochrome-conjugated monoclonal antibodies CD14-PerCP clone MΦP9 (BD Bioscience) and anti-CD163-PE clone GHI/61 (BD Bioscience) for surface staining for 15 min in the dark at room temperature. The samples were washed in 2 mL PBS, centrifuged, and fixed with 500 μL fixation buffer (PBS 4 % paraformaldehyde) for 30 min in the dark at 4 °C. After centrifugation, 50 μL permeabilization buffer (PBS 1 % FCS; 0.1 % saponin), anti-IL-6-APC clone MQ2-13A5 (BD Bioscience), and anti-TNF-PE-Cy7 clone Mab11 (BD Bioscience) were added to the tubes. The tubes were incubated for 30 min in the dark on ice. Then, the samples were washed with 2 mL permeabilization buffer, and the cells were suspended in Macs buffer for flow cytometric analysis [15].
Phagocytosis of monocytes in whole blood
Phagocytosis of monocytes was measured using Escherichia coli conjugated to FITC (Phagotest™, Glycotope Biotechnology, Heidelberg, Germany), accordingly to the manufacturer instructions.
Flow cytometry
Detection of phagocytosis and the production of ROS, NO, IL-6, and TNF-α by monocytes in whole blood was performed by multiparameter flow cytometry (LSRFORTESSA (BD Bioscience)). Events acquisition was performed using FACSDiva software (BD Bioscience). For detection of the production of ROS, NO, IL-6, and TNF-α by monocytes, 5000 events were acquired using forward- and side-scatter parameters combined with CD14-positive cells. For the detection of phagocytosis, 15,000 events were acquired using forward- and side-scatter parameters to determine the monocyte population. All events were acquired and stored, and the analysis was performed using FlowJo (Tree Star INC. Ashland, OR, USA).
Detection of the production of ROS, NO, IL-6, and TNF-α
Monocyte analysis was performed by assessing individual cells (singlets) combined with side-scatter parameters versus CD14 positiveness. Monocytes were further characterized as CD163+ or CD163− cells. The quadrant for CD163+ cells was established based on isotype control.
The productions of ROS and NO were analyzed in monocytes and in the subsets of CD163+ and CD163− monocytes in histogram charts. They were quantified by the geometric mean fluorescence intensity (MGIF) associated with the detection of DCFH and DAF, respectively (Fig. 1). Under the experimental conditions for oxidative metabolism measurement, the expression of CD163 on monocytes was 50.5 ± 17.7 % (mean ± SD) in septic patients and 21.3 ± 20.2 % in healthy volunteers.
Intracellular cytokine levels were analyzed both in monocytes and in the subsets of CD163+ and CD163− monocytes based on the quadrants established in the sample without stimulation and are expressed as the percentage of cytokine-producing monocytes (Fig. 2). Under the experimental conditions for intracellular cytokines detection, the expression of CD163 on monocytes was 41.6 ± 4.4 % (mean ± SD) in septic patients and 30.9 ± 18.9 % in healthy volunteers.
Co-location of gp91phox and p47phox by immunofluorescence
PBMCs were obtained using the Ficoll density gradient method (Ficoll-Paque PLUS; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and stored in liquid nitrogen until use. After defrosting, the cells were spun on glass slides. The cells were incubated overnight with the primary antibodies goat anti-Nox2 (1:200) and rabbit anti-p47 (1:100) and then incubated with red fluorescent Alexa Fluor 594 (donkey anti-goat; 1:400), and/or green fluorescent Alexa Fluor 488 (donkey anti-rabbit; 1:200). Nuclear material was stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, USA). Images of stained cells were captured using a confocal microscope SP5 (Leica, USA). The images were analyzed in the program ImageJ (National Institutes of Health, Bethesda, Maryland, USA) using the plugincolocalizationanalysis/colocalizationhighlighter (co-localized points—8 bit). That tool generated a new image that presented the points of co-localization of p47phox and gp91phox. Those points of co-localization were quantified from the average fluorescence intensity corresponding to two to four cells/randomly selected field.
Statistical analysis
The results were analyzed using SPSS (Statistical Package for Social Sciences v 19.0) (IBM, Armonk, NY, USA). The Shapiro-Wilk test was applied to determine the normality of the results. Comparisons between healthy volunteers and patients were performed using the Mann-Whitney U test, and comparisons between patient samples (D7 vs. D0) were performed using the Wilcoxon signed-rank test. Group comparisons were performed by using the Kruskal-Wallis test. The variables that showed differences among the three groups were compared group to group by the Mann-Whitney test.
The interactions of CD163 with ROS, NO, IL-6, and TNF levels were analyzed by two-way repeated measures analysis of variance (ANOVA) with the Bonferroni posttest. P values ≤0.05 were considered significant.