Abstract
Interferon-gamma (IFN-γ) is a key regulator of immune responses. A hallmark of the IFN-γ 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-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 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-γ 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-γ-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-γ 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-γ 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-γ induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-γ may regulate inflammatory responses and the implications of our findings are discussed.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Declaration of Competing Interest The authors have conflicts of interest to declare
Abbreviations
- DMEM
- Dulbecco’s modified minimal essential medium
- IFN-γ
- Interferon-gamma
- LNMA
- NG-Methyl-L-arginine acetate salt
- NO
- Nitric Oxide
- NOS
- Nitric Oxide Synthase