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Next Generation Sequencing analysis using splenic CD8 T cells isolated from Wild Type-OT-I (WT) and NIK -CD4Cre-OT-I (KO) mice

ABSTRACT: Metabolic reprograming towards aerobic glycolysis is a pivotal mechanism that shapes immune responses. While deregulated T cell metabolism is associated with autoimmune diseases, metabolic deficiency contributes to T cell exhaustion in tumor microenvironment. Here we describe a posttranslational mechanism of glycolysis regulation mediated by the NF-kB-inducing kinase (NIK). NIK deficiency impairs glycolysis induction, rendering CD8 effector T cells hypofunctional with features of exhaustion in tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8 T cell metabolism and prevents exhaustion, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. Interestingly, although NIK is known as a kinase mediating activation of noncanonical NF-kB, NIK regulates T cell metabolism via an NF-kB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK deficiency causes autophagic degradation of HK2, at least in part due to aberrant ROS accumulation. NIK phosphorylates, and maintains the activity of, glucose-6-phosphate dehydrogenase (G6PD), an enzyme mediating production of the antioxidant NADPH required for preventing ROS accumulation and oxidative stress. We provide genetic evidence that the G6PD-NADPH redox system has a vital role in regulating HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a posttranslational mechanism of metabolic regulation involving the G6PD-NADPH redox system.

ORGANISM(S): Mus musculus

PROVIDER: GSE155576 | GEO | 2020/10/02

REPOSITORIES: GEO

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Project description:Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB–inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease. This study compares nine t(11;18)-positive MALT lymphomas (8 from the stomach and 1 from lung) and eight translocation negative MALT lymphomas (all from the stomach) using gene set enrichment analysis (GSEA). All cases were subjected to Affymetrix U133A and U133B microarray analysis. The cases used in this study are the same cases used for the study by Hamoudi et al. (2010) entitled "Differential expression of NF-kB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism" with GEO reference number: GSE18736 and PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/20520640 All cases were subjected to non-specific filtering to eliminate non-variant probes, then the U133A and U133B probes were collapsed and the collapsed set was subjected to GSEA using the NF-kB target gene set as described in Hamoudi et al. (2010) study mentioned above. The 34 samples in this study are identical to the ones done in the previous series except that the gene set enrichment was done on just those 34 samples and not the complete set.

Project description:The NF-κB pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic beta cell dysfunction in the metabolic syndrome. While canonical NF-κB signaling is well studied, there is little information on the divergent non-canonical NF-κB pathway in the context of pancreatic islet dysfunction in diabetes. Here, we demonstrate that pharmacological activation of the non-canonical NF-κB inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. Further, we identify NIK as a critical negative regulator of beta cell function as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of non-canonical NF-κB components p100 to p52, and accumulation of RelB. Tumor necrosis factor α (TNFα) and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive beta cell intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the non-canonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to beta cell failure. These studies reveal that NIK contributes a central mechanism for beta cell failure in diet-induced obesity. We identify a role for Nuclear Factor inducing κB (NIK) in pancreatic beta cell failure. NIK activation disrupts glucose homeostasis in zebrafish in vivo and impairs glucose-stimulated insulin secretion in mouse and human islets in vitro. NIK activation also perturbs beta cell insulin secretion in a diet-induced obesity mouse model. These studies reveal that NIK contributes a central mechanism for beta cell failure in obesity. To uncover the role of NIK in pancreatic beta cells, we performed a gene expression microarray analysis comparing pancreatic islets with constitutive beta cell intrinsicNIK activation from the 16 week old mice (beta cell specific TRAF2 and TRAF2 knockout mice) to their controls (n=3 per group).

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Next Generation Sequencing analysis using splenic CD8 T cells isolated from Wild Type-OT-I (WT) and NIK -CD4Cre-OT-I (KO) mice

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