Figure 6. Parasite-induced host arg1 expression impairs macrophage anti-leishmanial activity.

A) Expression of hamster arginase mRNA in primary hamster splenic fibroblasts that were uninfected (Un) or infected in vitro with L. donovani stationary-phase promastigotes for 24 hrs (Inf). The mean and standard deviation (error bars) of the fold-increase of arg1 mRNA relative to normal BHK cells, determined by real time RT-PCR in groups of 6 animals, is shown from data pooled from 3 independent experiments. B) Arginase activity and NO production in BHK fibroblasts. Arginase activity was determined stimulated with IL-4 or IFN- γ respectively. Arginase activity was determined in BHK cells that were unstimulated (Uns) or stimulated for 48h with IL-4 (10% v/v), LPS (1 µg/mL), or both. The mean and standard deviation (error bars) of the arginase activity of 6 different stimulated samples compared to that of non stimulated cells determined by assay of urea production, is shown from a single experiment that is representative of 2 experiments. NO production was estimated by the measurement of nitrites + nitrates in supernatants of unstimulated cells (Uns) and cells stimulated with IFN-γ (10% v/v of hamster recombinant IFN-γ supernatants) plus 1 µg/mL LPS for 48h. The mean and standard deviation (error bars) of the nitrites/nitrates of 6 different samples determined by Griess assay is shown from a single experiment that is representative of 2 experiments. C) Expression of hamster arginase mRNA in hamster BHK cells that were uninfected (Un) or infected in vitro with L. donovani stationary-phase promastigotes for 24 hrs (Inf) and cultured with or without recombinant hamster IL-4 (10% v/v supernatant) or sham supernatant. The mean and standard deviation (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 animals, is shown from data pooled from 3 independent experiments. D) Expression of hamster arginase mRNA in hamster BHK fibroblasts that were uninfected (Un) or infected in vitro with L. donovani tissue-derived amastigotes for 24 hrs (Inf). The mean and standard deviation (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 animals, is shown from data pooled from 2 independent experiments. E) Effect of cycloheximide (CHX) on parasite-induced hamster arg1 mRNA expression in BHK cells was determined by exposing cells to CHX (20 µg/mL) early (4–12 hrs) and/or late (12–24 hrs) during a 24-hr L. donovani infection. The mean and SD (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 samples, is shown from data from a single experiment that is representative of 2 independent experiments. F) Induction of expression of hamster arg1 in peritoneal macrophages by soluble parasite factors was determined by culturing hamster peritoneal macrophages with stationary phase promastigotes (1∶10 ratio) separated by a 0.4 µ pore size membrane (Falcon). The mean and SEM (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 animals, is shown from data pooled from 3 independent experiments. G) miRNAi-mediated arg1 knockdown in BHK cells. Arg1 protein in BHK cells stably transfected with a miRNAi vector targeting arg1 (Arg-1 KD) or stably transfected with a miRNAi vector coding a control sequence (Control) determined by Western blot using a specific polyclonal antibody raised against hamster arg1. Representative of 2 different blots. H) Effect of miRNAi-mediated knockdown on parasite burden was determined in BHK cells that were transfected with a non-targeting miRNAi vector (control) or a vector specific to hamster arg1. The transfected cells were infected with L. donovani metacyclic promastigotes and the mean and standard deviation (error bars) of the parasite burden at 4, 24, 48 and 72 hrs post-infection, determined by luminometry, is shown from a single experiment that is representative of 2 independent experiments. There was no difference in parasite burden between the two groups at 4 and 24 hrs post-infection. I) Arg1 knockdown did not induce NO production in BHK cells infected with L. donovani. NO production in BHK cells stably transfected with a miRNAi vector targeting Arg1 (Arg1 knockdown) or transfected with an irrelevant miRNAi vector (Non targeted). The mean and standard deviation (error bars) of the nitrites/nitrates released in the supernatant of cells after 48h infection with L. donovani was determined by Griess assay. Data are from a single experiment that is representative of 3 independent experiments. (**p<0.001). The statistical significance of differences in each of the panels is identified by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).