Figure 6. Intranasal α-toxin Treatment Is Sufficient to Induce Increased Lethality in Atg16L1-Deficient Mice.

(A) Survival curve of WT and Atg16L1^HM mice inoculated intranasally with a 20μl suspension of filter-sterilized S. aureus culture supernatant containing α-toxin (+toxin) or lacking α-toxin (toxin). Supernatant were prepared from the exotoxin-deficient Newman Δhla/hlg/lukAB/lukED/pvl& strain +/− α-toxin overexpression. n=15–17 mice/group.

(B) Protein content of bronchoalveolar lavage fluid of WT and Atg16L1^HM mice 3 hours post-intranasal inoculation with α-toxin as in (A). n=12–13 mice/group. Data points represent mean and SEM.

(C) Quantification of TUNEL⁺ cells per field of WT and Atg16L1^HM mice 3 hours post-intranasal inoculation with α-toxin as in (A). n=3 mice/group. Data points represent mean and SEM.

(D–H) Flow cytometry analysis of single cell lung homogenates from WT and Atg16L1^HM mice 3 hours post-intranasal inoculation with α-toxin.

(D) Plots showing gating strategy to identify ICAM⁺PE-CAM⁺ cells within the CD45⁻ fraction. Representative WT mouse shown.

(E) Representative plots of PI⁺ cells gated on the CD45⁻ICAM⁺PE-CAM⁺ fraction as in (D) from lungs harvested from mice treated with α-toxin (bottom panels) or control suspension (top panels). Samples harvested from WT and Atg16L1^HM mice are represented in the left and right panels, respectively.

(F–H) Quantification of the proportion of (F) CD45⁺, (G) CD45⁻EpCam⁺, and (H) CD45⁻ICAM⁺PE-CAM⁺ cells that are PI⁺ 3 hours post-intranasal inoculation with α-toxin or control suspension. Values are expressed as fold-change over WT mice treated with control suspension. n=4–7 mice/group

Individual points in (B), (C), (F), (G), and (H) represent cells from individual animals and bar graphs in (B) represent mean and SEM from at least two independent experiments. *p<0.05, **p<0.01, ***p<0.001