Immune response evaluation in Balb/c mice after crude extract of Anisakis typica sensitization

Background and Aim: Anisakis is a global challenge for a fish product which may lead to a decrease in economic value and consumers’ preference. Skipjack (Katsuwonus pelamis) in Kupang, Nusa Tenggara Timur, Indonesia, have important economic value for local fisheries. Anisakis typica is one of the Anisakis species which potent to induce an allergic reaction. However, the study about A. typica involved in the dendritic cells (DCs), T helper 1 (Th1), T helper 2 (Th2), and regulatory T cells (Tregs) is still limited. This study aimed to analyze the dynamic changed of the immune system including DCs, CD4+ T cells, and Tregs after 1 week of A. typica sensitization. Materials and Methods: Twenty-four male Balb/C mice were randomly divided into four groups (n=6), mice treated with crude A. typica extract (CAE) 50, 75, and 100 mg/kg BW, respectively. CAE was given orally per day for a week. At the end of the experiment, the animals were sacrificed and the spleen was collected. DCs were labeled as CD11c+ interleukin-6+ (IL-6+); CD4+ T cells were distinguished as Th1 (CD4+ interferon-γ+ [IFN-γ+]) and Th2 (CD4+ IL-4+ and CD4+ IL-5+); Tregs were labeled as CD4+CD25+CD62L+. The expression of each cell was determined by flow cytometry. Results: Our result described that CAE elicits CD11c+ IL-6+, CD4+ IFN-γ+, CD4+ IL-4+, and CD4+ IL-5+ and reduces CD4+CD25+CD62L+ significantly (p<0.05) in dose-dependent manner in mice after A. typica infection. Conclusion: The Th1/Th2 ratio after A. typica crude extract treatment exhibits a mixed pattern rather than the classical model allergy to food antigens. Our study is expected as a basic understanding of the changes in immune response after A. typic a infection.


Introduction
Food safety and food security due to food-borne infections are gaining interest in the past decade [1,2]. Anisakiasis, the zoonotic disease caused by nematode larvae of the genus Anisakis is considered as one of the most important biohazards in the fish products [3]. The previous study reported that Anisakis spp. were found in commercially fish, particularly anchovies (Engraulis encrasicolus), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus) [4].
Anisakis spp. have a complex life cycle and reach maturation in the third stage (L3). Marine mammals are a final host to complete its life cycle [5]. A human can accidentally be infected by Anisakis after consumed the raw or half-cooked fish meats, which is strongly associated with acute gastrointestinal (GI) symptoms [6] and allergen reaction [7]. Both live and death larva of Anisakis could induce the allergic reaction due to its thermal-and pepsin-resistant properties [5,8]. Interestingly, the simple prepared crude extract of Anisakis is enough to induce the allergic reaction [9].
The allergic reaction due to Anisakis has been reported to elicit the host immune response which is characterized by T helper 2 (Th 2 ) response predominantly by secreting cytokines such as interleukin-4 (IL-4) and IL-5 [10]. Furthermore, T helper 1 (Th 1 ) maturation by dendritic cells (DCs) was suppressed by regulatory T cells [11] which assist Th 2 polarization during helminth infection. This regulatory network results in the decrease of interferon-γ (IFN-γ), the cytokine which secreted by Th 1 [12]. In contrast, the previous study reported that Anisakis allergy exhibits a mixed Th 1 /Th 2 pattern [13]. Meanwhile, prolonged nematode infection may lead to chronic infection predominantly by Th 1 [14].
Nowadays, there are nine Anisakis species which have been confirmed [15]. Among them, Anisakis Available at www.veterinaryworld.org/Vol.12/October-2019/3.pdf simplex sensu stricto (s.s) and Anisakis pegreffii are the best known caused the allergic reaction and other health problems [16]. However, as far as our knowledge, there is little information about allergen reaction which involved immune cells such as DCs, Th 1 , Th 2 , and regulatory T cells (Tregs) caused by Anisakis typica. A. typica have been found parasitizes Delphinidae, Phocoenidae, and Pontoporidae in Atlantic and Indian Oceans and in the Eastern Mediterranean Sea. Surprisingly, A. typica also found in Australian and Indonesia [17]. This finding increases the potential risk for anisakiasis frequency in Southeast Asia, mainly in Indonesia.
This study aimed to evaluate the immune response underlying host after A. typica infection. The present study is expected as a basic understanding of A. typica accidental infection due to marine products and, subsequently, develops the intervention strategies.

Ethical approval
This study was approved by the Ethical Committee of Brawijaya University (approval number 938-KEP-UB).

Animals
Male Balb/C mice aged 5 weeks were supplied from the Integrated Research and Testing Laboratory-Unit IV, Gadjah Mada University. Mice were housed in plastic cages for a period of acclimatization. Mice were given food and water ad libitum and maintained at room temperature with a 12 h light/dark cycle.

Crude A. typica preparation
Skipjack (Katsuwonus pelamis) were purchased from the traditional market in Kupang, Nusa Tenggara Timur, Indonesia. A. typica was manually harvested from the abdominal cavity of skipjack which naturally parasitized by A. typica. A. typica was identified by polymerase chain reaction according to Soewarlan et al. [18] using NC5 (forward; 5'-GTAGGTGAACCTGCGGAAGATCATT-3') and NC2 (reverse: 5'-TTAGTTTCTTTTCCTCCGCT-3') primer (data not shown). A. typica washed with distilled water then stored at 4°C. The crude extract of A. typica was made by crushed A. typica using porcelain mortar and pestle at 4°C. The protein content of milled A. typica then measured using nanodrop spectrophotometer (ND1000). The protein content then considered as a standard to determine the dosage given to animals.

Experimental design
Twenty-four male Balb/C mice weight 25 g were randomly and equally divided into four groups (n=6): Normal (unsensitized mice) and mice treated with crude Anisakis extract (CAE) 50, 75, and 100 mg/kg BW, respectively. Mice were intragastrically challenged per day for 7 days consecutively with CAE, except the normal group. At the 8 th day, mice were anesthetized through intraperitoneal injection the combination of ketamine and xylazine (90 mg/kg and 10 mg/kg, respectively) [19] followed by euthanized by cervical dislocation. The spleen was collected and washed 3 times in sterile phosphate-buffered saline (PBS) then crushed into single-cell suspensions. Single-cell suspensions then added with PBS until reached 10 mL and centrifuged at 2500 rpm for 5 min at 10°C. The supernatant then discarded and the pellet was homogenized with 1 mL PBS [20]. Homogenates then divided into several 1.5 mL tubes according to the staining used then centrifuged at 2500 rpm for 5 min at 10°C. no.: 421002) and centrifuged at 2500 rpm at 4°C for 5 min. Supernatant was discarded, and the pellet was stained with intracellular staining (50 μL) of PE anti-mouse IL-6 (BioLegend, clone: MP5-20F3) which combined with CD11c. Pellet which previously stained with anti mouse-CD4 were stained with PE-Cy7 anti-mouse IL-4 (BioLegend, clone: 11B11) and PE-anti-mouse IL-5 (BioLegend, clone: TRFK5) to identified T h 2 Besides, pellet which previously stained with anti mouse-CD4 were stained with PE anti-mouse IFN-γ (BioLegend, clone: XMG1.2) to identified T h 1 . Data were obtained using FACSCalibur ™ (BD Biosciences, San Jose, CA, USA). A total of 10,000 cell events were collected for each sample. The cell suspensions for each sample were collected immediately with low or medium flow rate. The single-cell populations were gated according to the staining used for further analysis. Data analysis was conducted using software BD CellQuest Pro ™ (BD Biosciences, San Jose, CA, USA).

Statistical analysis
Statistical analyses were performed using Microsoft Excel 2016. All data were expressed as mean±standard deviation. p<0.05 was determined using one-way ANOVA followed by Duncan's multiple range test.

Discussion
Marine food consumption, especially fish, is quite popular globally due to its nutritional content. Anisakis is one of the important biohazards in fishery products which may lead to a rejection by consumers and economic losses to the fish industry [17,21]. Nowadays, among nine Anisakis species, A. simplex (s.s) and Anisakis pegreffii are best known for causing infection in human. However, there is little information about A. typica causing infection. A. typica challenge in our research would greatly improve knowledge of anisakiasis besides A. simplex (s.s) and A. pegreffii epidemiology.
Our result suggests that the expression of CD11c + (DCs) was increased after A. typica treatment. DCs have a responsibility to present antigen then elicited immune response during parasite infection. Macrophage, other antigen-presenting cells have reported to secrete IL-6 through toll-like receptors activation and elicit Th 2 polarization after antigens, native carbohydrates derived from metacestode larvae parasites sensitization [22]. These results are in line with our study which suggests that A. typica challenge induces DCs maturation to secrete IL-6 as a pro-inflammatory cytokine. Another study reported that in vitro crude extract A. pegreffii elicits DCs to develop immune response by increase CCL3, CXCL4, , and granulocyte-macrophage colony-stimulating factor. DCs maturation in lymph node provokes pro-inflammatory IL-6 secretion and participates in Th 2 differentiation [23].
Tregs are known to work synchronize with Th 2 in the early phase of infection through TGF-β signaling. Furthermore, Tregs elicit Th 2 response counter worm infection [24] by attracting eosinophils, mast cells, basophils, and production of IgE [25]. Our study suggests that after 1 week challenged by A. typica, there is reduce of naive Tregs population. The previous study reported that A. simplex challenge represents the balanced between Th 1 /Th 2 responses [13]. IL-4 secretion triggers IgE production by B-lymphocyte, while IL-5 involved in eosinophilic production under anisakiasis [26,27]. Elevated Th 1 -mediated response during anisakiasis was associated with Th 17 activation [28] and severe GI symptoms, which displays the clinical manifestation in patients [29]. Furthermore, one of DCs subset (CD11c mid CD45RB high ) reported to activate CD4 + T cells to secrete the high levels of both IFN-γ and IL-4 in nematode-infected mice [30].

Conclusion
We have evaluated the immune profile after A. typica challenged, which have generated new possibilities to understand the role of A. typica after infected mice. In our present study, A. typica infection exhibits a mixed Th 1 /Th 2 pattern which more skewed to the pro-inflammatory state than the classical model of an allergic reaction to food antigens. Further studies are required to understanding the molecular mechanism of A. typica infection which may imply the human allergic reaction during parasite infection. Further experiments are needed to explain the detailed mechanism of Anisakis infection. More appropriate experiments such as different route of administration and sample preparation are expected to complete the detailed Anisakis infection mechanism.

Authors' Contributions
Concept: AA, ES, and AMH; Design: LH; Supervision: AA, ES, and AMH; Resources: LH; Materials: LH; Data collection and/or processing: LH; Analysis and/or interpretation: LH, AA, ES, and AMH; Literature search: LH; Writing manuscript: LH; Critical review: AA, ES, and AMH. All authors read and approved the final manuscript.