HEPES, penicillin, streptomycin, sodium bicarbonate, 2-mercaptoethanol (2-ME), bovine serum albumin (BSA), histopaque, fetal calf serum (FCS), RPMI 1640 medium, M 199 medium were purchased from Sigma–Aldrich. Anti-PI3K and anti-phospho-PI3K (p85) were obtained from Santa Cruz Biotech, Inc. All other antibodies [anti-phosphoinositide-dependent kinase 1 (PDK1) and anti-phospho-PDK1, anti-p38 mitogen-activated protein kinase (p38MAPK) and anti-phospho-P38MAPK] and LumiGlo reagents for chemiluminescence were obtained from Cell Signaling (Beverly, MA). Tritiated thymidine [3H]-thymidine (6.7Ci/mmol) was obtained from PerkinElmer®, deoxynucleoside triphosphates obtained from Promega, Taq polymerase and Moloney leukemia virus reverse transcriptase were purchased from Invitrogen and RNeasy minikit was purchased from Qiagen.

BALB/c mice 4–6 weeks old with almost equal weight and same sex, reared in Institute facilities, were used for experimental purposes with prior approval of the Animal Ethics Committee of the Indian Institute of Chemical Biology, Kolkata and all animal experimentations were performed at Indian Institute of Chemical Biology, Kolkata, India following the National Regulatory Guidelines issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India. L. donovani isolate, AG83 (MHOM/IN/1983/AG83), originally obtained from confirmed an Indian Kala-Azar patient was used for the preparation of the live-attenuated vaccine candidate. This isolate is maintained in Golden hamsters and the promastigotes obtained after transforming the amastigotes from infected animal spleen were cultured in medium M 199 supplemented with 10% FCS along with 100U/ml penicillin and 100μg/ml streptomycin, maintained at 22°C.32

L. donovani isolate, AG83, was attenuated by gamma irradiation from a 60Co gamma chamber with a rate of radiation of 10Gy/60s at Indian Institute of Chemical Biology, Kolkata, India. AG83 parasites (5×106/BALB/c mice) were irradiated and washed three times in sterile phosphate buffer saline (PBS) and irradiated in the gamma chamber at three different doses: 50, 100, and 150Gy respectively. The description of the animal groups for the study was as follows: Group 1, normal control animals without any infection and therapeutic immunization; Group 2, infected control animals without any immunization, while Group 3, Group 4 and Group 5 were animals that had been infected and received therapeutic immunizations with L. donovani isolate, AG83 (5×106 parasites/animal) attenuated at 50, 100 and 150Gy doses of gamma radiation, respectively, at 75 days post-infection. The treatment was repeated for three immunized groups at 15-day interval. The mice were sacrificed 15 days after the last treatment, for experimental purposes.

T-cell proliferation assay was performed as described previously.33 Briefly, single cell suspensions of splenocytes from different experimental groups of BALB/c mice were prepared after Ficoll density gradient centrifugation and then suspended in complete RPMI 1640. Cells were plated in triplicate at 105 cell/well concentrations in 96-well plates and allowed to proliferate for 72h at 37°C in 5% CO2 incubator in the presence of SLA concentration (5μg/ml). Eighteen hours before they were harvested in GFC membrane, cells were pulsed with 1μCi [3H]-thymidine/well. [3H]-Thymidine incorporation was measured by liquid scintillation counter (Tri-Carb 2100TR; Packard Instrument).

RNA isolation from the splenocytes was performed using the RNeasy minikit procedure (Qiagen).15 Forward and reverse primers were used to amplify the transcripts. Samples (1μg) of RNA from different experimental groups of mice were first utilized for cDNA synthesis with reverse primers (IDT; Sigma) using Mouse Moloney Leukemia Virus reverse transcriptase (Invitrogen) at 37°C for 90min. A common master mixture containing deoxynucleoside triphosphates (Promega) and Taq polymerase (Invitrogen), as well as gene specific primers and 0.25 volume of cDNA, was used for amplification with an Applied Biosystems thermocycler. The cycling conditions for genes of interest were 5min at 95°C, followed by 40 cycles of denaturation at 95°C for 30s, annealing at 56°C for 40s, and extension at 72°C for 40s. The identities of the PCR amplified gene products were verified by agarose gel electrophoresis. Identical aliquots were processed in parallel without reverse transcriptase to rule out the presence of residual genomic DNA contamination in PCR amplification preparations. Densitometric analyses were done using the Quantity One software (Bio-Rad), ethidium bromide staining, and visualization under a UV transilluminator. For densitometric calculations, the same band area was used to determine band intensity and normalized for HGPRT.

Splenocytes from different groups of animals were harvested, resuspended in chilled lysis buffer (containing 20mM Tris–HCl (pH 7.5), 0.5mM EGTA, 1mM EDTA, 0.1% (vol/vol) 2-mercaptoethanol)34 and rapidly freeze-thawed thrice and passed through a 26-gauge needle (10 times) for lysis. The lysates were centrifuged (800×g for 10min at 4°C), the supernatants were collected, and the proteins were estimated using Bradford reagent. The supernatants were mixed with Laemmli buffer, heated in a boiling water bath for five min and cooled to room temperature. About 20μg of protein was loaded in lanes of Sodium Dodecyl Sulfate Poly Acrylamide Gels (SDS-PAGE) for electrophoresis. After the run, the gel was transferred to polyvinylidene difluoride membrane. Immunoblotting was performed with appropriately diluted specific primary antibodies for PI3K (anti-phosphoinositide 3 kinase Mab) and phospho PDK1 (anti-phosphoinositide-dependent Kinase 1 Mab) and p38MAPK (anti-p38 mitogen-activated protein kinase Mab) respectively. Finally, chemiluminescence was performed with LumiGlo reagent as described by the manufacturer.35 Densitometry analyses were done using Quantity1 (Bio-Rad) software.15

Each experiment was performed three times and results are expressed as means±SD. Student's t-test was performed for significance and a p-value of <0.05 was considered significant. Western blots and RT-PCR show representative data from three independent experiments. * p<0.05, ** p<0.01, *** p<0.001.

Cell mediated immunity is impaired in Leishmania infection characterized by marked T cell anergy to specific Leishmania antigens.15 After observing the potential therapeutic effects of 100 and 150Gy attenuated parasites in terms of reduction in organ parasite burden,31 we became interested to see whether the T cell anergy observed during progressive infection could be reversed by the treatment. Splenocytes of infected animals (Group 2) failed to mount anti-leishmanial T cell proliferation to leishmanial antigens suggesting global immune unresponsiveness at the active stage of the disease. Though Group 3 animals were treated with 50Gy radio-attenuated parasites, they also failed to do so. On the other hand, the splenocytes from treated groups (Group 4 and Group 5) stimulated with SLA showed a significantly (p<0.001) higher level (3.5–4 folds) of T cell proliferation (Fig. 1).

Fig. 1.

T cell proliferation assay in BALB/c mice groups. Number of proliferated T cells is expressed as CPM (count per minute). Proliferations of T cells were checked and compared between infected control group and treated animal groups (50, 100 and 150Gy groups), respectively. Higher T-cell proliferations compared to infected animals were found in the treated animal groups, 100 and 150Gy, respectively. Left to right, bars represent N, healthy control; I, Infected: mice received infection only; 50, 100, 150Gy represent three groups of mice treated with attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150Gy absorbed doses of γ-radiation, respectively. Infected (5×106 parasites/animal) and treated (twice @ 5×106 parasites per animal in 15-day interval) mice were sacrificed at 120dpi. Splenocytes were isolated and cultured in medium RPMI-1640 in 24-well plates @ 2×106 splenocytes/well. After 72h the splenocytes were pulsed with H3 thymidine and after 18h of pulsing the count was taken in liquid scintillation counter. Data represent mean±SD of six animals per group and are representative of three independent experiments; paired two-tailed Student's t-test was performed. p<0.05 was considered significant.

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The mRNA expression levels of iNOS and TGF-β genes (Fig. 2) were checked in different animal groups by RT-PCR method. A significantly higher level of expression (six-fold, p<0.001) of iNOS mRNA was seen in animals of groups 4 and 5, whereas expression of TGF-β mRNA has been lowered by two-fold (p<0.001) in the same groups of animals compared to that of infected group of animals (Group 2). Treated animals of Group 3 did not show such increment or decrement.

Fig. 2.

RT-PCR in BALB/c mice groups for checking the expressions of iNOS and TGF-β genes. Levels of iNOS and TGF-β genes expression were compared between infected control group and treated animal groups (50, 100 and 150Gy groups), respectively. Left to right, bands and bars represent 1, healthy control; 2, Infected: mice received infection only; 3, 4, 5 represent three groups of mice treated with attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150Gy absorbed doses of γ-radiation, respectively. Infected (5×106 parasites/animal) and treated (twice @ 5×106 parasites per animal in 15 days interval) mice were sacrificed at 120dpi. Splenocytes were isolated from which RNA was prepared. cDNA was prepared from RNA and PCR experiments were carried out for iNOS and TGF-β gene expressions taking HGPRT gene as the housekeeping gene for internal control. Densitometry analysis was done and the ratio of iNOS and TGF-β expressed in the bar diagram. All data were compared to the infected control group. Data represent mean±SD of six animals per group and are representative of three independent experiments; paired two-tailed Student's t-test was performed. p<0.05 was considered significant. *, p<0.05; **, p<0.01; ***, p<0.001.

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In order to score the changes, if any, in the signaling mechanisms of splenocytes of the immunized animals, immunoblot studies were carried out with PI3K, PDK1, and P38MAPK molecules (Fig. 3). These are some of the most important molecules in triggering the production of free radicals like NO and ROS. In our previous intramuscular therapeutic studies, we identified a significant increase of nitric oxide (NO) and reactive oxygen species (ROS) levels in animals of different immunized/treated groups.30,31 We measured the phosphorylation level of these three molecules in splenocytes of the animals and observed from 3.5- to 4-fold increase in phosphorylation in 100 and 150Gy animal groups compared to the infected groups (Fig. 3). This indicates the participation of these molecules in activating the effector molecules so that protection could be achieved. Animals of Group 3 that received immunization with parasites attenuated with 50Gy dose of radiation did not show any higher expression in terms of phosphorylation of any of the three signaling proteins studied here. This corroborates our previous observations where we noticed that this group of animals had no protection and succumbed to the disease.30,31

Fig. 3.

Western blot analysis to evaluate PDK1, PI3K and p38MAPK phosphorylation levels in BALB/C mice groups. Levels of PDK1, PI3K and p38MAPK phosphorylation were checked and compared between infected control group and treated animal groups (50, 100 and 150Gy groups), respectively. Splenocytes from different groups of animals were taken and the cell lysates were prepared and run in 10% polyacrylamide gels and then immunoblotted with respective monoclonal antibodies (MAbs) against non-phosphorylated and phosphorylated forms with β actin as an internal control. Phosphorylation status of each of the above mentioned molecules were expressed as the densitometric ratio of the phosphorylated form (labeled phospho or P) versus the internal control (β-actin). Left to right, bars represent N, healthy control; I, infected: mice received infection only; 50, 100, 150Gy represent three groups of mice treated with attenuated Leishmania donovani parasites, doses of attenuation being 50, 100 and 150Gy absorbed doses of γ-radiation, respectively. Infected (5×106 parasites/animal) and treated (twice @ 5×106 parasites per animal in 15 days interval) mice were sacrificed at 75dpi. Splenocytes were isolated and cultured in medium RPMI-1640 in 24-well plates @ 2×106 splenocytes/well. Representative data of three similar experiments are presented here. Data represent mean±SD value of three repeat experiments with six animals in each group; paired two-tailed Student's t-test was performed. p<0.05 was considered significant. *, p<0.05; **, p<0.01; ***, p<0.001.

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