Coordinated chemokine expression defines macrophage subsets across tissues [scRNA-seq: mTumor]

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture. Eight C57BL/6 mice were injected intravenously via the tail vein with 4 × 10^5 B16F10 melanoma cells cultured under standard ATCC conditions to establish a lung metastatic model. At harvest, the mice were euthanized and the lung tissues pooled together to generate biological replicates. To differentiate intravascular and extravascular leukocytes, mice were injected intravenously with APC-Cy7–conjugated anti-CD45 antibody 5 minutes before organ harvest. Lung single-cell suspensions were prepared and subjected to fluorescence-activated cell sorting on a FACSAria Fusion (BD Biosciences) to enrich for extravascular monocyte–macrophage populations (CD64^+CD11b^+ cells). Approximately 30,000 cells per sample were loaded on the Chromium Next GEM Single Cell 3′ platform (10x Genomics) and sequenced on an Illumina NextSeq 500/550 with an average depth of ~50,000 reads per cell.