Anti cd14 antibody

Blood samples were obtained from six patients with sepsis caused by community-acquired pneumonia within 24 h after admission to the intensive care unit of Amsterdam UMC, location Academic Medical Center (AMC), University of Amsterdam, The Netherlands, and from four healthy subjects (Khan et al., 2020 (link)). Sepsis was defined on the basis of a “probable” or “definite” infection according to the Center for Disease Control and Prevention four-point scale (Garner et al., 1988 (link)) combined with at least one of general, inflammatory, hemodynamic, organ dysfunction, or tissue perfusion parameters derived from the International Sepsis Forum consensus definitions (Calandra et al., 2005 (link)), as previously described in detail (Klouwenberg et al., 2013 (link)). Monocytes were isolated using fluorescence-activated cell sorting (FACS) and stored in RNA protect cell reagent (Qiagen, Hilden, Germany). Total monocytes were identified as CD14⁺CD15⁻ cells in morphologically defined gates and sorted on FACS Canto II flow cytometer (BD Biosciences) with anti-CD14 antibody (Miltenyi Biotech), as previously described (Hoogendijk et al., 2017 (link); Khan et al., 2020 (link)). The Medical Ethics Committee of the Academic Medical Center approved the study (IRB no. 10-056C), and written informed consent was obtained from all patients (or legal representative) and healthy controls.

Anti-CD14⁺ antibody was purchased from Miltenyi Biotec Inc. (San Diego, CA). Oligomycin, FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone], antimycin A, ADP, pyruvate, malate, rotenone, succinate, ascorbate, TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine), and azide were purchased from Sigma-Aldrich (St. Louis, MO). The XF Plasma Membrane Permeabilizer reagent was from Agilent Technologies (Santa Clara, CA).

Mononuclear cells were isolated by Ficoll Hypaque (GE Healthcare) gradient density centrifugation. Monocytes were extracted from blood mononuclear cells by positive selection using magnetic microbeads coupled to anti-CD14 antibodies (Miltenyi Biotec) (34 (link)–36 (link)). Purity assessed by flow cytometry was >95%. Viability determined by trypan blue exclusion was >95%. Monocytes were cultured in RPMI medium 1640 supplemented with 10% (vol/vol) FCS (GE Healthcare) and GM-CSF (50 ng/ml) (Peprotech) or M-CSF (50 ng/ml) (Peprotech) for 1 week to induce M1 or M2 macrophages, respectively. Ultrapure E. coli O111:B4 LPS was purchased from List Biological Laboratories. Polyclonal and monoclonal antibodies (pAbs and mAbs) used for flow cytometry, Western blotting and cytometry by time of flight (CyTOF) are described in Table S1 in Supplementary Material. Unless specified otherwise, all other reagents were obtained from Sigma-Aldrich.

Placental macrophages were isolated as previously described (Mezouar et al., 2019a (link)). Briefly, entire placenta tissue was digested in Hank’s Balanced Salt Solution (HBSS), DNase I 2.5 mM, and 2.5% trypsin (Life Technologies). Cell suspension was filtered through 100-μm pores and deposited on a Ficoll cushion and centrifuged at 700 ×g for 20 min to collect mononuclear cells. Placental macrophages were isolated using magnetic beads coated with anti-CD14 antibodies (Miltenyi Biotec). The purity of isolated CD14⁺ placental macrophages was assessed by flow cytometry and was higher than 98%.

Isolation of monocytes and lymphocytes from peripheral blood of atopic individuals was performed as described before
[52 (link)]. Briefly, PBMC were isolated from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Europe, Freiburg, Germany). Monocytes were isolated with anti-CD14 antibodies (Miltenyi Biotech, Bergisch Gladbach, Germany) by magnetic cell separation of PBMC and cultured for 6 days in RPMI 1640 + Glutamax (supplemented with 7% of autologous serum and 800 U/ml GM-CSF). GM-CSF was chosen for differentiation as it leads to macrophages which resemble more closely the alveolar phenotype
[53 (link)]. Cells negative for CD14 were collected as lymphocytes and kept frozen until co-culture at -80°C. After differentiation to macrophages cells were alternatively activated with 10 ng/ml IL-4 for 24 h and afterwards stimulated with 2000 BU/ml grass allergen extract (SQ225, Alk-Albello Arzneimittel GmbH, Hamburg, Germany) for 48 h. Cells were harvested and splitted for either analysis of surface markers or M2 marker gene expression or further co-cultured with autologous lymphocytes.

PBMCs were prepared from leukopacks (Etablissement Français du Sang) or blood collected in ethylene-diamine-tetraacetic acid (EDTA) tubes from donors and patients after centrifugation through a Ficoll density cushion. Monocytes were isolated from PBMCs by CD14 positive selection using magnetic beads coated with anti-CD14 antibodies (Miltenyi Biotec). CD14⁺ monocytes were differentiated into macrophages by cell culture (Ghigo et al., 2010 (link)). To obtain M1 macrophages, macrophages were stimulated with 20 ng/mL recombinant human IFN-γ (Tebu-bio) for 18 h. M2 macrophages were obtained by incubating macrophages with 10 ng/mL IL-10 or 20 ng/mL IL-4 (R&D Systems) for 18 h.