Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components - PubMed (original) (raw)
Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components
Jhang Ho Pak et al. Korean J Parasitol. 2019 Aug.
Abstract
Clonorchis sinensis is a carcinogenic human liver fluke that promotes hepatic inflammatory environments via direct contact or through their excretory-secretory products (ESPs), subsequently leading to cholangitis, periductal fibrosis, liver cirrhosis, and even cholangiocarcinoma (CCA). This study was conducted to examine the host inflammatory responses to C. sinensis ESPs and their putative protein components selected from C. sinensis expressed sequenced tag (EST) pool databases, including TGF-β receptor interacting protein 1(CsTRIP1), legumain (CsLeg), and growth factor binding protein 2 (CsGrb2). Treatment of CCA cells (HuCCT1) with the ESPs or bacterial recombinant C. sinensis proteins differentially promoted the secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) as well as anti-inflammatory cytokines (IL-10, TGF-β1, and TGF-β2) in a time-dependent manner. In particular, recombinant C. sinensis protein treatment resulted in increase (at maximum) of ~7-fold in TGF-β1, ~30-fold in TGF-β2, and ~3-fold in TNF-α compared with the increase produced by ESPs, indicating that CsTrip1, CsLeg, and CsGrb2 function as strong inducers for secretion of these cytokines in host cells. These results suggest that C. sinensis ESPs contribute to the immunopathological response in host cells, leading to clonorchiasis-associated hepatobiliary abnormalities of greater severity.
Keywords: Clonorchis sinensis; excretory-secretory products; host immune response; inflammatory cytokine; recombinant Cs-driven protein.
Conflict of interest statement
CONFLICT OF INTEREST
The authors declare no conflict of interest related to this study.
Figures
Fig. 1
Bacterial expression and purification of recombinant Cs proteins. (A) Recombinant CsTrip1 purified using a Ni-NTA column in 10% SDS-PAGE. 2.5 μl of elute (lane 1), 1.25 μl of elute (lane 2). (B) Recombinant CsLeg in 10% SDS-PAGE. Uninduced total (lane 1), induced total (lane 2), induced soluble (lane 3), induced pellet (Lane 4), pass-through (lane 5), washing (lane 6), and elute fractions (lane 7 and 8). (C) On-bead cleavage of recombinant CsGrb2 in 10% SDS-PAGE. Uninduced total (lane 1), induced total (lane 2), induced soluble (lane 3), dialyzed soluble (lane 4), pass-through (lane 5), washing (lane 6), cleaved-off CsGrb2 (lane 7), and washed-off Sj26GST tag protein (lane 8).
Fig. 2
Proinflammatory cytokine profiling of HuCCT1 cells in response to C. sinensis ESPs. After 0–24 hr ESP treatment (800 ng/ml), culture supernatants were harvested and analyzed for the production of IL-1β (A), IL-2 (B), IL-6 (C), IL-10 (D), TGF-β1 (E), TGF-β2 (F), TNF-α (G), and INF-γ (H) by ELISA. Data are presented as mean±S.D. of 3 independent experiments. *P<0.05 compared with 0 hr.
Fig. 3
Differential expressions of IL-1β, IL-6, TGF-β1, TGF-β2, and TNF-α in recombinant C. sinensis protein-treated HuCCT1 cells. Cells were treated with 2 μg/ml of CsTrip1, CsLeg, or CsGrb2 proteins for 0–24 hr, and the production of IL-1β (A), IL-6 (B), TGF-β1 (C), TGF-β2 (D), and TNF-α (F) in culture supernatants was determined by ELISA. Data are presented as mean±S.D. of 3 independent experiments. *P<0.05 compared with 0 hr.
References
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