ABSTRACT: Interstitial macrophages (IMs) are increasingly recognized for their vital roles in maintaining tissue homeostasis and orchestrating immune responses. Here, we present a comprehensive transcriptomic analysis of murine lung IMs that highlights their diverse functional roles, specialized divisions of labor, and distinct spatial organization within the lung microenvironment. Building on earlier work showing that two overarching IM subsets—CD206hi and CD206lo—encompass ten unique chemokine-expressing subpopulations that regulate immune cell recruitment and tertiary lymphoid structures, we now demonstrate that these IM subsets also exhibit distinct cytokine and receptor gene profiles, along with an extensive autocrine network that influences their migration and cytokine-driven functions. Furthermore, we identify distinct innate immune signatures, including complement components, scavenger receptors, and pattern recognition pathways (e.g., Toll-like receptors and C-type lectins), underlining their broad contributions to innate immunity. Using Xenium spatial transcriptomics, we show that IMs predominantly localize to three lung regions: bronchovascular bundles, interstitium, and periphery. CD206hi and CD206lo IMs preferentially occupy specific sub-tissular niches, likely driven by differential integrin and metallopeptidase gene expression. Additionally, chemokine expression within IMs exhibits distinct spatial localization patterns, directing the recruitment of T cells and B cells. Two summary tables were provided to outline the differential gene expression across IM subsets in the dataset. Overall, our findings advance the understanding of IM heterogeneity and their multifaceted roles in chemoattraction, inflammation regulation, innate immune defense, and tissue maintenance, while providing a high-resolution framework for investigating their localization, interactions, and contributions to lung immunity and disease.
