Heparan sulfate regulates amphiregulin programming of tissue reparative lung mesenchymal cells during influenza A virus infection in mice

Amphiregulin is an immune cell-derived growth factor that plays critical roles in tissue repair. After influenza A infection, Treg cells in the lung produce amphiregulin, which signals to a specific Col14a1-expressing subpopulation of lung mesenchymal cells (Col14-LMC) to coordinate their repair activities towards the damaged lung epithelium. Heparan sulfate is a subtype of proteoglycan that can be produced by all cells in the body, and has been implicated in altering growth factor signaling in numerous contexts. Amphiregulin has a heparan sulfate-binding domain; we sought to investigate how the inhibition of heparan sulfate on primary Col14-LMC affects their response to amphiregulin at the transcriptional level. Overall design: We isolated Col14-LMC, a reparative subset of lung mesenchymal cells, from mice by flow cytometric sorting. Cells were allowed to adhere in culture for 24h, then dead cells were washed out/media was replaced. Cells were subsequently incubated with water vehicle or 3 mg/ml sodium chlorate (NaClO3) overnight (16-18h) to inhibit heparan sulfate. Then, cells were treated with varying concentrations of recombinant mouse amphiregulin (vehicle, 200 ng/ml, 500 ng/ml, 1000 ng/ml), incubated for 4h, then lysed for total mRNA for bulk RNA-seq.