Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Image of the intestine of Kaede mice before and after ex vivo photoconversion of the dissected organ by exposure to a 390 nm wavelength light for 2 minutes. (B) Representative flow cytometric analysis of T cells harvested from photoactivated (PA) PPs and BM of Kaede mice subjected or not subjected to in vivo photoconversion. Plots show the relative frequency of KaedeR total T cells, TNF+ T cells, and Th17 cells. Ten-week-old female SFB+ Kaede mice were subjected to surgical laparotomy to access the PPs in the distal SI. PP cells were photoconverted by exposing them to a 390 nm light for 2 minutes. Mice were sacrificed immediately after the photoconversion. (C–E) Relative frequency of KaedeR total T cells, TNF+ T cells, and Th17 cells in PPs from sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (F and G) Relative and absolute frequency of KaedeR total T cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (H and I) Relative and absolute frequency of KaedeR total TNF+ T cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. (J and K) Relative and absolute frequency of KaedeR total Th17 cells in the BM of sham-operated mice and mice that had undergone ovx 24 hours and 48 hours after photoconversion. For panels C–K, 10-week-old Kaede mice were subjected to either ovx or sham surgery. After 2 weeks, mice underwent surgical laparotomy and PP cells were photoconverted. Mice were sacrificed 24 or 48 hours later and the number of KaedeR T cells in PPs and BM measured by flow cytometry. n = 6–14 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: Ex Vivo, In Vivo, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Representative flow cytometry plot and frequency of EGFP+ Th17 cells in the BM of WT and Tnf–/– sham-operated mice and mice that had undergone ovx. (B) Relative and absolute frequency of EGFP+ Th17 cells in the BM of WT and Tnf–/– mice subjected to sham surgery or ovx 2 weeks before adoptive transfer of IL-17A-EGFP+ cells. (C) BM CD4+ T cell EGFP MFI. In these experiments, EGFP+CD4+ Th17 cells were injected i.v. into WT and Tnf–/– mice that had been subjected to sham operation or ovx 14 days before the T cell transfer. Twenty-four hours after transfer, EGFP+CD4+ T cells (EGFP+ Th17 cells) were enumerated by flow cytometry in BM of recipient mice. (D) Relative frequency of BM Vβ14+ Th17 cells in WT and Tnf–/– mice. (E and F) Relative and absolute frequency of BM of total Th17 cells in WT and Tnf–/– mice. (G) BM Ccl20 transcript levels in Tnf–/– mice. (H) Relative frequency of BM Vβ14+ Th17 cells in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. (I and J) Relative and absolute frequency of BM of total Th17 cells in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. (K) BM Ccl20 transcript levels in Tcrβ–/– mice reconstituted with WT T cells or Tnf–/– T cells. n = 5–6 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: Flow Cytometry, Adoptive Transfer Assay, Injection

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the number of PP and BM TNF+ T cells and on the level of Tnf transcripts in mice treated with FTY720. (B) Effects of ovx on the number of PP and BM Th17 cells and on the level of Il17a transcripts in mice treated with FTY720. (C) Effects of ovx on BV/TV, Tb.Th, Tb.N, and Tb.Sp in mice treated with FTY720. (D) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp in mice treated with FTY720. (E) Effects of ovx on serum CTX levels and serum osteocalcin levels in mice treated with FTY720. (F) Effects of ovx on femoral Ct.Ar and Ct.Th in mice treated with FTY720. n = 10 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the frequency of BM Th17 cells and Vβ14+ Th17 cells and on the level of Il17a transcripts. (B) Effects of ovx on the frequency of BM TNF+IL-17+ T cells. (C) Effects of ovx on the number of BM TNF+ T cells and on the level of Tnf transcripts. (D) Effects of ovx on femoral BV/TV, Tb.Th, Tb.N, and Tb.Sp. (E) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp. (F) Effects of ovx on serum CTX levels and serum osteocalcin levels. (G) Effects of ovx on femoral Ct.Ar and Ct.Th. Mice were treated with anti-CCL20 Ab or irrelevant (Irr.) Ab. n = 5 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF + T cells and Th17 cells

doi: 10.1172/JCI143137

Figure Lengend Snippet: (A) Effects of ovx on the number of BM TNF+ T cells and on the level of Tnf transcripts in WT mice and Cxcr3–/– mice. (B) Effects of ovx on the BM cell transcript levels of Ccl20 in WT mice and Cxcr3–/– mice. (C) Effects of ovx on the number of BM Th17 cells and on the levels of Il17a transcripts in WT mice and Cxcr3–/– mice. (D) Effects of ovx on femoral BV/TV, Tb.Th, Tb.N, and Tb.Sp in WT mice and Cxcr3–/– mice. (E) Effects of ovx on spinal BV/TV, Tb.Th, Tb.N, and Tb.Sp in WT mice and Cxcr3–/– mice. (F) Effects of ovx on serum CTX levels and serum osteocalcin levels in WT mice and Cxcr3–/– mice. (G) Effects of ovx on femoral Ct.Ar and Ct.Th in WT mice and Cxcr3–/– mice. n = 5 mice per group. Data are expressed as mean ± SEM. All data were normally distributed according to the Shapiro-Wilk normality test and analyzed by 2-way ANOVA and post hoc tests applying Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, compared with the indicated group.

Article Snippet: EGFP – naive CD4 + T cells were cultured under Th17 polarizing conditions for 4 days using the Mouse Th17 Cell Differentiation Kit (R&D Systems) to generate EGFP + CD4 + Th17 cells.

Techniques: