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

<p><b>A</b>) Expression of hamster arginase mRNA in primary hamster splenic fibroblasts that were uninfected (Un) or infected in vitro with <i>L. donovani</i> 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>B</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. <b>C</b>) Expression of hamster arginase mRNA in hamster BHK cells that were uninfected (Un) or infected in vitro with <i>L. donovani</i> 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. <b>D</b>) Expression of hamster arginase mRNA in hamster BHK fibroblasts that were uninfected (Un) or infected in vitro with <i>L. donovani</i> 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. <b>E</b>) 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 <i>L. donovani</i> 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. <b>F</b>) 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. <b>G</b>) 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. <b>H</b>) 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 <i>L. donovani</i> 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. <b>I</b>) Arg1 knockdown did not induce NO production in BHK cells infected with <i>L. donovani</i>. 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 <i>L. donovani</i> 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).