Study population
The Ethics Committees of the King Memorial Chulalongkorn Hospital (KMCH) approved the study (IRB No. 610/64) to use blood samples from the healthy volunteers and patients with sepsis with written informed consents according to the STROBE guideline. Then, patients with sepsis admitted to the intensive care unit (ICU) of KMCH between December 2021 and October 2023 and the healthy volunteers were recruited. The inclusion criteria were age > 18 years, diagnosed by at least 2 physicians, and sequential organ failure assessment (SOFA) scores higher than 2, while the exclusion criteria were pregnancy, hematologic diseases, neutropenia, the use of granulocyte-colony stimulating factor and immunosuppressive drugs, and organ transplantation history. The initial sepsis severity using the APACHE II and SOFA scores was performed at the enrollment. The catheter-related infection and major acute infectious events in the respiratory system, urinary tract, and digestive tract of each patient were noted during follow-up prospectively.
Isolation of peripheral blood mononuclear cells (PBMC) and neutrophils
Heparinized blood samples were collected within 24 h after fulfilling the sepsis criteria (Sepsis-3) and processed within 2 h after the collection without refrigeration. The normal- and low-density neutrophils, NDNs and LDNs, respectively, were isolated using double-layered-density gradient centrifugation containing the Lymphoprep® (Stem Cell Technologies, Vancouver, Canada) density separation (upper part) and PolymorphPrep® (Serumwerk, Bernburg, Germany) density separation (lower part) at a ratio of 1:1:1. Both fractions were centrifuged at 800 g for 30 min at room temperature with brakeless deceleration. The PBMCs at the interface of the plasma and Lymphoprep® layers were carefully isolated, and the neutrophils were recovered from the intermediary layer (the interface between Lymphoprep® and PolymorphPrep®). The isolated PBMC and neutrophil fractions were washed in 1X deionized phosphate buffer solution (D-PBS) and eradicated contaminating red blood cells (RBCs) by 1X RBC lysis buffer (Biolegend, San Diego, CA, USA) before resuspension in the complete RPMI-based media, RPMI1640 culture media (Gibco, Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). The cell viability was observed by Trypan blue dye staining (Thermo Fisher Scientific, Waltham, MA, USA).
Low-density neutrophil induction in vitro
The isolated neutrophils from gradient separation, as mentioned above, were incubated with the complete RPMI media. For low-density neutrophils (LDNs) in vitro induction, the isolated neutrophils (1 × 106cells) were incubated with lipopolysaccharide (LPS) from Escherichia coli 026:B6 (Sigma-Aldrich, St. Louis, MI, USA) at various concentrations following a previous report11. After activation, stimulated neutrophils were re-centrifuged using the density gradient separation method before the retrieval of NDNs and LDNs from the upper and lower parts of the Lymphoprep® and PolymorphPrep®, respectively. The percentage of the induced LDNs was counted under a microscope and the cell characteristics were analyzed by flow cytometry (mentioned later).
Flow cytometry
All analyses were measured using a FACS Canto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with the FlowJo V10 (Ashland, DE, USA). Neutrophils and PBMC (5 × 105 cells) were suspended in staining buffer (2% fetal bovine serum with 0.1% sodium azide in 1XD-PBS) and labelled with antibody panels for flow cytometric analysis. Antibodies used in the experiment were fluorescein isothiocyanate (FITC)-conjugated anti-CD66b (BD Biosciences, Franklin Lakes, NJ, USA), phycoerythrin (PE)-conjugated anti-CD63 (BD Biosciences, Franklin Lakes, NJ, USA), PE-conjugated anti CD279 (BD Biosciences, Franklin Lakes, NJ, USA), peridinin-Chlorophyll-Protein (Per-CP)-conjugated anti-CD14 (Biolegend, San Diego, CA, USA), PE-Cyanine 5 (cy5)-conjugated anti-CD184 (BD Biosciences, Franklin Lakes, NJ, USA), PE-Cyanine 7 (cy7)-conjugated anti-CD274 (BD Biosciences, Franklin Lakes, NJ, USA), PE-cy7-conjugated anti-CD182 (Biolegend, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-CD62L (BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated anti-CD45 (BD Biosciences, Franklin Lakes, NJ, USA), and APC-cy7-conjugated anti-CD11b (BD Biosciences, Franklin Lakes, NJ, USA). The cells were labeled for 30 min at 4 °C in the dark with antibodies before being fixed with 4.2% paraformaldehyde (BD Biosciences, Franklin Lakes, NJ, USA). Analysis was gated by the dot-plot analysis, and at least 25,000 cells were acquired per sample.
Apoptosis was quantified by annexin V-FITC and propidium iodide (PI) staining (BD Biosciences, Franklin Lakes, NJ, USA), with early apoptotic cells (annexin V + /PI-) and late apoptosis (annexin V + /PI +) using an apoptosis assay following the manufacturer’s instructions. After incubating for 20 min at 4 °C in the dark, cells were washed and measured. The reactive oxygen species (ROS) production was assessed by detecting fluorescence changes in the cells loaded with dihydroethidium (DHE) (Thermo Fisher Scientific, Waltham, MA, USA), following previous protocols12. Briefly, the cells (2.5 × 105 cells) were resuspended in the complete RPMI-based media containing 2.5 μM DHE and incubated for 15 min at 37 °C in the dark. Cells were washed with cold D-PBS and resuspended in the cold 4.2% paraformaldehyde (BD Biosciences, Franklin Lakes, NJ, USA). Cells were stored cold in the dark until analyzed by flow cytometry using 485 nm (excitation; blue laser) and 520 nm (emission; PE) filters, gated by dot-plot analysis (10,000 cells were acquired per sample), and reported as mean fluorescent intensity (MFI). To analyze the phagocytic capacity, all cells were mixed with pHrodo S. aureus Bioparticle™ (Thermo Fisher Scientific, Waltham, MA, USA) before labeling with anti-CD66b antibody (a neutrophil biomarker) and being fixed with 4.2% paraformaldehyde (BD Biosciences, Franklin Lakes, NJ, USA). Phagocytosis was demonstrated as a percentage of the CD66b-positive cells with positive pHrodo S. aureus Bioparticle™.
Human T cell isolation and proliferation assay
Isolated T cells in the suspension were captured by immunomagnetic negative selection using the EasySep™ Human T Cell Isolation Kit (Stem Cell Technologies, Vancouver, Canada) and the isolated CD3 + T cells were labeled with trypan blue (Thermo Fisher Scientific, Waltham, MA, USA) to detect cell viability. The T cell proliferation of PBMC or isolated CD3 + T cells was assessed by carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, MA, USA) dilution as previously described13. Briefly, the proliferation index was calculated from the total number of divisions divided by the number of cells that underwent cell proliferation processes (http://docs.flowjo.com/vx/experiment-based-platforms/proliferation/plat-prolif-protocols/). After CFSE labeling, the isolated CD3 + T cells (1.0 × 105 cells) or T cells in PBMC (containing 1.0 × 105 CD3 + T cells) were seeded into a 96-well plate and stimulated with CD3/CD28 dyna-bead (Thermo Fisher Scientific, Waltham, MA, USA) using the unstimulated T cells (no dyna-bead) as the control according to the manufacturer’s protocol. After activation for 4 days, the cells were harvested and were stained with APC-conjugated anti-CD3 antibody (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by flow cytometry as previously described13.
Wright–Giemsa stain assay
The PBMC and neutrophils portion after gradient separation were smeared, naturally dried, stained with the Wright-Giemsa solution (Merck, Darmstadt, Germany) at room temperature for 1–2 min, mixed with phosphate buffer solution (pH 7.2), and washed with sterile water. The results of cell staining were observed using a bright field microscope (Nikon, Shinagawa, Tokyo, Japan).
Statistical analysis
GraphPad Prism 8.0 (GraphPad Software, Inc.) was used for statistical analysis and graph presentation using the student’s t-test or Mann–Whitney U test and one-way analysis of variance (ANOVA) with Tukey’s analysis or Kruskal–Wallis for the 2- and 3-group comparisons, respectively. A p-value < 0.05 was considered a statistically significant difference. Kaplan–Meier analysis and receiver operating characteristic (ROC) curve were used to predict the efficacy of the circulating LDN for determination of secondary infection at 45 day after the enrollment. The agreement between the accumulation of LDN in PBMC measured by flow cytometry and microscopy was assessed by Bland Altman analysis14.
