Ara-LAM treated parasitized macrophages (figure 2G). In contrast SB203580 (p38 MAPK inhibitor) treatment of infected macro- phages almost completely abrogated Ara-LAM induced genera- tion of IL-12 (figure 2A,2B) and NO (figure 2E,2F) whilst enhancing IL-10 production (figure 2C,2D) both at the protein and mRNA level resulting in drastic increase in intracellular parasite load (figure 2G), compared to Ara-LAM treated parasitized macrophages. In contrast, treatment of cells with the drugs (PD098059 and SB203580) after adding Ara-LAM and parasite resulted in similar levels of IL-12 p40, NO and IL-10 (both at the protein as well as the mRNA level) as compared to only Ara-LAM treated parasitized macrophages (data not shown). Interestingly, PD098059 and SB203580 itself had no effect on the levels of NO, IL-12p40 and IL-10 in Leishmania infected macrophages both at the protein as well as the mRNA level (figure 2). 2. Ara-LAM stimulation of infected macrophages resulted in histone modifications and altered transcription factor binding at the IL-10 and IL-12 promoter region via reciprocal regulation of MAPK pathway To get the mechanistic insight by which Ara-LAM reduces IL- 10 and augmentes IL-12 synthesis during leishmanial pathogen- esis, we analyzed various histone modifications at the IL-10 and IL-12 locus by Chromatin immunoprecipitation assay (CHIP). High level of IL-10 production in leishmania-infected macro- phages was found to be associated with increased histone H3 phosphorylation (figure 3A) and acetylation (figure 3B) at the IL- 10 promoter which was abrogated in Ara-LAM pretreated parasitized macrophages. Interestingly, ERK inhibition prior to Ara-LAM treatment in parasitized macrophages slightly reduced both histone H3 phosphorylation (figure 3A) and acetylation (figure 3B) at IL-10 locus compared to only Ara-LAM treated parasitized macrophages. In contrast there was significant increase in histone H3 phosphorylation (figure 3A) and acetylation (figure 3B) at IL-10 locus in parasitized macrophages treated with both p38 inhibitor SB203580 and Ara-LAM, compared to only Ara-LAM pretreated parasitized macrophages. Because STAT3 is an obligate factor required for IL-10 gene transcription [20], we explored whether Ara-LAM could modulate the STAT3 expres- sion during leishmanial infection. Ara-LAM pretreatment of host macrophages, abrogated parasite induced phosphorylation (figure 3C) and nuclear translocation (figure 3D) of STAT3 at early time points. Strikingly, inhibiting ERK resulted in decreased phosphorylation (figure 3C) and subsequent nuclear translocation (figure 3D) of STAT3 in Ara-LAM treated parasitized macro- phages while p-38 inhibition reverses the phenomena and significantly increases the nuclear translocation of phosphorylated STAT3 compared to only Ara-LAM treated parasitized macro- phages (figure 3C,3D). CHIP assays were performed to examine the binding of STAT3 to the IL-10 promoter. Unstimulated control cells exhibited virtually no binding of STAT3 to the IL-10 promoter. Ara-LAM pretreatment significantly reduced the parasite induced binding of STAT3 to the IL-10 promoter (figure 3E). Furthermore, inhibiting ERK prior to Ara-LAM treatment in infected macrophages, caused further decrease in STAT3 binding to the IL-10 promoter while inhibition of p38 in Ara-LAM treated macrophages resulted in significant increase in the binding of STAT3 to the IL-10 promoter when compared to only Ara-LAM treated infected sets (figure 3E). Transcriptional induction of IL-12 gene requires histone modification of the promoter region, a mast for NF-kB recruitment, which plays a pivotal role in IL-12 transcription Ara-LAM Mediated Protection in Leishmaniasis and is regulated by STAT3 [13,15]. CHIP assay showed infected macrophages pretreated with Ara-LAM had higher levels of phosphorylated (figure 4A) and acetylated (figure 4B) H3 associated with IL-12p40 promoter relative to infected macro- phages. Moreover, ERK inhibition in presence of Ara-LAM caused further increase in histone H3 phosphorylation (figure 4A) and acetylation (figure 4B) at IL-12p40 locus while p-38 inhibition significantly attenuated such Ara-LAM mediated histone modifi- cation at this locus in infected macrophages compared to only Ara- LAM treated parasitized macrophages (figure 4A,4B). Ara-LAM induced nuclear translocation of NF-kB was p38 dependent as p38 inhibition showed marked reduction in the nuclear translocation of NF-kB in infected macrophages (figure 4C). Because, STAT3 plays a decisive role in the recruitment of NF-kB to the IL-12p40 promoter region [15], we investigated Ara-LAM mediated recruitment of NF-kB to the IL- 12p40 promoter region under STAT3 silenced condition. There was significant enhanced recruitment of NF-kB to the IL-12p40 promoter in STAT3 knocked down macrophages, as compared to only Ara-LAM treated parasitized macrophages (figure 4D), thereby pointing towards the critical role of STAT3 in down- regulating IL-12 gene expression in parasitized macrophages under Ara-LAM-treatment. 3. Ara-LAM induced attenuated SOCS3 expression is STAT3 dependent during VL STAT3 activation plays a critical role in SOCS3 gene expression which in turn suppresses macrophage function [16]. As Ara-LAM treatment reduces the parasite induced STAT3 phosphorylation, we further studied its effect on SOCS3 expression, a downstream signaling intermediate of STAT3 pathway. Pretreatment of parasitized macrophages with Ara- LAM resulted in significant abrogation of the expression of SOCS3 both at the protein (figure 5A) and m-RNA level (figure 5B, 5C) compared to infected macrophages. To further examine whether Ara-LAM suppresses the expression of SOCS3 via the effective regulation of STAT3 signaling, we knock down STAT3 genes using STAT3 specific small interfering RNA (siRNA). STAT3 silencing of Ara-LAM treated parasitized macrophages resulted in higher reduction of SOCS3 expression as compared to only Ara-LAM treated parasitized macrophages which is not statistically significant. (figure 5A, 5B, and 5C). These findings clearly suggest that Ara-LAM negatively regulates the STAT3 signaling leading to marked reduction in SOCS3 expression. 4. SOCS3 silencing significantly enhances host protective proinflammatory response and antigen presentation in Ara-LAM treated infected macrophages Previous study with Ara-LAM showed that it could confer protection against leishmanial pathogenesis via induction of the proinflammatory response [8]. As SOCS3 has been speculated to participate in inhibition of macrophage activation [17], we tested whether SOCS3 interferes with Ara-LAM -stimulated signal transduction events in Leishmania donovani infected macrophages. We observed that SOCS3 silencing could markedly increase the Ara-LAM induced production of proinflammatory mediators such as IL-12 (figure 6B, 6F), TNF-a, (figure 6A, 6F) NO (figure 6D, 6E, 6F) along with the downregulation of immunosupressive cytokine IL-10 (figure 6C, 6F) in parasitized macrophages both at the protein and mRNA level. These results suggest that Ara-LAM leads to the production of host protective proinflammatory response by supressing SOCS3 functioning. PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24141