Fig. 5: S. aureus induced apoptosis of neutrophils and macrophages in the nasal tissue enhances nasal colonization.

Mice were colonized with streptomycin resistant Newman (2 × 10⁸ CFU/nose) and culled 24 h post-colonization to excise the NT. Cells were isolated from the nasal tissue and sequentially stained with Apotracker, Zombie NIR and extracellular markers to identify neutrophils (CD11b⁺LY6G^high) and macrophages (CD11b⁺LY6G^low-midF4/80⁺) via flow cytometry. Apotracker/Zombie NIR double positive cells are deemed apoptotic cells. Results are shown as Zombie NIR vs apotracker gated on neutrophils and macrophages (A) with a representative plot and pooled absolute counts of apoptotic cells shown for neutrophils (B) and macrophages (C). At 24 h post-colonization, NT was excised, homogenized, and incubated with Sepharose beads to extract proteins for western blotting for Trail and total protein by Ponceau S staining (D). Densitometry of Trail was determined using Bio-Rad’s Image Lab software, normalized to the total protein, and expressed as relative expression (E). Bacterial burden within the NT was also assessed in mice that received the caspase-3 inhibitor Z-DEVD-FMK (2.5 mg/kg) compared to the vehicle control (F). Results are expressed as mean ± SD and statistically analyzed for animal experiments (10 mice per group) using an unpaired T test for CFU assessment and a Mann–Whitney U test for absolute counts of apoptotic cells (P value ** <0.01, *** <0.001 and **** <0.0001).