Ara-LAM Mediated Protection in Leishmaniasis Ara-LAM leads to significant protection during leishmania infection via a Th1 polarized anti-parasitic response [8], we further probed Ara-LAM induced leishmanicidal activity in the presence of p38MAPK and ERK inhibitors. Interestingly, preincubation of cells with PD098059 (an ERK inhibitor) followed by Ara-LAM treatment in parasitized macrophages caused slight increase in host protective IL-12 (figure 2A, 2B) and NO generation (figure 2E, 2F) along with concomitant decrease in IL-10 production (figure 2C, 2D) both at the protein and mRNA level resulting in reduction in intracellular parasite load compared to Figure 2. Ara-LAM mediated effector function depends on the reciprocal activation of p38MAPK and ERK–1/2. Peritoneal macrophages (26106cells/mL) were treated with SB203580 (SB) or PD098059 (PD) for 2 h, followed by Ara-LAM treatment for 3 hr. The cells were then infected with Leishmania parasite for 24 h and assayed for the levels of IL-12 (A) and IL-10 (C) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. Macrophages cultured in a 24-well plate (16106 cells/mL) were pretreated and infected as described above and assayed for the levels of extracellular NO as described in the Materials and Methods (E). Asterisks indicate statistically significant induction of nitrite generation, compared with Ara-LAM–pretreated infected macrophages. *P,.001. From a separate set of cells (26106 cells/mL) RNA was isolated and levels of mRNA expression for IL-12p40 (B), IL-10 (D), inducible nitric oxide synthase 2 (iNOS2) (F) were determined by quantitative RT-PCR. Results are presented as changes (nfold) relative to uninfected control cells. The data represent the mean values6 standard deviation of results from 3 independent experiments that all yielded similar results. In a separate experiment, the macrophages were cultured in coverglasses treated with SB or PD for 2 h, subsequently followed by 3 hr of Ara-LAM treatment and 4 h of Leishmania infection. After indicated time of incubation intracellular parasite number were assessed as described in methods. Pretreatment with SB significantly inhibited Ara-LAM – mediated parasite killing compared with levels in corresponding Ara-LAM pretreated infected controls (G). *P,.001 for SB. doi:10.1371/journal.pone.0024141.g002 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24141