N-acetyl Aspartate Induces a Pro-Inflammatory Phenotype in Peritoneal Resident Macrophages, Driving Metabolic and Transcriptional Changes 2309239

Abstract Introduction Peritoneal cavity (PC) tissue resident macrophages (TRM) are key players in immune defense and tissue homeostasis. The metabolite N-acetyl aspartate (NAA) is elevated in the PC. NAA is synthesized from acetate and aspartate by Nat8l and metabolized back to aspartate by Aspa. TRM depend on their tissue niche, PC TRM uniquely express Aspa, and NAA levels are associated with worse outcomes in ovarian cancer patients suggesting NAA is key in the biology of the PC. Methods The PC niche was examined by phenotype, metabolomics, 13C carbon tracing and single cell RNA sequencing in wild type and Aspa-/- or Nat8l-/- mice. In addition, multiple type 2 inflammatory models and orthotopic tumor models carried out in vivo and macrophage responses to NAA assessed in vitro. Results We reveal the expression of Nat8l by PC TRM, indicating the NAA metabolic loop can run within the PC. Moreover, PC NAA levels are dynamic in type 2 mouse models, however, there is low incorporation of metabolized NAA-derived aspartate into the TCA cycle. In vitro, NAA alters the intracellular metabolome and increases metabolic rates. These metabolic changes are also seen in vivo, where Aspa-/- mice display increased PC NAA levels and glycolytic intermediates following stimulation. The role of NAA is not only metabolic, as macrophages exposed to NAA have an increased pro-inflammatory gene signature. Lastly, in contrast with previous literature, genetic perturbation of the NAA metabolic loop within the PC leads to reduced growth in a murine intraperitoneal tumor challenge model. Conclusion This work shows a role for the NAA metabolic loop within the PC, anchored by the TRM. NAA within the PC serves as a metabolic and pro-inflammatory modulator rather than a fuel, leading to increased cellular energetics, and an altered metabolome. Therefore, the primary role of Nat8l and Aspa in PC TRM may be to modulate NAA levels and control the inflammatory potential of the peritoneal cavity. Funding Source NIH Project Number ZIA BC 010300 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)