Regulation of IL-33 alarmin production from lung epithelial cells via CLEC-2:Podoplanin interactions
Lung epithelial cells produce alarmins that promote allergic inflammation, however, the molecular mechanisms that regulate alarmin expression and production have not been fully elucidated. We constructed an integrated single-cell RNA-seq atlas of murine airways, lung, and vagal nerves, which we use to characterize novel lung cell subsets and infer intercellular interactions, including an interaction between podoplanin (PDPN) on alveolar epithelial cells and C-type Lectin-like Receptor-2 (CLEC-2/Clec1b) on alveolar macrophages that controls the expression of IL-33. Clec1b-/- mice spontaneously develop allergic airway inflammation with increased Il33 expression in alveolar epithelial type 2 (AT2) cells and airway-associated fibroblasts; this airway inflammation is ameliorated by co-deletion of the IL-33 receptor, Il1rl1 (ST2). Myeloid cell-specific deletion of Clec1b and acute blockade of endogenous CLEC-2:PDPN interactions in vivo also promote type 2 immune responses in airway inflammation. Organoid experiments reveal that the regulation of AT2 Il33 expression by CLEC-2:PDPN interactions is directly mediated by alveolar macrophages. Single-cell characterization of house dust mite (HDM)-induced asthma reveals a smaller population of Clec1b expressing alveolar macrophages and increased Il33 expression by AT2 cells.  Thus, our study has identified the CLEC-2:PDPN pathway as an important regulator of epithelial alarmin production and induction of type 2 immunity. Overall design: C57BL/6J, C57BL/6-Tg(Pf4-icre)Q3Rsko/J (PF4-Cre) and B6.129P2-Lyz2tm1(cre)Ifo/J (LysM-Cre) mice were from the Jackson Laboratory. Clec1b-/- mice on 129/Sv background were provided by Shannon Turley (Dana-Farber Cancer Institute, Boston, Massachusetts, USA) and backcrossed with C57BL/6J for 5 generations in house. Clec1b fl/fl mice on 129/Sv background were provided by Mark Kahn (University of Pennsylvania, Philadelphia, Pennsylvania, USA) and crossed with PF4-Cre or LysM-Cre in house. Il1rl1-/- mice on C57BL/6J background were originally generated by Andrew McKenzie and provided by Diane Mathis (Harvard Medical School, Boston, Massachusetts, USA). Littermates were used as controls for all experiments using genetically modified mice and experimental and control mice were co-housed. Mice were age-matched for experiments and between 6 weeks and 4 months of age. To induce allergic airway inflammation, mice were treated intranasally with 10 µg house dust mite (HDM) extract (Greer Laboratories) on day 0, 7, 8, and 9, and then euthanized on day 10. For some experiments 25 µg Podoplanin-Fc fusion protein (PDPN-Fc; Biolegend, 551704) or isotype control (Biolegend, 403502) was administered intranasally on the same day as HDM. All mice were maintained in specific pathogen-free conditions at the Hale Building of Transformative Medicine. All animal experiments were performed in compliance with the approved BWH IACUC protocols.