Th9/IL-9 profile in human echinococcosis: their involvement in immune response during infection by Echinococcus granulosus

Abstract

Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group), the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group), as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF- β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group) compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF- β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.

Figure 1

Quantitative RT-PCR. Total RNAs were extracted and subjected to reverse transcription, followed by determination by quantitative RT-PCR. The 2^−ΔΔCt method was used to determine the cycle number Ct value corresponding to a specific fluorescence threshold and quantify the target genes. β-Actin was used as an internal control. (a) The PU.1 and IL-9 mRNA expression in HC, CE, and PCE groups. (b) Relative expression values of PU.1 and IL-9 mRNA were calculated based on the values of β-actin. (c) The GATA-3 and IRF-4 mRNA expression in HC, CE, and PCE groups. (d) Relative expression values of GATA-3 and IRF-4 mRNA were calculated based on the values of β-actin. *P < 0.05; **P < 0.01, compared with the HC group. ^△ P < 0.05; ^△△ P < 0.01, compared with CE group. HC: healthy control; CE: CE pretherapy group; PCE: CE posttherapy group.

Figure 2

Percentages of Th9, Th1, Th2, Th17, and Tregs cells in peripheral blood. Subtypes of CD4⁺ T cells in peripheral blood were detected by flow cytometry analysis. The detected cell types included Th9 (IL-9⁺) cells, Th1 (IFN-γ ⁺) cells, Th2 (IL-4⁺) cells, Treg (Foxp3⁺) cells, and Th17 (IL-17⁺) cells. Cells were first gated on CD3⁺CD4⁺ T cells and then on IL-9⁺/IFN-γ ⁺/IL-4⁺/Foxp3⁺/IL-17⁺CD3⁺CD4⁺ T cells, respectively. (a) The representative flow cytometric dot-plots of Th9, Th1, Th2, Th17, and Tregs cells in the blood of the HC, CE, and PCE groups were shown. Numbers within each quadrant indicated percentages of cells within each dot-plot. (b) Comparisons of the percentages of Th9, Th1, Th2, Th17, and Tregs cells in the peripheral blood of the HC, CE, and PCE groups. Horizontal bars indicated means. The percentages of Th cells represented Th cell numbers in total CD4⁺ T-cell numbers were determined by flow cytometry. (c) Th9 cells were correlated negatively with Th1, Th17, and Tregs cells, while Th9 cells were positively correlated with the numbers of Th2 in peripheral blood in CE patients. Correlations were determined by Spearman rank correlation coefficients. HC: healthy control; CE: CE pretherapy group; PCE: CE post-therapy group.

Figure 3

Th9 related cytokines changed in CE patients as determined by the CBA experiment. (a) Comparisons of levels of IL-9, IFN-γ, IL-4, IL-10, IL-17, TGF-β, and IL-6 in HC, CE, and PCE groups. The horizontal bars indicated means. Comparison was performed using one-way analysis of variance. (b) In CE patients, IL-9 concentrations were correlated negatively with IFN-γ, IL-10, IL-17, TGF-β, and IL-6 in CE patients (r = −0.011, r = −0.021, r = −0.051, r = 0.101, and r = 0.116, resp.). IL-9 was positively correlated with the concentrations of IL-4 (P = 0.041, r = 0.397).