Role of Leishmania donovani and its lipophosphoglycan in CD4+ T-cell activation-induced human immunodeficiency virus replication

Abstract

Chronic immune activation by coinfecting pathogens has been suggested as a cofactor in human immunodeficiency virus (HIV) disease progression, particularly in the setting of developing countries. Here, we used in vivo-infected mononuclear cells to examine the role of the protozoan parasite Leishmania donovani and its major membrane constituent, lipophosphoglycan (LPG), in mediating CD4+ T-lymphocyte activation-induced HIV replication and CD4+ T-cell death. We found that Leishmania antigens upregulated HIV replication in CD8-depleted peripheral blood mononuclear cells from asymptomatic HIV-infected donors compared to unstimulated cells. L. donovani-induced viral replication was associated with cellular proliferation, increased expression of the cellular immune activation markers CD25 and HLA-DR within the CD4+ subpopulation, and enhanced secretion of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), and IL-6. LPG induced TNF-alpha secretion in the absence of increased expression of cellular activation markers. Moreover, in a few cases we observed that L. donovani induced HIV replication without significant cellular activation but with cytokine secretion. The rate of apoptosis was accelerated in these latently infected CD4+ T cells primed with Leishmania antigens compared to controls, and TNF-alpha production appeared to be the central event necessary for this effect. Furthermore, we demonstrate that thalidomide inhibited Leishmania-induced virus replication coupled with abrogated Leishmania-induced TNF-alpha secretion but not IL-2 or IL-6 production. Furthermore, thalidomide did not affect Leishmania-induced apoptosis. The results suggest that Leishmania and its product, LPG, up-regulate HIV replication in latently infected cells through distinct antigen-specific and non-antigen-specific cellular immune activation mechanisms and that TNF-alpha secretion is pivotal in this process. The immunomodulatory role of thalidomide raises interest as a potential adjuvant to reduce HIV disease progression in Leishmania-HIV coinfected individuals.

FIG. 1

Effects of Leishmania antigens on HIV replication in CD8-depleted PBMC. CD8-depleted PBMC were incubated in the presence or absence of PHA (5 μg/ml) (used as a positive control), LPG (12.5 μg/ml), or L. donovani (L.d.) promastigotes (at a 1:1 cell-to-parasite ratio). Half of the culture supernatants were harvested at the indicated time points and were replenished with complete RPMI medium. Levels of p24 antigen in culture supernatants were measured by ELISA. Leishmania- and LPG-treated CD8-depleted PBMC cultures had significantly higher p24 antigen levels between days 5 and 9 than did negative controls (P < 0.05). Data are expressed as means ± SEM from seven individual donors.

FIG. 2

Effects of Leishmania antigens on cellular activation and virus induction in an individual with a strong Leishmania-specific response (left panels) compared to an individual with a poor response (right panels). Methods are as described in the legends to Fig. 1, 3, 4, and 5.

FIG. 3

Effects of Leishmania antigens on cellular immune activation. For determination of cellular proliferation (A), an aliquot of cells was harvested at day 5, pulsed with [³H]thymidine for 18 h, and counted. Results are expressed as mean counts per minute ± SEM of triplicate cultures from eight independent experiments. For determination of cellular activation (B), an aliquot of cells was harvested between days 8 and 10 and assessed by FACScan for expression of CD25 or HLA-DR immune activation markers. IL-2 secretion (C) was measured by ELISA in culture supernatants harvested after 24 h of stimulation. Data in panels B and C represent mean values (± SEM) from four experiments done independently.

FIG. 4

Effects of Leishmania antigens on cell loss and apoptosis (APO) in CD4⁺ T cells (A) and effects of thalidomide on Leishmania-induced apoptosis (B). Cells were stimulated with LPG or L. donovani (L.d.) or were left untreated for 6 days in vitro. In some cultures, cells were also treated with thalidomide. CD4⁺ T-cell loss was determined by FACScan. LPG or L. donovani activation-induced apoptosis was determined by FACScan analysis by the TUNEL method. The data shown were obtained from an individual and are representative of three similar experiments.

FIG. 5

Effects of Leishmania antigens on cytokine induction. Culture supernatants collected after 48 h were analyzed for the presence of TNF-α (A) and IL-6 (B) by ELISA. L. donovani (L.d.), but not LPG, induced significant TNF-α and IL-6 secretion (P < 0.05). PHA-stimulated cultures, used as positive controls, resulted in 19-fold (P < 0.001) and 6-fold (P < 0.005) increases over negative controls in TNF-α and IL-6 production, respectively (data not shown). Data are means ± SEM from eight (A) or six (B) independent experiments.

FIG. 6

Kinetics of TNF-α production. Culture supernatants were harvested at the indicated time points, and TNF-α levels were determined by ELISA. LPG enhanced TNF-α secretion significantly (P < 0.05) at days 2, 5, and 9, and L. donovani (L.d.) increased TNF-α secretion significantly (P < 0.05) between days 2 and 15. Data are means ± SEM of five independent experiments.

FIG. 7

Effects of thalidomide (Thal) on Leishmania-induced cytokine production (A) and HIV replication (B). The effect of thalidomide on Leishmania antigen-mediated TNF-α and IL-6 production was assessed by ELISA at 48 h in culture supernatants of CD8-depleted PBMC. Levels of TNF-α, but not IL-6, in culture supernatants of cells treated with Leishmania antigen plus thalidomide were significantly lower than the levels in supernatants of cells treated with Leishmania antigen only. Virus replication was monitored by measuring p24 antigen levels in culture supernatants at day 9. Data are expressed as means ± SEM from three independent experiments. L.d., L. donovani.

FIG. 8

Effect of thalidomide (Thal) on Leishmania-induced IL-2 secretion. IL-2 production was assessed by ELISA at 24 h in culture supernatants. Levels of IL-2 in culture supernatants of cells treated with Leishmania antigen plus thalidomide were not significantly different (P > 0.05) from the levels in cells treated with Leishmania antigens only. L.d., L. donovani.