Figure 6: Anakinra protects Cftr^−/−mice from infections and NLRP3 inflammation.

C57BL/6 and Cftr^−/− mice (n=6 for all groups) were infected intranasally with live A. fumigatus conidia or P. aeruginosa and treated with anakinra (100 mg kg⁻¹ per day) throughout the infection. (a,g) Survival, (b,h) microbial growth (log CFU, mean±s.d.), (c,i) lung histology (periodic acid-Schiff staining) and reduced deposition of DNA on lung parenchyma cells by TUNEL; (d) caspase-1 cleavage by immunoblotting with specific antibodies (scanning densitometry was done with Image Lab 3.1.1 software. Representative of three independent experiments and corresponding pixel density ratio normalized against actin); (e,j) IL-1β levels in lung homogenates; NLRP3 protein expression by immunofluorescence staining (f) and immunoblotting (k) of lungs of anakinra-treated mice. Assays were done at 7 dpi. (l) Fungal growth (log CFU, mean±s.d.) and (m) lung histology (periodic acid-Schiff staining and NLRP3 immunofluorescence staining in the inset) of A. fumigatus-infected and anakinra-treated Il1ra^−/− mice. Representative images of two independent experiments were acquired using EVOS FL Color Imaging System with a × 40 objective for histology (Scale bar, 100 μm) and a high-resolution Microscopy Olympus DP71 using a × 20 objective for TUNEL and immunofluorescent staining (Scale bar, 200 μm, inset 50 μm). Data pooled from three experiments and presented as mean±s.d. for all bar graphs. *P<0.05, **P<0.01, ***P<0.001, anakinra treated versus untreated mice (none), One-way ANOVA (b,h), Two-way ANOVA (e,j) Bonferroni post hoc test and two-sides Student’s t-test. (l) For NLRP3 quantification, see Supplementary Fig. 1.