Fig. 8

Chronic alternative tobacco product aerosol-suppressed vaccination efficacy translates to diminished bacterial clearance from the lungs. All exposed-mice were given an intratracheal instillation of 1 million NTHI cfu 8 weeks after P6 vaccination. Animals were euthanized 0, 4 and 12 h after bacterial challenge and BAL and lungs were harvested. Lung tissue homogenates were prepared to measure %NTHI clearance, and the BAL was processed to quantify the markers of lung damage as shown in schema (A) and described in detail in Additional file 1. Lung bacterial burden was calculated as NTHI clearance from the lungs of mice and measured by bacterial colony-plating assay. Data are represented as %NTHI clearance (B). Levels of total proteins (C) and albumin (D) in the BAL were quantified as described in Additional file 1: supplementary methods. Two-way ANOVA was performed to determine statistically significant differences between two groups and p-values were calculated using Tukey’s post-test multiple comparison by GraphPad Prism 9.5.1 software (GraphPad; La Jolla, California, USA). Difference between two groups was considered significant at p < 0.05, and symbols ^μp < 0.05 vs. Air; ^£p < 0.01 vs. Air; ^Φp < 0.001 vs. Air; ^┬p < 0.0001 vs. Air; ^Ωp < 0.01 vs. CC; ^Ψp < 0.0001 vs. CC and ^Υp < 0.001 vs. IQOS are used to denote significant difference between two groups. Number of mice was n = 6 at 0 h, n = 8 at 4 h and n = 6 at 12 h per group; mean ± SEM