Vaping reorganizes the pulmonary macrophage landscape into multiple sex-dimorphic microenvironments

BACKGROUND: Pulmonary macrophages are central orchestrators of inflammatory resolution and tissue repair in the lung. Whether vaping-associated macrophage dysregulation is spatially organized, sex-dimorphic, or detectable by bulk transcriptomic approaches remains poorly understood. METHODS: We performed a targeted re-analysis of a published spatial transcriptomics dataset of vaping-exposed murine lung, isolating macrophage-associated spots and applying unsupervised Leiden clustering to identify transcriptionally distinct macrophage microenvironments. Macrophage polarization was assessed using SMaRTspot, a novel adaptation of the validated SMaRT framework enabling polarization scoring at the spatial microenvironment level. Cell-type-specific differential gene expression analysis was performed against a purpose-built curated macrophage gene universe. RESULTS: In the vaped animals, the conserved homeostatic macrophage program of non-vaped lung was replaced with multiple transcriptionally distinct microenvironments. These microenvironments exhibited strong sexual dimorphism and were undetectable by bulk transcriptomic analysis. Vaped male microenvironments showed progressive homeostatic identity loss, inflammatory polarization signatures, senescence-associated biology, and fibrotic remodeling potential. Suppression of IFN-γ-responsive genes, including Ciita was a consistent feature of all vaped male-containing microenvironments regardless of polarization state. Vaped female macrophage microenvironments showed preserved homeostatic identity with alternative activation marked by a de novo lipid synthesis signature. A shared cross-sex microenvironment exhibited TLR4-associated inflammasome priming, senescence-associated secretory phenotype activation, and estrogen receptor upregulation in both sexes. Spatially distinct distributions between sexes suggested convergent responses to distinct local stimuli rather than a shared paracrine mechanism. CONCLUSIONS: Vaping was associated with spatially organized, sex-dimorphic macrophage microenvironmental reprogramming invisible to bulk approaches. SMaRTspot extends validated macrophage polarization scoring to spatial transcriptomics and reveals opposing sex-specific polarization trajectories: male-associated suppression of IFN-γ responsiveness and antigen presentation capacity, and female-associated lipid-reprogrammed alternative activation with preserved homeostatic identity. These alterations may play a key role in vaping-associated pulmonary injury. As a re-analysis of a single-animal-per-condition dataset, these findings define testable hypotheses for confirmation in larger cohorts.