Th1/Th2/Th17 pattern in pregnant mice inoculated with live Echinococcus granulosus Protoscolex

Introduction

It has been shown that T and B cells are the major players of the immune system, forming innate and adaptive immune systems (Paciullo et al., 2017). Helper-T-lymphocytes (Th cells) are subtypes of CD₄⁺, which are divided into four categories according to their cytokine profile: Th1, Th2, T-regulatory and Th17 cells. Tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and IL-12 as well as interferon-γ (IFN-γ) are produced by Th1 cells, consequently resulting in cell-mediated immunity, whereas Th2 cells contribute to the production of IL-4, IL-5, IL-6, IL-10, and IL-13 to stimulate humoral immunity. Th17 cells, as potential pro-inflammatory cells, contribute to the secretion of IL-21, IL-22, and IL-10. Notably, Th17 cells are able to convert into Th1 cells (Muranski et al., 2013). For a host to establish immunity against parasites and other invading insults, Th cells trigger the production of some responses such as IFN-γ, leading to the removal of infection (von der Weid and Langhorne, 1993). However, the story is a bit different about the role of Th2 cells. It has been demonstrated that Th2-related cytokines such as IL-4, IL-5, and IL-6 are in favor in terms of the establishment of Fasciola gigantica infection and evasion. Notably, cytokines present a distinct profile during pregnancy (Sheng et al., 2019). In contrast to Th2 cytokines, Th1 cytokines lead to adverse impacts on the pregnancy and may alter the outcome of pregnancy (Zhang et al., 2015). Abortion has been observed due to the increased concentrations of Th1 cytokines (Zhang et al., 2015). Furthermore, different data demonstrate the potential role of increased levels of Th17 cells in the stimulation of pregnancy disorders such as preeclampsia, mental illness, recurrent spontaneous abortion, and so on (Tesmer et al., 2008).

Hydatid disease (HD), or cystic echinococcosis, is a globally distributed zoonotic disease caused by Echinococcus granulosus sensu lato complex (Craig et al., 2017; Thompson, 2017). During the recent decade, much attention has been made towards controlling this zoonotic disease, which is widely due to huge cost burden on nations, so that World Health Organization (WHO) has estimated a cost as much as $3 billion to control this infectious disease (Solomon et al., 2017; Harandi et al., 2012). Interestingly, the hydatid cyst of E. granulosus has sustainability as high as 50 years, resulting from two phenomena: passive escape and immunomodulation (Mihmanli et al., 2016). So, this parasite can be hidden from immune response, and also decreasing the effect of host responses. Canine definitive hosts and intermediate hosts such as sheep, cattle and camel involve in maintaining E. granulosus in our surrounding environment (Banks et al., 2006). The adult E. granulosus resides through the small intestine of the definitive host. Gravid proglottids release eggs in the feces, which are immediately infectious. After ingestion of the eggs by a suitable intermediate host, they hatch in the small intestine and then, release six-hooked oncospheres, which interpenetrate in the wall of the small intestinal and migrate through the circulatory system into different organs, particularly the liver and lungs. In the affected organs, the oncosphere develops into a thick-walled hydatid cyst that expands progressively, producing protoscolices (PSC) and daughter cysts that fill the cyst interior. The cyst-containing organs of the infected intermediate host can infect the definitive host. After ingestion, the protoscolices release, attach to the intestinal mucosa, and develop into adult form pending 32 to 80 days (please insert the reference).

Humans are known as the aberrant intermediate hosts who become infected by the ingestion of the eggs. As expected, oncospheres are released in the small intestine, and hydatid cysts develop in the various organs. If cysts rupture, the released protoscolices may produce secondary cysts in other organs (secondary echinococcosis)(Moro et al., 2009).

In the present study, we provided a pregnant mouse model E. granulosus infection to investigate the alteration in levels of Th1/Th2/Th17-related cytokines.

Materials and methods

Animal source:20 Balb/c mice were purchased from the laboratory animals rearing center of Pasteur Institute of Iran and divided into four groups, randomly, including A) control group; B) healthy pregnant group; C) pregnant group infected with PSCs; and D) non-pregnant group infected with PSCs. Each group of mice was kept in isolated cages and a clean room with a constant temperature of 25°C and a 12-hour cycle of light and darkness (L:D) and had access to adequate food and water. This study has been approved at the University of Tabriz ethics committee (Ref No. IR.TABRIZU.REC.1398.004).

PSCs preparation and viability assessment:In the fall of 2018, the fresh cysts of hydatid were provided from the naturally infected livers of sheep slaughtered in Tabriz industrial abattoir, East Azerbaijan province, Iran, and transferred to the laboratory of Parasitology at the Faculty of Veterinary Medicine, University of Tabriz. Initially, the hydatid cysts were surface disinfected twice with ethanol (70%). Then, cyst fluid was aspirated from each of the cysts by a 50 ml syringe, transferred into glass tubes and centrifuged (1500 rpm/3 min). The supernatant was elegantly discharged, and the deposited PSCs were washed with PBS solution (pH 7.2) several times. To determine the density of PSCs per ml, five microliters of the mixture containing PSCs were transferred on a microscopic slide, covered with a coverslip, and the prepared slide was then examined under a light microscope (40x). This step was performed in triplicates, and the mean was recorded as PSCs density/mL. Finally, the PSCs density was adjusted up to 500 PSCs/mL in 0.9% saline normal over 90% viability for inoculation to mice. Also, to evaluate the viability of PSCs, by using Smyth and Barrett’s method (Smyth and Barrett., 1980), 20 µl of PSCs mixture was mixed with an equal volume of eosin (Sigma-Aldrich, St. Louis, MO, USA) stain 0.1% (powder of eosin (1 g) in 1 L of distilled water) in a clean microtube. After 15 minutes of exposure to the eosin staining, the colorless PSCs (Fig. 1a) under the light microscope were considered as live while the colored red (Fig. 1b) ones were considered as dead. The viability percentage of PSCs was calculated by dividing the number of live PSCs to the total number of calculated ones by 100. This stage was also performed in triplicates (Mahmoudvand et al., 2017). To ensure, some mixture containing PSCs was heated (60°/30 min) in an oven (Fan Azma Gostar, Karaj, Iran) to kill the PSCs, and then stained with eosin 1% the same above mentioned.

Preparation of animal model:To induce E. granulosus infection, 500 PSCs (Yumin et al., 2019, Valizadeh et al., 2017) was injected into each mouse (mice in the groups of C and D) intraperitoneally (i.p.). The control group received only normal saline i.p. One week before PSCs induction, the male and female mice were kept together to adapt and provide pregnancy. The pregnancy was recognized through the pregnancy plaques formation in the vagina 10 days after keeping together.

Evaluation of cytokine levels:At 7, 14, 21, and 28 d.p.i., the blood samples were taken from the tail vein, and the TNF-α, IL-4, IFN-γ, and IL-17 levels were determined using commercial kits according to the manufacturer’s instructions Bender Med Co. Austria; R&D Systems, the United Kingdom). Briefly, each of the cytokines was individually assayed in a sandwich ELISA method, which described simultaneously due to the similarity of the work process. Two wells were considered for each sample, one pair of the cells as blank controls and seven pairs of cells as standard samples. Initially, the plate wells were washed twice with a buffer, and each time with a volume of approximately 300 μl before returning and discharging the content of the plates, it was given for 10 to 15 s, and then extra water of the plate was taken by a tissue. Standard dilutions with the assay of each cytokine were provided in wells. For this purpose, 225 μl of sample diluents were added to all seven pairs of wells. 225 μl from the cytokine standard stock solution was added to each of the first wells, and mixed completely with the contents of the wells several times. Then 225 μl of this mixture was added to the second well and the work proceeded in the same way; eventually 225 μl of this mixture discarded from the seventh wells. Test samples (conditioned media of spleen cells culture) were added to the wells at 50 μl. Following this, 50 μl of the dilution buffer was added to each of these wells and mixed with the test sample. 100 μl of the dilution buffer was added to the blank wells. In the next step, 50 μl of secondary antibody conjugated with biotin (x1) was added. Then the plates were covered and incubated at room temperature for 2 hours. Then, the plates were washed with washing buffer for three times, as in the first step. 100 μl of Streptavidin to the HRP (1x) solution were added to all wells and, after covering the plate surface, were incubated at room temperature for an hour. Similar to the first step, the plates were washed three times, and 100 μl of the HRP enzyme-substrate (TMB) was immediately added. Once again, the surface of the plates was covered and incubated at room temperature for about 10 minutes. The intensity of the color created at the 620 nm wavelength was evaluated by ELISA and, as soon as standard No. 1 showed an optical absorption of 0.9 to 0.95, the reaction stopped by adding 100 μl of 1M phosphoric acid, and plates were immediately read by an ELISA reader with 450 nm wavelength. The average OD was calculated, and the standard curve was plotted on logarithmic paper. By using the standard curve, the amount of each of the cytokines in the sample was determined as pg/ml, and the standard curve was depicted between 15.7-2000 pg/ml. The sensitivity of the test was 4.0 pg/ml for IL-17 and IL-4 and 1.4 pg/ml for TNF-α and 6.3 pg/ml for IFN-γ, according to the manufacturer. The criterion for ensuring work accuracy is a lack of difference of more than 20% of each sample with the average calculated. It should be noted that since the test sample is diluted in a 1 to 2 ratio in the third step, the final concentration of cytokines is determined by doubling the numbers obtained from the standard curve. The results from five subjects were expressed as a mean ± SEM (the standard error of the mean).

Data analysis:Statistical analysis was performed using SPSS software version 19 and ANOVA test and t-test with P< 0.05 as a significant level. All data were expressed as mean ± SEM.

Results           

The levels of cytokines at 14 d.p.i:After E. granulosus PSCs induction, the levels of TNF-α, IFN-γ, IL-4, and IL-17 increased in the C and D groups compared to the A and B groups, but the differences were not significant (P < 0.05) (Fig. 2).

The cytokines levels during the second week:TNF-α, IL-17, and IFN-γ serum levels in group D, remarkably increased compared to the others, but the difference concerning IL-17 was not significant (P < 0.05). Notably, in the second week, the level of IL-4 in group C was more than group D but the difference was not significant (Fig 3). Also, IFN-γ and TNF-α serum levels in group C were higher than group B, but the differences were not significant.

The cytokines levels during the third week:At the third week, TNF-α, IL-17 and IFN-γ serum levels were remarkably increased in group D compared with the other groups (P < 0.05). Besides, similar to the second week, the level of IL-4 was higher in group C compared to with others (P < 0.05) (Fig 4). Moreover, TNF-α, IL-17, and IFN-γ serum levels in group C were increased compared with group B, but the difference was not significant.

The levels of cytokines during the fourth week:In the fourth week, there was a trend like other weeks, and the levels of cytokines significantly increased. Furthermore, similar to the second and third weeks, the level of IL-4 in the fourth week was higher in group C compared with group D (Fig 5).