Schistosoma japonicum Infection Promotes the Response of Tfh Cells Through Down-Regulation of Caspase-3-Mediating Apoptosis

Abstract

CD4⁺ T follicular helper (Tfh) cells, a new subset of immune cells, have been demonstrated to be involved in granulomatous responses to Schistosoma japonicum (S. japonicum) infection. However, the role and underlying mechanisms of Tfh cell aggregation in S. japonicum infection remain incompletely understood. In this study, we provide evidence that S. japonicum infection enhances the accumulation of Tfh cells in the spleen, lymph nodes, and peripheral blood of C57BL/6 mice. Infection-induced Tfh cells exhibited more potent effects directly on B cell responses than the control Tfh cells (P < 0.05). Furthermore, reduced apoptosis of Tfh cells was found both in S. japonicum infected mice and in soluble egg antigen (SEA) treated Tfh cells (P < 0.05). Mechanistic studies reveal that caspase-3 is the primary drivers of down-regulated apoptotic Tfh cell death in S. japonicum infection. In summary, this study demonstrates that Tfh cell accumulation might have an impact on the generation of immune responses in S. japonicum infection, and caspase-3 signaling mediated apoptosis down-regulation might responsible for the accumulation of Tfh cell in this course.

Keywords: SEA; Schistosoma japonicum; T follicular helper (Tfh) cells; apoptosis; caspase-3.

Figure 1

S. japonicum-infection induce Tfh cells in vivo. (A–D) C57BL/6 mice were infected percutaneously with 40 ± 5 cercariae and sacrificed at 5–6 weeks after infection, and different tissues were harvested. (A) Representative images of livers. (B) Representative images of liver H&E staining; arrows indicate granuloma. The percentage (C,D) and absolute numbers (E) of CD4⁺CXCR5⁺PD1⁺ cells in the lymphocytes isolated from spleen (SP), lymph node (LN), peripheral blood (PB), and live of both normal and infected mice were evaluated by flow cytometry after staining with specific antibodies. Each group included 10 mice. (F) Spleens from infected mice were harvested; the dynamic proportions of CD4⁺CXCR5⁺PD-1⁺ Tfh cells in the S. japonicum infected mice was evaluated by flow cytometry. Data are shown as mean ± SEM of six samples in each group from one representative experiment, and repeat three times with similar results. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding controls (0W), One-way ANOVA was used.

Figure 2

Activation associated molecules and cytokines expressing on Tfh cells. Single-cell suspensions of spleens were isolated from normal and infected mice. The expression of CD25 and CD69 on Tfh cells was detected using cell surface staining. The expression of CD25, CD69 (A), and ICOS (B) was analyzed using FCM; average expression of CD25, CD69, and ICOS was calculated from FACS data. Three independent experiments with similar results were performed and mean± SEM values of six samples pooled from three experiments are shown. ^*P < 0.05, compared with the controls; unpaired t-tests were used. (C) Single-cell suspensions of spleen cells from normal and S. japonicum infected mice were stimulated with or without PMA and ionomycin. Expressions of IL-21, IL-10, IL-17, IFN-γ, IL-4, and IL-5 were detected in Tfh cells by FACS analysis. Numbers in quadrants are percentages of cells in each expression phenotype (n = 5 mice per group). A representative of two independent experiments is shown. ^*P < 0.05, compared with the controls.

Figure 3

Function of Tfh cells from S. japonicum infected mice. (A) CD4⁺CXCR5⁺PD-1⁺ Tfh cells from normal or infected mice and allogeneic CD19⁺ B cells from normal controls are incubated at a ratio of 1:1 in RPMI1640 complete medium. (B,C) After 3 and or days of culture, the B cell subpopulations are examined based on surface phenotype. (B) Representative staining of CD27 and CD69 on B cells from naive mice at days 3 and 10 following co-culture with Tfh cells from normal and S. japonicum infected mice. (D,E) Mean ± SEM of one representative experiment. IgM and IgG concentration in the supernatant at day 3 or 10. Data are shown as mean ± SEM of 4–6 samples in each group from one representative experiment. ^*P < 0.05, ^**P < 0.01 compared with the corresponding controls; unpaired t-tests were used.

Figure 4

S. japonicum infection reduces apoptosis via caspase-3 pathway in vivo. (A) In spleen Tfh cells purified from normal or S. japonicum infected mice, gene expression was determined by qRT-PCR. (B,C) Single-cell suspensions of spleens were isolated from in different time points infected mice, and apoptosis was detected using Annexin V-FITC/PI double staining on CD4⁺CXCR5⁺PD-1⁺ Tfh cells and CD4⁺CXCR5⁻PD-1⁻ non-Tfh cells by flow cytometry. (D) The percentage of caspase-3⁺ Tfh cells in the spleen of normal and S. japonicum infected mice. Representative images (left) and pooled data (right) were shown. (E) The caspase-3 activity of the spleen Tfh cells purified from normal or S. japonicum infected mice (^*P < 0.05). (F) In spleen Tfh cells purified from normal or S. japonicum infected mice, caspase-3 and caspase-8 expression was determined by Western blotting. (G) Quantitative RT-PCR analysis of Mcl-1, XIAP, BCL-2, BAX, and Survivin expression in spleen Tfh cells purified from normal or S. japonicum infected mice. The data are relative levels of expression compared with those of control cells after normalization with β-actin expression. Data are shown as mean ± SEM of six samples in each group from one representative experiment. ^*P < 0.05; ns, not significant (P > 0.05); unpaired t-test.

Figure 5

SEA reduces apoptosis via caspase-3 pathway in vitro. (A,B) Mouse spleen cells (A) or Tfh cells (B) purified from spleen in normal mice were cultured in medium containing IL-21 (10 ng/ml) with SEA (100 μg/mL) or PBS (control); vehicle was used as control. Apoptosis was detected using Annexin V-FITC/PI double staining with flow cytometry. (Left) Representative results from three independent experiments. (Right) Mean ± SEM values from three independent experiments. (C) Tfh cells were cultured in IL21 with the indicated treatments for 24 h; mRNA expression of Mcl-1, XIAP, BCL-2, BAX, and Survivin was determined by qRT-PCR. The data are relative levels of expression compared with those of control cells after normalization with β-actin expression. Data are shown as mean ± SEM of three to five samples in each group from one representative experiment. ^*P < 0.05; ns, not significant (P > 0.05); unpaired t-test.

Figure 6

SEA reduces apoptosis via caspase-3 pathway in vitro. (A,B) Splenocytes (A) was isolated from normal mice, and CD4⁺CXCR5⁺PD-1⁺ Tfh cells was purified by FACS. The splenocytes and Tfh cells were cultured in medium containing IL-21 (10 ng/ml) with SEA (100 μg/mL), PAC-1 (100 μM/mL); vehicle was used as control. Apoptosis was detected using Annexin V-FITC/PI double staining with flow cytometry. (Left) Representative results from three to five independent experiments. (Right) Data are shown as mean ± SEM of six samples in each group from one representative experiment, and repeat three times with similar results. ^*P < 0.05.