Inhibition of Granulomatous Inflammation and Prophylactic Treatment of Schistosomiasis with a Combination of Edelfosine and Praziquantel

Abstract

Background: Schistosomiasis is the third most devastating tropical disease worldwide caused by blood flukes of the genus Schistosoma. This parasitic disease is due to immunologic reactions to Schistosoma eggs trapped in tissues. Egg-released antigens stimulate tissue-destructive inflammatory and granulomatous reactions, involving different immune cell populations, including T cells and granulocytes. Granulomas lead to collagen fibers deposition and fibrosis, resulting in organ damage. Praziquantel (PZQ) is the drug of choice for treating all species of schistosomes. However, PZQ kills only adult Schistosoma worms, not immature stages. The inability of PZQ to abort early infection or prevent re-infection, and the lack of prophylactic effect prompt the need for novel drugs and strategies for the prevention of schistosomiasis.

Methodology/principal findings: Using in vitro and in vivo approaches, we have found that the alkylphospholipid analog edelfosine kills schistosomula, and displays anti-inflammatory activity. The combined treatment of PZQ and edelfosine during a few days before and after cercariae infection in a schistosomiasis mouse model, simulating a prophylactic treatment, led to seven major effects: a) killing of Schistosoma parasites at early and late development stages; b) reduction of hepatomegaly; c) granuloma size reduction; d) down-regulation of Th1, Th2 and Th17 responses at late post-infection times, thus inhibiting granuloma formation; e) upregulation of IL-10 at early post-infection times, thus potentiating anti-inflammatory actions; f) down-regulation of IL-10 at late post-infection times, thus favoring resistance to re-infection; g) reduction in the number of blood granulocytes in late post-infection times as compared to infected untreated animals.

Conclusions/significance: Taken together, these data suggest that the combined treatment of PZQ and edelfosine promotes a high decrease in granuloma formation, as well as in the cellular immune response that underlies granuloma development, with changes in the cytokine patterns, and may provide a promising and effective strategy for a prophylactic treatment of schistosomiasis.

Fig 1. Experimental design for in vivo experiments.

This scheme depicts schematically the studies conducted with S. mansoni-infected mice (n = 8) in this present work. Mice were treated daily (oral administration) with PZQ, EDLF or PZQ+EDLF since three days before animals were infected until eight days after infection. The untreated infected control group received only the vehicle solution used for 12 days. Animals that were untreated and uninfected (naive) were also run in parallel. Asterisks indicate when samples from animals were taken (sampling) to analyze the parameters indicated in the box. Animals were sacrificed at 8 weeks p.i., and the timeline of some major events in parasite life cycle and disease-related processes are indicated at the top of the scheme. See text for further details.

Fig 2. In vitro effects of EDLF on the viability of S. mansoni schistosomula.

Schistosomula were untreated (Control), heat-killed at 56°C, or treated with 10 or 20 μM EDLF for 24 h. Then, schistosomula viability was analyzed by propidium iodide (PI) incorporation and light microscopy morphology as shown in Materials and Methods. RFU, relative fluorescence units. Data are shown as means ± SEM of three separate experiments. Asterisks represent statistical significance with respect to control-live group. **, p<0.01; ***, p<0.001. Scale bar, 100 μm.

Fig 3. Effect on adult male and female worm burdens after PZQ, EDLF and PZQ+EDLF prophylactic treatments in mice infected with S. mansoni.

Mice were treated by oral administration of 100 mg/kg/day PZQ, 45 mg/kg/day EDLF or PZQ+EDLF as prophylactic treatments for S. mansoni infection as shown in Materials and Methods. Infected untreated (untreated) mice were treated with vehicle. Each point represents data from an individual treated or infected untreated mouse. Horizontal bars indicate mean values. Significance (p) values with respect to infected untreated mice are indicated. The means ± SEM (n = 8) for each experimental condition are as follows: female (untreated: 20.38 ± 1.36; PZQ: 13.75 ± 2.29; EDLF: 11.25 ± 2.23; PZQ+EDLF: 10.38 ± 2.26) and male (untreated: 21.75 ± 1.49; PZQ: 14.25 ± 2.44; EDLF: 10.88 ± 2.03; PZQ+EDLF: 8.62 ± 1.76).

Fig 4. Effects on granuloma size and parasite egg burdens in liver after prophylactic treatment of S. mansoni-infected mice with PZQ, EDLF and PZQ+EDLF treatments.

(A) Representative hepatic granulomas of 8-week-infected drug-untreated and drug-treated mice. Photographs were taken at 10 x. (B) Granuloma diameter. The average values of the diameters of 25 granulomas measured in liver sections from 5 infected mice per group (5 granulomas per mouse) are shown. Each point represents the value for an individual mouse. Significance (p) values with respect to infected untreated mice are indicated. Statistical significance between the PZQ and PZQ+EDLF groups is also included. (*) p<0.05. The means ± SEM (n = 5) for each experimental condition are as follows: untreated: 620.4 ± 28.70; PZQ: 526.9 ± 15.95; EDLF: 276 ± 14.41; PZQ+EDLF: 264.9 ± 5.41. (C) Parasite egg burden in liver. Infected mice were treated with 100 mg/kg PZQ, 45 mg/kg EDLF, or PZQ+EDLF. Infected untreated mice were run in parallel. Compounds were administered orally as described in Materials and Methods, and the number of eggs in liver was determined as eggs per gram (epg). Each point represents data from an individual treated- or infected untreated mouse. Horizontal bars indicate average values. Significance (p) values with respect to infected untreated mice are shown. Statistical significance between the PZQ and PZQ+EDLF groups is also included. (*) p<0.05. The means ± SEM (n = 5) for each experimental condition are as follows: untreated: 17411 ± 2805; PZQ: 17094 ± 2368; EDLF: 13796 ± 1804; PZQ+EDLF: 7126 ± 1279).

Fig 5. IL-2 and IL-4 plasma levels in drug treated- and untreated-mice.

The plasma levels of the indicated cytokines were determined in uninfected-untreated naive mice and the distinct untreated and treated infected mice, namely infected untreated (untreated), PZQ, EDLF and PZQ+EDLF as shown in Materials and Methods. Samples were taken at week 3 p.i. Data are shown as means ± SEM of eight mice. Asterisks represent statistical significance with respect to the infected untreated group. (***) p<0.001.

Fig 6. IL-10 plasma level in drug treated- and untreated-mice.

The plasma levels of IL-10 were determined in uninfected-untreated naive mice and the distinct untreated and treated infected mice, namely infected untreated (untreated), PZQ, EDLF and PZQ+EDLF as shown in Materials and Methods. Samples were taken at weeks 3 and 8 p.i. Data are shown as means ± SEM of eight mice. Asterisks represent statistical significance with respect to the infected untreated group. (*) p<0.05; (***) p<0.001.

Fig 7. IFN-γ, TNF-α, GM-CSF, IL-5, IL-6 and IL-17 plasma levels in PZQ+EDLF-treated- and untreated infected mice.

The plasma levels of the indicated cytokines were determined in infected untreated (untreated) and PZQ+EDLF-treated infected mice as shown in Materials and Methods. Samples were taken at week 8 p.i. Data are shown as means ± SEM of eight mice. Asterisks represent statistical significance with respect to the infected untreated group. (**) p<0.01; (***) p<0.001.

Fig 8. Granulocyte analysis in drug treated- and untreated-mice.

Blood samples were analyzed for neutrophil, eosinophil and basophil counts in uninfected-untreated naive mice, infected untreated (untreated) mice and infected mice treated with PZQ, EDLF or PZQ+EDLF. Samples were taken at weeks 3 (A) and 8 (B) p.i. Data are shown as means ± SEM of eight mice. Asterisks represent statistical significance with respect to the infected untreated group. (*) p<0.05; (**) p<0.01; (***) p<0.001.