2.2. In vitro endotoxin-tolerance assays

Recruitment of healthy volunteers for blood sampling was approved by the institutional ethics committee (CEP/UNIFESP 1172/2020) and all donors gave informed consent before inclusion in the study. The average age was 32.8 years (SD ±11.2) and 60% were female. Blood was diluted in PBS (1:1, v:v) and PBMCs were isolated by the Ficoll gradient method (Ficoll-Paque PLUS; GE Healthcare Bio-Sciences, Uppsala, Sweden). Isolated cells were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 55 µM 2-Mercaptoethanol (Gibco) and 10% Human AB serum (H6914, Sigma-Aldrich, Saint Louis, MO) in polypropylene tubes to avoid cell adhesion.

To induce endotoxin-tolerance in PBMCs 2.5x10⁶ cells/mL were incubated in the presence or absence (control) of lipopolysaccharide (LPS) from Salmonella abortus equi (kindly provided by C. Galanos, Max-Planck Institute of Immunobiology, Germany) at 10 ng/mL or 100 ng/mL for 48 hours (h) at 37 °C and 5% CO₂. Tolerant and naïve control cells were then washed twice in PBS and challenged with 100 ng/mL LPS for 24h at 37 °C and 5% CO₂; a group cultured only in supplemented RPMI (both incubation times) was included as a negative control.

For intracellular cytokine measurement, 10 µg/mL brefeldin A (Thermo Fisher Scientific, Waltham, MA) was added to the cell suspension after 30 minutes (min) of incubation with the challenge dose of LPS. After 24 hours, cells were washed and stained with anti-CD14-BV711, clone MφP9 (BD Biosciences, Franklin Lakes, NJ) for identification of monocytes. Intracellular staining with anti-TNF-α-PE-Cy7, clone Mab11 (BD Biosciences) was performed after fixation and permeabilization with 2× lysing solution (BD Biosciences) containing 0.05% Tween 20. The cells were then washed and suspended in a buffer solution (PBS, 0.1% BSA, 2 mM EDTA) before flow cytometric analysis; the gating strategy is described in Supplementary File 1 . Flow cytometry was performed in an LSR FORTESSA (BD Biosciences), and data were analyzed using the FlowJo software (FlowJo v10, BD Biosciences).

After 24h of LPS challenge, cells were spun down, supernatants were stored for lactate quantification and cell pellets were lysed for ATP measurement with ATP Assay Kit Colorimetric/Fluorimetric (Abcam, Cambridge, United Kingdom). Briefly, after lysis samples were centrifuged at 16000 g for 2 min at 4°C, and an aliquot of cell lysate was stored for total protein quantification using the Bradford method. Samples were then deproteinized by centrifugation in 10kD spin columns (Abcam) at 10000 g for 40 min at 4°C, snap-frozen in liquid nitrogen, and stored at -80°C. ATP measurement was carried out using the fluorometric method following the manufacturer’s instructions, fluorescence was measured in a Synergy H1 plate reader (Biotek, Winooski, VT) at Ex/Em of 535/587nm and results were calculated as pmol ATP/µg protein. Lactate was measured in supernatants using a colorimetric commercial kit, following the manufacturer’s instructions (Labtest, Lagoa Santa-MG, Brazil).

Endotoxin tolerance was induced as previously described: 2.5x10⁶ PBMCs/mL were incubated in the presence or absence of 100 ng/mL LPS for 48 h. Cells from each group were then washed, split in different tubes, and incubated with 2 mM 2-deoxy-D-Glucose (2-DG; Cayman Chemical, Ann Arbor, MI) to limit glycolysis, 1 µM Oligomycin A (Cayman Chemical) to inhibit OXPHOS, or 100 µM 6-Aminonicotinamide (6-AN; Cayman Chemical) to inhibit the pentose phosphate pathway (PPP). To evaluate cytokine production, 100 ng/mL LPS challenge were added to the cells 1h after the metabolic modulators. After 24 h incubation, supernatants were collected and stored at -80° C for further measurement of TNF-α and IL-6 using a Cytometric Bead Array (CBA; BD Biosciences) following the manufacturer’s instructions. To measure the phagocytic capacity, endotoxin-tolerant and control PBMCs were incubated for 22 h with the metabolic modulators before a 2.5 h incubation with 0.5mg/mL pHrodo Green E. coli BioParticles (Thermo Fisher Scientific). Cells were stained with anti-CD14-BV711 (BD Biosciences) for monocyte identification and pHrodo green median fluorescence intensity was determined by flow cytometry.

Normality was evaluated by the Shapiro–Wilk test. Differences between the distinct conditions were evaluated by repeated measures one-way ANOVA followed by Bonferroni posttest or paired Student’s t-test. A p-value < 0.05 was considered significant and all statistical analyses were performed using Graph Pad Prism 9 (GraphPad Software, San Diego, CA).