Background/Purpose: B cells contribute to disease pathophysiology through several mechanisms, including cytokine secretion. A wide variety of stimuli can activate B cells to produce cytokines including B cell receptor and Toll-like receptor engagement. Recently, numerous observations have established the role of metabolic pathways in the diverse array of immune cell functions. It is unknown though how these metabolic pathways influence B cell cytokine production. We sought to elucidate the metabolic programs required for B cell cytokine production

Methods: B cells were isolated from the spleens of C57Bl/6J mice and activated overnight individually by anti-μ antibody, anti-CD40 agonist antibody, poly(I:C), LPS, R484, and CpG. Supernatants were analyzed for the quantification of cytokines using the Milliplex cytokine kit (EMD Millipore). Real-time analysis of extracellular acidification rates and oxygen consumption rates of activated B cells were performed using the XF-96 Extracellular Flux Analyzer (Seahorse Bioscience). To assess 3-carbon sources for oxidative phosphorylation, inhibitors to fatty acid oxidation (etomoxir), pyruvate transfer to mitochondria (UK-5099), and glutamine usage (BPTES) were used. Amino acids, organic acids, and acylcarnitines were extracted using methanol for targeted metabolomics studies using mass spectroscopy.

Results: CpG stimulation of mouse splenic B cells increased both glycolysis and mitochondrial respiration to a larger extent that by other stimuli such as LPS or B cell receptor alone. These processes are highly dependent on glutamine, as inhibition of glutaminolysis with BPTES significantly reduced both processes. Furthermore, glutaminolysis activated mTOR, which was required for glycolysis, mitochondrial respiration, and ATP production in CpG-activated B cells. Quantitative targeted metabolomic studies confirmed CpG-stimulation drove increases in intracellular amino acid levels, aerobic glycolysis and TCA cycle intermediates, all of which were reversed in the presence of BPTES. Production of TNF-α, IL-6, and IL-10 by CpG-stimulated B cells also relied on glutaminolysis and mTOR activation. Interesting, inhibition of glycolysis alone selectively decreased IL-10 production.

Conclusion: B cells undergo metabolic reprogramming when stimulated with CpG, requiring glutaminolysis and mTOR activation, which drove aerobic glycolysis, TCA cycling, and ATP production. Cytokine production is intrinsically linked with this reprogramming, although glycolysis was required for B cell IL-10 production suggesting the possibility that various B cell cytokines differentially rely on specific metabolic pathways. These data are the among first to demonstrate a relationship between B cell effector function and metabolic reprogramming, and suggest that B cell cytokine secretion can be manipulated by altering the local metabolic environment. Manipulating metabolic pathways may represent an interesting therapeutic approach for modulating B cells in autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cytokine-production-by-cpg-activated-b-cells-require-glutaminolysis-to-drive-mtor-activation-aerobic-glycolysis-and-mitochondrial-respiration/