modifications at the IL-12p40 promoter loci, making it more accessible for the recruitment of NF-kB leading to transcriptional induction of IL-12 [13]. In contrast, enhanced IL-10 transcription is associated with ERK1/2 activation leading to phosphorylation and acetylation of histone H3 at the IL-10 promoter loci which facilitates the binding of Signal transducer and activator of transcription 3 (STAT3) to the IL-10 promoter resulting in enhanced IL-10 transcription [14]. Moreover activated STAT3 attenuates the transcription of proinfllammatory mediators with the help of Suppressor of cytokine signaling 3 (SOCS3) inductions [15,16, and 17]. Previous work from our laboratory has shown that Ara-LAM is involved in IL-12 induction and IL-10 attenuation during infection demonstrating the suitability of it as a potential candidate for immunotherapy to cure VL. But, how Ara-LAM treatment of parasitized macrophages leads to epige- netic modification at the locus of these two counteractive cytokine genes leading to their transcriptional regulation and the involve- ment of MAPK signaling in this regard is yet to be explored. In the present study, we have found that Ara-LAM, a TLR-2 ligand confers protection against leishmanial pathogenesis via reciprocal regulation of MAPK signaling. This Ara-LAM mediated regulation of MAPK signaling resulted in antagonistic regulation of IL-12 and IL-10 in host macrophages. Detailed investigation at the molecular level showed that Ara-LAM could induce IL-12 by selective phosphorylation and acetylation of histone H3 residues at the IL-12p40 promoter region while attenuated IL-10 production by abrogating such histone H3 Ara-LAM Mediated Protection in Leishmaniasis modification at IL-10 promoter in parasitized macrophages. This antagonistic regulation of effector response by Ara-LAM in the form of IL-10 and IL-12 was further linked to STAT3 and SOCS3 which were found to be crucial in regulating the host protective immune response in leishmania infected macrophages. Results 1. ERK and p38 MAP kinases differentially regulate Ara- LAM-mediated generation of macrophage effector molecules in Leishmania donovani infected macrophages Ara-LAM has been reported to confer protection against leishmanial pathogenesis via TLR2 signaling–mediated induction of the proinflammatory response [8]. However, it is unclear whether Ara-LAM can modulate the p38 and ERK1/2 MAPK signaling molecules which play differential role in the leishmanial pathogenesis [12]. We found that at an early time point, Ara-LAM stimulated phosphorylation of p38MAPK was much higher than infected macrophages; in contrast, ERK1/2 phosphorylation was abrogated in Ara-LAM treated parasitized macrophages com- pared to that in infected macrophages (figure 1A). Interestingly, gene silencing of TLR-2 in infected macrophages reverses the Ara- LAM mediated regulation of MAPK family (figure 1B). The MAPKs are key regulators of IL-10, IL-12 generation and NO production [18,19]; leishmanial parasite leads to impaired effector response by suppressing p38MAPK induced IL-12, NO secretion while augmenting ERK-1/2 induced IL-10 production [12]. As Figure 1. Ara-LAM mediated changes in MAPK signaling cascade in Leishmania donovani–infected macrophages. Macrophages were pretreated with Ara-LAM for 3 hr or in some cases followed by Leishmania infection for 15, 30, 60, and 120 min. The cells were then lysed and subjected to Western blot with anti-pp38MAPK, p38MAPK and pERK1/2, ERK1/2, as described in Materials and Methods (A). In separate experimental sets, cells were transfected with control siRNA or TLR2-specific siRNA for 24 hr washed and then treated with Ara-LAM followed by L. donovani for 30 min. Western blot analysis was performed to analyze the expression of anti-pp38MAPK, p38MAPK and pERK1/2, ERK1/2 (B). doi:10.1371/journal.pone.0024141.g001 PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24141