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. 2019 Apr 12;15(4):e1007694. doi: 10.1371/journal.ppat.1007694

Fig 5. LL-37 preferentially accumulates in infected epithelial cells, activating caspase-1 via a P2X7R- and FPRL-1-independent mechanism.

Fig 5

NHBE cells were exposed for 3 hours to media only (control), 20 μg/ml LL-37 only, PAO1/ heat killed PAO1/ P. aeruginosa strain T3SS at 10:1 MOI +/- 20 μg/ml LL-37, or PAO1 supernatant +/- 20 μg/ml LL-37. Some cells were pre-exposed for 1 hour to FPRL1 inhibitor WRW4 (10 μM) (A), or P2X7R inhibitor KN-62 (20 μM) (B). Caspase 1 activity was measured by FLICA assay (A–C). Data represent means +/- SEM from n = 3 independent experimental repeats, *** p < 0.001, ** p<0.01, * p<0.05, by 2-way ANOVA with Bonferroni Post-test. ns = no significant difference. D) Timelapse series of images (brightfield/fluorescence merge) taken by confocal microscopy showing GFP-PAO1 (green) adhering to NHBE cells (white arrows) with concentration of TAMRA-labelled LL-37 (red), on heavily infected cells. Scale bar = 100 μm. E) Representative maximum projection merged image of a field of NHBE cells stained with GFP-PAO1 (green), TAMRA-labelled LL-37 (red), and Brilliant Violet-YVAD (blue). Scale bar = 50 μm. Inset shows cropped single plane of one NHBE cell demonstrating GFP-PAO1 bacteria (white arrow heads) inside a TAMRA-LL-37 positive cell that is also positive for caspase 1 activation, indicated by Brilliant Violet-YVAD staining. Scale bar = 10 μm.