Fig 3. Ara-LAM reciprocally regulated IRF4 and IRF8 expression and corresponding immune response during L. donovani infection both in vitro and in vivo.

(A) L. donovani infected and uninfected control peritoneal macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. Data represent means ± SD for three experiments. Inset 1: Comparison between IRF4 and IRF8 expression in L. donovani infected and uninfected control macrophages. (B) Peritoneal macrophages (2x10⁶cells) from BALB/c mice were cultured and subjected to Ara-LAM pre-treatment (3μg/ml) for 3hrs, followed by L. donovani challenge for 6hrs. Both rIFN-γ stimulated (20ng/ml, 45mins) and unstimulated cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. Data are from one representative experiment, which was performed at least thrice. Inset 2: Reciprocal expression of IRF4 and IRF8 by Ara-LAM in L. donovani infected macrophages. (C-D) BALB/c derived peritoneal macrophages (2x10⁶cells) were treated with Ara-LAM (3μg/ml) for 3hrs, followed by L. donovani challenge for 6hrs. After 45 min of rIFN-γ stimulation immuno-precipitations were conducted using IRF8 (IP:IRF8) and IRF4 (IP: IRF4) specific Abs. Semi quantitative RT-PCR was performed for amplifying the putative IRF8 binding sites of the IL-12p40 promoter and IRF4 binding sites of the IL-10 promoter. Data represent means ± SD for three sets of experiments. Inset 3: Ara-LAM mediated up-regulation of IRF8 binding to the IL-12 promoter and down-regulation of IRF4 binding to the IL-10 promoter in infected macrophages. (E-F) BALB/c mice were injected with respective shRNAs (for 2 days) followed by treatment with either phosphate-buffered saline (PBS) (control) or Ara-LAM (30 μg intraperitoneally) for 2 days, after which mice were infected. 28 days later, mice were sacrificed and splenocytes (2x10⁶) were collected in Trizol for mRNA extraction and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis (see Methods). Data are from one of three representative experiments. Inset 4: Correlations (R-values) between IRF4 and IL-10 or IL-12 expression in Ara-LAM treated and untreated infected splenocytes. Inset 5: Correlations (R-values) between IRF8 and IL-10 or IL-12 expression in same set of mice. (G-H) A separate set of splenocytes (2x10⁶) were stimulated with soluble leishmanial antigen (SLA) at 5 μg/ml for 48 h. Release of Interleukin 12 (IL-12) p70 and Interleukin 10 (IL-10) culture supernatants were determined by enzyme-linked immunosorbent assay. Data represent means ± SD for 4 animals per group. ***P <.001 and **P < .01 for the comparison with infected mice.