ABSTRACT: Pulmonary resident macrophages consist of alveolar macrophages (AMs) and interstitial macrophages (IMs). While much is known about the role of AMs, the function of IMs, which are conserved across multiple organs and species, remains unclear. Using single-cell RNA sequencing (scRNA-seq) after inducing acute lung injury, we identified at least six distinct subtypes of pulmonary IMs, based on the chemokines they express. These subtypes are: IMck1 (Ccl2, Ccl7, Ccl12, a few Cxcl14); IMck2-4 (Ccl3, Ccl4, Ccl5, Cxcl2, Cxcl3); IMck5-7 (Ccl6, Ccl8, Ccl9); IMck8 (Cxcl9, Cxcl10); IMck9 (Cxcl13); and IMck10 (Ccl24). Based on the selective chemokine expression profiles observed, we hypothesized that pulmonary IMs play a critical role in recruiting leukocytes and forming inducible bronchus-associated lymphoid tissue (iBALT) in the lung. First, we examined multiple Cre-expressing mice (Lyve1, Nes, Pdpn, and Pf4) for IM specificity by crossing them with Rosa26EYFP. Only the Pf4Cre mice selectively induced EYFP expression in CD206hiCD64hi IMs, where no other immune cell in the lung expressed EYFP. We then created a CD206hiCD64hi IM-depleting mouse by crossing Pf4Cre with Rosa26DTR and CX3CR1_001DTR. In a bacterial infection and allergic airway disease, depletion of CD206hiCD64hi IMs resulted in diminished leukocyte recruitment and iBALT formation. Overall, our study highlights the increased granularity in IM populations and demonstrates a division of labor for cellular recruitment and iBALT formation in the lung. Furthermore, the study suggests that the specific combination of chemokines expressed by IMs are tightly regulated, like Th cytokine-producing cells.
