Global transcriptome analysis identifies nicotinamide metabolism to play key roles in IFN-gamma and nitric oxide modulated responses

Abstract

Interferon-gamma (IFN-{gamma}) is a key regulator of immune responses. A hallmark of the IFN-{gamma} response is inducible nitric oxide (NO) production, driven primarily by nitric oxide synthase 2 (NOS2). In this study, we investigated the influence of NO on the IFN-{gamma}-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-{gamma} activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed early by IFN-{gamma} that were either NO-independent or NO-dependent. Inhibition of NO modulated a minor subset of the transcriptome, notably affecting Klf6 (a tumor suppressor) and Zfp36 (a regulator of pro-inflammatory cytokines). Interestingly, both Klf6 and Zfp36 correlatively showed high expression in most cancers. The PPI network exhibited dense clustering with scale-free and small-world properties, identifying Stat1, Irf7, Irf1, Cxcl10, and Isg15 as top five hubs. The top IFN-{gamma} signalling genes exhibited deficient expression in the brain but were highly expressed in lung, spleen, and EBV-transformed lymphocytes. Gene-disease associations linked the IFN-{gamma}-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-{gamma} upregulated genes were involved in nicotinamide metabolism, suggesting a transcriptional basis for modulation of metabolic pathways. This novel aspect was experimentally tested to show that IFN-{gamma} induced NAD amounts. Importantly, the inhibition of purine nucleoside phosphorylase (using Forodesine hydrochloride) which is involved in the endogenous pathway for NAD generation, lowered IFN-{gamma} induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-{gamma} may regulate inflammatory responses and the implications of our findings are discussed.