Figure 5. Ly6C^hi inflammatory monocytes regulate neutrophil activation via production of PGE₂.

(a) Parasite localization visualized using RFP-expressing T. gondii in whole tissue mounts of jejunal sections from day 8 infection (Scale bar 2 mm). (b) Fixed jejunal tissue from day 8 infected CD11c^YFPxLysM^eGFP mice was whole mounted and sites of T. gondii invasion analysed by confocal microscopy (CD11c, white; LysM, Green; T. gondii, Red) (Scale bar 20 μm). Circle denotes foci of high parasite density. (c) Frequency of neutrophils (Ly6G⁺) and monocytes (MHCII^hi) within LysM^eGFP population from T. gondii infected SILp. (d) Bone marrow neutrophils (Ly6G⁺CD11b⁺) were sorted by FACS and stimulated by E. coli lysate with Ly6C^hi monocyte supernatant or control medium. Production of TNF-α was assessed by intracellular cytokine staining. (e) TNF-α production by neutrophils stimulated with E. coli lysate and treated with control medium, monocyte supernatant, or neutrophil supernatant. (f) Neutrophils were stimulated as in (d) and treated with Anti-IL-10R, Anti-IL-27R, Anti-TGF-β, or pan adenosine receptor (AdR) inhibitors in combination with monocyte supernatant and TNF-α production assessed. (g) Neutrophils were stimulated as in (d) and pre-treated with either inhibitors to the PGE₂-receptors EP2 and EP4 or vehicle. (h) Neutrophils were stimulated as in (d) and treated with monocyte supernatants that were generated in the presence or absence of indomethacin and TNF-α production assessed. (i) SILp Ly6C^hi monocytes, CX3CR1^hi resident macrophages, neutrophils and T cells were sorted by FACS and cultured at equal concentration for 18 hrs. PGE₂ was measured in triplicate supernatants by EIA. (j) Neutrophils were stimulated as in (d) and treated with increasing concentrations of purified PGE₂. TNF-α production was assessed by flow cytometry. Additionally, neutrophils were treated with formyl-peptide, fMLP in the presence of increasing concentrations of purified PGE₂. ROS production was assessed by flow cytometry. Histograms represent TNF-α production and ROS production ± SEM. Data are representative of three independent experiments, results are mean ± SEM. Statistical comparisons performed using the Student’s t test (*P<0.05, **P<0.01, ***P<0.001).