Date Published: July 24, 2017
Publisher: Public Library of Science
Author(s): Foojan Mehrdana, Per Walter Kania, Sasan Nazemi, Kurt Buchmann, Jyotshna Kanungo.
Excretory/secretory (ES) compounds isolated from third-stage larvae of the anisakid nematode Contracaecum osculatum parasitizing liver of Baltic cod were investigated for effects on immune gene expression in a zebrafish LPS-induced inflammation model. ES products containing a series of proteins, of which some had enzymatic activity, were injected solely or with LPS. ES proteins alone induced up-regulation of a number of immune-related genes, but generally to a lower degree compared to LPS. When co-injected with LPS, the worm products exacerbated merely expression of five genes affecting Th1, Th2, Th17 and innate responses compared to the LPS-injected group. However, the level of overexpression decreased in an inverse dose-dependent manner. The immune regulating action of C. osculatum ES products is interpreted as an important evolutionary ability of larval parasites in the transport host which makes it less susceptible to host immune responses whereby the probability of reaching the final host is increased.
Parasitic helminths produce a series of excretory/secretory (ES) compounds which have been suggested to play an important role in parasite-host interactions . In nematodes this complex of molecules originates from different organs of the parasite (oesophagus, ventricle, intestine, glands) and comprises various enzymes with different functions in the host including penetration and migration in the host tissues, alteration of host physiology, and immunomodulation with the purpose of favouring parasite survival [1, 2]. Enzymes such as acetylcholinesterase (AChE), glutathione-S-transferase (GST), and superoxide dismutase (SOD) secreted by the hookworm Necator americanus act as anti-inflammatory molecules creating a shielded pathway in order to protect the worm from immune reactions . Correspondingly, the filarial nematode Wuchereria bancrofti produces AChE in the human host circulation which degrades acetylcholine, inhibits lysosomal enzyme release and phagocytosis . ES compounds have been suggested as potential therapeutics for inflammatory disorders. Thus, in a murine experimental asthma model it was shown that the Ascaris suum ES protein (PAS-1) is able to suppress allergen-induced Th2 responses, inhibit cellular migration, suppress cytokine expression (IL-4, IL-5), and reduce chemokine production in bronchoalveolar tissues . We, therefore, hypothesize that ES products of third-stage nematode larvae of Contracaecum osculatum possess similar immunoregulatory properties. The life cycle of C. osculatum (Rudolphi, 1802) comprises adult worms in marine mammals, e.g. seals [6, 7], and infective third-stage larvae in invertebrates and teleosts serving as intermediate/transport hosts. Humans may accidentally obtain third-stage larvae of the parasite through consumption of raw or under-processed seafood which causes anisakidosis associated with gastrointestinal symptoms [8–10] and experimental C. osculatum infections of pigs elicit eosinophilic granuloma formation . Other ascarid nematodes, e.g. Ascaris lumbricoides, Toxocara canis, and Anisakis spp. [12–16] produce a series of immunogenic molecules, including allergens, which suggests that the immunogenicity of C. osculatum proteins should be addressed. The occurrence of C. osculatum is increasing in certain localities, e.g. the Baltic Sea [7, 17] and may have an increasing influence on health in fish and mammals including humans. It is, therefore, worthwhile to investigate the immunogenic properties of C. osculatum, and we here present data on immunoregulation by ES proteins from this parasite elucidated in a zebrafish inflammation model. This experimental fish represents some advantages (small size, ease of handling and breeding, and rapid life cycle) compared to rodent models . Therefore, zebrafish is currently being applied in biomedical research including immunology, as innate and adaptive immune responses are highly evolutionarily conserved. Thus, both fish and mammals share similar sets of immune signalling molecules and immune cells (e.g. cytokines, neutrophils, macrophages, dendritic cells, B and T cells)  and zebrafish models have been used in the studies of human inflammatory disorders such as hepatic inflammation and inflammatory bowel disease (IBD) [20, 21]. To date, no animal model has directly used ES compounds from C. osculatum for immunomodulation. Thus, in the present study we applied a lipopolysaccharide (LPS)-induced inflammation model in zebrafish aiming to elucidate whether C. osculatum ES compounds have any effects on the inflammatory responses.
The zebrafish, Danio rerio, was in this investigation applied as a model for inflammation and found useful for studies of inflammation regulatory molecules. This fish species showed to react strongly to injection of LPS and a long range of genes encoding inflammatory factors such as cytokines and acute phase reactants were subsequently up-regulated. We found that LPS triggered production of proinflammatory cytokines (IL-1β, IL-8, and TNFα) and modulated gene expression related to the Th2 (IL-4/13a, IL-4/13b) and Th17 (IL-17A/F1, IL-22) signalling pathways. A 3-fold increase in Myd88 gene expression and an extreme up-regulation of the acute phase protein SAA gene was observed due to LPS exposure. Mammals apply TLR4 for LPS recognition, while most fish lack this receptor [24–26], but it has previously been shown that zebrafish respond to LPS stimuli and produce an inflammatory reaction even from an early stage of 2 days post fertilization, although the responsible receptor has not yet been identified .
In the present study, we used adult wild-type zebrafish to establish an inflammation model using LPS and investigated modulation of immune genes expression after exposure to different concentrations of C. osculatum ES compounds. Inflammatory reactions were successfully induced in zebrafish not only by i.p. injection of LPS, but also by ES protein injection. The C. osculatum ES products exhibited in some cases a significant influence on expression of genes otherwise up-regulated by LPS exposure, but the composed nature of ES products can direct host reactions differently. Therefore, future work must isolate individual components in the ES mixture and precisely determine the pathways affected by individual proteins. The mixed responses described in this work may mimic the natural condition of host infection (mammalian or fish intermediate host) with the C. osculatum larvae; while parasite larvae penetrate into the host tissue and induce inflammatory immune responses, they also produce ES compounds with a potential to reduce inflammatory reactions. This may be interpreted as an evolutionary important ability, as this will minimize the host response towards the worm and thereby increase survival and subsequent reproduction.