Species-specific iNOS expression distinguishes epithelial and myeloid IFNγ responses in tuberculosis

Abstract Rationale Tuberculosis vaccines aim to elicit protective immunity, with IFNγ-producing CD4⁺ T cells considered central mediators of host defense. In murine models, IFNγ activates macrophages to induce inducible nitric oxide synthase (iNOS), resulting in nitric oxide–dependent control of Mycobacterium tuberculosis ( Mtb ). Objective To define the relevance of IFNγ-induced iNOS activity in humans and other natural host of virulent mycobacterial species. Methods We systematically compared IFNγ-induced effector responses in Mtb -infected myeloid cells across species. Using bulk RNA sequencing, functional infection assays, and nitric oxide measurements, we assessed IFNγ responsiveness in human and mouse macrophages. These analyses were extended to monocytes from seven mammalian species and complemented by reanalysis of publicly available single-cell RNA-sequencing datasets and spatial proteomic imaging of tuberculous granulomas. Measurements and Main Results Single-cell transcriptomic reanalysis revealed minimal NOS2 expression in myeloid cells from human and non-human primate granulomas. In vitro , IFNγ pretreatment failed to induce NOS2 transcription, iNOS activity, or Mtb growth restriction in human macrophages, in stark contrast to murine cells. Across species, iNOS activity was largely restricted to mice, with limited induction in cattle monocytes. Instead, human respiratory epithelial cells consistently expressed NOS2 , and multiplexed ion beam imaging localized iNOS protein to epithelial compartments adjacent to granulomatous lesions. Conclusion IFNγ signaling is uncoupled from iNOS induction in primate myeloid cells and epithelial compartments represent dominant sources of iNOS in human tuberculosis. These findings, challenge murine macrophage–centric paradigms and IFNγ-based correlates used in TB vaccine development and central to pathophysiology of tuberculosis and other pneumonias.