Fig. 4.

Fig. 4.

Pre-treatment with the immunosuppressant rapamycin before genetic induction of ubiquitous GSP-Il-1β^mat alleviates systemic inflammation. (A) Western blot showing dose-dependent response of ribosomal S6 phosphorylation to increasing rapamycin concentrations (0–250 nM) in zebrafish embryos. The level of total S6 was unaffected by rapamycin treatment. (B) Schematic of experimental approach. Embryos were treated with vehicle (DMSO) or 250 nM rapamycin (Rap) at 1 dpf (yellow arrowhead). At 2 dpf, embryos were induced with vehicle or 1 µM Teb (red arrowhead). Survival was determined daily over 10 days for vehicle, Teb (red arrow), and Teb plus rapamycin (orange arrow) cohorts. In a parallel experiment, larvae were imaged at 4 dpf (black arrowheads) and total neutrophil numbers determined. (C) Representative images of larvae at 4 dpf showing beneficial effects of rapamycin pre-treatment. Vehicle treated, 250 nM rapamycin (Rap) treated, 1 µM Teb, and 250 nM Rap+1 µM Teb-treated larvae are shown. Brightfield images are minimum intensity projections and mCherry images are maximum intensity projections of confocal stacks. (D) Ten-day survival curves showing increased survival for larvae pre-treated with 250 nM rapamycin (Rap) at 1 dpf (yellow arrowhead) followed by 1 µM Teb at 2 dpf (red arrowhead). Rapamycin treatment alone (yellow line) shows equivalent survival to vehicle controls (not shown); n=30 for each cohort. (E) Pre-treatment with 250 nM rapamycin (Rap) reduces total neutrophil number versus 1 µM Teb alone. Vehicle, Rap alone or Rap+Teb were not significantly different from each other; n=3 for each cohort (one way ANOVA with Tukey HSD). (*P<0.05, **P<0.01, ***P<0.001).