Regulation of anti-inflammatory cytokines IL-10 and TGF-β in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Abstract

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-β production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-β significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-β. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase 1/2.

Figure 1

Panel A shows Morphological changes of DC2.4 cells treated with (a) medium, (b) 20 μg ml⁻¹ of crude antigen (CA). Cells were plated in six-well plates (2 × 10⁵ cells per well). After incubation for 24 h, changes in cellular morphology in each group were determined using microscopy. Images were acquired using a digital camera at × 200 magnification. This figure shows representative features of four independent experiments. Panel B shows no differences in levels of cell surface markers (CD80, CD86, Toll-like receptor (TLR)2 and TLR4) after treatment with C. sinensis crude antigen.

Figure 2

Production of cytokines following treatment of DC2.4 cells with C. sinensis crude antigen (CA) and lipopolysaccharide (LPS). DC2.4 cells were incubated with phosphate-buffered saline, 20 μg ml⁻¹ CA and 100 ng ml⁻¹ LPS for 24 h, supernatants were collected and expression levels of interleukin (IL)-10, transforming growth factor (TGF-β), IL-4, IL-5, IL-6, IL-13, IL-12, interferon (IFN)-γ and TNF-α were analyzed by enzyme-linked immunosorbent assay (ELISA). Data represent the means of three independent experiments. *P<0.01 (t-test) was considered statistically significant.

Figure 3

Production of cytokines following treatment of bone marrow-derived DCs (BMDC) cells with C. sinensis crude antigen (CA). BMDC cells were incubated with either phosphate-buffered saline or 20 μg ml⁻¹ of CA for 24 h, and the supernatant was collected to analyze the expression levels of interleukin (IL)-10, transforming growth factor (TGF)-β, IL-4, IL-5, IL-6, IL-13, IL-12, interferon (IFN)-γ and TNF-α by enzyme-linked immunosorbent assay (ELISA). Data represent the means of three independent experiments. *P<0.01 (t-test) was considered statistically significant.

Figure 4

C. sinensis crude antigen (CA) regulates interleukin (IL)-10 and transforming growth factor (TGF)-β in dendritic cells (DCs) compared with other parasite antigens. DC2.4 cells were plated in six-well plates (2 × 10⁵ cells per well). After treatment with different parasite antigens for 24 h, including C. sinensis CA, P. westermani CA (PW), S. erinacei plerocercoid CA (SP) and E. granulosus hydatid cystic fluid (CY), supernatants were collected. Production of IL-10 and TGF-β were determined using enzyme-linked immunosorbent assay (ELISA). Data represent the means of three independent experiments. *P<0.05 and ^**P<0.01 (t-test) were considered statistically significant.

Figure 5

ERK1/2 phosphorylation in DC2.4 cells treated with C. sinensis crude antigen (CA). After treatment with C. sinensis CA, whole-cell lysates were collected at 10, 30 and 60 min. Phosphorylated ERK1/2, p38, JNK and total-ERK1/2, p38 and JNK levels were analyzed via western blot. Densitometry values were normalized to t-ERK, t-p38 or t-JNK, and then to the nontreated controls. Data represent the means of three independent experiments. *P<0.05 and ^**P<0.01 (t-test) were considered statistically significant.

Figure 6

Effects of ERK activation inhibitor on crude antigen (CA)-induced interleukin (IL)-10 and transforming growth factor (TGF)-β levels. DC2.4 cells were cultured with 2 or 5 μM of ERK activation inhibitor peptide I in phosphate-buffered saline and collected after incubation with 20 μg ml⁻¹ of CA for 24 h. Data represent the means±s.e. of three independent experiments. *P<0.05 and ^**P<0.01 (t-test) were considered statistically significant.