Global transcriptomic profiling reveals nicotinamide metabolism as a key regulator of nitric oxide-modulated interferon-γ responses in macrophages

Interferon-gamma (IFN-γ) is a key regulator of immune responses. A hallmark of IFN-γ responses is inducible nitric oxide (NO), driven primarily by nitric oxide synthase (NOS)2. In this study, we investigated the influence of NO on the IFN-γ-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-γ activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed early by IFN-γ 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 protein-protein interaction (PPI) network exhibited dense clustering with scale-free and small-world properties, identifying Stat1, Irf7, Irf1, Cxcl10, and Isg15 as top five hubs. Interestingly, the RNA seq analysis identified IFN-γ upregulated genes, Nampt, Pnp and Pnp2, to be involved in nicotinamide metabolism. This novel aspect was experimentally tested to show that IFN-γ induced NAD⁺ amounts in a NO-dependent manner. Importantly, the inhibition of purine nucleoside phosphorylase (PNP) which is involved in the endogenous pathway for NAD⁺ generation, lowered IFN-γ-induced nitrite and increased cell survival, demonstrating biological relevance of this study. Interestingly, NAMPT and PNP are expressed in multiple organs in humans and Epstein Barr Virus (EBV)-transformed lymphocytes. In addition, polymorphisms in NAMPT and PNP are associated with several diseases. Functionally, enriched nicotinamide metabolism by IFN-γ may regulate inflammatory responses and the implications of our findings are discussed.