Journal: Aging (Albany NY)

Article Title: Let-7f-5p suppresses Th17 differentiation via targeting STAT3 in multiple sclerosis

doi: 10.18632/aging.102093

Figure Lengend Snippet: Let-7f-5p expression is decreased during MS and downregulated in Th17 cells. ( A ) CD4 + T cells were sorted from whole blood of multiple sclerosis (MS) patients (n = 6) and healthy controls (HC) (n = 6). The expression (let-7f-5p vs. U6) of let-7f-5p was analyzed by RT-PCR. ( B ) Naive CD4 + T cells were sorted from mouse spleens and maintained in Th17 differentiation medium. After 24, 48 and 96 h, the expression (let-7f-5p vs. U6) of let-7f-5p was analyzed by RT-PCR. (* p < 0.05, ** p < 0.01)

Article Snippet: Subsequently, a mouse Th17 staining kit (MULTI SCIENCES, China) was used to analyze the cells after 96 h by FCM (Beckman Coulter, USA) following the manufacturer’s recommendations.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Aging (Albany NY)

Article Title: Let-7f-5p suppresses Th17 differentiation via targeting STAT3 in multiple sclerosis

doi: 10.18632/aging.102093

Figure Lengend Snippet: Let-7f-5p suppresses Th17 differentiation. ( A ) RT-PCR analysis of the expression of let-7f-5p in induced mouse Th17 cells transfected with 100 pmol of let-7f-5p mimic (pre-let-7f-5p), let-7f-5p inhibitor (anti-let-7f-5p) or negative control RNA (pre-miR-control or anti-miR-control). ( B ) The effect of let-7f-5p on Th17 differentiation. Naive CD4 + T cells were sorted from C57BL/6J mice, maintained in Th17 differentiation medium and transfected with pre-let-7f-5p, anti-let-7f-5p or negative control RNA for 96 h. The frequency of CD3 + CD4 + IL17A + T cells is shown. The statistical analysis of the percentage of CD3 + CD4 + IL17A + T cells is shown in ( D ). Data represent means ± SD from three independent experiments. ( C , E ) The effect of let-7f-5p on Th17 apoptosis. Induced mouse Th17 cells were cotransfected with pre-let-7f-5p, anti-let-7f-5p or negative control RNA. Cells were collected after 96 h and analyzed by flow cytometry. ( F ) Th17-specific gene expression in the transfected cells in ( A ) was analyzed by RT-PCR, and presented relative to GAPDH expression. ( G ) IL-17A expression in the culture supernatants of transfected cells in ( A ) was analyzed by ELISA. (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Subsequently, a mouse Th17 staining kit (MULTI SCIENCES, China) was used to analyze the cells after 96 h by FCM (Beckman Coulter, USA) following the manufacturer’s recommendations.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control, Control, Flow Cytometry, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Let-7f-5p suppresses Th17 differentiation via targeting STAT3 in multiple sclerosis

doi: 10.18632/aging.102093

Figure Lengend Snippet: STAT3 is a direct target of let-7f-5p. ( A ) RT-PCR analysis of the expression of let-7f-5p in Jurkat cells transfected with 100 pmol of pre-let-7f-5p, anti-let-7f-5p or negative control RNA. ( B ) Western blotting analysis to detect STAT3 and p-STAT3 protein levels in Jurkat cells and induced mouse Th17 cells (Th17 cell) transfected with 100 pmol of the pre-let-7f-5p, anti-let-7f-5p or negative control RNA. ( B ): representative image; ( C , D ) quantitative analysis. ( E ) RT-PCR analysis of STAT3 mRNA levels in Jurkat cells and induced mouse Th17 cells transfected with 100 pmol of the pre-let-7f-5p, anti-let-7f-5p or negative control RNA. ( F ) Luciferase plasmids contain the wild-type (Wt) and mutant 3’-UTR of STAT3 and sequences of Wt and Mut target sites for let-7f-5p in the 3’-UTR of STAT3 are shown. ( G ) Dual luciferase reporter assay was used to confirm the direct recognition of the STAT3 3’-UTR by let-7f-5p. Wt and Mut luciferase plasmids were cotransfected into 293T cells with 100 pmol of pre-let-7f-5p, anti-let-7f-5p or negative control RNA. The β-galactosidase (β-gal) expression plasmid was used as a transfection control. ( H ) The working model of the role of let-7f-5p in Th17 differentiation. (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Subsequently, a mouse Th17 staining kit (MULTI SCIENCES, China) was used to analyze the cells after 96 h by FCM (Beckman Coulter, USA) following the manufacturer’s recommendations.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control, Western Blot, Luciferase, Mutagenesis, Reporter Assay, Plasmid Preparation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 1. TH17 cells derived in vitro show different metabolic states. (A) RT-PCR analysis of key glycolytic pathway genes in TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; gene expression was normalized to Actb. (B) ECAR of TH17 (b) and TH17 (23) cells differentiated for 96 h assessed by a glycolytic stress test. ECAR was measured under basal conditions and in response to glucose (10 mM), oligomycin (1.0 mM), and 2-deoxyglucose (2-DG) (50 mM). (C) Principal component analysis (PCA) analysis of identified metabolites of TH17 (b) and TH17 (23) cells (n = 5) differentiated in vitro for 48 h by metabolomics. (D) Metabolomics analysis of TH17 (b) and TH17 (23) cells differentiated in vitro for 48 h; the top differentially observed metabolites are shown in heat map. Data are representative of three independent experiments (A and B). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 2. TH17 cells show differential metabolic pathway gene activation in vivo. (A) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with differential TH17 signature genes highlighted (black dot). differentially-expressed genes are filtered as false discovery rate (FDR) , 0.05 and FC $ 1.5 from DESeq2. (B) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glycolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (C) Scatter plot shows differential expressed genes between CNS TH17 (red) and ileum TH17 (blue) cells with glutaminolysis pathway genes highlighted (black dot) and differential genes labeled. DEGs are filtered as in (A). (D) CNS TH17 and ileum TH17 cells depend on distinct metabolic pathways. Numbers indicate the differentially expressed genes and total genes for each selected pathway. (E) KEGG pathway enrichment for CNS TH17 cell highly expressed genes. (F) KEGG pathway enrichment for ileum TH17 cell highly expressed genes. Color represents log-transferred FDR, and dot size represents the number of differentially expressed genes observed for this pathway (E and F).

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Activation Assay, In Vivo, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 3. TH17 cells derived in vitro show discrete chromatin states. (A) Scatter plot for differentially opened/closed regions (OCRs) between TH17 (23) (red) and TH17 (b) cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between TH17 (23) and TH17 (b) cells. (C) The enriched transcription factor motifs identified from top 1000 differentially OCRs between TH17 (23) cells and TH17 (b) cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals for key glycolytic genes in TH17 (23) and TH17 (b) cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Derivative Assay, In Vitro, Labeling, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 4. TH17 cells show discrete chromatin states in vivo. (A) Scatter plot for differentially opened/closed regions (OCRs) between CNS TH17 (red) and ileum TH17 cells (blue) with differential numbers labeled. (B) ATAC-seq reads density heatmap around the OCRs (shows the peak summit 6 1 kb region) between CNS TH17 and ileum TH17 cells. (C) The enriched transcription factor motifs identified from top 1000 differential OCRs between CNS TH17 cells and ileum TH17 cells; color bar represents log-transferred p values. Right panel shows the identified motif sequences for key regulators. (D) WashU Epigenome Browser visualization of differentially OCR signals with corresponding gene expression levels for key TH17 signature genes and metabolic genes in CNS TH17 and ileum TH17 cells. Arrows represent the NF-kB binding motif sites.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: In Vivo, Labeling, Gene Expression, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 5. Identification of a miR-21–Peli1–c-Rel pathway controlling pathogenic TH17 cell glycolysis. (A) WashU Epigenome Browser visualization of ATAC-seq signals across selected microRNA genome loci in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells. (B) Relative expression of microRNA in ileum homeostatic and CNS-infiltrated pathogenic TH17 cells (n = 3). (C) KEGG pathway enrichment for genes downregulated in miR-212/2

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of Glycolysis in Pathogenic T H 17 Cells through Targeting a miR -21-Peli1 -c-Rel Pathway Prevents Autoimmunity.

doi: 10.4049/jimmunol.2000060

Figure Lengend Snippet: FIGURE 6. Pharmaceutical inhibition c-Rel–mediated glycolysis in pathogenic TH17 cells prevents autoimmunity. (A) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (B) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (C) Western blot results for c-Rel and RelA from TH17 cells differentiated under indicated conditions. (D) Normalized RT-PCR results for key glycolytic pathway genes in TH17 (23) cells differentiated in vitro for 48 h, treated with H2O or 500 mg/ml PTXF for 16 h. (E) ECAR of TH17 (23) cells differentiated for 48 h and then were treated with H2O or 500 mg/ml PTXF for 16 h, assessed by a glycolysis stress test. (F) Basal and maximal glycolytic capability of TH17 (23) cells differentiated for 48 h and then were treated with H2O or PTXF for 16 h. (G) EAE development in C57BL/6 mice, i.p. PBS or 100 mg/kg PTXF per mouse (n = 6) from day 7 to day 14. (H) EAE development in recipient C57BL/6 mice (sublethal irradiation; n = 6) i.p. with pathogenic TH17 (23) cells differentiated from naive 2D2 CD4+ T cells for 96 h and then treated with H2O or 500 mg/ml PTXF for 16 h. Data shown are one experiment representative of three independent experiments (A–C, G, and H). Data are representative of three independent experiments (D–F). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined by two-tailed unpaired t test.

Article Snippet: Naive CD4+ T cells were stimulated with plate-bound anti-CD3 mAb (catalog no. 16-0038-85, 5 mg/ml; Thermo Fisher Scientific) in the presence of antiCD28 mAb (catalog no. 16-0289-85, 2 mg/ml; Thermo Fisher Scientific) in a 48-well plate under neutral condition (catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), TH17 (b) condition (catalog no. 7666-MB-005, 2 ng/ml TGF-b1; R&D Systems; catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific), and TH17 (23) condition (catalog no. 406-ML-025, 20 ng/ml IL-6; R&D Systems; catalog no. 401-ML-025, 20 ng/ml IL-1b; R&D Systems; catalog no. 1887-ML-010, 20 ng/ml IL-23; R&D Systems; catalog no. 16-7041-95, 10 mg/ml anti–IL-4 mAb; Thermo Fisher Scientific; and catalog no. 16-7311-38, 10 mg/ml anti–IFN-g mAb; Thermo Fisher Scientific).

Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Irradiation, Two Tailed Test

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: