Background 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 (BCR) and Toll-like receptor (TLR) 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.
Objectives Delineate the array of cytokines generated by B cells and their metabolic requirement following activation.
Methods B cells were isolated from the spleens of C57Bl/6J mice and activated overnight individually by the following agents: anti-μ antibody (B cell receptor), anti-CD40 agonist antibody, poly(I:C) (TLR3), LPS (TLR4), ssPolyU (TLR7/8), and CpG (TLR9). Supernatants were collected and analyzed for the quantification of cytokines using the Milliplex Multiplex cytokine kit (EMD Millipore). Real-time analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) of activated B cells were performed using the XF-96 Extracellular Flux Analyzer (Seahorse Bioscience). Three or more consecutive measurements were obtained under basal conditions and after the sequential addition of 1 μM oligomycin, to inhibit mitochondrial ATP synthase; 1.5 μM FCCP (fluoro-carbonyl cyanide phenylhydrazone), a protonophore that uncouples ATP synthesis from oxygen consumption by the electron-transport chain; and 100 nM rotenone plus 1 μM antimycin A, which inhibit the electron transport chain. To assess 3-carbon sources for oxidative phosphorylation, inhibitors to fatty acid oxidation (etomoxir, which irreversibly inhibits carnitine palmitoyltransferase-1), pyruvate transfer to mitochondria (UK-5099), and glutamine usage (BPTES, which inhibits mitochondrial glutaminases) were used.
Results Both LPS and CpG activation of B cells independently generated the highest levels of cytokine secretion. In addition, these stimuli generated high OCR and ECAR values, reflecting the need for oxidative phosphorylation and lactate production respectively. Both LPS and CpG required free fatty acids and glucose as a carbon sources, as addition of etomoxir and UK-5099 abrogated both cytokine secretion and oxidative phosphorylation.
Conclusions We catalogued the breadth of cytokines secreted by B cells via various stimulatory agents. High-dose LPS and CpG required free fatty acids and glucose for the elaboration of LPS or CpG-induced cytokine production. These data suggest that B cell cytokine secretion can be manipulated by altering the local metabolic environment, and may represent an interesting therapeutic approach for modulating B cells in autoimmune diseases.
Disclosure of Interest D. Wu: None declared, M. Dunlap: None declared, D. Huang: None declared, E. Pearce: None declared, A. Kim Grant/research support from: Rheumatology Research Foundation, Kypha Inc.