Fig 2: Effect of NuP-3 on lung disease after IL-13 challenge in minipigs using histology readouts.a, Immunostaining for MUC5AC plus CLCA1 and MUC5AC plus MUC5B with DAPI counterstaining in lung sections from the protocol scheme in Fig. 3b. Images are representative of 3 minipigs per condition. b, Quantitation of mucosal and submucosal immunostaining for conditions in (a). Values represent mean ± S.E.M. for 3 minipigs per condition with 2 technical replicates per minipig. *P <0.05 by one-way ANOVA with Tukey correction.

Fig 3: Effect of NuP-3 on mucus production in human airway epithelial cell culture.a, Protocol scheme for MUC5AC inhibition for vehicle versus compound: NuP-43 (BIRB-796) or NuP-3 in human tracheobronchial epithelial cells (hTECs) cultured under air-liquid interface (ALI) conditions without or with IL-13 (50 ng/ml). b, MUC5AC levels for vehicle versus NuP-43 or NuP-3 (each at 10 nM x 8 doses) for conditions from (a) from one subject representative of three subjects. c, Corresponding values for live cell numbers based on resazurin assay for vehicle versus NuP-43 or NuP-3 (each at 100 nM x 9 doses) for conditions in (a) from 1 subject representative of three subjects. d, Corresponding values for conditions in (b) derived from 6 subjects. e, Corresponding values for conditions in (c) derived from 6 subjects. f, Protocol scheme for inhibition of MUC5AC and CLCA1 mRNA for vehicle versus NuP-3 (10 nM x 4 doses) in hTECs cultured under organoid conditions without or with IL-13 (1 ng/ml). FC, fold-change. g, Corresponding levels of MUC5AC and CLCA1 mRNA for conditions in (f). Values derived from an individual subject representative of 5 subjects. FC, fold-change. h, Corresponding levels of organoid size and number for conditions in (f). Values derived from an individual subject representative of 3 subjects. All bar graphs depict mean ± S.E.M.; box and whisker plots depict upper and lower quartile, range, and median. *P <0.05 by ANOVA with Dunnett’s or Tukey correction; ns, not significant. Each sample condition included 4–8 technical replicates per subject.

Fig 4: Effect of NuP-3 on lung disease after SeV infection in minipigs using histology readouts.a, Immunostaining for MUC5AC plus CLCA1 and MUC5AC plus MUC5B with DAPI counterstaining in lung sections at 7 d after SeV infection from the protocol scheme in Fig. 5a. Images are representative of 3 minipigs per condition. b, Quantitation of mucosal and submucosal immunostaining for conditions in (a). Values represent mean ± S.E.M. for 3 minipigs per condition with 2 technical replicates per minipig. *P <0.05 by ANOVA with Tukey correction.

Fig 5: Effect of NuP-3 on IL-13 challenge in minipigs using mucus production and immune cell infiltration readouts.a, Pharmacokinetic analysis of NuP-3 in minipigs with plasma concentrations determined after a single oral dose of 2 mg/kg (n=2 minipigs). b, Protocol scheme for IL-13 challenge of right lung segments on Study Days 0 and 14 with BAL sample of right and left lung on indicated study days and either vehicle or NuP-3 treatment (2 mg/kg twice per day) on Study Days 12–17. c, Levels of MUC5AC and CLCA1 in BAL fluid for protocol scheme in (b) using treatment with vehicle control (left column) or NuP-3 (right column). d, Corresponding levels of eosinophil (Eos), neutrophil (Neu), lymphocyte (Lymph), and macrophage (Mac) counts in BAL for conditions in (a). For (c), each plot represents mean ± S.E.M. for 3 minipigs per condition with 3 lung segments and 2 technical replicates per segment per minipig; for (d), values represent 3 minipigs with 3 lung segments and a single assay per segment per minipig. *P <0.05 by one-way ANOVA with Tukey correction.