No abstract

No abstract

BACKGROUND Significant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL-21), which is known to support T helper (Th) cell function, is highly expressed within inflamed tissues of IBD patients compared to healthy controls. Also, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD, and the complete induction of IBD in mice has been shown to require IL-21. Given that healthy Tregs are critical for self-tolerance and prevention of IBD, we investigated the metabolic role of IL-21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolic pathways during IBD. METHODS Human Tregs as well as relevant control Th cells were generated from naïve CD4+ T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted transcriptional profiling. Immune phenotyping was assessed by fluorescence-activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry-based targeted metabolomics. Colitis was induced in Rag1-/- (T and B cell deficient) mice by adoptive transfer of pathogenic naïve CD4+ T cells. RESULTS Acute IL-21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed IL-21-induced alterations in the expression of genes associated with glycolysis and OXPHOS metabolism. Prolonged IL-21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of inflammatory Th cytokines such as interferon γ, tumor necrosis factor, IL-17A, and IL-17F. Exploring the mechanisms underlying IL-21-induced effects, we observed reduced expression of inactive glycogen synthase kinase 3 (GSK3) β, a protein known to prevent pyruvate entry into the mitochondria. This reduction in inactive GSK3β in response to IL-21 was accompanied by high abundance of intracellular pyruvate and lactate, as assessed by targeted metabolomics. Importantly, GSK3 inhibition or exogenous membrane-permeable methyl pyruvate abrogated IL-21-induced metabolic rewiring and Treg inflammatory response. Moreover, GSK3 inhibition diminished the metabolic and inflammatory function of Th cells. Lastly, GSK3 inhibition prevented pathogenic CD4+ T cell-induced colitis in mice, as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines. CONCLUSIONS IL-21 potently engages human Tregs in a metabolic state that subsequently augments an inflammatory phenotype. Therefore, desensitizing CD4+ T cells to detrimental cues, such as IL-21, may augment Treg function during human IBD.

Background Significant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL‐21), which is known to support T helper (Th) cell function, is highly expressed within inflamed intestinal tissues of IBD patients compared to healthy controls. In addition, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD. Given that healthy Tregs are critical for self‐tolerance and prevention of IBD, we investigated the metabolic role of IL‐21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolism pathways during IBD pathogenesis. Methods Human Tregs as well as relevant control effector Th cells were generated from naïve CD4+ T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted metabolic transcriptional profiling. Immune phenotyping was assessed by fluorescence‐activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry‐based metabolomics. Ultrastructural analysis of mitochondria was performed by confocal and transmission electron microscopy. Intestinal inflammation was induced in Rag1‐/‐ (T and B cell deficient) mice by the adoptive transfer of pathogenic naïve CD4+ T cells. Results Acute IL‐21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed an IL‐21‐mediated increase in the expression of genes associated with glycolysis and pathways known to support anabolic and OXPHOS metabolism, thus resembling a hypermetabolic state. Furthermore, IL‐21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of effector Th cell‐associated cytokines such as interferon γ, tumor necrosis factor, IL‐17A, and IL‐17F. Exploring the mechanisms underlying IL‐21‐induced effects, we found significant disruption of mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent pyruvate entry into the mitochondria. IL‐21‐induced GSK3β activation was accompanied by a marked increase in intracellular and extracellular metabolites such as pyruvate and lactate, as assessed by metabolomics. Importantly, GSK3 inhibition or supplementation with mitochondrial membrane‐permeable methyl pyruvate broadly abrogated metabolic wiring of and inflammatory responses by IL‐21‐stimulated Tregs and effector Th cells. Collectively, these results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4+ T cells. Lastly, GSK3 inhibition prevented pathogenic CD4+ T cell‐induced colitis in mice as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines. Conclusions IL‐21 potently engages human Tregs in a hypermetabolic state tha...

No abstract