Ara-LAM Mediated Protection in Leishmaniasis Figure 7. Ara-LAM induced MHC-II expression, antigen presentation ability of infected macrophages increased under SOCS3 silenced condition. Peritoneal macrophages (26106 cells/mL) were transfected with control siRNA or SOCS3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 4 hr as mentioned above. After 24 hr of incubation treated macrophages were analyzed by flow cytometry for MHCII (FL2-H) expression as described in material method (A). Data are from 1 of 3 experiments conducted in the same way with similar results. In a separate set, normal and treated macrohages either uninfected or infected were pulsed with lR12–26, and then were incubated with T-cell hybridoma 9H 3.5. The culture supernatants were analysed for the presence of IL-2 by ELISA as described in the methods (B). Incorporation of 3H-Thymidine in the IL-2 dependent cell line HT-2 was assessed in presence of the cultured supernatant. Results are expressed as mean 6 SD of 5 replicate experiments (C). *P,.001, **P,.005 compared with infected macrophages. doi:10.1371/journal.pone.0024141.g007 significantly enhances Ara-LAM mediated recruitment of NF-kB to the IL-12 p40 promoter (figure 4D) indicating that STAT3 negatively regulates Ara-LAM mediated transcription of IL-12 gene during leishmanial pathogenesis. Moreover, STAT3 activation leads to the induction of SOCS3, which plays a decisive role in inactivation of macrophage function [16]. SOCS3 is critical for the survival strategy of mycobacteria [29], leishmania [30], listeria [31] and RNA viruses [32] in the host macrophages. Moreover silencing of the SOCS3 gene resulted in a marked reversal of the immunosuppressive effect of IL-10 on many inflammatory mediators including cytokines, chemokines, and IFN- inducible, apoptotic and TNF ligand/receptor genes [33]. Ara-LAM treatment lead to significant reduction in SOCS3 expression by inactivation of STAT3 (figure 5A, 5B, 5C). These changes resulted in significant induction of host protective proinflammatory responses (figure 6), MHC-II expression, and antigen presentation capability of infected macrophages (figure 7A, 7C) along with enhanced T cell effector functioning as evident from higher IL-2 production from co-cultured T cells (figure 7B). Further confirmation about the role of SOCS3 in the generation of host protective immune responses in Ara-LAM treated infected sets was done by SOCS3 silencing which was suggestive of SOCS3 hindrance in Ara-LAM mediated induction of proinflammatory response (figure 6, 7A, 7B, 7C). In summary, our current findings provide a detailed molecular mechanistic insight of Ara-LAM mediated IL-12 and IL-10 production in Leishmania donovani infected macrophages where MAPK signaling via differential regulation of p38 and ERK plays an important role in generating host protective immune response during leishmanial pathogenesis. Materials and Methods Reagents and Chemicals RPMI-1640 medium, M-199 medium (M199), penicillin and streptomycin, SB203580 (p38MAPK inhibitor), PD098059 (ERK inhibitor) and TRI Reagent were from Sigma (St Louis, MO, USA). Fetal calf serum (FCS) was obtained from Gibco BRL PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24141