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

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. Three groups of C57BL/6 mice (n=2) were treated for cell collection. LPS was intranasally administrated to mice with 10 ug in 50 ul sterile PBS 24 hours before harvest. a-Gr1 Ab was intraperitoneally administrated to mice with 300 ug in 300 ul also 24 hours before harvest. Mice in group 1 are naïve mice without any treatment, where the collected cells are expected to contain only steady-state IMs; mice in group 2 are treated with LPS i.n. to introduce acute inflammatory lung injury, where the collected cells are expected to contain both stimulated IMs and recruited monocytes; mice in group 3 are treated with LPS i.n. and a-Gr1 Ab to introduce injury and block the recruitment of blood monocytes, where the collected cells are expected to contain only stimulated IMs. To differentiate intravascular and extravascular leukocytes, mice were injected intravenously with APC-Cy7-conjugated anti-CD45 antibody 5 minutes before organ harvest. The mice were euthanized using CO2 inhalation.