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. 2020 Mar 20;13(1):147.
doi: 10.1186/s13071-020-04015-3.

B Cells Induced by Schistosoma Japonicum Infection Display Diverse Regulatory Phenotypes and Modulate CD4 + T Cell Response

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Free PMC article

B Cells Induced by Schistosoma Japonicum Infection Display Diverse Regulatory Phenotypes and Modulate CD4 + T Cell Response

Junli Xiao et al. Parasit Vectors. .
Free PMC article

Abstract

Background: The increased activity of regulatory B cells (Breg) is known to be involved in immunosuppression during helminth infection, which is characterized by inducing IL-10-producing Breg cells. However, the current knowledge of B cell subsets differentiation and IL-10-independent immunoregulatory mechanisms of B cells in schistosomiasis is insufficient.

Methods: BALB/c mice were percutaneously infected with cercariae for investigating the profile of B cell subsets during Schistosoma japonicum infection. B cells isolated from the spleen or peritoneal cavity were analyzed for the regulatory phenotype after stimulation with soluble egg antigens (SEA) in vitro. CD4+ T cells were then cocultured with B cells pretreated with or without anti-PD-L1 antibody for investigating the role of B cells from infected mice on regulating CD4+ T cells. Furthermore, the in vivo administration of anti-PD-L1 antibody was conducted to investigate the role of PD-L1 in regulating host immunity during infection.

Results: The percentages of peritoneal and splenic B-1a cells, as well as marginal zone B (MZB) cells were decreased at eight and twelve weeks after infection compared to those from uninfected mice. In splenic B cells, TGF-β expression was increased at eight weeks but declined at twelve weeks of infection, and PD-L1 expression was elevated at both eight and twelve weeks of infection. In addition, SEA stimulation in vitro significantly promoted the expression of IL-10 in peritoneal B cells and CD5 in splenic B cells, and the SEA-stimulated splenic and peritoneal B cells preferentially expressed PD-L1 and TGF-β. The splenic B cells from infected mice were able to suppress the function of Th1 and Th2 cells in vitro but to expand the expression of Tfh transcription factor Bcl6, which was further enhanced by blocking PD-L1 of B cells before co-cultivation. Moreover, Th2 response and Bcl6 expression in CD4+ T cells were also increased in vivo by blocking PD-L1 after infection, although the hepatic pathology was slightly influenced.

Conclusions: Our findings revealed that S. japonicum infection modulates the differentiation of B cell subsets that have the capability to affect the CD4+ T cell response. This study contributes to a better understanding of B cells immune response during schistosomiasis.

Keywords: IL-10; PD-L1; Regulatory B cells; Schistosoma japonicum; Th responses.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Infection with Schistosoma japonicum alters splenic and PerC B-cell composition. Splenic and PerC lymphocytes were isolated from mice at zero (uninfected mice, 0W), three (3W), eight (8W), and twelve weeks (12W) post-infection. Gating schemes for PerC B-1a cells (a) and splenic B-cell subsets (b) within the CD19+ gated B-cell population are shown. Splenic B-cell subpopulations (b) are shown: B-1a cells (CD19+CD5+), T1 B cells (CD19+CD5CD21CD23), T2-MZP cells (CD19+CD5CD21+CD23+), MZB cells (CD19+CD5CD23CD21+) and FOB cells (CD19+CD5CD23+CD21). The percentages of PerC B-1a cells (c) were measured. The percentages (d) and the absolute numbers (e) of splenic B cell subsets were measured from at least five mice of each time point after infection. All data are representative of at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
Infection with Schistosoma japonicum elevates PD-L1 expression and modulates cytokines profile of splenic B cells. a Flow cytometric histograms represent cell surface expression of PD-L1 in splenic and PerC B cells from uninfected mice (black line) and infected mice (red line) at each time point. The shaded histograms represent the FMO control. Significance levels from statistical analysis of mean fluorescence intensity (MFI) of PD-L1 in splenic and PerC B cells are indicated. b The MFI of PD-L1 in splenic B cell subsets are summarized. The splenic (c) and PerC (d) lymphocytes were stimulated with PMA and ionomycin in the presence of Brefeldin A for 4 h and followed by extracellular and intracellular staining. The percentages of IFN-γ+, TGF-β+ or IL-10+ cells gated on CD19+ B cells are shown. All data are representative results of two independent experiments with at least 5 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
SEA drives CD5 and PD-L1 expression of B cells in vitro. Splenic B cells and PerC washout cells from uninfected mice were cultured for 24 h in the presence of PBS or indicated stimuli. a Representative dot plots indicate the expression of CD5 on gated CD19+ B cells from spleen and peritoneal cavity and significance levels from statistical analyses are indicated. b Flow cytometric histograms represent cell surface expression of PD-L1 in splenic and PerC B cells, and significance levels from statistical analyses of PD-L1 MFI of splenic and PerC B-cell are indicated. The expression of CD23 (c) and CD21 (d) in splenic B cells was measured at the end of culture. The shaded histograms represent the FMO control. In e and f, splenic B cells were cultured for 24 h in the presence of PBS or SWA (20 μg/ml). e Representative dot plots indicate the expression of CD5 on gated CD19+ B cells from spleen and significance levels from statistical analyses are indicated. f Flow cytometric histograms represent cell surface expression of PD-L1 in splenic B cells, and significance from statistical analyses of PD-L1 MFI of splenic B-cell are indicated. All data are representative results of two independent experiments with at least 4 mice per group. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
SEA-stimulated B cells acquire elevated expression of IL-10 and TGF-β in vitro. Splenic B cells and PerC washout cells from uninfected mice were stimulated with SEA for 24 h. Intracellular IL-10 and IFN-γ staining were performed after the restimulation with PMA and ionomycin in the presence of Brefeldin A. Representative flow cytometric plots and significance from statistical analyses of the percentages of IL-10+ B cells and IFN-γ+ B cells in splenic B cells (a) and PerC B cells (b) are shown. c Representative plots of CD5 and TGF-β expression on CD19+ B cells and the percentages of B cell subsets expressing TGF-β. All data are representative results of two independent experiments with at least 4 mice per group. *P < 0.05, **P < 0.01
Fig. 5
Fig. 5
SEA induces expression of PD-L1 and TGF-β on B cells in vivo. Flow cytometry histograms represent cell surface expression of PD-L1 in splenic B cells (a) and PerC B cells (b), and significance levels from statistical analysis of PD-L1 MFI of splenic (a) and PerC B cells (b) are shown. The shaded histograms represent the FMO control. c Representative plots of TGF-β surface expression in CD19+ B cells and the percentages of TGF-β expressing B cells. The data are representative results of two independent experiments with at least 4 mice per group. *P < 0.05
Fig. 6
Fig. 6
B Cells from infected mice modulate the phenotype of CD4+ T Cells in vivo. Purified splenic B cells from uninfected mice and infected mice (eight weeks) were treated with isotype antibody or anti-PD-L1 antibody respectively prior to co-culture with CD4+ T cells. a Representative dot plots of T-bet, Gata3, and FoxP3 in CD4+ T cells and significance levels from statistical analysis. b Representative flow cytometric plots of IL-10, IL-4 and IFN-γ in CD4+ T cells and significance levels from statistical analyses. c Representative flow cytometric histograms of Bcl6 expression in CD4+ T cells and significance levels from statistical analysis. The shaded histograms represent the FMO control. d Representative dot plots of CD44 versus CD62L gated on CD4+ T cells (CD44+CD62L T effector memory, TEM) and significance levels from statistical analysis. All data are representative results of two independent experiments with at least 4 mice per group. *P < 0.05, **P < 0.01
Fig. 7
Fig. 7
PD-L1 blocking fails to alter the regulatory molecules in splenic B-cell during infection. a Overlay of representative histograms show PD-L1 expression in splenic CD19+ B cells. Representative dot plots show the expression of CD5 (b), IFN-γ, IL-10 and TGF-β (c) in splenic CD19+ B cells. Bar graphs indicate the results from one of two independent experiments with ≥ 5 mice in each group. *P < 0.05, **P < 0.01
Fig. 8
Fig. 8
In vivo blocking of PD-L1 partially expands Th2 response after S. japonicum infection. a Flow cytometric dot plots represent expression of T-bet, Gata3, FoxP3 and Ki67 in splenic CD4+ T cells. b Flow cytometric histograms represent Bcl6 expression in splenic CD4+ T cells from uninfected mice (black line) and infected mice treated with isotype antibody (red line) and infected mice treated with anti-PD-L1 antibody (dashed line). The shaded histograms represent the FMO control. c Flow cytometric dot plots showing expression of IFN-γ, IL-4, IL-10 and TGF-β in splenic CD4+ T cells. All bar graphs indicate results from one of two independent experiments with ≥ 5 mice in each group. Experiments were performed two times with n = 10–12 per group. *P < 0.05, **P < 0.01
Fig. 9
Fig. 9
PD-L1 blocking fails to affect hepatic pathology and serum cytokines during infection. a Representative images of Masson’s trichrome staining for hepatic fibrosis analysis (original magnification of 200×). b Quantification of hepatic collagen deposition and egg burden are shown, and total RNA was extracted from livers and analyzed by RT-PCR for the expression of Col1a1 and a-SMA. In a and b, the data are representative results of two independent experiments with at least 4 mice per group. c The serum levels of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-γ were assayed by CBA. The data represent the cumulative results of two independent experiments. *P < 0.05. Scale-bars: a, 100 μm

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