Fig 1.
S. japonicum infection drove bone loss in mice.
Male C57BL/6 mice were infected with 12 cercariae of S. japonicum per mouse. (A) Mice were sacrificed at 3, 5, 8 or 13 weeks post-infection. Femurs were isolated from male age-matched normal and S.japonicum-infected mice, and analyzed by X-ray. Representative of X-ray images of femurs; (B-E) Femurs from mice 13 weeks after infection and age-matched normal mice were analyzed by microcomputed tomography (μCT) scan. (B) Representative μCT images of distal femurs (top, longitudinal view; second, axial view of metaphyseal region; third, 3D view of metaphyseal region; bottom, axial 3D view of the cortical region); (C) Quantitative assessment of trabecular and cortical bone mineral density (BMD); (D) Average quantifications of tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), number (Tb.N.), space (Tb.Sp.), the structure model index (SMI), and connectivity density (Conn.D.); (E) Average quantifications of cortical bone thickness (Ct.Th), total cross-sectional area (Tt.Ar), bone area (Ct.Ar), and cortical bone fraction (Ct.Ar/Tt.Ar). Data are representative of three independent experiments, and expressed as the mean ± SD of 6 mice from each group. *, P<0.05; **, P<0.01; ***, P<0.001, NS indicating not significant.
Fig 2.
Aberrant osteoclastogenesis resulted in bone loss in chronic S.japonicum-infected mice.
OCs and bone resorption in mice 13 weeks after schistosome infection. (A) C-terminal telopeptide (CTx) and osteocalcin were quantified in the serum of normal and S.japonicum-infected mice; (B) Histology and histomorphometry for OCs (purple) were performed in tibias. Representative TRAP-stained tibia sections are shown for normal and S.japonicum-infected mice at magnifications of 200×; (C) OC surface per bone surface area (Oc.S/BS) was calculated according to TRAP-stained tibia sections for normal and S.japonicum-infected mice; (D) H&E-stained tibias for normal and S.japonicum-infected mice at 50× magnification; (E) Trabecular areas were measured using Image-Pro Plus software. Values are given as mean ± SD of 6 mice from each group. Data are representative of three independent experiments. *, P<0.05; ***, P<0.001, NS indicating not significant.
Fig 3.
RANKL blockade ameliorated schistosome infection-induced bone loss in mice.
(A-B) Mouse BMs from mice 8 weeks infected with S. japonicum and normal mice were harvested and the cells were surface stained with CD3-PerCP-Cy5.5, B220-PerCP-Cy5.5, NK1.1-PerCP-Cy5.5, CD11b-FITC, CD115-PE, and CD117-APC and analyzed by flow cytometry. Representative flow cytometry data plots and statistics show the percentage and absolute number of OCPs in BM. Data are representative of three experiments with 6 mice in each group; (B) The concentrations of RANKL and OPG in the BM plasma (BMp) of normal mice and mice 8 weeks after infection were quantified by ELISA. Data are representative of three experiments with 6 mice in each group; (C,D) Mouse mesenteric lymph nodes and spleens from mice at 0, 3, 5, 8, or 13 weeks post-infection or age-matched normal mice were harvested and the cells were surface stained with RANKL-PE and analyzed by flow cytometry. Representative flow cytometry data plots and statistics show the frequencies and absolute numbers of RANKL+ cells in mesenteric lymph nodes (C) and spleens (D). Data are representative of three independent experiments with 6 mice in each group; (E) Femurs were isolated from male age-matched normal and S.japonicum-infected mice treated with or without anti-RANKL blocking antibody, and analyzed by X-ray. Representative of X-ray images of femurs. (F) Representative μCT images of distal femurs (top, longitudinal view; second, axial view of metaphyseal region; third, 3D view of metaphyseal region; bottom, axial 3D view of the cortical region); (G) Quantitative assessment of tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV) (n = 4–5, pool of two independent experiments). *, P<0.05, **, P<0.01, ***, P<0.001, NS indicating not significant.
Fig 4.
CD4+ T cells contributed to the increased level of RANKL during schistosome infection.
Mouse mesenteric lymph nodes and spleens from mice at 0, 3, 5, 8, or 13 weeks post-infection or age-matched normal mice were harvested and the cells were surface stained with CD3- PerCP-Cy5.5, CD4-APC, CD19-FITC, and RANKL-PE, and analyzed by flow cytometry. (A, B) Representative flow cytometry data plots and statistics show the distribution of B cells (red), CD4+ T (blue), CD4- T (green), and the other cells (black) within total RANKL+ cells in infected and normal mice. Areas represent the means of percentages of B cells, CD4+ T, CD4- T, and the other cells within total RANKL+ cells. Gated to RANKL+ cells; (C-F) Flow cytometry data plots and statistics show the frequencies and absolute numbers of RANKL+ cells in mesenteric lymph nodes (C-D) and spleens (E-F), Gated to CD3+ cells; The percentages of RANKL+ cells of CD4+ T cells in the graphs were calculated by RANKL+CD4+ / (RANKL+CD4+ + RANKL-CD4+) in the plots. (G-J) Flow cytometry data plots and statistics show the frequencies and absolute numbers of RANKL+ cells with CD19+ cells mesenteric lymph nodes (G-H) and spleens (I-J), Gated to total lymphocytes. Data are representative of three independent experiments with 6 mice in each group. *, P<0.05, **, P<0.01, ***, P<0.001.
Fig 5.
Tfh cells were identified as a predominant cellular source of RANKL during schistosome infection.
Spleens (SP) and mesenteric lymph nodes (LN) were from wildtype (WT) or ICOSL knockout (KO) mice at 8 or 13 weeks post-infected with or without Schistosoma japonicum. (A, B) Representative flow cytometry data plots and statistics show the distribution of Tfh (red), Treg (blue), Th1 (green), Th2 (purple), Th17 (black) and the other CD4+ T cells (orange) within total RANKL+ CD4+ T cells in mice 13 weeks after infection. Areas represent the means of percentages of Tfh, Treg, Th1, Th2, Th17, and the other CD4+ T cells within total RANKL+ CD4+ T cells in infected mice; (C) Flow cytometry data statistics show the frequencies of RANKL+ Tfh cells with CD4+ T cells in LN and SP of normal or infected mice; (D) Representative flow cytometry data plots show the percentages of Tfh cells within CD4+ T cells in SP and LN in WT or ICOSL KO mice infected with or without Schistosoma japonicum; (E) Flow cytometry data statistics show the frequencies of RANKL+ cells within CD4+ T cells in LN and SP in WT or ICOSL KO mice infected with or without Schistosoma japonicum; (F) Flow cytometry data statistics show the frequencies of RANKL+ cells in LN and SP in WT or ICOSL KO mice infected with or without Schistosoma japonicum; (G) Femurs were isolated from WT or ICOSL KO mice infected with or without Schistosoma japonicum, and analyzed by X-ray. Representative of X-ray images of femurs; (H) Representative μCT images of distal femurs from WT or ICOSL KO mice infected with or without Schistosoma japonicum (top, longitudinal view; second, axial view of metaphyseal region; third, 3D view of metaphyseal region; bottom, axial 3D view of the cortical region); (I) Quantitative assessment of tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV). Data are representative of two independent experiments with 4 mice in each group. *, P<0.05, **, P<0.01, ***, P<0.001, NS indicating not significant.