9-O-acetylated sialoglycoproteins are important immunomodulators in Indian visceral leishmaniasis

Abstract

Overexpression of disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on peripheral blood mononuclear cells (PBMC) of visceral leishmaniasis (VL) patients (PBMC(VL)) compared to their levels of expression in healthy individuals has been demonstrated using a lectin, achatinin-H, with specificity toward 9-O-acetylated sialic acid derivatives alpha2-6 linkage with subterminal N-acetylgalactosamine (9-O-AcSAalpha2-6GalNAc). The decreased presence of disease-associated 9-O-AcSGPs on different immune cells of parasitologically cured individuals after successful treatment relative to the levels in patients with active VL prior to treatment was demonstrated. However, their contributory role as immunomodulatory determinants on PBMC(VL) remained unexplored. Accordingly, 9-O-AcSGPs on PBMC(VL) were sensitized with achatinin-H, leading to their enhanced proliferation compared to that observed with different known mitogens or parasite antigen. This lymphoproliferative response was characterized by evaluation of the TH1/TH2 response by intracellular staining and enzyme-linked immunosorbent assay for secreted cytokines, and the results were corroborated by their genetic expression. Sensitized PBMC(VL) evidenced a mixed TH1/TH2 cellular response with a predominance of the TH1 response, indicating the ability of 9-O-AcSGPs to modulate the host cell toward a favorable response. Interestingly, the humoral and cellular responses showed a good correlation. Further, high levels of anti-9-O-AcSGP antibodies with an order of distribution of immunoglobulin M (IgM) > IgG1 = IgG3 > IgG4 > IgG2 > IgE could be explained by a mixed TH1/TH2 response. A good correlation of enhanced 9-O-AcSGPs with both the cell-mediated (r = 0.98) and humoral (r = 0.99) response was observed. In summary, it may be concluded that sensitization of 9-O-AcSGPs on PBMC(VL) may provide a basis for the modulation of the host's immune response by their controlled expression, leading to a beneficial immune response and influencing the disease pathology.

FIG. 1.

Enhanced lymphoproliferation of PBMC_VL upon sensitization of disease-associated 9-O-AcSGPs by achatinin-H. (A) SI as demonstrated by the radiometric method. PBMC_VL (1 × 10⁵ cells/ml) before (▪) and after (□) sensitization were cultured separately with achatinin-H (0.1 to 12 μg) for 72 h at 37°C in an atmosphere of 5% CO₂. Cultures were pulsed with [³H]thymidine (1 μCi/well) for 18 h, and its uptake was measured. Results are expressed as the SI as described in Materials and Methods. The data show a profile representative of the results for 25 patients. The points represent the means ± SD of the results for triplicate determinations. (B) Maximal lymphoproliferation demonstrated by colorimetric MTT assay. PBMC_VL (n = 25) before (▪) and after (□) treatment were sensitized through 9-O-AcSGPs by using different concentrations of achatinin-H (0.1 to 10 μg) for 72 h at 37°C in an atmosphere of 5% CO₂ and assessed by MTT assay. The formazan crystals that formed were dissolved in dimethyl sulfoxide (450 μl), and the absorbance at 570 nm was recorded with a spectrophotometer as a quantitative measure of cell proliferation (or viability). In parallel, PBMC_H (n = 25) (▴) were processed similarly. The points represent the means ± SD of the results for triplicate determinations. (C) Comparison of levels of lymphoproliferation induced by different mitogens versus the level induced by achatinin-H. PBMC_VL (1 × 10⁵ cells/ml; n = 25) were sensitized with two well-known mitogens, namely, PHA and ConA, and with crude parasite antigen and achatinin-H (0.1 μg) under identical assay conditions. Subsequently, the viability of the cells was assessed by MTT as described for panel B. Unsensitized PBMC_VL served as controls for the assay. The points represent the means ± SD the results for triplicate determinations. *, P value of <0.01 for the observed lymphoproliferation with achatinin-H versus that in unsensitized controls; #, P value of <0.05 versus the lymphoproliferation with other mitogens (PHA and ConA). (D) SI of PBMC_VL sensitized with achatinin-H (0.1 μg) before (▪) and after (□) treatment plotted against different culture time points (0 to 96 h). The data show a profile representative of the results for 25 patients. Each point represents the mean ± SD of the results for triplicate determinations. (E) 9-O-AcSGPs serve as an important glycotope for lymphoproliferation after sensitization with achatinin-H. PBMC_VL were cultured in the absence (□) or presence of achatinin-H (0.1 μg) before (▪) and after preincubation with BSM (+) and were de-O-acetylated (+ O-acetyl esterase), and their viability was assessed by MTT assay as described in Materials and Methods. The data show a profile representative of the results for 25 patients. The points represent the means ± SD of the results for triplicate determinations. +, present; −, absent.

FIG. 2.

Enhanced secretion of TH1 cytokines by 9-O-AcSGP-sensitized PBMC_VL. PBMC_VL were cultured after sensitization in the absence (▪) or presence (□) of achatinin-H (0.1 μg) for 0 to 96 h. The concentration of the released cytokines, namely, IFN-γ (A), IL-12 (B), IL-2 (C), IL-10 (D), IL-4 (E), and TNF-α (F), in the culture supernatant was detected by ELISA as described in Materials and Methods. In parallel, PBMC_PT were sensitized in the absence (▴) or presence (▵) of achatinin-H under identical conditions, where unsensitized cells served as controls. The data show a profile representative of the results for VL patients (n = 25). The points represent the means ± SD of the results for triplicate sets.

FIG. 3.

Representative profiles of cell-mediated responses upon sensitization of 9-O-AcSGPs on PBMC_VL. 9-O-AcSGPs of PBMC_VL (n = 15) were sensitized with achatinin-H (0.1 μg) for 72 h and cultured at 37°C in an atmosphere of 5% CO₂. Subsequently, the cells were stained for intracellular cytokines with PE-conjugated, cytokine-specific antibodies for 30 min at 4°C in the dark, processed, and analyzed by flow cytometry using CellQuest software. Lymphocytes were gated on the basis of their light scatter properties and were checked for the presence of cell surface 9-O-AcSGPs using FITC-achatinin-H to determine the percentages of 9-O-AcSA-positive cells that were stained for the respective intracellular cytokines. In parallel, PBMC_PT were processed similarly. Unsensitized PBMC served as negative controls.

FIG. 4.

Enhanced mRNA expression of the TH1 cytokines in sensitized PBMC_VL. The levels of different cytokines along with GAPDH (housekeeping gene) were evaluated by reverse transcription of RNA (0.1 μg) extracted from achatinin-H (0.1 μg) sensitized PBMC_VL (n = 10) at different time points (0 to 96 h) as described in Materials and Methods. The mRNA expression of unsensitized cells served as the control. The values were compared by densitometric scores and are represented as the increase in band intensity compared with the expression of GAPDH at the respective time points.

FIG. 5.

Distribution of anti-O-AcSGP-specific antibody subclasses in VL patients before and after treatment. The levels of anti-O-AcSGP-specific IgM (A), IgG1 (B), IgG2 (C), IgG3 (D), IgG4 (E), and IgE (F) in VL patients before treatment (n = 25) were determined by subclass ELISA as described in Materials and Methods. The sera of individuals after treatment (n = 25) were tested similarly. Horizontal lines indicate mean values. *, P value of <0.01 for difference from results for successfully treated individuals; #, P value of <0.05, and **, P value of <0.01 for difference from results for healthy controls.