Bleomycin

Mice underwent intraperitoneal injection with 0.018 U·g⁻¹ bleomycin (Thermo Fisher Scientific) or vehicle (PBS) twice weekly for 4 weeks (Pi et al., 2018 (link)). For diphtheria toxin studies, mice were injected intraperitoneally for three consecutive days with 100 ng of diphtheria toxin (Sigma Aldrich) before initiation of bleomycin protocol, with PBS injection used as control (Goren et al., 2009 (link)), and twice weekly thereafter. Euthanasia and data collection are performed 5 days after final injection of animal with vehicle or experimental agent (diphtheria toxin and bleomycin), Day 33 of the intraperitoneal bleomycin injection protocol. Anti‐PD‐L1 (500 μg, intraperitoneally) and rat IgG2b, both purchased from BioXcell, were administered on Day 0 and then once weekly for three additional doses (Celada et al., 2018 (link)) throughout bleomycin exposure.

Mice were fed alcohol or control diet for 15 days (day 15) and then lightly anesthetized with isoflurane gas. PBS control or bleomycin sulfate (Sigma, B1141000, 2 mg/kg body weight) or was administered intranasally at 30 μl per mouse. Mice were continually on control diet or alcohol diet. Lungs were collected 14 days after bleomycin treatment. Mice received nasally 6.65 μg CGRP for 5 days on day 8 after administration of alcohol. Mice were also given capsaicin (M2028, 50 mg/kg, s.c.; Sigma) one time every three days before and after intratracheal instillation of bleomycin to deplete endogenous CGRP. For ILC depletion, mice were intraperitoneally injected with 200 μg anti-Thy1.2 per mouse (BioXCell) or IgG1 isotype control antibody every three days after administration of bleomycin.