Anti gm csf

Mice were given antibiotics in drinking water as described previously^{10 (link), 15 (link)}. Mice were given broad-spectrum antibiotics (ampicillin 1 g/L, neomycin sulfate 1 g/L, metronidazole 1 g/L, and vancomycin 0.5 g/L) in drinking water for 10–14 days. Antibiotic therapy was stopped 3 days prior to infection. PRR ligands were prepared as described previously and administered by oral gavage^{10 (link)}. Anti-GM-CSF (BioLegend), anti-CXCL2 (R&D Systems), anti-CXCL1 (R&D Systems), and isotype control, rGM-CSF (BioLegend) or rIL-17A (Thermo Fisher Scientific), were administered intranasally concomitant with S. pneumoniae or K. pneumoniae inoculation, unless stated otherwise. Anti-IL-17A (R&D Systems) was administered 3 days prior to infection and concomitant with infection via the intraperitoneal route.

Freshly prepared or PMA/Ionomycin-stimulated tissue homogenates in FACs tubes were washed once with PBS and stained at 1:60 with 10 ul live dead blue viability stain for 5 mins on ice, stained for an additional 5 mins with 50 ul Fc block at 1:25 (anti-CD16/32, clone 2.4G2) and then for 20 mins on ice with 50 ul of cell surface stains. Cells were then fixed overnight (~16 hours) with the foxp3 ebioscience fix/perm kit and stained for intracellular proteins for 45 mins on ice. Flow cytometry data were acquired on an LSR Fortessa and analyzed using Flowjo software (Tree Star). Events analyzed were: dye-negative cells; FSC-A by FSC-H singlet cells; SSC-H by SSC-W singlet cells; and FSC A by SSC A lymphocytes. The following antibodies were used for flow cytometry: anti CD4 (ebioscience, GK1.5); anti IL-17A (ebioscience, eBio17B7,), anti CD3 (Biolegend, 17A2); anti-CD44 (Biolegend, IM7); anti TNFα (BD, MP6-XT22), anti CD25 (ebioscience, PC61.5); anti CD45.2 (Biolegend, 104); anti CD45.1 (Biolegend, A20), anti Ly6G (BD, 1A8), anti Ly6C (Biolegend, HK1.4); anti Ki67 (Biolegend, 16A8); anti GM-CSF (Biolegend, MP1-22E9); anti RORγT (ebioscience, B2D); anti Siglec F (BD, E502440); anti CD11b (Biolegend, M1/70) and anti IL-10 (ebioscience, JES5-16E3).

All subjects gave informed consent before their participation in the current study. All human studies were approved by the Institutional Review Board (IRB) of Thomas Jefferson University, and all methods were performed in accordance with the relevant guidelines and regulations. Whole blood samples were collected from healthy donors and peripheral blood mononuclear cells (PBMCs) were enriched by gradient centrifugation in Ficoll. CD14⁺ monocytes were isolated by positive selection using magnetic beads following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of cells was above 90%, measured by flow cytometry. Monocytes were seeded (1 × 10⁶/ml) in 24-well plates and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS, 1% penicillin/streptomycin antibiotic (Gibco), 2 mM glutamine and 2β- Mercaptoethanol (50 µg/ml, Gibco). Monocytes were activated with lipopolysaccharide (100 ng/mL, Sigma-Aldrich) for 18 h at 37 °C in the presence of recombinant human GM-CSF (10 ng/mL, R&D Systems, Minneapolis, MN, USA) or anti-GM-CSF (10 µg/mL, Biolegend, San Diego, CA). LPS-activated cells (mature monocytes) cultured with PBS were used as controls and culture of monocytes without LPS stimulation were considered as immature cells.