L 902 688

30,000 THP-1-derived macrophages were seeded on a black 96-well plate and treated with EP4 agonist L-902688 (Cayman Chemicals) or vehicle control (0.01% [vol/vol] DMSO) in RPMI lacking antibiotics for 30 min prior to infection with GFP-expressing wild-type and YopJC172A Y. pseudotuberculosis at an MOI of 15:1 for 1 h. At 1 hpi, media were removed, and cells were washed twice with PBS to remove extracellular bacteria. RPMI media lacking antibiotics but supplemented with gentamicin (100 μg/ml) and with or without 10 μM EP4 agonist L-902,688 or 10 μM JUN inhibitor T-5224 were added to the cells, and the cells were incubated further for another hour. At 2 hpi, the media were removed again, and the cells were washed twice with PBS and resuspended in media containing a lower concentration of gentamicin (10 μg/ml) for the remainder of the infection. For bacterial CFU counts, the cells were lysed with 0.1% Triton X-100, serially diluted in sterile PBS, plated on LB, and allowed to grow overnight at 37°C. Colonies were counted and calculated as CFU/ml. For fluorescence imaging, the THP-1 cells were stained with Hoechst stain at the indicated times (2 and 24 hpi). The cells were then washed with PBS, fixed in 4% PFA, and visualized by Cytation 5 (BioTek).

Bone marrow was collected from the hind limbs of mice (femur and tibia) and single cell suspensions were prepared followed by red blood cell lysis [155 mM ammonium chloride (Merck, Darmstadt, Germany), 10 mM potassium bicarbonate (Carl Roth, Karlsruhe, Germany) and EDTA (0.1 mM) in ddH2O]. Cells were then cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, 50 µM 2-mercaptoethanol (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA) together with 20 ng/ml GM-CSF and 20 ng/ml IL-6 (BioLegend, San Diego, USA) at 37°C, 5% CO2 v/v. The following compounds were tested: PGE2, EP2 receptor agonist Butaprost, EP1/EP3 receptor agonist Sulprostone and EP4 receptor agonist L-902,688 (all from Cayman Chemical, Ann Arbor, USA). Cells were harvested after three days and subtyped for MDSCs by flow cytometry (BD FACSCanto II, BD Biosciences, Franklin Lakes, NJ, USA). Both PMN-MDSCs (based on positive selection of the Ly6G marker) and M-MDSCs (based on positive selection of the Gr1 marker of the Ly6G depleted cell suspension) were isolated from the cell suspension using the magnetic-activated cell sorting (MACS) MDSC isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to manufacturer’s instruction.