Semaphorin3A Rewires CD4+ T-Cell Metabolism via AKT/mTORC1 Inhibition in Health and Rheumatoid Arthritis

Figures

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  Figure 1. Sema3A downregulates OXPHOS and mitochondrial membrane potential. (A) Shows the measurement of three parameters: basal and maximal respiration as well as ATP production. Results were obtained from 11 healthy donors. (B) Measuring ATP production using an ATP determination kit after 12 h of activation. Results were obtained from 11 healthy donors. (C) Mitochondrial staining using TMRE is seen in red, and double-stranded DNA staining using PicoGreen is seen in green. Yellow is the overlap between red and green. Images were captured using a 60× oil objective lens. Scale bar = 20 µm (D) Statistical analysis of fluorescence intensity/area was obtained from 200 cells per group from 6 healthy donors. Mann–Whitney and Kruskal–Wallis tests. p value * < 0.05, ** < 0.005, *** < 0.0005.

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  Figure 2. Sema3A downregulates glycolysis and increases FAO in activated T cells. (A) Shows the measurement of glycolysis and glycolytic capacity. Results were obtained from 11 healthy controls. (B) Demonstrates a representative ECAR graph of activated T cells treated with NSPI-CM, Sema3A-CM, or acute injection. (C) Shows the measurement of glycolysis and glycolytic capacity. Results were obtained from 8 healthy control. (D) Fatty acid oxidation assay shows the FAO of unstimulated and activated T cells cultured with or without Sema3A. Results were obtained from 9 healthy donors. Kruskal–Wallis test. p value * < 0.05, ** < 0.005, *** < 0.0005.

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  Figure 3. Sema3A inhibits glucose uptake and glycolysis but does not induce oxidative stress. (A–F) Glycolysis and TCA cycle metabolites and intermediates. (G,H) NAD+/NADH and GSH/GSSG ratios. Results were obtained from 9 healthy donors from 3 independent repetitions. Kruskal–Wallis test was used. p value * < 0.05, ** < 0.005, *** < 0.005, ns—not significant.

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  Figure 4. Sema3A inhibits AKT/mTORC1 signaling pathway. (A–D) Sema3A’s effect on the intracellular signaling pathway was assessed by immunoblotting for the indicated phosphoproteins in unstimulated and 24 h-activated T cells treated with EB or purified Sema3A protein. Results were obtained from 3 independent experiments. Kruskal–Wallis. p value * < 0.01, ** < 0.001, ns—not significant.

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  Figure 5. Sema3A inhibits OXPHOS, glycolysis, and ATP production while increasing FAO in CD4+ T cells of RA patients. (A) Shows the measurement of the basal and maximal respiration. (B) Shows the spare respiratory capacity. (C) ECAR at baseline and after sequential treatment with oligomycin and FCCP. GR, glycolytic reserve. Results were obtained from 10 RA patients. (D) Shows the measurement of the glycolysis and glycolytic capacity. Results were obtained from 5 RA patients. (E) The graph shows the absolute and relative ATP production of unstimulated and stimulated T cells after a 12 h activation period with or without Sema3A. Results were obtained from 7 RA patients. (F) Fatty acid oxidation assay shows the FAO of unstimulated and activated T cells cultured with or without Sema3A. Results were obtained from 10 RA patients. Mann–Whitney and Kruskal–Wallis tests. p value * < 0.05, ** < 0.005, *** < 0.0005.