Fig 1: T Cell-Specific Gfm1 Deletion Attenuates Th17 Cell-Associated Cytokine Production and Mitigates the Development of EAE in MOG-Immunized Mice(A) Generation of the CD4-specific, tamoxifen-inducible Cre mouse line. The loxP-flanked exon 4 of the Gfm1 gene is deleted after exposure to tamoxifen.(B and C) Sorted naive CD4+ T cells from T-Gfm1? mice and littermate haplosufficient controls were pre-treated with 300 nM tamoxifen (4-OHT) or DMSO and maintained in a naive state 12 days prior to being differentiated under Th17 cell skewing conditions for 4 days. Intracellular COX1 and IL-17 were quantified by flow cytometry. (B) Representative histograms (left) of COX1 expression amongst CD4+ T cells. Bar graph (right) shows COX1 median fluorescence intensity (MFI) relative to DMSO. (C) Representative dot plots (left) show the percentage of IL-17+ amongst live CD4+ T cells. Bar graph (right) shows pooled means of the percentage of IL-17+ CD4+ T cells.(D and E) T-Gfm1? mice and haplosufficient controls were immunized with MOG35–55 in CFA and pertussis toxin to induce EAE. Tamoxifen was administered twice to every mouse, 3 and 5 days after induction of EAE. The graphs show the (D) mean clinical scores and (E) CNS-infiltrating cells for T-Gfm1? mice and T-Gfm1het haplosufficient controls.Bar graphs (B and C) contain the pooled means from the technical replicates of three individual experiments, with error bars representing the SD of the pooled means. Plots are obtained from the pooled data of two (D) and three (E) individual experiments, with error bars showing the (D) SEM of the pooled scores. Statistical significance was determined (D) using two-way ANOVA with Bonferroni multiple corrections test or with (E) an unpaired t test per condition. *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figure S6.
Fig 2: Linezolid and Other RAbos Disrupt Th17 Effector Cell Function by Targeting Mitochondrial Translation(A) Naive mouse CD4+ T cells cultured under Th17 cell polarizing conditions with Linezolid, Tigecycline, or Thiamphenicol. After 96 h of culture, cells were stained for intracellular IL-17. Bar graphs represent % of IL-17+ cells amongst live CD4+ T cells (top) and % viable CD4+ T cells (bottom).(B) Human naive T cells were stained for intracellular IL-17 (right) after being differentiated for 6 days in the presence of the indicated concentrations of Linezolid. Graphs contain the data for each of six individual donors, indicated by connected dots: % of IL-17+ cells amongst live CD4+ T cells (left) and % of viable CD4+ T cells (right).(C) Mitochondrial translation products in EL-4 cells pre-incubated for 1 h with Linezolid or DMSO and radioactively labeled with [35S]-methionine.(D) SDHA, COX1, ND1, and ß-actin quantified by western blot in mouse Th17 cells after 96 h of culture.(E) Naive mouse CD4+ T cells cultured under Th17 cell polarizing conditions in the presence of Linezolid, with and without the NLRP3 inflammasome inhibitor MCC950 (10 µM).Plots are representative of two (C) or three (D) experiments, and bar graphs are pooled means of technical replicates from three (A, middle and right and E) or four (A, left) independent experiments, with error bars representing the SD of the pooled means. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S1.
Fig 3: Arg C Treatment Selectively Affects High Proliferating Cells, Leading to Mitochondrial DysfunctionNaive CD4+ T cells were cultured under Th17 cell polarizing conditions in the presence of Arg C (60 nM), Arg F (60 nM), Linezolid (100 µM), or vehicle (DMSO).(A) SDHA, ND1, and COX1 were quantified by western blot at the indicated time points.(B) NAD+/NADH ratio measured at the indicated time points.(C and D) (C) Representative plot of oxygen consumption rate (OCR) at 72 h of culture and (D) respective basal (before drug addition) and maximal (after FCCP addition) respiratory activity of the cells. OCR is reported as picomoles (pmol) of O2 per minute. Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A.(E) Representative electronic microscopy images show mitochondria morphology of Arg C- (60 nM) or DMSO-treated naive T cells from 48 h of culture.(F) Cells were stained for intracellular IL-17. The graph shows % of IL-17+ cells amongst total live CD4+ T cells after 48, 72, and 96 h of culture.Statistical significance was determined using two-way ANOVA with Bonferroni multiple corrections test. Representative results of three (A), five (C), and one (E) experiments are shown. Results are pooled means of technical replicates from three (B and F) and five (D) independent experiments, with error bars representing the SD of pooled means. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figures S4 and S5.
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