iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

Journal article

Nitric oxide (NO) has fundamental roles in numerous physiological and pathophysiological processes. In macrophages, NO produced by inducible nitric oxide synthase (iNOS) modulates metabolic changes that are essential to macrophage activation and plasticity, driving the characteristic metabolic switch from oxidative phosphorylation to glycolysis1, 2. Itaconate, derived from the TCA cycle by decarboxylation of cis-aconitate by IRG1 (also referred to as CAD, ACOD1), is one of the most upregulated metabolites during the inflammatory response3. Itaconate regulates macrophage polarization by electrophilically modifying cysteines of key enzymes that control inflammatory states (such as ATF3, Jak1, IFNβ), participate in glycolysis (for example, GAPDH, LDHA) and limit oxidative stress through structural competitive inhibition of succinate dehydrogenase4, 5, 6, 7, 8–9. We recently reported that macrophages that are deficient in iNOS, and subsequent NO generation, produce strikingly higher levels of intracellular itaconate (up to ~15-fold) compared to wild-type cells when stimulated with inflammatory cytokines1, 2, 10. Here we show that iNOS inhibits IRG1 activity and itaconate levels through a conformation-dependent protein–protein interaction rather than through the production of NO. Using a variety of biochemical and computational approaches, we show that a direct interaction between iNOS and IRG1 occurs within mitochondria, in mouse and human cells, and that it depends on binding of the cofactor BH4 to iNOS but does not require its capability to produce NO. Our findings reveal a non-canonical cellular function for iNOS that places it at the centre of a signalling hub, linking redox signalling and metabolism to modulation of the inflammatory response in macrophages.

Authors: Diotallevi, M; Outeiral, C; Patel, P; Purvis, GSD; Chuaiphichai, S

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Nature Metabolism
• Volume: 8
• Issue: 4
• Pages: 855-868
• Publication date: 2026-04-10
• Acceptance date: 2026-02-23
• DOI: 10.1038/s42255-026-01492-1
• EISSN: 2522-5812
• ISSN: 2522-5812
• Language: English
• Keywords: Nitric oxide; Glycolysis; Mitochondrion; Intracellular; Nitric oxide synthase; Oxidative phosphorylation; Inflammation; Downregulation and upregulation; Oxidative stress; Macrophage