Fig 1.
Mice lacking B cells exhibit more severe liver pathology than WT mice after S. japonicum infection.
(A) Schematic representation of the model of S. japonicum infection. WT mice and μMT mice were infected with 18–20 cercariae of S. japonicum, and liver samples from these mice were harvested at the times indicated after infection. Comparisons were made with uninfected control mice. (B, C) Representative graphs of H&E staining (B) and picrosirius red staining (C) of liver specimens. (B) All images were taken at 200 × magnification. The red outlined areas indicate granulomas. (C) All images were taken at 40 × magnification. (D-G) Statistical analysis of granuloma sizes (D), proportion of collagen areas (E), amount of hepatic hydroxyproline (F), and levels of serum ALT (G). Data represent mean ± SD; n = 8–10 per time point from two independent experiments. *p < 0.05, versus WT mice.
Fig 2.
B cell deficiency results in increased number of macrophages in the liver.
(A) The infiltration of hepatic leukocytes (CD45+), macrophages (CD45+CD11b+F4/80+), neutrophils (CD45+CD11b+Ly6G+), T cells (CD45+CD3+NK1.1−), NK cells (CD45+CD3−NK1.1+), and NKT cells (CD45+CD3+NK1.1+) after infection were quantified by flow cytometric analysis. Controls (Ctrl) were uninfected mice. (B) Graphical summary showing percentages of KCs (CD11bloF4/80hi), Ly6Chi monocytes (CD11bhiF4/80loly6Chi), and Ly6Clo monocytes (CD11bhiF4/80loly6Clo) out of total hepatic macrophages (CD45+CD11b+F4/80+). (C) Absolute numbers of KCs, Ly6Chi monocytes, and Ly6Clo monocytes in WT mice and μMT mice. Data represent mean ± SD; n = 8–10 mice per time point from two independent experiments. *p < 0.05, **p < 0.01, versus WT mice.
Fig 3.
F4/80 and Ly6C expression are increased in the liver of μMT mice after S. japonicum infection.
WT mice and μMT mice were infected with 18–20 cercariae of S. japonicum, and liver samples from these mice were harvested at the times indicated after infection. Comparisons were made with uninfected control mice. (A, C) Liver sections were stained with F4/80 (A), and Ly6C (C). Original magnification, × 200 for F4/80, × 400 for Ly6C. (B, D) Numbers of F4/80- and Ly6C-positive cells per field are counted. (E) Relative mRNA expression of F4/80 and Ly6C in liver tissues was determined by quantitative real-time PCR. Genes were normalized to the housekeeping gene Actb as an internal control. Data represent mean ± SD; n = 8–10 mice per time point from two independent experiments. *p < 0.05, **p < 0.01, versus WT mice.
Fig 4.
Inflammatory cytokines and chemokines are increased in the liver of μMT mice after S. japonicum infection.
(A) Relative gene expression of chemokines (Ccl1, Ccl2, Ccl3, Ccl4, and Ccl5) and inflammatory cytokines (Tnfa, Il1b, Il6, Il10, Il12a, and Il12b) in the livers of WT mice and μMT mice uninfected and 6 weeks after infection. Genes were normalized to the housekeeping gene Actb as an internal control. (B) Protein levels of CCL2, CCL3, CCL4, CCL5, TNF-α, IL-6, and IL-12p40 in the livers of WT mice and μMT mice uninfected and 6 weeks after infection. Data represent mean ± SD; n = 8–10 mice per group from two independent experiments. *p < 0.05, **p < 0.01.
Fig 5.
Hepatic B1 cells increase whereas PC B1 cells decrease after S. japonicum infection.
(A) Number of B cells (CD3−CD19+) in WT mice at the times indicated after infection. (B) Representative flow cytometry plots of hepatic B1a (CD3−CD19+CD5+CD23−IgMhiIgDlo), B1b (CD3−CD19+CD5−CD23−IgMhiIgDlo), and B2 (CD3−CD19+CD5−CD23+IgMloIgDhi) cells in WT mice 6 weeks after infection. (C) Graphical summary showing the percentages of B1a, B1b, and B2 cells out of total B cells (top panel) and the number of indicated subsets (bottom panel) in the livers of WT mice without infection (Ctrl) and 6 weeks after infection. (D) Representative flow cytometry plots of PC B1a (CD3−CD19+CD5+CD11b+), B1b (CD3−CD19+CD5− CD11b+), and B2 cells (CD3−CD19+ CD5−CD11b−) in WT mice without infection and 6 weeks after infection. (E) The percentages of B1a, B1b, and B2 cells out of total B cells (left panel) and number of indicated subsets (right panel) in the PC of WT mice without infection and 6 weeks after infection. Data represent mean ± SD; n = 8–10 mice per group from two independent experiments. *p < 0.05, **p < 0.01.
Fig 6.
Adoptive transfer of B1 cells attenuates S. japonicum-induced liver pathology in μMT mice.
(A) Schematic representation of the model of mice treatment. The μMT mice were infected with 18–20 cercariae of S. japonicum. FACS-sorted PC B1 cells (2 × 106 cells) or B2 cells (2 × 106 cells) from uninfected WT mice were adoptive transferred into μMT mice 4 weeks after infection. Mice were sacrificed 6 weeks after infection. (B) Representative images of H&E, F4/80, and Ly6C stained liver tissues. Original magnification, × 200 for F4/80, × 400 for Ly6C. (C-E) Statistical analysis of hepatic granuloma sizes (C), numbers of F4/80- and Ly6C-positive cells per field (D), and serum ALT levels (E). (F) Graphical summary showing percentages of indicated cell subsets out of total hepatic macrophages in infected μMT mice after cell transfer assayed by flow cytometry. (G) Numbers of indicated cell subsets in the liver of infected μMT mice after cell transfer analyzed by flow cytometry. (H) Quantitative PCR analysis of chemokine and inflammatory cytokine gene expression levels in the liver. (I) Hepatic chemokine and inflammatory cytokine protein expression levels were examined by CBA and ELISA. Data represent mean ± SD; n = 5–7 per group from two independent experiments. *p < 0.05, **p < 0.01.
Fig 7.
IL-10 expression is increased in the liver and in B cells after S. japonicum infection.
(A) Relative Il10 gene expression in the livers of WT mice and μMT mice 6 weeks after infection. (B) IL-10 protein levels in the livers of WT mice and μMT mice 6 weeks after infection. (C) Quantitative PCR analysis of Il10 in sorted hepatic B cells. (D) The frequency of IL-10-positive cells out of total B cells in the livers of WT mice was examined by flow cytometry. (E) Graphical summary showing percentage of IL-10-positive cells out of indicated cell subsets in WT mice 6 weeks after infection. Data represent mean ± SD; n = 8–10 per group from two independent experiments. *p < 0.05, **p < 0.01.
Fig 8.
Adoptive transfer of WT PC B1 cells, but not Il10−/− PC B1 cells, attenuates S. japonicum-induced liver pathology in μMT mice.
μMT mice were infected with 18–20 cercariae of S. japonicum. The adoptive transfer of B1 cells (2 × 106 cells) purified from the PC of WT or Il10−/− mice into μMT mice was performed 4 weeks after infection. Mice were sacrificed 6 weeks after infection. (A) H&E staining and immunohistochemical staining for F4/80 and Ly6C of liver tissues. Original magnification, × 200 for F4/80, × 400 for Ly6C. (B) Statistical analysis of hepatic granuloma sizes. (C) Numbers of F4/80- and Ly6C-positive cells per field. (D) Serum ALT levels. (E) Graphical summary showing percentages of indicated cell subsets out of total hepatic macrophages in infected μMT mice after cell transfer assayed by flow cytometry. (F) Numbers of indicated cell subsets in the liver of infected μMT mice after cell transfer analyzed by flow cytometry. (G) Quantitative PCR analysis of chemokine and inflammatory cytokine gene expression levels in the liver. (H) Hepatic chemokine and inflammatory cytokine protein expression levels were examined by CBA and ELISA. Data represent mean ± SD; n = 5–7 per group from two independent experiments. *p < 0.05, **p < 0.01.