Introduction: The molecular mechanism by which effector CD4⁺ T helper (Th) cells remain activated to possibly mediate resistance to biologics and consequent refractory human inflammatory bowel disease (IBD) is not clear. We recently demonstrated a role for glycogen synthase kinase 3(GSK3)β in promoting the development of inflammatory-like regulatory T cells (Tregs) through metabolic rewiring; however, the role of GSK3β in effector Th cells is largely undefined. Here we investigated the metabolic role of GSK3β in sustaining effector Th Type 1 (Th1) and interleukin (IL)-17-producing (Th17) cell-mediated chronic intestinal inflammation. Methods: We utilized a Seahorse XF analyzer to examine the metabolic state of human Th1 and Th17 cells in real-time in the presence or absence of GSK3α/β inhibitor (LY2090314). Effector cytokine production was assessed and analyzed by fluorescence-activated cell sorting (FACS). Analysis of publically available single-cell RNA sequencing (scRNA-seq) dataset from inflamed ileal tissue of IBD patients responsive or non-responsive to biologic therapy (anti-TNF) was performed. Colitis was induced in immunodeficient (RAG1^-/-) mice via intraperitoneal injection of pathogenic CD4⁺ CD45RB⁺ T cells, followed by the injection of vehicle or GSK3α/β inhibitor (LY2090314). Murine serum cytokines were measured by FACS analysis using a cytometric bead array. Results: We found that inhibition of GSK3β enzymatic activity with LY2090314 reduced the glycolytic capacity of Th1 and Th17 cells, while concomitantly increasing the oxygen consumption rate of these cells. Notably, This LY2090314 treatment impaired the production of pathogenic effector cytokine by Th1 and Th17 cells. This suggests that GSK3β promotes glucose-derived pyruvate into lactate (glycolysis), which then results in effector T cell response in vitro (inflammatory cytokine production), and this glycolytic function was impaired by LY2090314. Moreover, ileal CD4⁺ T helper cells of non-responsive IBD patients display an enhanced glycolytic transcriptional program compared to responders. Lastly, treatment of Th1 and Th17-induced colitis mice with the GSK3β inhibitor (LY2090314) reduced clinical symptoms as well as colonic tissue inflammation and damage compared to vehicle-treated, as evidenced by a reduction in disease activity index and mouse colon histology index. Conclusions: GSK3β inhibition impairs effector CD4⁺ T cell activity and function in vitro and in vivo, potentially via metabolic reprogramming towards mitochondrial respiration. GSK3β is a metabolic checkpoint associated with T cell-driven inflammation.

Leena Abdelrahman

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