Figure 6. PGE₂ modulates neutrophil activation in vivo.

(a-b) C57BL/6 mice were intraperitoneally (i.p.) inoculated with 20 T. gondii cysts. Monocytes, sorted from bone marrow of orally infected mice, were stimulated with E. coli lysate in the presence of indomethicin (Indo), celecoxib (Celex), or vehicle (Veh). Treated monocytes were transferred i.p. at 4 days p.i. and mice subsequently administered E. coli lysate i.p. (a) Cells were isolated by peritoneal lavage and cultured in the presence of BFA and stained for neutrophil markers (Ly6G⁺CD11b^hi) and TNF-α examined by intracellular cytokine staining. (b) Histograms represent TNF-α production ± SEM of two separate experiments. (c-e) Wild-type (WT), or Ccr2^−/− mice were orally infected with T. gondii. Ccr2^−/− mice were treated with 16,16-dimethyl (diMe) PGE₂ or vehicle from days 6-8 post infection. (c) At day 8 p.i. cells were isolated from the small intestine lamina propria (SILp) and neutrophil production of TNF-α assessed. Numbers represent percentage of cells in each gate ± SEM. (d) Histogram represents the percentage of ROS producing SILp neutrophils assessed by flow cytometry ± SEM. (e) Representative H&E staininig of small intestine from infected Ccr2^−/− mice treated with diMePGE₂ or vehicle (Scale bar 100 μm). Graph depicts histological scoring. Amounts of aspartate aminotransferase (AST) were assessed in sera of animals (n=4). (f-i) WT mice were orally infected with T. gondii. On days 6–8 p.i. mice were treated intraperitoneally with indomethacin or vehicle. (f) Representative plot of TNF-α production gated on neutrophils from vehicle or indomethacin treatment. (g) SILp neutrophil production of ROS was assessed by flow-cytometry. Histograms represent mean production of ROS ± SEM (n=3). (h) Representative H&E stained section of small intestine from vehicle or celecoxib (Celex) treated groups (Scale bar 100 μm). Graph depicts histological scoring. Amounts of AST were assessed in sera of animals (n=4). (i) Survival curve of animals treated with indomethicin or celecoxib on days 6–8 post infection. Data are representative of at least two independent experiments. Data presented as mean ± SEM (n=3). Statistical comparisons performed using the Student’s t test (*P<0.05, **P<0.01, ***P<0.001).