Animals
For the experimental studies, a total of seven 6–8 week-old female BALB/c mice were used from Razi Institute (Mashhad, Iran). The mice were fed standard mouse chow ad libitum throughout the study and maintained under the standard conditions according to the protocol.
Leishmania promastigote culture and FML extraction
Leishmania infantum promastigotes (MCAN/IR/07/Moheb-gh) were grown at 26 °C in brain heart infusion broth (37 g/l; Himedia, Mumbai, India) supplemented with 10% of fetal bovine serum (Gibco, Paisley, UK), hemin (0.01 g/l) and folic acid (0.02 g/l; Sigma-Aldrich, St. Louis, MO, USA).
The stationary phase growth medium was centrifuged at 6000× g for 10 min to collect the promastigotes. The pellet containing promastigotes was washed with cold phosphate-buffered saline (PBS) and was stored at -20 °C until further analysis. Aqueous extraction of FML was carried out as previously described by Foroughi-Parvar et al. [25]. Briefly, frozen pellets of the parasite were mixed with cold distilled water and centrifuged at 6000× g for 10 min to collect the supernatant. This step was repeated once again, and both the supernatants were combined and boiled for 15 min at 100 °C. The sample was then centrifuged at 6000 for 20 min, and the supernatant was lyophilized and subjected to chromatography by loading 2 ml of lyophilized sample in cold deionized distilled water (10 mg/ml) on 100 × 1.6 cm column of P10 Bio-Gel (Bio-Rad, Watford, UK) to purify the FML. The collected FML samples were analyzed further for their carbohydrate content and for the presence of 10–96 kDa bands corresponding to FML glycoprotein on 10% SDS-PAGE. The purified FML samples were lyophilized and stored at -20 °C until further use.
Isolation of peritoneal macrophages
Seven female BALB/c mice were sacrificed by CO2 euthanasia. Isolation of peritoneal macrophages was performed based on the procedure described by Bibak et al [26]. Briefly, murine peritoneal cells were harvested by lavage of the peritoneal cavity with 10 ml of RPMI 1640 medium (Invitrogen, Darmstadt, Germany). The cells were centrifuged at 200× g for 10 min and washed in PBS/cold ddH2O. The cells were then cultured in RPMI 1640 medium in petri dishes at 37 °C for 4 h. In this experiment, we considered three control groups: (i) PBS group: activated macrophages were cultured in the presence of PBS alone at the same volume as the other additions (10 μl/ml); (ii) GL group: activated macrophages treated with GL (5 μg/ml); and (iii) FML group: activated macrophages treated with FML (5 μg/ml). Petri dishes were carefully washed using Hanks’ solution to remove the non-adherent cells. The cells adhered to the Petri dishes were trypsinized and the cell concentration was adjusted to 1 × 106 cells/ml in RPMI medium (RPMI medium with 10% FCS (Invitrogen) containing 50 IU/ml penicillin/streptomycin (Sigma-Aldrich).
Treatment of peritoneal macrophages with a combination of FML and GL
Isolated peritoneal macrophages were stimulated with 10 μg/ml of LPS at 37 °C and 5% CO2 for 4 h. The activated macrophages were treated with FML together with varying concentrations of GL to assess the immunomodulatory effects of the combination of FML and GL. To prepare activated macrophages, 2 × 105 macrophage cell suspensions (200 μl/well in 96-well flat-bottom plates) in each well were treated with 10 μg/ml of LPS from Escherichia coli O111: B4 (Sigma-Aldrich) in complete RPMI 1640 medium. Thereafter, 5 μg/ml of FML and 1, 10 and 20 μg/ml of GL (Sigma-Aldrich) mixtures were added to the activate cells in the wells in triplicate, as previously described [25, 27]. The cells were then cultured at 37 °C and 5% CO2 for 48 h. In the control or PBS group, activated macrophages were cultured in the presence of PBS alone using the same volume as the other additions (10 μl/ml). For the NO assay, we used only complete RPMI 1640 medium as the blank control group. After 48 h, culture supernatants from each well of the 96-well plate were collected and stored at -80 °C until further analysis. Each experiment was performed in triplicate. The different study groups are shown in Table 1. The MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (Merck, Darmstadt, Germany) assay was performed to evaluate the macrophage viability after 48 h of incubation at 37 °C with different concentrations of GL (0.1, 1, 10, 20, 50 and 100 μg/ml).
Nitric oxide and cytokine assay
The culture supernatants were evaluated for stable end-products of NO, nitrates, and nitrites, using Standard Griess Reagent according to the instructions provided in the manual (Cayman Chemical, Michigan, USA). Levels of NO in different treatment groups were determined by reading the absorbances at 540 nm in a microplate reader (BioTek, Winooski, Vermont, USA). The mean optical density (OD) values of the blank were subtracted from the mean OD values of the test groups. The concentration of the nitrite was calculated from the standard curve obtained with serial dilutions of sodium nitrite as the standard. Presence of TNF-α, IL-10 and IL-12p70 were measured in the cell culture supernatants using sandwich ELISA kits according to the instructions of the manufacturer (eBioscience, San Diego, CA, USA). The minimum detectable concentration was 5 pg/ml for both IL-12p70 and IL-10, and 1 pg/ml for TNF-α.
Statistical analysis
GraphPad Prism software version 5.0 (GraphPad Software, San Diego, USA) was used for statistical analysis of the data. Data distribution was analyzed by a Kolmogorov-Smirnov test. According to the results of the normality test, a one-way ANOVA followed by Dunn’s or Tukey’s post-hoc test or a non-parametric Kruskal-Wallis test were used for statistical comparisons. Data are shown as the mean ± SD of three independent experiments. P-values < 0.05 were considered statistically significant.
t-test: t (44)=1.23, P=0.09