Ara-LAM Mediated Protection in Leishmaniasis Figure 4. Hstone H3 modifications at the IL-12 promoter in Ara-LAM treated infected macrophages. Murine macrophages (16106cells/ mL) were treated and infected as described in the Figure 3 legend. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (A) or IP acetyl-H3 (B), and conventional RT-PCR or quantitative real-time PCR was performed using primers specific to the IL-12p40 promoter. *P,.001 compared with Ara-LAM–pretreated infected macrophages. In a separate experiment peritoneal macrophages were treated and infected by Leishmania for 15, 30 min as described in Figure 3 legend. Cytosolic and nuclear protein extracts were analyzed for the nuclear translocation of NF-kB (C). The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (i,ii) (inset). Peritoneal macrophages (16106 cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 45 min. Immunoprecipitations were conducted using NF-kB specific Abs. Conventional RT-PCR or quantitative real-time PCR was performed for amplifying the putative NF-kB binding sites of the IL-12p40 promoter. *P,.001 compared with Ara-LAM–pretreated infected macrophages. doi:10.1371/journal.pone.0024141.g004 SOCS3 has also been reported to down regulate major histo- compatibility -II (MHC-II) expression resulting in decreased antigen presentation by macrophages [21], a hallmark of leishmanial pathogenesis [22]. Fascinatingly, Ara-LAM treatment prominently restores MHC-II expression in L. donovani–infected macrophages (figure 7A). We also investigated the role of Ara- LAM on the antigen presentation capability of infected macro- phages by measuring their ability to activate l peptide stimulated T cell hybridoma 9H3.5 cells in terms of IL-2 production which is important for T effector cell functioning. In case of infected macrophage, there was a significant impairment of antigen presentation compared with uninfected macrophages (figure 7C) along with lower IL-2 secretion from T cell hybridoma 9H3.5 when co-cultured with infected macrophages (figure 7B). Inter- estingly the release of IL-2 from 9H3.5 cells and antigen presentation ability could be effectively increased under Ara- LAM pretreatment in infected macrophages. (figure 7B,7C). Moreover, parasitized macrophages in which SOCS3 was knocked PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24141