Ara-LAM Mediated Protection in Leishmaniasis Figure 5. STAT3 silencing potentiate Ara-LAM mediated abrogation of SOCS3 expression in Leishmania-donovani infected macrophages. Peritoneal macrophages (26106 cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 hr. The cells were then lysed and subjected to Western blot with anti-SOCS3 antibody as described in Materials and Methods (A). In a separate experiment macrophages were transfected with either control-siRNA or STAT3 specific siRNA, subsequently followed by Ara-LAM treatment for 3 hr and Leishmania infection for another 3 h. RNA was isolated and semi quantitative RT-PCR analyses for SOCS3 and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (B). Changes in expression of SOCS3 mRNA were also determined by quantitative real-time PCR. Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results (C). *P,.001 compared with infected macrophages. The comparison of SOCS3 expression between Ara-LAM treated infected macrophages and STAT3 siRNA group did not show any statistical significance. doi:10.1371/journal.pone.0024141.g005 down prior to Ara-LAM treatment resulted in enhanced MHC-II expression (figure 7A) as reflected by greatly increased antigen presentation (figure 7C) and higher IL-2 production from 9H3.5 cells (figure 7B) demonstrating that SOCS3 interferes with antigen presentation and decreased SOCS3 was further potentiating Ara-LAM mediated antigen presentation ability of infected macrophages. Discussion Leishmania-induced deactivation of macrophage functions are due to defects in the signaling pathways, which facilitates the parasite to infect and propagate within the cell [23]. Recently the capacity of Leishmania to suppress macrophage function has been linked to alterations in MAPK signaling cascades [12,24,25]. To revert this parasite induced changes in MAPK signaling, novel immunomodulators are being evaluated for their functionality in VL. Ara-LAM, a potential immunomodulator strongly initiates the host protective immune response during VL via TLR-2 mediated proinflammatory responses [8]. But its role in regulating the MAPK signaling pathway has not been explored till date. Recently it has been demonstrated that the differential activation of the MAPKs regulates cytokine production. CD40, a costimulatory molecule with host-protective anti-leishmanial function, could induce both IL-10 and IL-12 by differential activation of p38 MAPK and ERK-1/2, respectively. This reciprocal regulation of p38 MAPK and ERK1/2 has been shown to be responsible for the protection versus susceptibility to infection [12,24]. Ara-LAM pretreatment restored the impaired p38MAPK phosphorylation in parasitized macrophages and abrogated the ERK1/2 phosphorylation via TLR-2 mediated downstream signaling (figure 1) thus shifting the macrophageal microenviron- ment from anti-inflammatory (IL-10) towards proinflammatory (IL-12) host protective immune response (figure 2). Our finding is consistent with other studies which have also highlighted the involvement of this novel molecule, Ara-LAM, in inducing proinflammatory responses (IL-8, TNF-a) in macrophages via interacting with TLR-2 [26,27]. Additionally, Ara-LAM has been demonstrated as a major component responsible for the pro- inflammatory activity of the whole bacteria (M. smegmatis and M. fortuitum) [28]. Having identified the reciprocal alteration in Ara-LAM mediated production of IL-12 and IL-10 during VL, we studied whether Ara-LAM could modulate the chromatin (specifically histone H3) at the locus of these two counteractive cytokine genes since covalent modification to histones of chromatin can lead to differential gene expression. Our study revealed that Ara-LAM pretreatment leads to IL-12 induction via histone H3 phosphor- ylation (figure 4A) and acetylation (figure 4B) of chromatin at IL- 12 locus along with IL-10 attenuation by abrogating the parasite induced histone H3 phosphorylation (figure 3A) and acetylation (figure 3B) at IL-10 locus during leishmanial pathogenesis. Thus Ara-LAM mediated gene-specific chromatin modifications are found to be associated with transient silencing of IL-10 genes and priming of the IL-12 gene. To further ascertain the role of MAPKs in the regulation of counteracting cytokines IL-10 and IL-12, we investigated Ara- LAM mediated transcriptional alteration of these cytokine genes in the presence of their respective pharmacological inhibitors. Our results implicated that the inhibition of p38 MAPK pathway in Ara-LAM treated parasitized macrophages abrogates chromatin remodeling and nuclear translocation of NF-kB thereby attenuat- ing IL-12 transcription (figure 4A, 4B, 4C) and simultaneously reverses Ara-LAM mediated attenuated IL-10 production in parasitized macrophages via increased histone H3 phosphoryla- tion, acetylation and enhanced STAT3 recruitment to the IL-10 promoter leading to the induction of IL-10 transcription (figure 3A,3B,3D,3E). In contrast ERK inhibition of Ara-LAM treated parasitized macrophages leads to further increase in IL-12 transcription (figure 4A, 4B, 4C) along with significant reduction of the IL-10 (figure 3A,3B,3D,3E) compared to Ara-LAM treatment only. Therefore, this study suggested a crucial role of PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24141