Growth Factor and Th2 Cytokine Signaling Pathways Converge at STAT6 to Promote Arginase Expression in Progressive Experimental Visceral Leishmaniasis
Figure 5
Inhibition of IGF-1R signaling decreases arg1 expression and parasite burden in L. donovani infected macrophages.
(A–C) Hamster BMDMs were treated with an IGF-1R inhibitor (Picropodophyllin, PPP) or an equivalent concentration of vehicle (DMSO) and infected in vitro with L. donovani for 24 or 48 hrs. (A) arg1 mRNA expression determined by qRT-PCR at 24 hrs post-treatment. B) Intracellular parasite burden determined by luminometry from luciferase-transfected L. donovani at 48 hrs post-treatment. C) Viability of BMDMs determined by luminometry (Cell titer Glo) at 48 hrs post-treatment. D–J) Splenic macrophages from L. donovani infected hamsters (21–28 days p.i.) were isolated by adherence and treated with an IGF-1R inhibitor, AKT inhibitor, or JAK inhibitor, or an equivalent concentration of vehicle control (DMSO) for 24 of 48 hrs. D) arg1 mRNA expression determined by qRT-PCR at 24 hrs post-treatment. E) arg1 protein expression determined at 48 hrs post-treatment. Bars represent the percent of expression with reference to control (DMSO treated) cells calculated by densitometry analysis of immunoblot bands from 3 independent experiments. A representative immunoblot is also shown. F) Intracellular parasite burden determined by luminometry from luciferase-transfected L. donovani at 48 hrs post-treatment. G) Viability of splenic macrophages determined by luminometry (Cell titer Glo) at 48 hrs post-treatment. H) arg1 mRNA expression determined by qRT-PCR at 24 hrs post-treatment with AKT inhibitor (AKTi; CAS# 612847-09-3, Calbiochem), JAK inhibitor (JAKi; CAS# 457081-03-07) or DMSO control. I–J) Intracellular parasite burden determined by luminometry from luciferase-transfected L. donovani at 48 hrs post-treatment with AKT inhibitor (I) or JAK inhibitor (J), compared to DMSO treated controls. In each of the panels the mean and SEM from a single experiment that was representative of 2–3 independent experiments is shown. *p<0.05; ***p<0.001.