A hepatocyte-specific transcriptional program driven by Rela and Stat3 exacerbates experimental colitis in mice by modulating bile synthesis.

Hepatic factors secreted by the liver promote homeostasis and are pivotal for maintaining the liver-gut axis. Bile acid metabolism is one such example wherein, bile acid synthesis occurs in the liver and its biotransformation happens in the intestine. Dysfunctional interactions between the liver and the intestine stimulate varied pathological outcomes through its bidirectional portal communication. Indeed, aberrant bile acid metabolism has been reported in inflammatory bowel disease (IBD). However, the molecular mechanisms underlying these crosstalks that perpetuate intestinal permeability and inflammation remain obscure. Here, we identify a novel hepatic gene program regulated by Rela and Stat3 that accentuates the inflammation in an acute experimental colitis model. Hepatocyte-specific ablation of Rela and Stat3 reduces the levels of primary bile acids in both the liver and the gut and shows a restricted colitogenic phenotype. On supplementation of chenodeoxycholic acid (CDCA), knock-out mice exhibit enhanced colitis-induced alterations. This study provides persuasive evidence for the development of multi-organ strategies for treating IBD and identifies a hepatocyte-specific Rela-Stat3 network as a promising therapeutic target.

Non-canonical NF-κB signaling limits the tolerogenic ß-catenin-Raldh2 axis in gut dendritic cells to exacerbate intestinal pathologies.

Dendritic cell (DC) dysfunction is known to exacerbate intestinal pathologies, but the mechanisms compromising DC-mediated immune regulation in this context remain unclear. Here, we show that intestinal dendritic cells from a mouse model of experimental colitis exhibit significant levels of noncanonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviates intestinal pathologies in mice suffering from colitis. Deficiency of RelB:p52 diminishes transcription of Axin1, a critical component of the ß-catenin destruction complex, reinforcing ß-catenin-dependent expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid synthesis. DC-specific impairment of noncanonical NF-κB signaling leads to increased colonic numbers of Tregs and IgA+ B cells, which promote luminal IgA production and foster eubiosis. Experimentally introduced ß-catenin haploinsufficiency in DCs with deficient noncanonical NF-κB signaling moderates Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, inflammatory bowel-disease patients also display a deleterious noncanonical NF-κB signaling signature in intestinal DCs. In sum, we establish how noncanonical NF-κB signaling in dendritic cells can subvert retinoic acid synthesis to fuel intestinal inflammation.

Intracellular Acetyl CoA Potentiates the Therapeutic Efficacy of Antitumor CD8+ T Cells.

Effector CD8+ T cells rely primarily on glucose metabolism to meet their biosynthetic and functional needs. However, nutritional limitations in the tumor microenvironment can cause T-cell hyporesponsiveness. Therefore, T cells must acquire metabolic traits enabling sustained effector function at the tumor site to elicit a robust antitumor immune response. Here, we report that IL12-stimulated CD8+ T cells have elevated intracellular acetyl CoA levels and can maintain IFNγ levels in nutrient-deprived, tumor-conditioned media (TCM). Pharmacological and metabolic analyses demonstrated an active glucose-citrate-acetyl CoA circuit in IL12-stimulated CD8+ T cells supporting an intracellular pool of acetyl CoA in an ATP-citrate lyase (ACLY)-dependent manner. Intracellular acetyl CoA levels enhanced histone acetylation, lipid synthesis, and IFNγ production, improving the metabolic and functional fitness of CD8+ T cells in tumors. Pharmacological inhibition or genetic knockdown of ACLY severely impaired IFNγ production and viability of CD8+ T cells in nutrient-restricted conditions. Furthermore, CD8+ T cells cultured in high pyruvate-containing media in vitro acquired critical metabolic features of IL12-stimulated CD8+ T cells and displayed improved antitumor potential upon adoptive transfer in murine lymphoma and melanoma models. Overall, this study delineates the metabolic configuration of CD8+ T cells required for stable effector function in tumors and presents an affordable approach to promote the efficacy of CD8+ T cells for adoptive T-cell therapy. SIGNIFICANCE: IL12-mediated metabolic reprogramming increases intracellular acetyl CoA to promote the effector function of CD8+ T cells in nutrient-depleted tumor microenvironments, revealing strategies to potentiate the antitumor efficacy of T cells.

A Kinase Assay for Measuring the Activity of the NIK-IKK1 Complex Induced via the Noncanonical NF-κB Pathway.

Nuclear factor-kappa B (NF-κB) inducing kinase (NIK), a key component of the noncanonical NF-κB pathway, directs a range of physiological processes, such as lymphoid organogenesis, immune cell differentiation, and immune responses. Aberrant noncanonical NF-κΒ signaling also causes human ailments, including autoimmune and neoplastic diseases. As such, NIK is constitutively degraded in resting cells, and accumulates upon noncanonical NF-κB signaling. NIK then associates with and phosphorylates IkappaB kinase 1 (IKK1, alternately IKKα). Subsequently, the NIK-IKK1 complex mediates the phosphorylation of p100 that triggers partial proteolysis of p100 into p52. Typically, accumulation of NIK or processing of p100 is estimated by immunoblot analyses, and these indirect measurements are used as a surrogate for cellular NIK activity. However, studies involving knockout and cancerous cells indicated that the activity of NIK-IKK1 might not always correlate with the abundance of NIK or with the relative level of p52 and p100. In this report, we describe a specific and sensitive assay for direct evaluation of cellular NIK-IKK1 activity. Here, NIK immunoprecipitates are examined for the presence of IKK1-dependent kinase activity toward p100. The NIK-IKK1 assay captured selectively noncanonical NF-κB activation in the context of multiple cell activating stimuli and cell types, including patient-derived myeloma cells. We suggest that our assay may help advance our understanding of the role of NIK in health and diseases.

An epithelial Nfkb2 pathway exacerbates intestinal inflammation by supplementing latent RelA dimers to the canonical NF-κB module.

Aberrant inflammation, such as that associated with inflammatory bowel disease (IBD), is fueled by the inordinate activity of RelA/NF-κB factors. As such, the canonical NF-κB module mediates controlled nuclear activation of RelA dimers from the latent cytoplasmic complexes. What provokes pathological RelA activity in the colitogenic gut remains unclear. The noncanonical NF-κB pathway typically promotes immune organogenesis involving Nfkb2 gene products. Because NF-κB pathways are intertwined, we asked whether noncanonical signaling aggravated inflammatory RelA activity. Our investigation revealed frequent engagement of the noncanonical pathway in human IBD. In a mouse model of experimental colitis, we established that Nfkb2-mediated regulations escalated the RelA-driven proinflammatory gene response in intestinal epithelial cells, exacerbating the infiltration of inflammatory cells and colon pathologies. Our mechanistic studies clarified that cell-autonomous Nfkb2 signaling supplemented latent NF-κB dimers, leading to a hyperactive canonical RelA response in the inflamed colon. In sum, the regulation of latent NF-κB dimers appears to link noncanonical Nfkb2 signaling to RelA-driven inflammatory pathologies and may provide for therapeutic targets.