Hirsova, Petra

Abstract Background Inflammatory regulatory T cells (Tregs) are one of the hallmarks of therapy resistance. Yet mechanisms leading to inflammatory Treg development and its contribution to inflammatory bowel disease (IBD) are unexplored. Methods Tregs derived from healthy PBMCS were characterized by immune and mitochondrial phenotyping. Transcripts were validated in dataset from IBD intestinal lesions. Wildtype (WT) and IL21 receptor-deleted (Il21r−/−) Tregs were examined in murine models of CD4+ T cell-induced colitis. Results Through in vitro cytokine challenge of Tregs and subsequent analysis, we found that IL21 induces Tregs co-expressing TNF, IFNγ and IL17. Transcriptomic and metabolomic integration revealed upregulation of mRNA and metabolites associated with glycolysis, and amino acid, nucleotide and lipid metabolism. Moreover, IL21-induced metabolic transcripts were more enriched in Treg cluster within scRNA-seq dataset from intestinal lesions of anti-TNF non-responders vs. non-responders. Mechanistically, cell imaging and real-time bioenergetic analysis revealed disruption to mitochondria-ER ultrastructure and function, coinciding with glycogen synthase kinase 3β (GSK3β) activation and glycolytic switch rather than pyruvate-mediated oxidative phosphorylation. GSK3β inhibition or methyl pyruvate supplementation rescued IL21-induced metabolic and inflammatory phenotypes. In contrast to WT Tregs, murine Il21r−/− Treg transfer efficiently prevented and rescued CD4+CD45Rbhigh T cell-induced colitis. Conclusions IL21-induced metabolic dysfunction can incapacitate Treg immunosuppressive function. Desensitizing Tregs to IL21 through various therapeutic approaches can alleviate IBD. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) K01DK124358, the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery Career Development Award, and David F. and Margaret T. Grohne Cancer Immunology and Immunotherapy (to AOB).

Abstract Background Significant proportion of inflammatory bowel disease (IBD) patients respond inconsistently to therapies, underscoring disease complexity. Interleukin 21 (IL21) is highly expressed during IBD. In addition to T helper (Th) cells, inflammatory regulatory T cells (Tregs) have been linked to refractory IBD. Thus, we explored the metabolic role of IL21 in inducing Treg dysfunction and IBD. Methods Human Tregs and Th cells were subjected to transcriptional profiling and metabolic phenotyping. Colitis was induced in Rag1−/− mice by naïve CD4 T cell adoptive transfer. Results IL21 stimulation of human Tregs induced glycolysis and OXPHOS. In agreement, IL21 enhanced the expression of genes associated with glycolysis, anabolism, and OXPHOS, leading to inflammatory cytokine production. Mechanistically, we found disruption to mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent mitochondrial pyruvate metabolism. IL21-induced GSK3β activation was accompanied by pyruvate and lactate buildup. Notably, GSK3 inhibition or supplementation with membrane-permeable methyl pyruvate abrogated metabolic wiring of and inflammatory responses by IL21-stimulated Tregs and effector Th cells. 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 Mouse Colon Histology Index and serum inflammatory cytokines. Conclusions IL21 engages Tregs in a hypermetabolic state that augments inflammatory cytokine production. Therefore, desensitizing CD4 T cells to IL21 may also augment Treg function during IBD. Supported by NIDDK award K01DK124358, the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery.

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.