Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

doi:10.1016/j.parint.2011.07.002

Parasitology International

Volume 61, Issue 1, March 2012, Pages 118-123

https://doi.org/10.1016/j.parint.2011.07.002 Get rights and content

Abstract

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host–parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.

Graphical abstract

Highlights

► We evaluate six mice strain for Clonorchis sinensis susceptibility. ► We examine histopathological changes in liver, worm recovery and immune response. ► Mice are not susceptible to C. sinensis infection. ► C57BL/6, BALB/c and ICR strain mice are relatively favorable among the strains. ► Elevated IgE, IFN-γ, and IL-13 may be related with the host susceptibility.

Introduction

Clonorchis sinensis, is a carcinogenic liver fluke infecting about 20 million people in endemic East Asia. C. sinensis infection in humans leads to cholangitis, bile duct obstructions, cholangiofibrosis, and cholangiocarcinoma [1], [2], [3], [4], [5]. Infections usually result from the consumption of raw cyprinoid fish containing C. sinensis metacercariae. Clonorchiasis is a major public health issue in Korea and is comparable to the problem of Opisthorchis viverrini infection in Thailand [6], [7].

A number of animal models have been used to investigate the host–parasite interaction of C. sinensis. Among the in vivo models, hamsters serve as good candidates regarding the development of cholangiocarcinoma [8], [9]. However, studies of the immune response and pathogenesis in hamsters have been limited because of the commercial unavailability of antibodies and other reagents against hamsters. The absence of gall-bladders in rats made them a non preferable host in previous studies [10]; however, mice may serve both research purposes in having a gallbladder as well as commercially available antibodies and other reagents. Therefore, it is essential to develop a suitable and susceptible mouse model for C. sinensis in order to understand the mechanisms of carcinogenesis and the immune response.

Yoon et al. [11] compared two immunologically competent (BALB/c and FVB/NJ) and two immunodeficient (Nude and SCID) mice for the study of C. sinensis pathogenesis and described FVB/NJ as a suitable model for the host parasite relationship study. However, they suspected that the mouse haplotype could have some relation in regards to susceptibility. The relatively susceptible FVB/NJ mice have the H2q haplotype while BALB/c mice have the H2d haplotype that is resistant to C. sinensis infections.

We studied herein host susceptibility of mice through the evaluation of six mice strains of different haplotypes (H2q, H2d and H2k) for the infectivity of C. sinensis, worm recovery, pathological and immunological changes. Among the strains, ICR and DDY have at least two antigens controlled by H2q genes that were previously used by Kim et al. [12] to examine the egg production of C. sinensis. BALB/c and C57BL/6 mice have H2d haplotype. In addition, two strains of mice (CBA/N and C3H/HeN) with the haplotype H2k were also included in this study.

Section snippets

Preparation of C. sinensis metacercariae

Metacercariae of C. sinensis were collected from naturally infected fish Pseudorasbora parva caught in Shenyang, Liaoning Province, China. The metacercariae were isolated following the procedure described by Li et al. [13]. After collection, they were preserved in 1× PBS with antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml) and stored at 4 °C until use.

Animal and experimental design

Six strains (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) of five-week-old male mice were infected with 30 metacercariae of C.

Gross observations

In the gross view, mice infected with C. sinensis showed the dilated extrahepatic bile duct compared with the uninfected control (Fig. 1). Severe dilation of the duct was observed in CBA/N and C3H/HeN mice reaching about 1 mm whereas it was similar to the uninfected controls in C57BL/6 and DDY strains.

Worm recovery

The higher numbers of worms were recovered from C3H/HeN (20.7%) and BALB/c (16.0%) mice than from other strains, whereas ICR showed no worm recovery at the 4th week of infection. At the 8th week,

Discussion

The gross view of the liver indicated evidence of C. sinensis infection in most of the strains. Dilated bile ducts were marked in most of the strains, except the DDY strain. There were no cysts or tumor-like masses in all strains of mice, whereas cystic dilatation of the intrahepatic bile duct was noticed in FVB mice [10], [11]. Instead, the liver had small white spots, especially in the median lobe in most of the strains. Those spots were supposedly made by inflammation and edema around the

Acknowledgments

The present study was supported by a research grant from Seoul National University Hospital (21-2005-0330).

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Bile duct epithelial hyperplasia was observed at 2 dpi, and fibrotic hyperplasia began to emerge at 12 dpi. Therefore, the white spots in the gross liver observation are likely related to the pathologic changes of fibrotic hyperplasia, worms mechanical obstruction, and severe inflammation caused by juvenile C. sinensis infection (Uddin et al., 2012). These pathologic changes persistently exist in chronic infection and lead to cholangitis, cholelithiasis, cholecystitis and cholangiocarcinoma in the host.
Clonorchiasis is a zoonotic disease that can result in chronic infection in humans. The causative agent, Clonorchis sinensis (C. sinensis), is believed to primarily induce a Th2 immune response in infected mice. However, few studies have profiled host immune responses to C. sinensis infection during the juvenile phase. In the present study, the dynamics of select cellular responses and cytokine expression profiles during juvenile C. sinensis infection were investigated. The flow cytometry results showed that the CD4+ T cells percentage was significantly decreased between 12 days post-infection (dpi) and 24 dpi in the peripheral blood, and the CD8+ T cells percentage was significantly elevated after 3 dpi. The ratio of CD4+/CD8+ T cells was also significantly decreased after 3 dpi. Furthermore, we observed that the proportion of CD14+ monocyte-macrophages in the peripheral blood was significantly increased between 1 dpi and 12 dpi and peaked at 6 dpi. The percentage of classically activated macrophages (M1) and alternatively activated macrophages (M2) in the liver was significantly increased between 18 dpi and 30 dpi. qRT-PCR results showed that the expression levels of iNOS in the liver were significantly elevated after 3 dpi, and Arg-1 expression was significantly increased beginning at 12 dpi. ELISA results showed that the serum levels of the Th1 cytokines IFN-γ and IL-2 peaked at 6 dpi and decreased thereafter. Furthermore, the Th2 cytokines IL-4 and IL-13 began to be expressed and peaked at 24 dpi and 30 dpi, respectively. In addition, the levels of the Treg cytokines IL-10 and TGF-β₁ were significantly increased beginning at 6 dpi until 30 dpi. In the liver homogenate, the expression of IFN-γ, IL-2, and IL-4 mainly occurred before 6 dpi. IL-13 expression was significantly increased at 30 dpi. IL-10 and TGF-β₁ levels were significantly increased at 12 dpi and 24 dpi, and expression peaked at 24 dpi and 30 dpi, respectively. This study provides a fundamental characterization for the future analysis of host-parasite interactions and immune responses in hosts infected with juvenile C. sinensis.
Membrane-bound IgE on B cells is increased during Clonorchis sinensis infection
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Unlike mIgE, other BCR (mIgM or mIgD) that were expressed by most CD19+ cells did not show such a dramatic decrease, though they may have shown a slight change after 8 h of cultivation (Fig. 5C). Elevated production of IgE is a characteristic immune response during infection with helminths such as C. sinensis (Uddin et al., 2012). The traditional view is that IgE production is a consequence of the broader Th2 response, which is one of the key factors of parasite elimination (Oettgen, 2016).
A high level of serum IgE is a hallmark of helminthic disease. Secretory IgE can bind FcεRI or FcεRII/CD23. The combination of IgE and FcεRI, a high-affinity interaction, has long received attention and is believed to facilitate helminth control, while the properties of CD23-bound IgE have long been unexplored. Here, we established a Clonorchis sinensis (C. sinensis) infection model with different mouse strains and investigated membrane-bound IgE on B cells during infection. We show that after infection, the increase in CD23 expression on B cells was obvious, even in relatively resistant C57BL/6 mice, as well as in susceptible BALB/c and FVB mice. Although the serum IgE amount was lower in C57BL/6 mice than in BALB/c and FVB mice, the level of IgE binding to peripheral B cells was also elevated. Additionally, the IgE on B cells was soon undetectable in vitro due to dissociable binding. The results of the present study demonstrate the dramatic increase in CD23-bound IgE on B cells after C. sinensis infection. The significance of CD23-bound IgE in Ag transport and presentation has gained consideration in allergy development for its potential ability to promote the Th2 response. Therefore, even though the association of IgE and CD23 is not as substantial as that of IgE and FcεRI, membrane-bound IgE on B cells may be worth further study regarding clonorchiasis and other parasitic infections.
Clonorchis sinensis and Clonorchiasis: The Relevance of Exploring Genetic Variation
2018, Advances in Parasitology
Parasitic trematodes (flukes) cause substantial mortality and morbidity in humans. The Chinese liver fluke, Clonorchis sinensis, is one of the most destructive parasitic worms in humans in China, Vietnam, Korea and the Russian Far East. Although C. sinensis infection can be controlled relatively well using anthelmintics, the worm is carcinogenic, inducing cholangiocarcinoma and causing major suffering in ~ 15 million people in Asia. This chapter provides an account of C. sinensis and clonorchiasis research—covering aspects of biology, epidemiology, pathogenesis and immunity, diagnosis, treatment and control, genetics and genomics. It also describes progress in the area of molecular biology (genetics, genomics, transcriptomics and proteomics) and highlights challenges associated with comparative genomics and population genetics. It then reviews recent advances in the sequencing and characterisation of the mitochondrial and nuclear genomes for a Korean isolate of C. sinensis and summarises salient comparative genomic work and the implications thereof. The chapter concludes by considering how advances in genomic and informatics will enable research on the genetics of C. sinensis and related parasites, as well as the discovery of new fluke-specific intervention targets.
Local immune response to primary infection and re-infection by Clonorchis sinensis in FVB mice
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Because the resistance disappeared in immune-suppressed and nude rats, an increase in serum and bile levels of C. sinensis-specific antibodies was suggested to be correlated with the resistance [5]. Since most mouse strains are not susceptible to C. sinensis [6], FVB mice have commonly been used as a model [7,8]. In FVB mice, C. sinensis infection elevated levels of Th2 cytokines (IL-4, IL-5), anti-inflammatory cytokines (IL-10, TGF-β), as well as Th1 cytokines (IFN-γ) [8].
Although Clonorchis sinensis lives in the bile duct, few studies have investigated the local immune response in the liver and bile duct. To investigate the local immune response to C. sinensis, we investigated the activation and recruitment of various immune cells and cytokine levels in the liver and bile duct lymph nodes (BLN) in FVB mice after primary infection and re-infection. Male 4-week-old FVB mice were divided into 6 experimental groups: uninfected controls, primary infection lasting 1 week (PI 1w), primary infection lasting 4 weeks (PI 4w), praziquantel treatment after PI 4w (Tx), re-infection lasting 1 week after Tx (RI 1w), and re-infection lasting 4 weeks after Tx (RI 4w). Recovery rates were 80.0% and 73.0% in PI 1w and PI 4w mice, respectively, but significantly decreased during re-infection to 26.6% in RI 1w and 13.3% in RI 4w. This result suggested that the mice were resistant to re-infection. In the liver, Kupffer cells were augmented 70-fold in PI 1w mice (P < 0.001). Kupffer cells expressed Th2-related cytokines (IL-10 and IL-13) during primary infection. In addition, serum levels of C. sinensis-specific IgG1 and IgG2a strongly increased in RI 1w mice. Secretion of C. sinensis-specific IgE reached a plateau at 4 weeks after primary infection, and remained elevated in all infected groups. In conclusion, during infection with C. sinensis, Kupffer cells likely act as antigen-presenting cells, stimulating the Th2 cytokine production system.
Ultrasonic diagnosis of patients with clonorchiasis and preliminary study of pathogenic mechanism
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Adult liver fluke damages the liver tissue and leads to the change of the patient's cellular immunity and humoral immunity function [3]. It has reported that the clonorchiasis patient may produce specific immune response [4] which is often accompanied by complications like jaundice, cholangitis, cholelithiasis, liver cirrhosis, biliary duct cancer, liver cell cancer and other liver and gall diseases [5–8]. This seriously threatens the life of patients.
To discuss the liver function damage mechanism of patients with clonorchiasis by analyzing the ultrasound characteristics, liver function, change of the serum inflammatory factors and cell apoptosis factors.
Color Doppler ultrasound technique was adopted to detect the portal vein and blood flow change of patients with clonorchiasis; ELISA was used to determine the level of different serum inflammatory factors. The levels of serum total bilirubin, serum albumin and glutamic-pyruvic transaminase were detected by automatic biochemical analyzer. Western blot was used to determine the expression of proteins relevant to apoptosis.
Compared with the health control group, the trunk diameter of portal vein and the thickness of spleen, as well as the hepatic artery pulsation index of clonorchiasis patients increased obviously, the mean blood flow velocity of portal vein (P < 0.05 or P < 0.01) decreased. The content of total bilirubin and transaminase in plasma increased significantly, but albumin decreased (P < 0.05). Levels of TNF-α, IL-6 and IFN-γ increased remarkably, and the level of every factor was significantly different among patients with Child-Pugh , Child-Pugh II and Child-Pugh III classification of liver function (P < 0.05 or P < 0.01). With the exacerbation of liver dysfunction, levels of TNF-α, IL-6 and IFN-γ gradually increased (P < 0.05). Compared with the healthy control group, the expression quantity of apoptosis protein Fas, FasL, Bax and Caspase-3 increased significantly (P < 0.05 or P < 0.01), but Bcl-2 decreased (P < 0.05).
Changes of ultrasonic characteristics and liver dysfunction, caused by liver fluke infection, may be related to that both inflammatory response and apoptosis response have participated in the pathogenic process and liver damage course of clonorchiasis.
Clonorchiasis
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However, high infection intensity and reinfection rates in populations in endemic areas suggest that these immune responses have little protective effect. Notably, mice exhibit only low susceptibility to C sinensis infection, which may be related to both Th1 and Th2 responses, as suggested by raised concentrations of IgE, interferon γ, and interleukin 13.110 Although rats are susceptible to C sinensis infection, protective immunity appears in reinfection.111,112
On Aug 21, 1875, James McConnell published in The Lancet his findings from a post-mortem examination of a 20-year-old Chinese man—undertaken at the Medical College Hospital in Calcutta, India—in whom he found Clonorchis sinensis in the bile ducts. Now, exactly 140 years later, we have a sound understanding of the lifecycle of this liver fluke, including key clinical, diagnostic, and epidemiological features. Developments in the so-called -omics sciences have not only advanced our knowledge of the biology and pathology of the parasite, but also led to the discovery of new diagnostic, drug, and vaccine targets. C sinensis infection is primarily related to liver and biliary disorders, especially cholangiocarcinoma. Clonorchiasis mainly occurs in east Asia, as a result of the region's social-ecological systems and deeply rooted cultural habit of consuming raw freshwater fish. The Kato-Katz technique, applied on fresh stool samples, is the most widely used diagnostic approach. Praziquantel is the treatment of choice and has been considered for preventive chemotherapy. Tribendimidine showed good safety and therapeutic profiles in phase 2 trials and warrants further investigation. Still today, the precise distribution, the exact number of infected people, subtle morbidities and pathogenesis, and the global burden of clonorchiasis are unknown. Integrated control strategies, consisting of preventive chemotherapy; information, education, and communication; environmental management; and capacity building through intersectoral collaboration should be advocated.

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Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Preparation of C. sinensis metacercariae

Animal and experimental design

Gross observations

Worm recovery

Discussion

Acknowledgments

J Hepatol

Parasitol Today

Parasitol Int

Exp Parasitol

Clonorchiasis: an update

J Helminthol

Relation of cholangiocarcinomas to clonorchiasis and bile duct stones

Abdom Imaging

Clonorchis sinensis infection and increasing risk of cholangiocarcinoma in the Republic of Korea

Am J Trop Med Hyg

Parasitic infestations of the biliary tract

Curr Gastroenterol Rep

Descriptive epidemiology of cholangiocarcinoma and clonorchiasis in Korea

J Korean Med Sci

Opisthorchis viverrini infection in Thailand: studies on the morbidity of the infection and resolution following praziquantel treatment

Am J Trop Med Hyg

Clonorchiasis in Korea

Korean J Parasitol

Effect of Clonorchis sinensis infection and dimethylnitrosamine administration on the induction of cholangiocarcinoma in Syrian golden hamsters

Korean J Parasitol

Cytokine responses in mice infected with Clonorchis sinensis

Parasitol Res

Infectivity and pathological changes in murine clonorchiasis: comparison in immunocompetent and immunodeficient mice

J Vet Med Sci

Egg production of Clonorchis sinensis in different strains of inbred mice

Korean J Parasitol

The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment

Parasitol Res

Cytokine responses in mice infected with Angiostrongylus cantonensis

Parasitol Res

The host-parasite relations of Clonorchis sinensis in the mouse: studies on the development of C. sinensis in mouse

Korean J Parasitol

Susceptibility of experimental animals to reinfection with Clonorchis sinensis

Korean J Parasitol