In vitro model of postoncosphere development, and in vivo infection abilities of Taenia solium and Taenia saginata

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue.


Introduction
Taenia solium and T. saginata are two taeniid cestodes that cause the diseases taeniasis and cysticercosis [1]. These are zoonotic diseases, and swine and bovine act as intermediate hosts, causing porcine and bovine cysticercosis, respectively. Humans act as the definitive hosts in both T. solium and T. saginata infection leading to taeniasis. In the case of T. solium, humans can also act as accidental intermediate hosts causing human cysticercosis [2]. However, only T. solium causes human cysticercosis, while T. saginata does not [3].
When cysticercosis involves the central nervous system in humans, it is called neurocysticercosis (NCC). NCC is common throughout Latin America, sub-Saharan Africa, most of Asia, and parts of Oceania. Human NCC is believed to be the leading cause of acquired epilepsy worldwide [4,5].
The eggs of T. solium and T. saginata contain a six-hooked larva called the oncosphere [6]. When the eggs hatch, this oncosphere is released into the intestine. Intestinal fluid dissolves the oncospheral membrane, releasing and activating the oncosphere. The T. solium activated oncosphere can then penetrate the intestinal wall. Once in the tissue, usually in the muscle or the central nervous system, the oncosphere can transform into a postoncospheral form, and completely develop into cysticerci-a larval stage that consists of a fluid-filled sac containing an invaginated scolex. When this happens, the parasite produces a variety of molecules, which modulate the host immune response in order to evade parasite destruction [7].
The postoncospheral (PO) form is an intermediate stage between an oncosphere and a fully developed cysticercus in tissue [8]. The PO form of T. solium and T. saginata can be obtained in vitro by co-culture of oncospheres with a monolayer of mammalian feeder cells [9,10]. T. solium oncosphere and in-vitro generated PO forms can develop into cysticercus in rats causing NCC [9,11]. However, little is known regarding the in vitro development of the oncosphere to PO form of T. solium and T. saginata, specifically the immunological events that occur at the host/parasite interface. Also, it is not known if the development of the T. saginata oncosphere to PO form could cause NCC in the rat model as T. solium does. In vitro and in vivo models could lead to a better understanding of host-parasite relationships.
Host immune cells such as macrophages, lymphocytes, and polymorphonuclear leukocytes can produce cytokines and metalloproteinases (MMPs) in order to prevent the development of the parasite [12,13]. Because of this, the parasite has developed mechanisms to evade or modulate the host immune response.
Comparative studies on the oncosphere and the PO form of T. solium and T. saginata are limited. This study focused on the in vitro development of the oncosphere to the PO form for T. solium and T. saginata. These in vitro-developed larvae were then tested for infectivity in rats. Later, we obtained antigens from the T. solium and T. saginata oncosphere and PO forms to stimulate the production of cytokines and MMPs in healthy human peripheral blood mononuclear cells (PBMCs).

Cell culture preparation
HCT-8 and INT-407 cells, obtained from the American Tissue Culture Collectionper AU: (ATCC, Manassas, VA), were used to obtain T. solium and T. saginata PO forms. Cells were incubated at 37˚C in 5% CO 2 and grown in a specific medium as recommended by ATCC (EMEM media for INT-407, and RPMI for HCT-8; all medium was supplemented with 10% fetal bovine serum). The medium was changed every two days. Once cell confluency was obtained, cells were harvested using trypsin-EDTA (Sigma Chemical Co). Cells were placed into 24-well plates (1x10 5 cells per well) for maturation assay. The assays described below were performed when cells formed a monolayer.

Taenia solium and T. saginata oncosphere preparation
Tapeworms were collected after medical treatment of newly diagnosed patients, as described by Jeri et al [14]. Hatching of eggs and oncosphere activation were performed, as described by Verastegui et al [15]. The eggs were obtained from gravid proglottids of adult tapeworms by gentle homogenization in a 2.5% potassium dichromate solution (Sigma, St. Louis, Missouri). Eggs were then washed three times in distilled water with centrifugation steps to collect the eggs between washes in 2500g for 5 minutes. The eggs were hatched, and the oncospheres were released using a solution of 0.75% sodium hypochlorite in water for 10 minutes (Mallinckrodt Baker, Inc, Phillipsburg, NJ). Oncospheres were then washed three times in RPMI medium (Sigma, St. Louis, Missouri), and activated by incubation at 37˚C for 45 minutes (in the case of T. solium) or 90 minutes (in the case of T. saginata) with artificial intestinal fluid (1 g pancreatin (Sigma Chemical Co., St. Louis, MO), 200 mg Na 2 CO 3 , and 1 ml of fresh porcine bile (for T. solium), or 1 ml of fresh bovine bile (for T. saginata), with enough RPMI 1640 medium (pH 8.04) to make 100mL). After activation, the oncospheres were washed three times with RPMI medium and counted using a Neubauer chamber.

In vitro maturation assay
Parallel in vitro maturation assays with INT-407 and HCT-8 monolayer cells, using T. solium and T. saginata activated oncospheres, were conducted in order to compare the morphological characteristics during development of each species using the methodology reported by Chile et al [9]. Ten thousand activated oncospheres were cultured in confluent INT-407 and HCT-8 monolayer cells for two weeks. During that time, the medium was changed every three days. At day 15 of culture, the postoncospheral forms were collected and rinsed twice with fresh medium, then transferred to another well containing a confluent of monolayer cells. This process was repeated every three days for up to six months to allow the postoncospheral forms to continue to develop. Cultures were inspected daily using an inverted microscope (Leitz labovert FS). Parasites were collected at 15, 30, 60, 120, and 180 days of incubation.

Infection of rats with T. solium and T. saginata activated oncosphere (AO) and PO forms
To determine if T. saginata AO and PO forms can develop into viable cysts in vivo, 15-day old Holtzman rats, purchased from Universidad Peruana Cayetano Heredia, Lima, Peru, were infected intracranially (in the bregma) with oncospheres and 15-day old PO forms from either species following the methodology reported by Verastegui et al., 2015 [11]. Rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight) before infection.
Six rats were inoculated with 180 T. solium AO in 100 μL of saline solution; seven rats were inoculated with 180 T. saginata AO in 100 μL of saline solution. The negative control was 2 rats inoculated with saline solution.
Eight rats were inoculated with ten T. solium 15-day PO forms in 100 μL of saline solution, eight rats were inoculated with ten T. saginata 15-day PO forms in 100 μL of saline solution, and five rats were inoculated with 100 μL of saline solution as a control. A 24-gauge syringe needle was used.
After four months, necropsy was performed. Rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Anaesthetized rats were perfused with 200 ml of PBS and then with 100 ml of 4% paraformaldehyde in PBS. Brains were carefully removed, post-fixed for 24 hours at 4˚C with 4% paraformaldehyde in PBS, and stored in 70% ethanol. Brains were observed macroscopically to identify extraparenchymal cysticerci. Five millimeter coronal brain sections were cut until the intraparenchymal cysticerci were observed.

Antigen preparation
Antigens were obtained from AO and 30 day-PO forms of both parasites. AO and PO were obtained as described above. Parasites were rinsed three times with PBS buffer, sonicated, and centrifuged at 10,000g for 15 min at 4˚C. The supernatant (total soluble antigens) was separated, and proteins were quantified using Bradford Protein Assay (Bio-Rad) and stored at -70˚C until ready for use.

In vitro stimulation of peripheral blood mononuclear cells (PBMCs)
Healthy volunteers (n = 13) with negative serology for NCC were invited to participate in this study. After the volunteers signed an informed consent form, 10 mL of venous blood was collected from each non-infected donor. PBMCs were collected from 10 ml EDTA blood. The blood samples were centrifuged at 400g on a Ficoll-Hypaque gradient (Ficoll-Paque TM PLUS, GE Healthcare) for 10 minutes at room temperature for the separation of mononuclear cells. Cells were again suspended in RPMI medium plus 5% of inactivated human serum. Cell viability was evaluated with trypan blue and counted in a Neubauer chamber. Each well contained 2x10 5 group of cells was cultured in a 96-well plate at 37˚C with 5% CO 2 for 48 hours in an RPMI medium containing 5% of inactivated human serum. Cells were stimulated with 5 μg/ ml of phytohemaglutinin (PHA) as the positive control, 20 μg/ml of AO antigens (both species, separately), and 20 μg/ml of antigens from PO forms at 30 days of maturation (both species, separately). At the end of the incubation period, PBMCs were harvested and centrifuged for 10 min at 400g, and supernatants were collected and stored at −70˚C until tested for cytokine and metalloproteinase content by multiplex analysis.

Ethical statement
Experiments were done according to the Guide for care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics Animals of the Universidad Peruana Cayetano Heredia, Lima, Peru (Permit Numbers: 61242).

Data analysis
For cytokines and MMP assays, the median fluorescent intensities of the beads with the cytokines or MMP now bound were converted to concentrations (pg/ml), using a five-parameter logistic model using the Bio-Plex Manager™ 6.0 software (Bio-Rad Laboratories, Hercules, CA). Each MMP´s or cytokine's concentration was normalized by subtracting the data of medium alone (control). The Mann Whitney test was used to compare two unpaired groups, and ANOVA followed by Tukey's post-test was used for three or more group using the Prism V 6.0 statistical program (GraphPad). A P-value of <0.05 was considered statistically significant.

In vitro development of T. saginata and T. solium from oncosphere
T. saginata and T. solium developed from the oncosphere to the PO form in both cell lines, INT-407 and HCT-8 cells (Table 1). In HCT-8 cells, both species of PO forms developed until 60 days of culture, and then began to die. However, after 60 days in INT-407 cells, the T. saginata PO forms continued to develop to cysticerci, while the T. solium PO forms began to die. At 180 days of culture 0.25% of T. saginata oncospheres developed to cysticerci.
The morphological characteristics of the T. saginata and T. solium PO forms obtained from culture in INT-407 monolayer cells were compared. At 15 days of incubation, both species were morphologically similar (Fig 1A), ranging between 60 to 250 μm in size. Both had an oval form without hooks and shared characteristic movement. For both species at 30 days of incubation, PO forms increased in size (between 200 to 1000 μm), maintained their oval form, and formed a protuberance at one end of the body (Fig 1B).
At day 60 of culture, the T. solium PO form had a spherical shape, similar to a cyst without a scolex, and cells accumulated in the protuberance of one end of the body (Fig 1C). The T. saginata PO form had similar characteristics but also showed a neck-like prolongation at one end of the body (Fig 1C), reaching up to 3000 μm. Additionally, at day 60, T. solium PO forms began to die while T. saginata PO forms continued growing until 180 days of culture.
At day 120 of culture, T. saginata developed into a cysticercus with a spherical form and an evaginated pre-scolex containing four characteristic suckers and measuring 4 mm in diameter (Fig 2A). At day 180 of culture, T. saginata cysticerci increased in size compared to 120 day, reaching up to 6 mm and also displayed a well-defined scolex and suckers (Fig 2B).

T. solium oncosphere and PO form resulted in NCC in rat infection
We infected rats with T. saginata oncosphere and PO forms in order to evaluate if T. saginata can develop into cysticercus in vivo, as T. solium does. Table 2 shows rats that were infected with T. solium oncosphere or 15-days PO form developed NCC, while rats that were infected with T. saginata oncosphere or 15-days PO form did not develop NCC.

T. solium and T. saginata forms stimulate cytokines and MMP-9 in PBMC culture
Given that T. saginata does not develop to the cysticerci form in humans, we investigated the differences in host immune response to infection by T. solium and T. saginata. To determine this, we evaluated the production of cytokines during stimulation with AO and 30-day PO antigens of both species.
When compared by stages (AO and PO form) species, both T. solium and T. saginata PO forms stimulated a higher production of the majority of cytokines evaluated than the AO forms (S1 Fig). Nevertheless, when compared by species stages (AO and PO form), we observed a higher production of IL-4, IL-5, IL-13, IFN-γ and IL-2 cytokines stimulated by the T. solium AO form (Fig 3A and 3B) compared to the T. saginata AO; but in PO form, the T. saginata 30-day PO form stimulated a higher production of a variety of cytokines, including, IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 compared to the T. solium 30-day PO form (Fig 4A, 4B and 4C).
To evaluate whether MMPs are involved in the host immune response against the parasites, we stimulated PBMCs with T. solium and T. saginata forms. The T. solium AO stimulated higher MMP-9 production compared to the T. saginata AO, while in PO form there was no significant difference between both species (Fig 5). There was no significant difference in the production of MMP-2 by PBMCs stimulated with different antigens of both parasites (S2 Fig).

Discussion
The present study demonstrates that T. saginata activated oncospheres can develop into cysticerci in vitro in a human cell line, while T. solium AO do not. In contrast, in the rat model, the T. saginata oncosphere and PO forms did not develop to cysticerci in vivo as T. solium does. We suspect the differences in development between the two species in the in vitro model are due in large part to the environment of the growth media and the specific cell line, whereas in vivo it is the host immune response that plays a predominant role in regulating parasite development and survival. For instance, Heath and Smyth noted that serum used for in vitro culture contains factors unique to each host-parasite system, which can stimulate development of the parasite [16]. In our study, both species were cultured in media containing fetal bovine serum In the in vivo, T. solium oncosphere and PO forms developed to cysticerci in brain while T. saginata forms did not. This finding is likely due to T. solium's ability to evade the host immune response, either through binding of host plasma proteins or by synthesizing surface proteins that are antigenically similar to those of the rat [18]. Previous work has demonstrated that T. taeniaeformis oncospheres are able to initiate PO development after acquiring a component of rat serum on their surface, a component that presumably protects the oncosphere in the newly invaded host from recognition as a foreign antigen [17]. In the same study, T. saginata failed to bind this component and was subsequently attacked by the host immune system. A similar observation has been noted in pigs after infection with T. saginata eggs [19]. Another explanation may be genetic factors that play a role in the rat's resistance to infection from T. saginata [20].
Although the rat is not a natural host of T. solium, we observed in previous studies that the cysticerci that develop in the rat brain are morphologically equal to those that develop in humans, as is the observed inflammatory response and subsequent pathology [11,21]. Therefore, we believe the rat, like the human, has molecules that prevent the development of T. saginata.
The cytokines response plays an important role in survival of the oncosphere at the time of initial infection. We suspect T. solium stimulates a different cytokines profile than T. saginata allowing the T. solium oncosphere to survive, while T. saginata is destroyed by the host. To probe this hypothesis, we stimulated cytokine and MMP production in healthy human PBMCs using antigens of oncosphere and PO forms from both species.
We observed that T. solium oncospheres stimulate a higher production of IL-4, IL-5, IL-13, IFNγ and IL-2 compared to T. saginata oncospheres. The IL-4, IL-5, IL-13, are cytokines typically associated with a T helper 2 (T H 2) response, the predominant protective immune response against helminthic infections [22]. On the other hand, IFNγ and IL-2 are associated with a T helper 1 (T H 1) response, which is thought to be responsible for destruction of the parasite [23]. The T H 2 response is important in protecting against extracellular helminthic parasites through suppression of the T H 1 response, neutralization of toxins, and defense of the host against damage [22,24]. Inflammatory reactions are dependent on a delicate balance between T H 1 and T H 2 type responses. In the case of the T. solium oncosphere, it appears a mix immune reaction of T H 1/ T H 2 type response that aids survival of the parasite. Perhaps in doing so, T. solium oncospheres are able to migrate from the vasculature to the brain, where they develop into the PO form. Similar mixed T H 1/T H 2 phenotypes have been observed in patients with NCC [7,13] whereas a predominantly T H 2-type response is associated with asymptomatic disease [25,26]. In both species, the 30-day PO form generated an overall greater inflammatory response than the AO form. Levels cytokines increased when exposed to equal concentrations of PO vs AO antigen, suggesting that antigen composition changes as the parasite is maturing [9].
However, the T. saginata 30-day PO form stimulated a profile of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, TNF-α), plus a mix of T H 1 and T H 2 related cytokines (IL-4, IL-5, IL-13 and IFN-γ) that was stronger than the response produced by the T. solium 30-day PO form. IL-6, a pro-inflammatory cytokine, plays a role in the death of microorganisms by stimulating the behavior of neutrophils [27]; and TNF-α is strongly expressed at the sites of parasite and cell destruction [28]. Together with the overproduction of IL-4, IL-5, and IL-13, these cytokines may mount a response that could destroy the T. saginata PO form and prevents development of the cyst in vivo. Although these cytokines do have the suggested properties mentioned, they also could be involved in complex networks with mixed effects with respect to inflammation, for example IL-6 has been shown to exhibit anti-inflammatory properties [29].
T. solium oncospheres stimulated increased MMP-9 production in PBMCs compared to production by T. saginata oncospheres. MMP-9 is an endopeptidase produced by neutrophils, macrophages, monocytes, and intestinal epithelial cells [30,31]. It can degrade components of the blood brain barrier (BBB) as well as the extracellular matrix, increasing intestinal epithelial permeability [30,32,33]. MMP-9 has been associated with the breakdown of the BBB in a murine model of NCC [34,35] and is present in high concentrations in sera of symptomatic NCC patients [36]. We hypothesize that T. solium stimulates the production of MMP-9 as a means of enhancing epithelial permeability in order to pass restrictive biological barriers like the intestine and the BBB during early stages of infection.
In conclusion, we have found novel evidence to suggest that T. saginata PO forms are capable of developing into cysticerci in the human cell line INT-407 while not in HCT-8 cells. In vivo, T. saginata fails to develop into cysticerci in the rat brain, suggesting there are factors in the host immune system (that are not present in the in vitro culture) that destroy the parasite. In the oncosphere stage, T. solium stimulated a strong mix of T H 1 and T H 2-related cytokines and MMP-9 production in healthy PBMCs, which may mediate the inflammatory response and promote oncosphere survival in the vasculature, aiding the entrance of oncospheres into the brain. In the PO form, T. saginata stimulated a strong pro-inflammatory and mix of T H 1/ T H 2-related cytokines, responses that could be causing destruction of the parasite in the tissue. These differences between both species of Taenia found in vitro and in vivo could explain why