Phytoecdysteroids as modulators of the Toxoplasma gondii growth rate in human and mouse cells

Dzitko et al. Parasites & Vectors (2015) 8:422 DOI 10.1186/s13071-015-1019-7 RESEARCH Open Access Phytoecdysteroids as modulators of the Toxoplasma gondii growth rate in human and mouse cells Katarzyna Dzitko1*, Marcin Mikołaj Grzybowski1, Jakub Pawełczyk2, Bożena Dziadek1, Justyna Gatkowska1, Paweł Stączek3 and Henryka Długońska1 Abstract Background: Searching for new effective drugs against human and animal toxoplasmosis we decided to test the anti-Toxoplasma potential of phytoecdysteroids (α-ecdysone and 20-hydroxyecdysone) characterized by the pleiotropic activity on mammalian organisms including the enhancement of host’s anti-parasitic defence. This objective was accomplished by the in vitro evaluation of T. gondii growth in phytoecdysteroid-treated immunocompetent cells of selected hosts: humans and two strains of inbred mice with genetically determined different susceptibility to toxoplasmosis. Methods: Peripheral mononuclear blood cells were isolated from Toxoplasma-positive and Toxoplasma-negative women (N = 43) and men (N = 21). Non-infected mice (C57BL/6, N = 10 and BALB/c, N = 14) and mice (BALB/c, N = 10) challenged intraperitoneally with 5 tissue cysts of the T. gondii DX strain were also used in this study as a source of splenocytes. The effects of phytoecdysteroids on the viability of human PBMC and mouse splenocytes were evaluated using the MTT assay. The influence of phytoecdysteroids on PBMCs, splenocytes and T. gondii proliferation was measured using radioactivity tests (the level of 3[H] uracil incorporation by toxoplasms or 3[H] thymidine by PBMCs and splenocytes), which was confirmed by quantitative Real-Time PCR. Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH). The best-fit IC50 curves were plotted using GraphPad Prism 6.0 (GraphPad Software, Inc.). Results: Our results showed that phytoecdysteroids promote the multiplication of Toxoplasma in cultures of human or murine immune cells, in contrast to another apicomplexan parasite, Babesia gibsoni. Additionally, the tested phytoecdysteroids did not stimulate the in vitro secretion of the essential protective cytokines (IFN-γ, IL-2 and IL-10), neither by human nor by murine immune cells involved in an effective intracellular killing of the parasite. Conclusions: Judging by the effect of phytoecdysteroids on the T. gondii proliferation, demonstrated for the first time in this study, it seems that these compounds should not be taken into consideration as potential medications to treat toxoplasmosis. Phytoecdysteroids included in the food are most likely not harmful for human or animal health but certain nutrients containing ecdysteroids at high concentrations could promote T. gondii proliferation in chronically infected and immunocompromised individuals. In order to assess the real impact of ecdysteroids on the course of natural T. gondii invasion, in vivo research should be undertaken because it cannot be ruled out that the in vivo effect will be different than the in vitro one. However, taking into account the possible stimulating effect of ecdysteroids on some opportunistic parasites (such as Toxoplasma or Strongyloides) further studies are necessary and should focus on the mechanisms of their action, which directly or indirectly enhance the parasite growth. Since ecdysteroids are considered as potential drugs, it is essential to determine their effect on various parasitic pathogens, which may infect the host at the same time, especially in immunocompromised individuals. * Correspondence: 1 Department of Immunoparasitology, Faculty of Biology and Environmental Protection, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland Full list of author information is available at the end of the article © 2015 Dzitko et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dzitko et al. Parasites & Vectors (2015) 8:422 Background Toxoplasma gondii is a cosmopolitan intracellular protozoan that causes toxoplasmosis in birds, mammals and humans. Statistical data concerning the prevalence of toxoplasmosis indicated that at least one-third of human population has had contact with this parasite. The high prevalence of T. gondii infection (e.g., in some parts of Europe 58–90 %) is related to sex, age, culinary habits, personal hygiene, condition of the immune system and dysfunctions of the endocrinal network [1–3]. Despite the significant progress in the research on the biology of T. gondii invasion, the proteome of this protozoan and the immunity it induces, no preventive vaccines for humans have been developed yet. T. gondii infection leads to congenital or acquired postnatal toxoplasmosis characterized by diverse forms and symptoms. In immunocompetent humans, the infection is usually asymptomatic as the primary immunological response quickly limits the parasite replication and its spreading. Thus, symptomatic toxoplasmosis occurs infrequently. Quickly replicating tachyzoites are converted into bradyzoites and become enclosed in tissue cysts, which are a sign of chronic toxoplasmosis [4]. Recent studies demonstrated that a long-term presence of the parasite is not neutral to the host. It is increasingly often postulated that there is a causal connection between the T. gondii carriage and the increased risk of neurologic diseases, such as schizophrenia [5], Parkinson’s disease [6] or epilepsy [7]. Moreover, a very significant clinical problem is posed by congenital toxoplasmosis, resulting from the primary infection of a mother during pregnancy and transmission of the parasite to a fetus with an immature immune system. In chronically infected women there is usually no placental parasite transmission to a fetus, thus, an especially interesting group for clinicians and immunoparasitologists are seronegative women at a child-bearing age, in whom the development of a primary infection may cause a miscarriage or congenital defects of the offspring, such as hydrocephaly or microcephaly, chorioretinitis and intracranial calcification, which constitute the so-called “Sabin-Feldman triad”. This parasite may also cause serious medical disorders in humans related to the unchecked proliferation of the protozoan in immunocompromised patients. A weakened immune system is not able to inhibit the parasite replication during the reactivation of a latent infection or the reinvasion by a new strain of a different genotype. Uncontrolled T. gondii invasion in such ca (...truncated)