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Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

ABSTRACT: We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1? decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

SUBMITTER: Hongsrichan N

PROVIDER: S-EPMC3993434 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

Hongsrichan Nuttanan N Intuyod Kitti K Pinlaor Porntip P Khoontawad Jarinya J Yongvanit Puangrat P Wongkham Chaisiri C Roytrakul Sittiruk S Pinlaor Somchai S

Infection and immunity 20140310 5

We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, ...[more]

PMID: 24614660

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Project description:BackgroundCholangiocarcinoma (CCA)--cancer of the bile ducts--is associated with chronic infection with the liver fluke, Opisthorchis viverrini. Despite being the only eukaryote that is designated as a 'class I carcinogen' by the International Agency for Research on Cancer, little is known about its genome.ResultsApproximately 5,000 randomly selected cDNAs from the adult stage of O. viverrini were characterized and accounted for 1,932 contigs, representing ~14% of the entire transcriptome, and, presently, the largest sequence dataset for any species of liver fluke. Twenty percent of contigs were assigned GO classifications. Abundantly represented protein families included those involved in physiological functions that are essential to parasitism, such as anaerobic respiration, reproduction, detoxification, surface maintenance and feeding. GO assignments were well conserved in relation to other parasitic flukes, however, some categories were over-represented in O. viverrini, such as structural and motor proteins. An assessment of evolutionary relationships showed that O. viverrini was more similar to other parasitic (Clonorchis sinensis and Schistosoma japonicum) than to free-living (Schmidtea mediterranea) flatworms, and 105 sequences had close homologues in both parasitic species but not in S. mediterranea. A total of 164 O. viverrini contigs contained ORFs with signal sequences, many of which were platyhelminth-specific. Examples of convergent evolution between host and parasite secreted/membrane proteins were identified as were homologues of vaccine antigens from other helminths. Finally, ORFs representing secreted proteins with known roles in tumorigenesis were identified, and these might play roles in the pathogenesis of O. viverrini-induced CCA.ConclusionThis gene discovery effort for O. viverrini should expedite molecular studies of cholangiocarcinogenesis and accelerate research focused on developing new interventions, drugs and vaccines, to control O. viverrini and related flukes.

Project description:The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size.A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Project description:BACKGROUND: The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens. METHODOLOGY/PRINCIPAL FINDINGS: The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. CONCLUSIONS/SIGNIFICANCE: This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.

Dataset Information

Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

Publications

Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

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