(Grand Island, NY, USA) and ELISA Assay Kit (Quantikine M) for tumour necrosis factor (TNF)-a, IL-12, IL-10 were from R&D Systems (Minneapolis, MN, USA). dNTPs, RevertAidTM M- MuLV Reverse Transcriptase, oligo dT, RNase inhibitor and other chemicals required for cDNA synthesis were from Fermentas (USA). Anti-phospho-H3 and acetyl-H3 Abs were purchased from Abcam and chromatin immunoprecipitation (ChIP) assay kits were purchased from Millipore. SOCS3, STAT3 siRNA were procured from Santa Cruze biotech. T-cell hybridoma and lambda repressor peptide 9H3.5 (I-Ad restricted T-cell hybridoma) specific for lambda repressor N-terminal sequence 12–26 [LEDARRLKAIYEKKK, defined as lR 12–26]. T-cell hybridomas and peptides were kind gifts of Professor M. L. Gefter, Massachusetts Institute of Technology, Cambridge, MA, USA. The IL-2 dependent cell line HT-2 (mouse T helper cell line) was obtained from American Type Culture Collection. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 2-ME (561025 M) at 5% CO2 in humidified atmosphere. Animals and parasites BALB/c mice were purchased from the National Centre for Laboratory Animal Sciences, India. For each experiment 8–10 mice (4–6 weeks old) were used, regardless of sex. L. donovani organisms (strain MHOM/IN/1983/AG-83) were maintained in Medium 199 (Sigma) plus 10% fetal calf serum (Gibco). Amastigotes were prepared as described elsewhere [34]. Station- ary-phase promastigotes obtained by suitable transformation were used for experiments. All experimental protocols were given prior approval by the institutional animal ethics committee. Isolation and purification of Ara-LAM Ara-LAM was isolated as described elsewhere [35] Lipopoly- saccharide contamination was checked by the Limulus test and was ,25 ng/mg in Ara-LAM. The noncytotoxic dose of Ara- LAM was 3 mg/mL [36]. Peritoneal macrophage preparation Macrophages were isolated by peritoneal lavage with ice-cold phosphate-buffered saline at 48 h after intraperitoneal injection of 1.0 mL of sterile 4% thioglycolate broth (Difco). Cells were cultured as described elsewhere [37].The adherent cell population was cultured for 48 h prior to any treatment, to achieve the resting state. Uptake and intracellular multiplication For assessing the activity of Ara-LAM against the amastigote stage of parasite, peritoneal macrophages cultured on glass cover slips were pretreated with Ara-LAM (3 mg/ml) for 3 h, followed by infection with L.donovani promastigotes at a ratio of 1:10 for the indicated time periods; macrophages were then fixed and stained as described elsewhere [38] for calculation of the number of intracellular parasites. When indicated, the uptake and multipli- cation of L.donovani was studied in the presence and absence of the p38MAPK inhibitor SB203580 (10 mg/mL), or ERK inhibitor PD098059 (100 mmol/L; Sigma) [12]. Cytokine enzyme-linked immunosorbent assay (ELISA) The conditioned medium of macrophage culture was assayed for mouse cytokines and chemokines with use of the sandwich ELISA kit (Quantikine M; R&D Systems). The assay was performed according to the manufacturer’s instructions. Ara-LAM Mediated Protection in Leishmaniasis Preparation of cell lysate and immunoblot analysis Cell lysates were prepared as described elsewhere [39]. Equal amounts of protein (50 mg) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and were subsequently transferred to a nitrocellulose membrane. The membrane was blocked overnight with 3% bovine serum albumin in Tris-saline buffer (pH, 7.5), and immunoblotting was performed to detect Inducible Nitric Oxide Synthase 2 (iNOS2) and phosphorylated or dephosphorylated forms of p38MAPK, ERK1/2, STAT3 and b-Actin as described elsewhere [40]. Nitrite generation Nitrite level in culture was measured using the Nitric Oxide Colorimetric Assay kit (Boehringer Mannheim Biochemicals) [41]. Cell-free supernatants were collected from different experimental sets at different time points of infection, and nitrite levels were estimated in accordance with the manufacturer’s instructions. Data were expressed in micromoles of nitrite. Antigen presentation assay Antigen presenting ability of infected macrophages was studied by their ability to present lR12–26 to T-cell hybridoma (9H 3.5). The antigen presenting cells (APC) were incubated for 24 h with specific peptide and T-cell hybridoma in complete RPMI medium in a 37uC incubator. The culture supernatants were analysed for the presence of IL-2 by growing an IL-2 dependent cell line HT-2 in the supernatants. HT-2 (104 cells per well) was incubated with a 50% concentration of culture supernatant for 48 h. The cells were then pulsed with 1 mCi of 3H-Thymidine [6.7 Ci/mmole, New England Nuclear, Boston, NE, USA)] for the last 18 h [42]. The incorporation of radioactive thymidine was then assessed by a scintillation counter (Packard). Flow cytometry BALB/c-derived macrophages were stained with phycoerythrin (PE) labeled anti-MHCII antibodies. The cells were analyzed by a FACSVantageTM flow cytometer (Becton Dickinson). Preparation of nuclear and cytoplasmic extracts The nuclear extracts were prepared from normal and infected macrophages as described elsewhere [43]. Briefly, sedimented cells were resuspended in hypotonic buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, and 0.5 mM DTT) and allowed to swell on ice for 10 min. Cells were homogenized in a homogenizer. The nuclei were separated by spinning at 33006g for 5 min at 4uC. The supernatant was used as the cytoplasmic extract. The nuclear pellet was extracted in nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) for 30 min on ice and centrifuged at 12,0006g for 30 min. The supernatant was used as nuclear extract. CHIP assay CHIP assays were conducted using the CHIP Assay kit following the manufacturers Protocol (Millipore). Briefly, 16106 peritoneal macrophages were plated overnight in six-well plates. Cells were stimulated as described in figures, and then fixed for 10 min at 37uC in 1% paraformaldehyde. Cells were washed on ice with ice-cold HBSS containing 1 mM PMSF, harvested and then lysed in SDS lysis buffer. DNA was sheared by ultrasonica- tion using a High Intensity Ultrasonic Processor (hielscher) for 3610 s pulses at 20% amplitude. Lysates were cleared by centrifugation and diluted in ChIP dilution buffer. Lysates were PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24141