Therapeutic Potential of NRF2 activating drug RTA-408 in suppressing T-cell effector responses and inflammatory bowel disease

Targeting T-cells has emerged as a promising therapeutic strategy for treating inflammatory diseases. Triterpenoids, classified as antioxidant inflammatory modulator (AIM) compounds, are known to influence immune cell functions. RTA-408 is an AIM drug that was approved last year for the treatment of neurological disorders. In this study, we demonstrate the anti-inflammatory effects of RTA-408, on both mouse and human T-cells. At picomolar concentrations, RTA-408 robustly induces NRF2 activation in T-cells and reduces T-cell activation, proliferation, and cytokine functions. In in vitro activated CD4 and CD8 T-cells isolated from mice and human blood, we observed a significant decrease in proliferation, and expression of inflammatory cytokine IFN-γ, and cytotoxic granules (perforin and granzyme B). Mechanistically, treatment with RTA-408 significantly suppressed glycolysis and mitochondrial respiration in human T-cells, while increasing the expression of pentose phosphate pathway genes, such as transketolase (TKT) and transaldolase (TALDO). These findings suggest that RTA-408 modulates T-cell metabolism. Furthermore, treatment of circulating T-cells from inflammatory bowel disease (IBD) patients with RTA-408 caused a reduction in expansion and the expression of the inflammatory cytokine IL-17. Our results highlight the therapeutic potential of RTA-408 in treating chronic inflammatory conditions like IBD by modulating T-cell activity. Human T-cells were activated in vitro in presence of anti-CD3 and anti-CD28 to mimic T-cell activation with 0 and 100pM of RTA-408 (MedChemExpress). After 72 hours, total RNA was extracted using RNeasy Plus Micro kit (Qiagen) and processed at the Genomics Core Facility at the University of Kansas Medical Center. 100ng of the RNA was used to perform Agilent TapeStation QC analysis to verify RNA integrity and generate RNA Integrity number (RIN). Using Nova-Seq 6000 S1 200 cycle reagent kit, 25 million pair-ended reads were recorded for construction of mRNA libraries. For bioinformatic analysis, following quality check through fastQC data were aligned to human reference genome using RSEM (RNA-Seq by Expectation Maximization) resulting in the generation of gene count matrix. Further differential expression analysis was performed between untreated human T-cells and 100pM RTA-408 treated human T-cells for gene ontology (GO) enrichment using ClusterProfiler R-package, to identify significant biological processes, molecular pathways and metabolic pathways associated with the samples. The enrichment results were prioritized based on significance and the top GO terms were visualized using dotplots. Box-plots for genes were also generated using ggplot2 to illustrate the variability of the genes between the 2 groups with mean P-values annotated on each plot, respectively.