Ruxolitinib

Human small airway epithelial cells (SAECs) obtained from Lifeline Technology (FC-0016) were expanded using the complete BronchiaLife^TM media kit (Lifeline Technology, LL-0023). All culture wares were pre-coated in 30 μg/ml of Fibronectin (ThermoFisher Scientific, 33016015) for at least 1 h at room temperature. Calu-3 line (ATCC, HTB-55^TM) were cultured using Eagle’s Minimum Essential Medium (ATCC, 30-2003^TM) containing 10% fetal bovine serum (Cytiva, SH3007103) in 5% CO₂ atmosphere at 37 °C.
Cytokines (10 ng/ml; Human IFNβ, 300-02BC; Human IFNγ, 300-02; Human IL6, 200-06; Human IL7, 200-07; Human Growth hormone, 100-40, Peprotech; Human IFNα2b, 78077.1, Stem Cell Technologies; Human IFNλ3, 5259-IL-025, R&D systems) were added in respective culture media after serum starvation for 2 h, and the cells were incubated for 12 h in 5% CO₂ atmosphere at 37 °C. The cells were washed with PBS (Gibco, 14190144) twice and harvested.
Jak inhibitors, 10 μM of either Baricitinib (HY-15315A, MedChemExpress) or Ruxolitinib (HY-50856A, MedChemExpress), were added to BronchiLiafe^TM media with or without IFNβ. SAECs and incubated for 12 h and then washed with PBS twice and harvested.

Ruxolitinib (MedChemExpress LLC, Princeton, NJ, USA) was used as a JAK 1,2 inhibitor. The studied dose calculations for laboratory animals were based on the range of drug doses for human usage. For various indications, this was from 10 to 50 mg per day. For conversion, the following ratio was used: (drug dose/average human weight (70 kg)) × 5.9 according to which we determined a following range of 0.86–4.28 mg/kg per day [31 (link)]. Similar doses of Ruxolitinib have been used in other experimental studies in rats to demonstrate the renoprotective effect of Ruxolitinib in a model of diabetic nephropathy [32 (link)]. In light of the potential dose-dependence of the Ruxolitinib effect, we evaluated its effects at three different daily doses within the above range, specifically, 0.86, 2.58, and 4.28 mg/kg. The duration of Ruxolitinib administration was 4 weeks, starting from the second week after the last injection of MSs.

IL-4, IL-13, IL-22, and IFNγ (Cat No.: 574004, 571104, 571304, 570204, respectively) were purchased from Biolegend (San Diego, CA, USA, and IL-17A (Cat No.: 317ILB) was purchased from R&D Systems (Minneapolis, MN, USA). These cytokines were used at concentrations ranging from 3.1 to 200 ng/mL. IL-4 and IL-13 were used at a 1:1 ratio in all experiments. The JAKi used in these experiments—ruxolitinib, abrocitinib, fedratinib, ritlecitinib, and deucravacitinib (Cat No.: HY-50856, HY-107429, HY-10409, HY-100754, HY-117287, respectively)were purchased from MedChemExpress (Monmouth Junction, NJ, USA), and pyridone 6 (Cat No. 420097) was purchased from Millipore Sigma (Saint Louis, MO, USA). All JAKi were used at concentrations ranging from 50 to 400 nM. The JAKi vehicle was dimethyl sulfoxide (DMSO) diluted to the highest concentration (0.4%) used in JAKi experiments.

Ruxolitinib (MedChem Express, cat#HY-50856) was dissolved in DMSO for a stock concentration of 60 mg/ml. Prior to injection, 10 µl of this stock solution was dissolved in 200 µl saline. 10 µl DMSO dissolved in 200 µl saline was used in controls. 200 µl of the solution was delivered daily through oral gavage followed by dmPGE₂ by intradermal injection.