Metabolic reprogramming is implicated in the differential response of the CAL-1 plasmacytoid dendritic cell line to...

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I Interferons (IFN) and pro-inflammatory cytokines, playing crucial roles in antiviral and anticancer immunity as well as in autoimmune diseases. Understanding the mechanisms that regulate pDC function is therefore essential. Autophagy, a process responsible for recycling intracellular components, influences cellular metabolism and modulates immune responses. Here, we used the human CAL-1 pDC cell line, a validated model for primary pDCs, to investigate the functional impact of autophagy inhibition and the contribution of metabolism to these effects. CAL-1 cells were treated with two autophagy inhibitors, Spautin-1 and VPS34-IN1, and cytokine production was assessed by RT-qPCR and ELISA, while cellular metabolism was analysed by untargeted metabolomics of cell extracts and of medium supernatants. VPS34-IN1, but not Spautin-1, induced robust expression of IFN-β and TNF-α. The two inhibitors also elicited distinct metabolic responses: Spautin-1 enhanced glycolysis, promoted an anabolic phenotype with increased utilization of amino acids, and upregulated mTOR signaling. In contrast, VPS34-IN1 decreased glycolysis, increased intracellular amino acids, reduced TCA intermediates, and induced energy stress, reflected by an increased AMP/(ADP + ATP) ratio and decreased NAD⁺. These changes were consistent with AMPK activation, and pharmacological inhibition of AMPK with dorsomorphin (compound C) abolished cytokine production in VPS34-IN1-treated cells. Together, these results indicate that differential cytokine responses to autophagy inhibition are driven primarily by metabolic rewiring rather than autophagic flux per se, highlighting the interplay between metabolism, mitochondrial ROS, and signaling pathways in pDC activation.