Pretreatment with PTEN inhibitor SF1670 enhances the recruitment of transfused neutrophils to the inflamed peritoneal cavity, as well as the bactericidal capability of the host. (A) Enhanced neutrophil recruitment to inflamed peritoneal cavity in SF1670-treated mice. Mice were intraperitoneally challenged with 1 mL of 3% TG before intravenous injection of the indicated dose of SF1670. Mice were euthanized 4 hours after the challenge. Total neutrophil content in the lavage fluid was calculated by microscopic examination. Data shown are mean ± SD of 3 mice. **P < .01 vs control. (B) Recruitments of adoptively transferred neutrophils to the inflamed peritoneal cavities in neutropenic mice. The neutropenic mice were prepared as described in supplemental Figure 7. Purified mouse neutrophils were pretreated with or without SF1670 (250nM) for 30 minutes at 37°C and then labeled with CMTMR or CMFDA. Labeled cells were mixed (1:1) as indicated and then intravenously injected to neutropenic mice that had been challenged with 3% TG for 2.5 hours. Peritoneal lavage was harvested 1.5 hours after the injection of cell mixture. The amount of adoptively transferred neutrophils recruited to the peritoneal cavity was analyzed using an FACSCanto II flow cytometer and FACSDiva software. (C) Relative recruitment of neutrophils was calculated as the ratio of indicated populations in the peritoneal lavage. Data shown are mean ± SD of 3 mice. *P < .05 vs DMSO-treated neutrophils. (D) In vivo bactericidal assay. Live E coli particles (1.0 × 105 CFU) were injected intraperitoneally to neutropenic mice 4 hours before neutrophil-adoptive transfer. Mouse neutrophils were pretreated with or without SF1670 (250nM) for 30 minutes at 37°C and then washed twice before adoptive transfer. Two hours after transfer, mice were euthanized, and peritoneal lavage was collected to check bacteria loads. **P < .01, ***P < .001 vs control; ##P < .01 vs DMSO.
