Parasitol Res (2015) 114:2371–2379 DOI 10.1007/s00436-015-4435-x

ORIGINAL PAPER

CsRNASET2 is an important component of Clonorchis sinensis responsible for eliciting Th2 immune response Yanquan Xu 1,2 & Jinsi Lin 1,2,3 & Meng Bian 1,2 & Wenjun Chen 1,2 & Pei Liang 1,2 & Xiaoyun Wang 1,2 & Mei Shang 1,2 & Hongling Qu 1,2 & Zhongdao Wu 1,2 & Yan Huang 1,2 & Xinbing Yu, PhD 1,2

Received: 26 February 2015 / Accepted: 11 March 2015 / Published online: 2 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Many parasites can trigger the host immune response by releasing excretory/secretory proteins (ESPs). CsRNASET2, a glycosylated T2 ribonuclease present in ESPs of Clonorchis sinensis (C. sinensis, Cs), has recently been reported to possess potent effects in regulating mouse dendritic cells (DCs). However, it is unclear whether CsRNASET2 can induce adaptive immune response. In this study, we carried out further investigations on biochemical features of CsRNASET2. Besides, we immunized Balb/c mice with CsRNASET2 and orally infected Balb/c mice with C. sinensis, respectively. Sera of immunized mice were collected and evaluated for specific antibody titers by ELISA. Splenocytes of experimental mice were isolated and stimulated in vitro. The expression levels of IL-4 and IFN-γ in splenocytes of immunized mice and infected mice were detected by ELISA and flow cytometry. Our results showed that the sequence of CsRNASET2 had close relationship with the homologue from Echinococcus multilocularis. The conserved active site (CAS) motifs, active histidine residues, and N-

Yanquan Xu and Jinsi Lin contributed equally to this work. * Yan Huang [email protected] * Xinbing Yu, PhD [email protected] 1

Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China

2

Key Laboratory for Tropical Diseases Control, Ministry of Education, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China

3

Zhongshan Center for Disease Control and Prevention, Zhongshan 528405, People’s Republic of China

linked glycosylation region of CsRNASET2 were close to each other in the three-dimensional structure. In addition, sera of CsRNASET2 immunized mice had obviously higher levels of specific antibody titers. Splenocytes from both CsRNASET2 immunized mice and C. sinensis infected mice expressed increased levels of IL-4, while the production of IFN-γ exhibited no significant difference. Immunization with CsRNASET2 elicited Th2 immune response by promoting the synthesis of IL-4, consistent with the immune response initiated by infection of C. sinensis. Taken together, these data suggested that CsRNASET2 was important for C. sinensis to trigger Th2 immune response.

Keywords Clonorchis sinensis . CsRNASET2 . Immune response . Cytokine

Introduction Clonorchiasis, induced by infection of Clonorchis sinensis (C. sinensis, Cs), is one of the most common food-borne parasitic diseases (Keiser and Utzinger 2009; Rim 2005; WHO 1995). Clonorchiasis is predominantly epidemic in several Asian countries, such as China, Korea, and Vietnam (Huang et al. 2014). Approximately, there are 35 million people currently infected with C. sinensis worldwide (Bian et al. 2014; Chen et al. 2010; Lun et al. 2005). C. sinensis infection occurs when humans take raw or inappropriately cooked freshwater fish containing metacercariae (Chen et al. 2012). C. sinensis adult worms can inhabit for more than 10 years in bile duct system of the host. During the long chronic parasitism, C. sinensis adult worms continuously release excretory/ secretory proteins (ESPs), which are the fist helminthderived molecules to interact with the host. Most proteins present in ESPs of helminth are reported to harbor diverse

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biological activities (Cass et al. 2007; Everts et al. 2009). In addition, previous studies have revealed that some components of ESPs from C. sinensis (CsESPs) are strongly immunogenic and can initiate immune response of the host (Lei et al. 2013). CD4 T cells play a crucial role in immune protection. After encountering antigens, CD4 T cells undergo differentiation into different effector T helper (Th) cells. There are mainly two subsets of Th cells (Th1 and Th2) that play a series of distinct roles in host defense and autoimmunity (Fernandez-Botran et al. 1988; Mosmann et al. 1986; Mosmann and Coffman 1989; Weaver et al. 1988). To identify the molecule, driving differentiation of naïve CD4 T cells, is important for controlling specific Th responses by induction or ablation of the relative signaling pathway. Ribonuclease T2 is a family of enzymes that distribute broadly in the organism kingdom (Deshpande and Shankar 2002; Luhtala and Parker 2010). Ribonuclease T2 family members have been revealed to be implicated in diverse biological activities, including clearance of nucleic acids, degradation of RNA, serving as cytotoxins, and modulation of host immune responses. In our previous study, ribonuclease (RNase) T2 enzyme of C. sinensis (CsRNASET2) has been cloned and expressed in Pichia pastoris (Xu et al. 2013). In addition, CsRNASET2 has been identified to be a glycoprotein with RNase activity and be a component of CsESPs. Under in vitro conditions, CsRNASET2 can markedly inhibit the expression levels of IL-12p70 in mice dendritic cells (DCs), while it enhances the production levels of IL-10 in mice DCs. Moreover, CsRNASET2 can suppress maturation of mice DCs by decreasing the synthesis of CD11C, CD40, CD80, and CD86. In vivo, CsRNASET2 dramatically promotes the expression levels of IgG1 in sera of immunized mice. These results suggest CsRNASET2 to be an important immuneregulatory molecule in CsESPs. IL-12 plays an important role in the differentiation of Th1 cells (Hsieh et al. 1993; McKnight et al. 1994), while IL-10 is belonging to the Th2 subset, which has been revealed to suppress the synthesis of the Th1 cytokine IFN-γ (Ouyang et al. 2011). In addition, the elevated production of IgG1 in sera is generally correlated with Th2 polarization (Maassen et al. 2003; Mountford et al. 1994). Our previous work suggested that CsRNASET2 might be crucial for the initiation of Th2 immune response in the host. In the present report, we carried out further sequence analysis and structural modeling of CsRNASET2. In addition, we firstly examined the ability of CsRNASET2 to regulate the synthesis of IL-4 and IFN-γ in immunized mice. Moreover, we assessed expression levels of IL-4 and IFN-γ in splenocytes of C. sinensis infected mice. Our study may provide a better understanding about how C. sinensis associated immune responses were initiated.

Parasitol Res (2015) 114:2371–2379

Materials and methods Animals and parasites Female BALB/c mice (6–8 weeks old) were purchased from the animal center of Sun Yat-Sen University (Guangzhou, China) and housed in a pathogen-free condition. All animal experiments were carried out according to Animal Care and Use Committee of Sun Yat-sen University (Permit No: SCXK (Guangdong) 2009–0011). Living metacercariae of C. sinensis were derived from infected freshwater fish maintained in our laboratory ecologic pool following our established procedures (Wang et al. 2009). Subsequently, each mouse was orally infected with 30 metacercariae. Sequence analysis of CsRNASET2 Multiple sequences of RNASE T2 family from different species were downloaded from GenBank in NCBI [http://www. ncbi.nlm.nih.gov/]. The phylogenetic tree was constructed by using MEGA6 software with the neighbor-joining method. Bootstrap proportions were used to evaluate the robustness of the tree with 1000 bootstrap replications. In addition, three-dimensional structure of CsRNASET2 sequence was predicted by SWISS-MODEL program and analyzed by SPDBV 4.10 software. CsRNASET2 treatment Recombinant CsRNASET2 was produced in Pichia pastoris and purified as previously described (Xu et al. 2013). Subsequently, each mouse was subcutaneously (s.c.) injected with 100 μg CsRNASET2 emulsified in complete Freund’s adjuvant (Sigma-Aldrich, USA) at the first injection, while control mice were similarly administered with an equal volume of PBS (six mice/group). Two booster injections at 2 weeks of intervals were performed with 50 μg of CsRNASET2 or equal volume of PBS emulsified in incomplete Freund’s adjuvant (Sigma-Aldrich, USA). Anti-sera were harvested and mice were sacrificed for isolation of splenocytes at week 4 and week 6, respectively. Splenocytes preparation Experimental mice were sacrificed 4 and 6 weeks after the first immunization or infection, respectively. Spleens were removed from individual mouse, and single-cell suspensions were prepared by using cell grinders and filtering. Red blood cells were lysed by using red blood cell lysing buffer (SigmaAldrich, USA). The remaining splenocytes were then washed twice with Hanks’ balanced salt solution (Gibco, USA) and resuspended into 2×106/ml with complete RPMI1640 (Gibco, USA) supplemented with 10 % FCS. Splenocytes were

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stimulated with or without phorbol 12-myristate 13-acetate (PMA, 20 ng/ml; Sigma-Aldrich, USA) plus ionomycin (1 μg/ml; Sigma-Aldrich, USA) in triplicate and incubated at 37 °C in a humidified atmosphere including 5 % CO2. ELISA Cell-free culture supernatants were collected 48 h later for cytokines evaluation. Expression levels of IL-4 and IFN-γ of stimulated splenocytes in the culture supernatants were measured by commercially available ELISA kits (eBioscience, USA) according to the manufacturer’s protocol. The detection limit of the IL-4 and IFN-γ ELISA kits were 7.8 and 31.25 pg/ml, respectively. In addition, levels of specific antibody titers of total IgG in immunized mice sera were also measured by ELISA. Briefly, ELISA plates were coated with 100 μl CsRNASET2 (5 μg/ml) each well and placed at 4 °C overnight. Next, the plates were blocked with 5 % skimmed milk at 37 °C for 2 h and incubated with different dilutions of immunized mice sera at 37 °C for 2 h. Subsequently, the plates were incubated with HRP-conjugated goat anti-mouse IgG (Invitrogen, USA) at 37 °C for 1 h. Finally, the plates were reacted with 100 μl TMB substrate solution (BD Biosciences, USA), and 2 M H2SO4 was added to stop the reaction 10 min later. The optical density was tested at 450 nm. Intracellular cytokine staining and flow cytometric analysis For evaluating levels of intracellular cytokines, splenocytes of immunized mice were stimulated with PMA plus ionomycin for 6 h in the presence of brefeldin A (10 μg/ml; SigmaAldrich, USA). Splenocytes were thereafter washed with PBS supplemented with 0.1 % BSA and 0.05 % NaN3, and fixed with 4 % paraformaldehyde. Subsequently, cells were permeabilized in PBS containing 0.1 % BSA, 0.05 % NaN3, and 0.1 % saponin at 4 °C overnight. Next, cells were stained with mAbs (eBioscience, USA) for surface marker (CD4) and intracellular cytokines (IL-4 and IFN-γ) at 4 °C in the dark for 30 min. Followed by staining, cells were washed twice and resuspended into PBS. Flow cytometry was performed by using a Beckman Coulter Gallios cytometer (Beckman Coulter, USA) and analyzed with FlowJo software (TreeStar, USA). Statistical analysis Data were reported as means±SDs, and statistical significance of differences between two groups was analyzed by using the Mann–Whitney test in GraphPad Prism 5 software. p values