Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti

Macháček et al. Parasites & Vectors (2016) 9:579 DOI 10.1186/s13071-016-1869-7 RESEARCH Open Access Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti Tomáš Macháček* , Lucie Panská, Hana Dvořáková and Petr Horák Abstract Background: Helminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice. Results: Primary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-β1. Conclusions: Both astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens. Keywords: Astrocytes, Microglia, Trichobilharzia regenti, Avian schistosome, Neuroinfection, Nitric oxide, Proinflammatory cytokines, Anti-inflammatory cytokines, Cathepsin B * Correspondence: Department of Parasitology, Faculty of Science, Charles University, Viničná 7, Prague 2 12844, Czech Republic © The Author(s). 2016 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. Macháček et al. Parasites & Vectors (2016) 9:579 Background Invasion of the central nervous system (CNS) of mammals, including humans, by parasitic helminths is a wellrecognized phenomenon. Apart from recruitment of peripheral leukocytes, astrocytes and microglia (i.e. CNSresident glial cells) can be activated during the infection and exhibit antiparasitic effects [1, 2]. In this study, we examined the response of astrocytes and microglia to the neuropathogenic bird schistosome Trichobilharzia regenti. Trichobilharzia regenti is widely distributed in Europe, e.g. Czech Republic [3], Denmark [4], France [5], Iceland [6] or Russia [7], and was also detected in Iran [8]. It uses anatid birds, e.g. ducks, as definitive hosts. They become infected by cercariae, freely swimming larvae emerging from lymnaeid snails which serve as intermediate hosts [3]. Apart from birds, T. regenti cercariae are able to penetrate the skin of accidental mammalian hosts, e.g. mice or humans. This may result in a skin allergic reaction known as cercarial dermatitis which is regarded as a re-emerging disease [9–11]. To penetrate the host’s skin, cercariae are equipped with proteases present in their excretory/secretory products (ESP; [12]), such as cysteine protease cathepsin B2 from postacetabular glands that was shown to cleave skin proteins like collagen, keratin and elastin [13]. Contrary to human schistosomes, the newly transformed schistosomula of T. regenti avoid penetration into skin blood capillaries and rather enter peripheral nerves in host‘s limbs where they appear 1–1.5 day postinfection (dpi). Parasite migration in definitive hosts continues towards and via the spinal cord and the brain, and adult worms occur in nasal mucosa of ducks 13–14 dpi and lay eggs there [14, 15]. The invasion of the nervous system by T. regenti schistosomula is often accompanied by serious neurological malfunctions in birds that suffer from leg paralysis and balance disorders [16]. A different course of the infection is observed in mice. Although schistosomula are found in the lumbar spinal cord as early as two dpi and medulla oblongata may be invaded the day after in some individuals, most parasites stay localized in the thoracic and cervical spinal cord and the migration to the brain is exceptional [14, 16]. As recently demonstrated, schistosomula feed on the nervous tissue when they pass through the spinal cord [17]. A cysteine protease, cathepsin B1, the intestinal enzyme of schistosomula, may be responsible for digestion since it was shown to degrade myelin basic protein [18]. However, the development of T. regenti is suppressed in mice and schistosomula do not reach maturity. It was hypothesized that this is possibly due to the host immune response and/or the absence of some essential nutritional or stimulatory factors [19]. The supposed role of the host’s immunity in regulation of parasite Page 2 of 10 migration is supported by experiments with immunocompetent and immunodeficient mouse strains. Immunodeficient mice display higher schistosomulum burden, the parasites also migrate faster in their CNS and reach brain hemispheres more often [14, 20]. Furthermore, the damaged schistosomula can be detected in the CNS from seven dpi in immunocompetent mice whereas in immunodeficient ones the parasite destruction appears two weeks later [17]. Research on the host immune response revealed a strong inflammatory cellular infiltration consisting of mononuclear cells, granulocytes, plasma cells and histiocytes, observed especially around the damaged schistosomula [20, 21]. Mononuclear cells present in the lesions were characterized as macrophages and CD3+ lym (...truncated)