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

RationaleTuberculosis vaccines aim to elicit protective immunity, with IFN{gamma}-producing CD4 T cells considered central mediators of host defense. In murine models, IFN{gamma} activates macrophages to induce inducible nitric oxide synthase (iNOS), resulting in nitric oxide-dependent control of Mycobacterium tuberculosis (Mtb). ObjectiveTo define the relevance of IFN{gamma}-induced iNOS activity in humans and other natural host of virulent mycobacterial species. MethodsWe systematically compared IFN{gamma}-induced effector responses in Mtb-infected myeloid cells across species. Using bulk RNA sequencing, functional infection assays, and nitric oxide measurements, we assessed IFN{gamma} 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 ResultsSingle-cell transcriptomic reanalysis revealed minimal NOS2 expression in myeloid cells from human and non-human primate granulomas. In vitro, IFN{gamma} 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. ConclusionIFN{gamma} 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 IFN{gamma}-based correlates used in TB vaccine development and central to pathophysiology of tuberculosis and other pneumonias.