Kinetics of serum IL-17A, IL-6, IL-2, and TGF-β₁.

<p>Serum from each mouse was screened with cytometric bead array (CBA) to detect IL-6 (<b>A</b>), IL-17A (<b>B</b>), and IL-2 (<b>C</b>); enzyme-linked immunosorbent assays (ELISA) was used to measured TGF-β₁ (<b>D</b>). Data are expressed as mean ± S.E.M. Asterisks indicate statistically significant differences between infected mice and NC mice at a particular time point, *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001, compared with normal control mice.</p

Quantitative changes in splenic Treg and Th17 cell percentages and the relationship between Treg and Th17 subsets.

<p>Single-cell spleen suspensions from <i>C</i>. <i>sinensis</i>-infected and control mice (NC) were used to analyze Th17 and CD4⁺CD25⁺Foxp3⁺regulatory T cell percentages among CD4⁺ T cells by flow cytometry (<b>A</b>). Representative blots of IL-17⁺ and CD25⁺Foxp3⁺cells in splenic CD4⁺cell populations. <b>B</b> and <b>C</b> show quantitative changes in splenic Treg and Th17 cell percentages in CD4⁺T cells. The Treg/Th17 ratio was calculated (<b>D</b>). Data are expressed as mean ± S.E.M. Asterisks indicate statistically significant differences between infected and NC mice, **<i>P</i><0.01, ***<i>P</i><0.001, compared with normal control mice.</p

Hepatic expression levels of Foxp3 and RORγt in the mice infected by <i>Clonorchis sinenis</i>.

<p>Liver sections from <i>C</i>. <i>sinesis</i>-infected and NC mice were homogenized; and the expression levels of RORγt (<b>A</b>) and Foxp3 (<b>B</b>) were determined by western-blot. Data are expressed as mean ± S.E.M. Asterisks indicate statistically significant differences between infected and NC mice; **<i>P</i><0.01, compared with normal control mice.</p

Collagen depositions in the liver of <i>Clonorchis sinensis</i>-infected BALB/c mice.

<p>Compared to NC mice (<b>A</b>), collagen fibers were slightly stained blue by Masson’s trichrome staining on day 14 post-infection (PI) (<b>B</b>), and the degree of staining were increased on day 28 PI (<b>C</b>) and 56 PI (<b>D</b>). Correlations were calculated between the degree of Treg/Th17 imbalance and liver fibrosis caused by <i>C</i>. <i>sinensis</i> (<b>E</b>).</p

The Dynamics of Treg/Th17 and the Imbalance of Treg/Th17 in <i>Clonorchis sinensis</i>-Infected Mice

<div><p>Clonorchiasis, caused by the liver fluke <i>Clonorchis sinensis</i>, is a chronic parasitic infection regulated by T cell subsets. An imbalance of CD4⁺CD25⁺ Foxp3⁺regulatory T (Treg) and interleukin (IL)-17-secreting T cells (Th17) may control inflammation and play an important role in the pathogenesis of immune evasion. In the present study, we assessed the dynamics of Treg/Th17 and determined whether the Treg/Th17 ratio is altered in <i>C</i>. <i>sinensis</i>-infected mice. The results showed that the percentages of splenic Treg cells in CD4⁺ T cells were suppressed on day 14 post-infection (PI) but increased on day 56 PI, while Th17 cells were increased on day 56 PI compared with normal control (NC) mice. The Treg/Th17 ratio steadily increased from day 28 to day 56 PI. The hepatic levels of their specific transcription factors (Foxp3 for Treg and RORγt for Th17) were increased in <i>C</i>. <i>sinensis</i>-infected mice from day 14 to 56 PI, and significantly higher than those in NC mice. Meanwhile, serum levels of IL-2 and IL-17 were profoundly increased in <i>C</i>. <i>sinensis</i>-infected mice throughout the experiment; while the concentrations of IL-6 and transforming growth factor β₁ (TGF-β₁) peaked on day 14 PI, but then decreased on day 28 and 56 PI. Our results provide the first evidence of an increased Treg/Th17 ratio in <i>C</i>. <i>sinensis</i>-infected mice, suggesting that a Treg/Th17 imbalance may play a role in disease outcomes of clonorchiasis.</p></div

Representative histology of liver sections stained with hematoxylin and eosin (H&E).

<p><b>A</b> and <b>a</b> show liver sections from NC mice. In infected mice, massive inflammatory cells were infiltrated around the portal areas accompanied by cholangiocyte proliferation on day 14 post-infection (PI). (<b>B</b> and <b>b</b>). The numbers of inflammatory cells decreased, but bile duct hyperplasia was exaggerated on day 28 PI. (<b>C</b> and <b>c</b>) and 56 PI. (<b>D</b> and <b>d</b>). Capitals and lowercase letters indicate magnifications of ×100 and ×400 times respectively.</p

The percentages of hepatic Th1 and Th17 cells in normal and <i>Clonorchis sinensis</i>-infected mice.

<p>Data shown are gated on CD4⁺T cells. Numbers represent the ratios of Th1 (A) and Th17 (B) in CD4⁺T cells. (C) & (D) indicated the changes of Th1 and Th17 in three strains of mice, respectively. Data are mean±SEM of four or more independent experiments. * = <i>P</i> <0.05, ** = <i>P</i> <0.01, *** = <i>P</i> <0.001.</p

Correlations between Th2, Treg and hydroxyproline concentrations during <i>Clonorchis sinensis</i> infection within different strains of mice.

<p>Pearson’s correlation was used to analyze the associations between these two CD4⁺T cell subsets and hydroxyproline contents. The blue, yellow and red dots represent C57BL/6 infected mice, BALB/c infected mice and FVB infected mice, respectively.</p

Gross observation and histopathological changes of livers in normal and <i>Clonorchis sinensis</i>-infected mice.

<p>Each strain of mice was randomly divided into normal group (n = 5) and <i>C</i>. <i>sinensis</i>-infected group (n = 5) which were orally infected with 45 metacercariae. All the mice were sacrificed on 28 days post infection. (A) Gross observation of livers. A-1, A-2, A-3 and A-4 represent the livers in normal mice, <i>C</i>. <i>sinensis</i>- infected C57BL/6 mice, <i>C</i>. <i>sinensis</i>- infected BALB/c mice and <i>C</i>. <i>sinensis</i>- infected FVB mice, respectively. (B) The histopathological changes of livers in C57BL/6, BALB/c and FVB mice were examined by H&E staining (×100).</p

The percentages of hepatic Th2 and Treg cells in normal and <i>Clonorchis sinensis</i>-infected mice.

<p>Data shown are gated on CD4⁺T cells. Numbers represent the ratios of Th2 (A) and Treg (B) in CD4⁺T cells. (C) &(D) indicated the changes of Th2 and Tregs in three strains of mice, respectively. Data are mean±SEM of four or more independent experiments. * = <i>P</i> <0.05, ** = <i>P</i> <0.01, *** = <i>P</i> <0.001.</p