In a breakthrough that could redefine therapeutic strategies for chronic inflammatory diseases, researchers have uncovered a novel function of the protein inducible nitric oxide synthase (iNOS), long recognized solely for its role in producing nitric oxide (NO) during inflammatory responses. This newly identified role reveals that iNOS physically interacts with the mitochondrial protein IRG1, directly regulating immune cell metabolism and inflammation independent of NO production. Published in the prestigious journal Nature Metabolism, this discovery opens the door to innovative drug targets aimed at precisely modulating the immune response in diseases such as cardiovascular disorders, arthritis, and Crohn’s disease.
The immune system responds to infection or injury by activating inflammation, a complex biological process geared towards eliminating threats and initiating tissue repair. While essential for survival, unrestrained or chronic inflammation underpins the pathogenesis of numerous debilitating diseases. Therefore, unraveling the molecular mechanisms controlling the intensity and duration of inflammation represents a critical challenge in modern medicine. The newly reported interaction between iNOS and IRG1 adds a significant piece to this puzzle, presenting a specialized molecular checkpoint that could be harnessed therapeutically.
Until now, the immunological role of iNOS was predominantly attributed to its enzymatic generation of nitric oxide, a reactive molecule involved in killing pathogens and signaling to immune cells. However, researchers from the University of Surrey and the University of Oxford have demonstrated that iNOS’s regulatory effects extend beyond its catalytic activity. Their findings show that iNOS directly binds to IRG1 within mitochondria, inhibiting IRG1’s ability to produce itaconate—a metabolite that functions as a critical brake on inflammatory responses. This physical blockade curtails itaconate synthesis, effectively modulating the inflammatory cascade.
This paradigm-shifting discovery challenges the long-held assumption that iNOS governs immune cell behavior exclusively through NO synthesis. Instead, it is the structural conformation of iNOS—stabilized by the cofactor tetrahydrobiopterin (BH4)—that facilitates its interaction with IRG1. Intriguingly, this interaction occurs independently of NO production, implying that iNOS’s shape and binding capacity are primary determinants of its immunoregulatory function. This insight reframes our understanding of iNOS’s multifaceted role within immune cells, highlighting shape-driven protein-protein interactions as a novel regulatory mechanism.
The investigative team employed cutting-edge biochemical and biophysical techniques to interrogate this complex. Co-immunoprecipitation paired with mass spectrometry definitively confirmed that iNOS and IRG1 form a direct binding partnership in living immune cells. Complementary computational approaches, including molecular dynamics simulations, predicted and validated the three-dimensional structure of the iNOS-IRG1 interface. Moreover, surface plasmon resonance analyses revealed that this interaction is both stable and high-affinity in mouse and human models. Notably, the related enzyme endothelial NOS (eNOS) does not engage in such binding, underscoring the specificity and evolutionary conservation of the iNOS-IRG1 interaction.
Functional studies further illuminated the biological significance of this interaction. Immune cells deficient in iNOS exhibited dramatically elevated itaconate production—over 15 times greater than their normal counterparts—upon inflammatory stimulation. Even more compelling is the observation that iNOS mutants incapable of synthesizing nitric oxide retained the ability to suppress IRG1 activity, unequivocally showing that NO generation is not requisite for this inhibitory effect. The essential factor is iNOS’s capacity to assume the correct conformation via BH4 binding. Conversely, disruption of BH4 binding, and thus iNOS structural integrity, abolished its regulatory impact on IRG1.
The implications of this interaction extend beyond itaconate production. In the absence of iNOS, IRG1 was found to associate with an alternative set of proteins linked to glycolysis and broader cellular metabolism. This indicates that iNOS not only represses itaconate synthesis but also sequesters IRG1 away from pathways involved in energy management during inflammation. Thus, iNOS emerges as a master regulator orchestrating both immunometabolic and inflammatory networks, shaping how immune cells adapt their energy usage in response to stress and damage.
This study’s revelations pave the way for a more nuanced approach to immune modulation in chronic inflammatory conditions. Conventional therapies targeting iNOS and inflammation often focus on inhibiting NO production, a strategy plagued by lack of precision and unwanted side effects due to the broad biological roles of nitric oxide. By contrast, targeting the physical interaction interface between iNOS and IRG1 offers a refined therapeutic avenue. Selectively disrupting this protein-protein interaction could enhance itaconate-mediated anti-inflammatory mechanisms without compromising other vital functions of nitric oxide, thus offering higher specificity and reduced collateral damage.
Dr. Mark Crabtree of the University of Surrey, lead author of the study, emphasized the transformative potential of these findings: “Our work highlights a fundamentally different way to influence inflammation—by modulating protein interactions within cells rather than just their enzymatic products. This could provide a much more precise handle on immune regulation, which is critical given the immune system’s dual capacity to protect and to cause harm.”
The study was supported by the British Heart Foundation, ensuring a robust framework for translating these molecular insights into clinical advances, particularly for cardiovascular diseases where inflammation plays a pivotal role. The data provide a compelling rationale for drug development programs focused on designing molecules that selectively interfere with iNOS-IRG1 binding, potentially revolutionizing treatment paradigms for a wide spectrum of inflammatory disorders.
In conclusion, the identification of iNOS as a direct modulator of IRG1 and itaconate production through a nitric oxide-independent mechanism offers a fresh perspective on immune regulation. This discovery challenges existing dogmas, highlights the importance of protein structure and interaction networks, and suggests innovative therapeutic possibilities that harness the cell’s intrinsic checks and balances. As inflammatory diseases continue to impose a heavy global health burden, such groundbreaking insights are critical to paving the way for future precision medicines that can better balance immune activation and resolution.
Subject of Research: The novel molecular mechanism by which inducible nitric oxide synthase (iNOS) regulates inflammation through direct interaction with IRG1 in mitochondria, independently of nitric oxide production.
Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria
News Publication Date: 10-Apr-2026
Web References: http://dx.doi.org/10.1038/s42255-026-01492-1
Keywords: iNOS, inducible nitric oxide synthase, IRG1, itaconate, inflammation, immune regulation, protein-protein interaction, tetrahydrobiopterin (BH4), immunometabolism, mitochondrial signaling, nitric oxide-independent regulation, chronic inflammatory diseases, cardiovascular disease, arthritis, Crohn’s disease
