Expression of Toll-like Receptor (tlr) 2 and Tlr4 in the Livers of Mice Infected by Clonorchis Sinensis

Introduction: Clonorchis sinensis is one of the most important foodborne pathogens in humans, and can cause biliary diseases such as gallstones, cholecystitis, cholangitis, and cholangiocarcinoma. Toll-like receptors (TLRs) as sensors are crucial to initiating both innate and adaptive immune defenses against pathogens. However, little is known about the hepatic expression of TLRs of hosts induced by C. sinensis infection. Methodology: In the present study, the expression and distribution of TLR2 and TLR4 were investigated in a mouse model of clonorchiasis on days 28, 56, 84, and 112 post-infection (PI) using real-time quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemically staining, respectively. The levels of cytokines that are mediated by TLR2 and TLR4 were also evaluated using a cytometric bead array. Results: Results showed that the transcripts of TLR2 and TLR4 were upregulated on day 28 PI in C. sinensis-infected mice compared with non-infected ones (p < 0.01). In addition, their proteins were strongly immunohistochemically positive in the cytoplasm and membrane of endothelial cells, fibroblasts, and biliary epithelium cells of C. sinensis-infected mice. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were increased with activation of TLR2 and TLR4. Conclusions: The expression of TLR2 and TLR4 is upregulated against C. sinensis infection, which suggests that TLR2 and TLR4 might be involved in immune responses during C. sinensis infection.


Introduction
Clonorchiasis, caused by the liver fluke Clonorchis sinensis, is a foodborne parasitic zoonosis affecting approximately 15 million people in endemic Asia including China, Korea, Vietnam, and Japan [1].It has been claimed that about 85% of these people are in China [1][2][3].Such disease is characterized by cholangitis with remarkable recruiting inflammatory cells in the early stage of infection, followed by hyperplasia of the intrahepatic bile ducts, cholangiofibrosis, and cholelithiasis in the host liver during the chronic stage [4].Moreover, as a group I carcinogen, C. sinensis can induce human cholangiocarcinoma in chronically infected people [5][6][7].
The family of Toll-like receptors (TLRs) is one of the most important pattern recognition receptors (PRRs) that plays a key role in recognition of pathogen-associated molecular patterns (PAMPs) derived from a variety of pathogens, as well as in mediating the innate immune response and adaptive immune response of the host [8][9][10].TLRs are members of type I transmembrane proteins divided into three domains: ectodomains containing 18-31 leucine-rich repeats recognizing PAMPs derived from a wide range of viral, bacterial, and parasite ligands; transmembrane domains; and intracellular Tollinterleukin 1 receptor (TIR) domains that induce downstream signal transduction.Once PAMPs are recognized by leucine-rich domains, the signal cascades are initiated, and remarkable proinflammatory cytokines and chemokines, as well as costimulatory molecules, are ultimately induced through the activation of a variety of transcriptional complexes including NF-κB and AP-1 [11][12][13][14][15]. Additionally, host susceptibility to infectious diseases may be associated with the TLRs' polymorphisms [16][17][18].
Like other helminthiases, clonorchiasis is characterized by the predominant Th2-associated cytokine production with a suppression of Th1associated cytokines during chronic infection.However, the factors and molecular mechanisms underlying polarization of the immune response are largely unknown [19][20][21].Recent studies have shown that the expression of TLR2 and TLR4 is upregulated in other helminth infections, such as Fasciola spp., Schistosoma spp., Taenia spp., and Brugiamalayi, suggesting that TLRs might play an important role during helminth infections [22][23][24][25][26][27].For example, Gao et al. demonstrated that TLR2 but not TLR4 is critical to mediating interleukin (IL)-10 secretion from dendritic cells in response to Schistosome japomicum antigens [28].The expression of TLR2 and TLR4 can be upregulated by excretory/secretory products derived from another liver fluke, Opisthorchis viverrini, suggesting that TLR2 and TLR4 might participate in immune response against O. viverrini [29,30].However, the expression and distribution of TLR2 and TLR4, which are probably engaged in immune responses against C. sinensis infection, are not clear.In view of this background, the objective of present study was to identify such TLR expression and distribution in the livers of mice following infection with C. sinensis.

Ethics statement
This study strictly followed the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China.All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China.The study was approved by the animal ethics committee of Xuzhou Medical College (No. SCXK<SU>2010-0003).The animals used in this study were euthanized by overdose of MS-222 and sodium pentobarbital anesthesia, and all efforts made were to minimize the suffering of the animals.

Preparation of C. sinensis metacercariae
Metacercariae of C. sinensis used for infection were obtained from naturally infected fresh fish, Pseudorasbora parva.The fish in this experiment, which were not an endangered or protected species, were collected under the permission of the government of Guangxi Autonomous Region, China, and there was no specific permission required during this collection.The fish containing C. sinensis metacercariae were immediately homogenated and digested through artificial gastric juice (0.7% pepsin in 1% HCl solution, PH 2.0) as described by Chen [31].C. sinensis metacercariae were morphologically identified under a dissecting microscope by experienced staff before intragastric administration.

Mice, experimental infection, and sampling
C3H/HeN mice between six and eight weeks of age at the commencement of the experiment were purchased from the Animal Center of Xuzhou Medical College, China, and were provided food and water ad libitum.One hundred and ten mice were inoculated with 35 metacercariae of C. sinensis suspended in approximate 0.3 mL phosphate-buffered saline (PBS) by gavage intragastric intubation.Tissue samples in the portal area of liver removed from six to eight mice at each time point were immediately sampled, fixed in PBS with 10% formaldehyde for 24 hours, and paraffin embedded for hematoxylin and eosin (H&E) staining and specific immunohistochemistry.The segments of left lobe of the liver were also immediately placed in a 2 mL microcentrifuge tube containing 1 mL RNAstore (Tiangen Inc., Beijing, China) and stored at -80°C until RNA extraction.The same protocol was applied in six non-infected control mice, which were used for the basal TLR2 and TLR4 levels.

RNA extraction
About 50 mg of mice specimens were removed from RNAstore reagent (Tiangen Inc., Beijing, China), and total RNA was extracted using commercially TRiZoL reagent (Invitrogen, San Diego, USA) according to the manufacturer's recommendations.Total RNA was dissolved with 50 μL of RNase-free water and stored at -80°C.The RNA integrity was examined by electrophoresis on 2% fresh agarose gel.Total RNA concentration of each sample was adjusted to 1 μg/μL.
PCR reactions (20 μL) of the gene TLR2, TLR4, and β-actin were performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol, including 0.40 μM of each primer and 1 μL of cDNA under the following conditions: after an initial denaturation at 94°C for 5 minutes, 40 cycles of 94°C for 30 seconds (denaturation), 60°C for 30 seconds (annealing), and 72°C for 30 seconds (extension).Before analysis of the relative expression level of each gene, the threshold cycle at which the fluorescent signal reached an arbitrarily set threshold near the middle of the loglinear phase of the amplification for each reaction was calculated, and the relative quantity of mRNA was determined.Quantification of target gene expression was evaluated in the terms of the comparative cycling threshold (C t ) normalized by β-actin with the 2 -△△ Ct method.The primers used in this study are given in Table 1.

Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues from each mouse were prepared for immunohistochemical staining following the conventional procedure.Briefly, serial 4-μm thick sections were incubated with 3% H 2 O 2 in PBS for blocking endogenous peroxidase.Nonspecific antigenic sites in sections were subsequently hampered by 4% bovine serum albumin in PBS-T for 20 minutes.Immunostaining for TLR2 and TLR4 protein was performed using specific TLR2 and TLR4 primary antibodies (Abcam, Cambridge, USA, cat.no.ab47093 and ab24192) for 4°C overnight at concentrations of 2 μg/mL and 10 μg/mL, respectively.Immunostaining was then detected with the Polink-2 Plus Polymer HRP Detection System with 3,3-diaminobenzidine (Gold Bridge International, Inc., Mukilteo, USA) according to the procedural instructions.Sections were then washed in distilled H 2 O and counterstained with hematoxylin.Negative control sections were processed with IgG isotype instead of primary antibodies for evaluating their specificity.Immunohistochemistry-stained sections were checked by an inverted microscope (Olympus, Tokyo, Japan), and positive staining was identified when a brown pigmentation was microscopically observed and the images were analyzed using Image-Pro Plus software.

Cytometric bead array
The concentrations of IL-4, IL-10, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) cytokines in the sera from C. sinensis-infected mice and normal control mice were determined using a cytometric bead array (CBA) kit (BD Biosciences, Franklin Lakes, NJ, USA).In brief, 50 μL capture beads were mixed with 50 μL of mouse Th1/Th2 PE detection reagent II and 50 μL of sera from C. sinensis-infected mice.Simultaneously, a 10-point calibration curve was obtained using cytokine standards and beads for each cytokine.The samples were incubated for three hours at room temperature and kept away from light.They were then washed and centrifuged at 200 g for five minutes and analyzed in a flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA).Positive and negative serum controls, a standard reference curve (mouse inflammation standard or mouse Th1/Th2 cytokine standards) provided in the cytometric bead array kit was used to interpolate picograms per microliter levels of each cytokine from the sera.

Histological observation
All mice were assayed for histological observation

Statistical analysis
Data obtained from 6~8 mice were expressed as mean ± standard error of the mean (SEM); differences between infection groups and non-infected control group were analyzed by one-way analysis of variance (ANOVA).SPSS version 16.0 and GraphPad Prism software performed the statistical analyses.A statistically significant difference was observed when p < 0.05.

Expression and distribution of TLR2 and TLR4 in livers of normal and C. sinsensis-infected mice
In present study, mRNA transcripts of TLR2 and TLR4 at different time points during C. sinensis infection were investigated by relative quantitative PCR. Figure 1 shows the relative expression of TLR2 and TLR4 at different time points during C. sinensis infection.Upon infection, the expression of both TLR2 and TLR4 mRNA in C. sinensis-infected mice were significantly upregulated on day 28 postinfection (PI) compared with those of non-infected mice (p < 0.01, Figure 1).However, both the level of TLR2 and TLR4 gene expression subsequently returned to almost normal baseline levels until the experiment concluded (Figure 1).
To observe the distribution of TLR2 and TLR4 protein in the livers of C. sinensis-infected mice, immunohistochemical staining was employed to exhibit the localization of TLR2 (Figure 2) and TLR4 (Figure 3) protein specifically in the livers of C. sinensis-infected mice and non-infected ones.In noninfected control mice, TLR2 and TLR4 proteins were slightly stained in the hepatocytes (indicated as up arrow in Figure 2A and Figure 3A).In contrast, both TLR2 and TLR4 were strongly stain ed in cytoplasm and membrane of fibroblasts (indicated as △) and on the lateral and luminal surfaces of biliary epithelium cells (indicated as →) in C. sinensisinfected mice.Among these inflammatory cells, Kupffer cells appeared to express TLR2 and TLR4 proteins in responses to C. sinensis infection during the whole infection, but TLR2 and TLR4 in other cells, such as eosinophils and lymphocytes, were expressed weakly or negatively in the infection.As expected, most TLR2 and TLR4 proteins were located in the cytoplasm and membrane of the cells, suggesting that they might be processed en route to the apical cell membrane.

The dynamics of cytokines in sera from C. sinensisinfected C3N/HEN mice
To underline the significance of activation of TLR2 and TLR4 in the host immune system, cytokines in the sera of C. sinensis-infected mice such as IL-4, IL-10, IFN-γ, and TNF-α, which are closely relative with TLR2 and TLR4, were further detected.As shown in Figure 4, the levels of these cytokines were all increased as the TLR2 and TLR4 were activated on days 28 PI and 56 PI, and the levels of IL-4 (Figure 4D) and IFN-γ (Figure 4B) were still higher than in control mice on day 84 PI; however, IL-10 (Figure 4A), IFN-γ (Figure 4D), and TNF-α (Figure 4C) appeared to be normal levels on day 112 PI.

Histological findings
To further explore the potential roles of TLR2 and TLR4 in the disease outcomes of clonorchiasis, histological changes in the livers of C. sinensisinfected mice were examined.As shown in Figure 5 and Figure 6, extensive inflammatory cell infiltrations in portal areas of livers were observed accompanied by part of the liver cell degeneration, and bile duct obstruction of the infected liver appeared on day 28 PI (Figure 5).However, inflammatory cell infiltration in the livers of infected animals appeared to be reduced, and collagen fibers gradually increased with the infection prolonged from day 56 PI; these pathological changes did not disappear until the experiment concluded (Figure 5 and Figure 6).

Discussion
Among a number of animal models, hamsters are considered good candidates for the study of the relationship between host immune response and C. sinensis infection [32,33].However, this animal model is restricted for use because commercial antibodies and other reagents against hamsters are not available.In addition, rabbits and rats are not appropriate hosts for this purpose because they have no gallbladders.Therefore, mice are substitutable for hamsters to study host-parasite interaction of C. sinensis since they have gallbladders, and their antibodies or other reagents are commercially available [19][20][21].Importantly, C3H/HeN together with C3H/HeJ mice (a deficient function of TLR4) will provide contrastive data for further exploring the role of TLR4 in C. sinensis infection.Thus, C3H/HeN mice were chosen for investigating the expression and distribution of TLR2 and TLR4 in the liver of C3H/HeN mice following C. sinensis infection.

Specific
bacterial PAMPs such as lipopolysaccharides could stimulate innate immune responses in the hemocoel of Biomphalaria glabrata, which subsequently promotes cell proliferation in the amebocyte-producing organ (APO) and may contribute to the resistance to infection with larval trematodes [34].Furthermore, previous studies also have shown that up-regulation of TLR4, TLR2, or both are critical to recognition of PAMPs derived from helminths and for induction of innate immune response, ultimately leading to a protective Th2promoting phenotype through several signal pathways [35].However, the expression and distribution of TLR2 and TLR4 in response to the infection of C. sinensis in vivo have not yet been reported so far.The results of present study showed that the transcripts of both TLR2 and TLR4 gene were increased on day 28 PI, which is interestingly consistent with the increased in cytokines including Th1 and Th2 types immune responses, suggesting that TLR2 and TLR4 might be engaged in the production of cytokines such as IL-4, IL-10, IFN-γ, and TNF-α in mice with C. sinensis infection [36].In addition, TLR2-mediated immune response via inflammatory/anti-inflammatory cytokines may be involved in the imbalance of oxidants/antioxidants in patients re-infected by O. viverrini [37].
Several studies demonstrated that TLRs could be expressed by almost cell types in murine livers, even under normal physiological conditions, but different expression levels of TLRs can be regulated depending on the cell type in response to various exposures to PAMPs.The distribution of TLR2 and TLR4 in the liver of mice infected by C. sinensis is not clear.In our study, we found that both TLR2 and TLR4 proteins were mainly located in the cytoplasm and membrane of endothelial cells, fibroblast, and biliary epithelium cells (BEC) in C. sinensis-infected mice, although eosinophils, lymphocytes, and hepatic cells were slightly stained during the infection of C. sinensis compared to normal-control mice.Notably, the great density of TLR2 and TLR4 proteins were constantly stained in the fibroblast of fibrotic tissues, suggesting that the TLR signaling pathway might provide a new mechanism to account for persistent fibrogenesis [14,38].

Conclusions
The present study is the first report on TLR expression in a mouse model of clonorchiasis; it showed that expression of TLR2 and TLR4 were upregulated during the infection of C. sinensis, indicating that both TLR2 and TLR4 probably participate in the stimulation of the innate immune response during C. sinensis infection.The roles that TLR2 and TLR4 play in C. sisensis infection require further investigation.

Figure 1 .
Figure 1. mRNA expression of TLR2 and TLR4 in livers of C3H/HeN mice followed by C. sinensis infection.

Figure 2 .
Figure 2. Immunohistochemical staining of TLR2 in livers of normal and C. sinensis infected mice.

Figure 3 .
Figure 3. Distribution of TLR4 in livers of normal and C. sinensis infected mice.

Figure 5 .
Figure 5. Representative pathologic changes in the livers of C. sinensis-infected C3H/HeN mice at different points in time.

Figure 6 .
Figure 6.Masson's trichrome-stained sections for detection of collagen during the time course for the progression of liver damage caused by C. sinensis.