Sirolimus

Six-week-old C57BL/6 mice (Jackson Laboratories) were treated intraperitoneally (i.p.) daily (d) for the duration of the experiment with 500 μL of phosphate-buffered saline (PBS) containing 12 μg (high dose) or 1.5 μg (low dose) rapamycin (Rapamune oral solution, 1 mg/mL sirolimus; Pfizer) as described previously (1 (link),11 (link),17 (link)). Three days after treatment was initiated, mice were inoculated intranasally (i.n.) with 1.5 LD₅₀ of A/Puerto Rico/8/1934 (A/PR8) (H1N1) under avertin anesthesia (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/vim). Mice were monitored daily postinoculation for morbidity (weight loss) and mortality. All mice that lost 25% or greater body weight as determined by preinfection weight were humanely euthanized as per CDC-IACUC guidelines. Lungs were collected 6, 9, and 12 days and sera were collected 2 and 5 weeks postinoculation. Five to six weeks later, mice were challenged with a heterosubtypic virus, A/Hong Kong/1/68 (A/HK68) (H3N2), at 5 LD₅₀. All animal research conducted in this study was approved by the CDC-IACUC and was conducted in an AAALAC-accredited facility.

After 24 h of lining the vessel structures with primary cells, vessels were washed three times with primary cell media. Nintedanib (Selleckchem, S1010) or Sirolimus (Pfizer®, Selleckchem, S1039) were resuspended at 10 mM in DMSO and diluted to final concentrations of 1 or 10 μM in primary cell media. 4 ml of drug solution was added per vessel plate, to ensure full coverage of the vessel microdevices. Drug conditions were compared to DMSO vehicle control (DMSO at 0.4% in primary cell media). The drugs were incubated for 72 h, and vessel permeability was then tested. Vessels were washed afterward and fixed for immunofluorescence staining.
Toxicity on Patient B TEnC was tested via CellTiterGlo assay (Promega, G7570), as per manufacturer’s instructions. 5000 TEnC cells from patient B were seeded in a white glass-bottom 96 wellplate. Nintedanib or Sirolimus was added at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 μM) and incubated for 72 hours before reading. Luminescence readouts were normalized to vehicle control (DMSO at 0.4% in primary cell media).

INK128 (Cayman Chemical, Ann Arbor, MI) was dissolved in dimethyl sulfoxide (DMSO) and then diluted with PBS to 0.1 mg/ml. Mice were gavaged daily with 1 mg/kg INK128. Sirolimus (Pfizer, New York, NY) was suspended in PBS at a concentration of 0.5 mg/ml. Mice were gavaged daily with 5 mg/kg Sirolimus.