Interleukin 21-induced Mitochondrial Dysfunction Drives Regulatory T Cell Inflammatory Response during Intestinal Inflammation

Adebowale O. Bamidele, Mary R. Sagstetter, Petra Hirsova, Guilherme P. Ramos, Emily Klatt, Maria Westphal, William Faubion

Research output: Contribution to journalArticlepeer-review

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 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 that augments inflammatory cytokine production via induction of mitochondrial dysfunction. Therefore desensitizing CD4+ T cells to detrimental cues, such as IL-21, may also augment Treg function during human IBD.

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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