Date Published: July 20, 2015
Publisher: Public Library of Science
Author(s): Edward Yepes, Rubén E. Varela-M, Julio López-Abán, Jose Rojas-Caraballo, Antonio Muro, Faustino Mollinedo, Michael H. Hsieh. http://doi.org/10.1371/journal.pntd.0003893
Abstract: BackgroundSchistosomiasis 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 FindingsUsing 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/SignificanceTaken 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.
Partial Text: Schistosomiasis is caused by blood flukes (trematodes) belonging to the genus Schistosoma. Schistosoma spp. parasites need two hosts for their survival, namely an intermediate snail host, where asexual reproduction takes place and a definitive mammalian host, where the sexual reproduction occurs [1, 2]. Schistosomiasis is the most important water-borne disease, being the main human helminth infection in terms of global mortality and the third most devastating tropical disease in the world, following malaria and intestinal helminthiasis, and causing both significant morbidity and mortality on several continents [3–7]. The bulk of morbidity due to schistosomiasis results from cellular immune responses and the generation of cytokine patterns, elicited during the different stages of the parasite’s life cycle in the course of infection, that eventually lead to chronic immune response-based inflammation against Schistosoma eggs lodged in tissues, and subsequent granuloma formation and fibrosis [8, 9]. Symptoms and signs of the disease depend on the number and location of eggs trapped in the tissues, leading first to a reversible inflammatory reaction and then to the pathology associated with collagen deposition and fibrosis, resulting in organ damage [9, 10]. Most human schistosomiasis is caused by Schistosoma haematobium, S. mansoni, and S. japonicum [6, 11–13]. The World Health Organization (WHO) estimates that schistosomiasis is endemic in 74 developing countries, infecting at least 230 million people in rural and peri-urban areas worldwide (80% in sub-Saharan Africa). Of these, ~120 million have symptoms of the disease, and ~20 million have severe disease, resulting in approximately 280,000 deaths annually [2, 4, 7, 14]. Human infection occurs by direct contact with S. mansoni cercariae-contaminated water. Following penetration of cercariae through the skin, they lose their tails and transform into schistosomula. The schistosomula then enter the venous system and reach the lungs, where they mature to pre-adult stages. About 8–10 days after infection, the pre-adult forms reach the portal system, where they mature to adult males and females [1, 5, 9]. Both male and female S. mansoni parasites achieve sexual maturity in the bloodstream, and then sexual reproduction occurs with the deposition of hundreds of eggs per day [12, 15, 16], predominantly in the liver and intestine. Deposition of schistosome eggs in the tissues is an important stimulus to the influx of immune cells that leads to the development of a granulomatous reaction. This immunological reaction protects the host by neutralizing the schistosome eggs antigens and destroying eggs. However, schistosome eggs elicit a CD4+ T-helper (Th) cell-mediated hepatic granulomatous inflammation, which is the major pathological consequence of the disease [15, 16]. Nevertheless, paradoxically, the development of granulomatous inflammation around parasite eggs has an essential host-protective and facilitates the successful excretion of the eggs from the host [14, 16, 17]. Two main clinical conditions are recognized in S. mansoni-infected individuals: acute schistosomiasis and chronic schistosomiasis. Acute schistosomiasis in humans is a debilitating febrile illness (Katayama fever) that can occur before the appearance of eggs in the stool and generally peaks around six to eight weeks after infection . Cytokine production by peripheral blood mononuclear cells after stimulation with parasite antigen reflects a dominant T helper 1 (Th1) response, with production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) . Thus, during the acute phase of the disease there is a predominance of a Th1 response, producing elevated levels of Th1 cytokines in the plasma . Then, in the natural progression of the disease, after parasites mature, mate and start to produce eggs at the fifth-sixth week, the initial Th1 response is followed by a developing egg antigen-induced regulatory T cell (Treg cell) and T helper 2 (Th2) response that downregulates the production and effector functions of the pro-inflammatory Th1 mediators with accompanying granuloma formation [15, 20, 21]. Treg and Th2 cells share some features, notably their ability to synthesize interleukin-10 (IL-10) through which suppress the development of Th1 responses to schistosome egg antigens, thus cooperating both cell types to enforce the Th2 polarization that characterizes the immune response in schistosome-infected mice . The production of IL-10 during this latter period seems to have an important role in hepatic granuloma formation and in the regulation of CD4+ T cell responses in schistosomiasis, as well as in the transition from acute to chronic disease state [17, 23–25]. In the mouse model, both Th1 and Th2 cytokines can orchestrate granuloma development [16, 25, 26]. Th2–type responses are typically characterized by increases in the levels of interleukin-4 (IL-4) and other cytokines (including IL-5, IL-6, IL-9, and IL-13), activation and expansion of CD4+ Th2 cells, plasma cells secreting IgE, eosinophils, mast cells and basophils [16, 27]. IL-17 is the signature cytokine of the proinflammatory Th17 cell population [28, 29], and a subsequent Th17 response is elicited during infection that plays a major role for full deployment of inflammation  and for the development of severe schistosome egg-induced immunopathology . Elucidation of the actual determinants of immunomodulation in human or murine schistosomiasis could lead to the development of drugs or vaccines for disease control or to spin-off benefits for other granulomatous diseases . Praziquantel (PZQ) is currently the only available antischistosomal drug and it is distributed through mass administration programs to millions of people every year, thus increasing the risk for drug resistance, and therefore search for new antischistosomal drugs and therapeutic approaches is urgently needed [7, 32]. Adult worms are highly sensitive to PZQ, but unfortunately this drug has minor activity against juvenile stages like schistosomula, pre-adults and juvenile adults . Despite the paucity of a concerted effort to develop novel antischistosomal drugs, with a lack of dedicated drug discovery and development programs pursued for schistosomiasis, a number of compounds with promising antischistosomal properties have been recently identified, such as the alkylphospholipid analogs (APLs) [33–35]. APLs are a class of structurally related synthetic lipid compounds, including edelfosine (EDLF) and miltefosine, which act on cell membranes rather than on DNA [36, 37]. EDLF (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), considered as the prototype APL molecule, is a promising antitumor ether phospholipid drug [38, 39], that acts by activating apoptosis through its interaction with cell membrane domains [36, 37, 40–42]. Interestingly, the APL miltefosine is currently being used in the clinic for the treatment of human and animal leishmaniasis [43, 44], and the APL EDLF has been reported to display anti-inflammatory properties  and to modulate cytokine production, including IFN-γ, IL-2 and IL-10 [45–47]. EDLF has also been shown to cause interruption of oviposition in a preliminary in vitro screening, and a significant reduction in worm burden in vivo, with a preferential activity against male worms . Here, using both in vitro and in vivo approaches, we have found that EDLF is able to kill juvenile stages as schistosomula, and the combination of PZQ and EDLF behaves as a promising prophylactic treatment against schistosomiasis, showing a significant reduction in adult worm burden, number of parasite eggs in liver and intestine tissues and granuloma size, as well as exerting an anti-inflammatory action, through modulation of cytokine production in infected mice, that might be of special importance for the treatment and/or prevention of schistosomiasis.
In the present study we have found that the combination of PZQ and the ether phospholipid EDLF behaves as a potent and promising prophylactic treatment for schistosomiasis. This prophylactic effect was significantly greater than those observed in the single drug treatment groups. Our results represent the first evidence that EDLF kills immature forms of S. mansoni using both in vitro and in vivo assays. Thus, we have found here that EDLF kills schistosomula, and both PZQ [59, 60] and EDLF  have been previously shown to be effective drugs against S. mansoni adult worms. Recently, we have shown that EDLF reduces worm burden in a murine model , and we report here a statistically significant decrease in worm count in an in vivo assay following a prophylactic treatment with EDLF or the PZQ+EDLF combination treatment that was higher than that detected following PZQ prophylactic treatment. PZQ is less active against the juvenile stages of S. mansoni than the adult schistosomes [59, 60]. The minor activity of PZQ against juvenile schistosomes is believed to be a key factor explaining the observed treatment ‘failures’ in areas highly endemic for schistosomiasis and that require frequent retreatments [32, 68]. The relative resistance of the larval stages of S. mansoni to schistosomicide drugs may result in a therapeutic failure because of the presence of migrating, drug-resistant, immature forms of the parasite . On the other hand, although the existing antischistosomal drugs are highly effective against adult worms, they do not prevent against re-infection or granuloma formation [70, 71]. In this context, the present results on the killing activity of EDLF on S. mansoni schistosomula are of major importance in the development of effective schistosomicide drugs. It is worth mentioning that very recent evidence shows that EDLF elicits a selective and direct killing on soil-dwelling nematode Caenorhabditis elegans embryos . Taken together, these data suggest that EDLF is able to kill helminths at both early and late developmental stages. The mechanism of action of EDLF on schistosomes is still unclear. In this regard, it is worth mentioning that EDLF is a proapoptotic agent in cancer cells [38, 41, 42] affecting processes at the membrane level [37, 40, 73, 74], and recent evidence suggests that EDLF promotes an apoptosis-like process in Leishmania spp. and S. mansoni adult worms [35, 75]. APLs, including miltefosine and EDLF, have been recently shown to exert schistosomicide activity on various species and strains of schistosomes [33–35, 76, 77], by interfering with membrane stability and structural integrity of worms’ tegument, and resulting in marked alterations of the digestive tract and the reproductive system of the worms. In addition, it has been very recently shown that Akt inhibition induces profound alterations in S. mansoni adult worm pairing and egg laying as well as affects the viability of schistosomula larvae , and EDLF also inhibits Akt signaling in cancer cells .