Abstract 2822 Elucidating the role of HPRT in regulation of immune response in macrophages

Macrophages are critical innate immune cells that dynamically reprogram their metabolism in response to environmental stimuli.Previous work in our lab revealed a distinct switch from de novo purine synthesis to purine salvage after stimulation with lipopolysaccharide and interferon-g (LPS+IFNg).Genetic ablation of the key enzyme that enables purine salvage flux, hypoxanthine-guanine ribosyl transferase (HPRT), results in significantly reduced migration towards chemoattractant, cytokine release, and intracellular parasite clearance without significant defects in viability.However, the mechanism by which HPRT regulates these functions is not yet known.Therefore, this study seeks to establish the role of HPRT and purine metabolism in regulating macrophage immune responses.We utilized RNAsequencing to profile the transcriptomics of wild-type and HPRT KO cells, then completed gene ontology analysis (GO).This identified regulation of cell migration and cell motility as the most significantly downregulated, which we then validated transcriptional analysis of downstream genes and immunoblotting for key activators of these pathways.By analyzing the most significantly downregulated genes with the transcriptional factor Enrichr software, we identified STAT3 signaling networks as significantly affected.We hypothesized that HPRT either directly interacts with STAT3 to facilitate activity or its metabolic activity somehow supports activation to affect STAT3 activity.Immunoblotting revealed STAT3 phosphorylation was significantly downregulated in HPRT KO cells after LPS+IFNg stimulation.Investigation of Il10 (an activator of STAT3) by qPCR and ELISA analysis has revealed significantly reduced Il10 transcription, leading to significantly reduced Il10 secretion in HPRT KOs relative to wild-type RAW cells.While the mechanism by which HPRT interacts with STAT3 is unclear, further work to elucidate this is ongoing.Current literature suggests HPRT can bind STAT3 to modulate migration in invasive cancer cell lines, therefore, as future methods we will employ co-immunoprecipitation of HPRT followed by proteomics to determine potential binding partners, allowing us to investigate direct interactions HPRT may engage in.This work serves to establish a role for HPRT in macrophage immune response as well as potentially shedding light on a previously unknown moonlighting role of HPRT.