Ribosome-Targeting Antibiotics Impair T Cell Effector Function and Ameliorate Autoimmunity by Blocking Mitochondrial Protein Synthesis

Abstract

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.

Keywords: Argyrin; Linezolid; NAD+; T cells; antibiotics; autoimmunity; elongation factor G1; mitochondria; mitochondrial translation; ribosome-targeting.

Graphical abstract

Figure 1

Linezolid Inhibits the Onset of T Cell-Mediated Autoimmune Diseases EAE in MOG-Immunized Mice (A) EAE clinical score of mice treated daily with Linezolid, Vancomycin, or untreated. (B) Distribution of disease severity: no EAE: score < 1, mild EAE: 1 ≤ score < 3, severe EAE: score ≥ 3. (C) Frequency (top) and number (bottom) of CD4⁺ T cells isolated from the central nervous system of Linezolid-treated, Vancomycin-treated, and untreated mice. Plots are obtained from the pooled data of three independent experiments. (A–C) individual experiments, with error bars showing the SEM of the pooled scores (A). Statistical significance was determined using two-way ANOVA with Bonferroni multiple corrections test compared to untreated mice. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 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 β-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.

Figure 3

Inhibiting Mitochondrial Translation with Arg C Inhibits Th17 Cell Function (A) Chemical structure of Arg C. (B) Left: mouse naive CD4⁺ T cells cultured for 4 days under Th17 cell polarizing conditions in the presence of Arg C or vehicle (DMSO). Cells were stained for intracellular IL-17 and gated on live CD4⁺ T cells. Bar graphs show % of IL-17⁺ cells (top left) amongst total live CD4⁺ T cells (bottom left). Right: human CD4⁺ CD45RO⁻ naive T cells isolated from cord blood were cultured for 6 days under Th17 cell polarizing conditions and treated with Arg C or vehicle (DMSO). Cells were stained for intracellular IL-17, gated on live CD4⁺ T cells. Graphs contain the data of five individual donors, indicated by connected dots. The values represent % of IL-17⁺ cells amongst live CD4⁺ cells (above) and the % of viable CD4⁺ cells (below). (C) Mitochondrial translation products in EL-4 cells pre-incubated for 1 h with Arg C, Arg F, or vehicle (DMSO) and radioactively labeled with [³⁵S]-methionine. (D–F) Mouse naive CD4⁺ T cells were cultured under Th17 cell polarizing conditions in the presence of Arg C (60 nM), Linezolid (100 μM), or DMSO. (D) Venn diagrams showing the number of genes differentially up (left) or downregulated (right) by Linezolid versus Arg C, DMSO versus Arg C, and DMSO versus Linezolid. The overlapping genes are those found in both conditions (E). Top canonical pathways up or downregulated by both Arg C and Linezolid. (F) Heat maps showing expression of genes among three selected relevant pathways. Results (B, left) are the pooled means of technical replicates from three independent experiments, with error bars representing the SD of the pooled means. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Representative results of two (C) experiments. Genes (E) are included if they were found to be up or downregulated in both independent experiments performed. See also Figures S2 and S3.

Figure 4

Arg C Treatment Selectively Affects High Proliferating Cells, Leading to Mitochondrial Dysfunction Naive 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.

Figure 5

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-Gfm1Δ 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-Gfm1Δ mice and haplosufficient controls were immunized with MOG_35–55 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-Gfm1Δ mice and T-Gfm1^het 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.

Figure 6

The Availability of Electron Acceptors Restores Cytokine Production in Th17 Cells with Impaired Mitochondrial Respiration (A) Image depicting the pyruvate-lactate cycling and consequent intracellular regeneration of NAD⁺ enabled by exogenous addition of Lox (25 mU) and Cat (50 U). (B–E) Naive CD4+ T cells were cultured under Th17 cell polarizing conditions in the presence of the indicated drugs. The culture medium was supplemented with (B and C) Cat alone (Cat) or in combination with Lox (LoxCat), (D) pyruvate or a-ketobutyrate (both 10 mM), or (E) DS18561882, and (B, D, and E) cytokine production and (c) NAD⁺/NADH ratios were measured. Statistical significance was determined using multiple t tests (A, B, D, and E) or a two-way ANOVA with Bonferroni multiple corrections test (C). Bar graphs contain the pooled means from the technical replicates of three (D), four (B and C), and six (E) individual experiments, with error bars representing the SD of pooled means. Significance was determined as follows: ^∗p < 0.05,^∗∗p < 0.01, and ^∗∗∗p < 0.001.