Fig. 7.

TdT-mediated dUTP nick-end labeling (TUNEL) and lung AT₂ cell death markers after S. aureus inoculation. A: scatterplots showing an increase in alveolar cell death by TUNEL staining after S. aureus inoculation. Cell death increased by 7- to 8-fold at days 1 and 2 before falling to near control levels by day 3. B: cleaved to native caspase-3 ratio follows a similar pattern as the TUNEL staining. Peak apoptosis occurs on day 2 and subsides by day 3. C: scatterplots showing increased levels of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) on day 1 followed by a decline on day 2. D: scatterplots for mixed lineage kinase domain-like protein phosphoprotein (pMLKL) show a dramatic increase on day 1 followed by normalization on days 2 and 3 postinoculation. C and D: necroptosis on day 1 consistent with failing mitophagy after inoculation. E: photomicrograph of immunochemical stains for sufactant protein-C (S-C; green) and TUNEL (red) on formalin-fixed control lung section. Nuclei are stained with DAPI (blue). Control lung shows little or no evidence of alveolar cell death. F: the same 3 stains (SP-C, TUNEL, and DAPI) on formalin-fixed lung sections obtained at day 1 indicate dying AT₂ cells (long arrows). Extensive neutrophil and macrophage influx is noted, and the occasional macrophage has collected SP-C, perhaps from surfactant clearance (short arrow). G: the 3 stains applied to day 3 lung indicate neutrophil clearance and near resolution of AT₂ cell death. Some flat TUNEL-positive cell nuclei are consistent with dying alveolar type I (AT₁) cells (arrows). Scatterplot bars are means ± SE for n = 6 per time point; *P < 0.05 vs. time 0 by one-way ANOVA.