Fig. 5: NOX2 inhibition downregulates CAP-dependent ROS production in macrophages and abrogates anti-staphylococcal activity of CAP.

a, b mBMDM and c, d RAW264.7 macrophages were infected with S. aureus Xen36 or S. aureus USA300 and treated with helium (control) or CAP. a, c Cells were lysed at 0, 2, and 5 h. p. i., and released bacteria were counted by CFU enumeration. The effect of CAP treatment on cellular proliferation was measured in b mBMDM and d RAW264.7 macrophages infected with S. aureus Xen36 or S. aureus USA300. e, f RAW264.7 macrophages pre-treated or not with gp91 ds-tat (NOX2 inhibitor), infected with S. aureus Xen36 and exposed to helium (control) or CAP. e ROS production and f relative number of living bacteria in cells. g Three-dimensional bioengineered human skin treated or not with gp91 ds-tat or GSK2795039 (NOX2 inhibitors) was inoculated with S. aureus Xen36 and exposed to helium (control) or CAP. The number of living bacteria was determined by counting CFU at 24 h. p. i. CFU average in control group was normalized to 100. n = 17 for control, n = 19 for CAP, n = 11 for CAP + gp91 ds-tat, n = 8 CAP + GSK2795039. Values are expressed as mean ± s.e.m. of three independent experiments. Two-tailed unpaired Student’s t test. In a, c, f time zero average was normalized to 100. For each time point and for each strain, the number of CFU is shown relative to the number of CFU at time zero. Values are expressed as mean ± s.e.m. of three independent experiments with at least six biological replicates in each condition. Absolute CFU values, precise number of replicates in each group and exact p are reported in Supplementary Data 5. For a–d significance was determined by Mann–Whitney U test, and e, f by one-way ANOVA with Tukey’s multiple comparison post hoc test, *p < 0.05, **p < 0.01, ***p ≤ 0.001.