RNA-Seq of RAW 264.7 macrophages treated with Interferon gamma and Nitric Oxide Synthase Inhibitor LNMA

Interferon-gamma (IFN-?) is a key regulator of immune responses. A hallmark of the IFN-? 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-?-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-? activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed by IFN-? 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). 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-? 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-g-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-? 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-? 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-? induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-g may regulate inflammatory responses and the implications of our findings are discussed. Overall design: RAW 264.7 macrophage cells were treated with IFN-? under conditions with and without NO inhibition by NG-methyl-L-arginine (LNMA) for 6 hours, and transcriptomic changes were assessed using bulk RNA sequencing.