Figure 1.

Effect of S. pneumoniae on interleukin-36γ (IL-36γ) induction and secretion in the lung. (a) Wild-type (WT) mice were infected with an intrathecal (i.t.) injection of Streptococcus pneumoniae (Sp) (5 × 10⁴ colony-forming unit (CFU)), and lungs were harvested at the specified time points. (*P<0.01 and ^†P<0.05 as compared with uninfected mice by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test, n=4 per time point, representative of three experiments). (b) Representative western immunoblot of whole lung homogenates from Sp-infected WT mice were assessed for IL-36γ with β-actin as a housekeeping control. Bands are representative of two separate experiments of n=3 replicates per group. Densitometry represents relative density compared with β-actin from all replicates pooled from both experiments. (*P<0.01 compared with uninfected mice by one-way ANOVA with Dunnett’s multiple comparisons test). (c) bronchoalveolar lavage fluid (BALF) from Sp-infected WT mice was assessed by IL-36γ ELISA (enzyme-linked immunosorbent assay) with or without sonication. (*P<0.05 as compared with untreated mice by one-way ANOVA with Dunnett’s multiple comparisons test, n=6 per group, representative of two experiments). (d) Microparticles (MPs) from infected mice were assessed by flow cytometry for size relative to submicron size calibration beads (panel 1). MPs were then gated to select DAPI (4',6-diamidino-2-phenylindole)-negative particles, corresponding to viable MPs (not shown). MPs were stained for either Annexin V-phycoerythrin (PE) or a PE-labeled isotype control immunoglobulin G (IgG) (panel 2). Within 95% confidence intervals as compared with isotype controls, 80% of particles were Annexin V-positive (representative of three separate experiments). (e) Representative western immunoblot of isolated MPs from Sp-infected WT mice were stained for IL-36γ with β-actin as a housekeeping control (n=2 per group, representative of five experiments). Densitometry represents relative density compared with β-actin from all replicates pooled from all experiments. (*P<0.05 compared with MPs from uninfected mice by two-tailed Student’s t-test).