Designing therapies against experimental visceral leishmaniasis by modulating the membrane fluidity of antigen-presenting cells

Abstract

The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-gamma signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.

FIG. 1.

TEM micrographs of liposomes and lipid emulsions and their size distributions. (A and E) Chemical structures of cholesterol (A) and 4-cholest-3-one (E). A mixture of cholesterol and PC at a molar ratio of 1.5:1 was either sonicated to prepare liposomes (cholesterol liposomes) (B) or not sonicated (emulsion) (D). (C) Section of liposomes showing lamellation. (F) Similarly, 4-cholesten-3-one, the cholesterol analogue, was mixed with PC at a molar ratio of 1.5:1 and sonicated to prepare analogue liposomes. (F and G) The size distributions of cholesterol liposomes (G) and analogue liposomes (H) were analyzed by using TEM and were determined at random by measuring the diameters of 300 individual liposomes. Magnification, ×43,500.

FIG. 2.

Parasite burdens in the spleen and liver of hamsters infected with promastigotes. The groups and their treatments are indicated at the top and bottom, respectively. Hamsters infected for 45 days (group I) received the following treatments: cholesterol liposomes (group II), analogue liposomes (group III), emulsion (group IV), sub-SAG (group V), and a combination of cholesterol liposomes and sub-SAG (group VI). SAG was administered intramuscularly at a dose of 50 mg/kg (body weight) on days 7, 14, and 21. All the hamsters were sacrificed 30 days after liposome treatment, and the parasite burdens in the spleen and liver were determined. Each data point indicates the parasite burden in a hamster either in the spleen or in the liver. The results of three representative experiments (experiments i, ii, and iii) are shown.

FIG. 3.

Parasite burdens in the spleen and liver of hamsters infected with amastigotes. The groups and their treatments are indicated at the top and bottom, respectively. Hamsters infected for 60 days (group I) received the following treatments: cholesterol liposomes (group II), analogue liposomes (group III), sub-SAG (group IV), and a combination of cholesterol liposomes and sub-SAG (group V). SAG was administered as described in the legend to Fig. 2. All the hamsters were sacrificed 30 days after liposome treatment, and the parasite burdens in the spleen and liver were determined. The results of two representative experiments (experiments i and ii) are shown.

FIG. 4.

Lymphoproliferation of splenocytes in response to SLA at a concentration of 5 μg/ml (A) and to the nonspecific mitogen ConA (B). The proliferative response was analyzed by using [³H]thymidine incorporation. The background counts for medium alone varied between 500 and 1,000 cpm. Two experiments were conducted separately, and the data show the results of one representative experiment. The groups indicated at the top are the groups described in the legend to Fig. 2.

FIG. 5.

Quantification of MΦ membrane cholesterol. MΦs (about 90% of the cells were phagocytic with SRBC) from five or six hamsters were pooled. The status of cholesterol in the MΦ membrane was assessed by direct chemical measurement using an Amplex Red assay kit. The bars indicate averages of triplicate measurements, and the error bars indicate standard deviations.

FIG. 6.

Expression of the endogenous leishmanial antigen KMP-11 in MΦs derived from the splenocytes of I-hamsters and L-I-hamsters and their ability to form synapses with an anti-KMP-11 T-cell line (T^KMP cells). MΦs were stained with anti-KMP-11 MAb and were probed with FITC-labeled anti-mouse IgG. MΦs from normal hamsters did not show any staining with anti-KMP-11 Ab. To study synapse formation, MΦs were stained with CTXB-FITC, mixed with PKH26-labeled T^KMP cells, and kept at 37°C for 30 min. Conjugate formation was analyzed with a confocal microscope (Zeiss model LSM 510), and the results were expressed as percentages of synapse formation. Cells found in joint couplets by phase-contrast microscopy and exhibiting a zone of colocalization as determined by confocal microscopy were considered to have formed a synapse. The number of synapse-forming couplets per 100 APCs is expressed as the percentage of synapse formation. To study the extent of true synapse formation, fura-2-loaded T^KMP cells were mixed with MΦs, and fluorescence was monitored in real time to determine the intracellular Ca²⁺ mobilization. In a separate experiment using a similar setup, MΦs were mixed with T^KMP cells, and the resulting IL-2 production in the supernatant was monitored for the IL-2-dependent cell line HT-2. Growth of HT-2 cells was monitored by using [³H]thymidine incorporation.

FIG. 7.

Semiquantitative RT-PCR cytokine (TGF-β, TNF-α, IL-4, IFN-γ, and IL-10), iNOS, and HGPRT profiles for the splenocytes of I-hamsters and L-I-hamsters (n = 6). Equivalent amounts of RNA from splenic tissues of two groups of hamsters were used as the input for RT-PCR analysis, where the HGPRT gene was used as the housekeeping control gene. Expression of each cytokine transcript was expressed as a ratio of cytokine mRNA to HGPRT mRNA. The values obtained from densitometric analysis of each cytokine were expressed as means ± standard deviations instead of individual values. The ratios of IFN-γ to IL-10 and IFN-γ to TGF-β were analyzed for I-hamsters and L-I-hamsters. Two experiments were conducted separately, and the data show the results of a representative experiment.

FIG. 8.

Production of leishmanicidal effector molecules (NO and ROS) by splenocytes derived from different experimental groups of hamsters. The splenocytes were stimulated with SLA (5 μg/ml) and assayed for NO as described in Materials and Methods. ROS generation was measured by 2′,7′-dichlorodihydrofluorescein diacetate staining of splenocytes from different experimental groups. Three experiments were conducted separately, and the data show the results of a representative experiment. The groups indicated at the top are the groups described in the legend to Fig. 2. MFI, mean fluorescence intensity.

FIG. 9.

Antileishmanial IgG1 and IgG2 Ab titers in different groups of hamsters (normal hamsters, I-hamsters, and L-I-hamsters). Sera of individual hamsters were assayed to determine the Ab titer by ELISA. The insets show the median IgG2/IgG1 ratios at a 10⁻³ dilution of sera between I-hamsters and L-I-hamsters. O.D., optical density.

FIG. 10.

Long-term survival of I-hamsters, L-I-hamsters, and L-I-sub-SAG-hamsters. (A) Percentages of survival. The asterisks indicate when hamsters were sacrificed. (B) Organ weight (spleen and liver) for I-hamsters, L-I-hamsters, and L-I-sub-SAG-hamsters (three hamsters were chosen at random from each group). Three-month-old hamsters were used as the normal group as there was not much variation in organ weight after this. The organ weights (spleen and liver) were recorded 30 days after the hamsters received either cholesterol liposomes or cholesterol liposomes and sub-SAG and were compared to the data for the corresponding infected groups. The organ weights (spleen and liver) were also recorded at the time that the experiment was terminated. Bars a and b indicate organ weights for L-I-hamsters and L-I-sub-SAG-hamsters, respectively. Since all the I-hamsters were dead by 6 months postinfection, there was no infected control for groups that survived until termination of the experiment. (C and D) The antileishmanial IgG1 (C) and IgG2 (D) levels were determined at the termination of the experiment and were compared to the data for age-matched controls. Representative data for three hamsters are shown. The statistical significance for comparisons of normal and infected groups and of infected and protected groups are indicated. The error bars indicate standard deviations. OD, optical density.