Fig. 4.

S. aureus-derived exotoxins induce 5-LOX activation in human neutrophils accompanied by elevation of [Ca²⁺]_i. a 5-LOX product formation of human neutrophils was detected upon stimulation with 1% unconditioned medium vs. 1% BCM of S. aureus (LS1, LS1∆hla or LS1∆agr/sarA) or 100 µg/mL α-hemolysin vs. vehicle (water) for 10 min at 37 °C. Data are expressed in pg/5 × 10⁶ cells as mean ± SEM of three different donors. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 LS1 or α-hemolysin vs. vehicle control or *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the corresponding LS1 wild-type strain using one-way ANOVA with Tukey multiple comparison post hoc test. b Lipid mediator formation of neutrophils shown in a as percentage of LS1 wild type in a heat map. c Fura-2AM-labeled neutrophils were stimulated with 2 µM ionomycin, 100 µg/mL α-hemolysin, 1% unconditioned medium, or 1% BCM of S. aureus (LS1, LS1∆hla, or LS1∆agr/sarA) for 10 min at 37 °C and intracellular Ca²⁺ concentrations were monitored by continuous fluorescence reading. Data are given as mean ± SEM, n = 4 donors; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 LS1 or ionomycin vs. vehicle control or *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the corresponding LS1 wild-type strain using one-way ANOVA with Tukey multiple comparison post-hoc test. d 5-LOX translocation was monitored by immunofluorescence microscopy in neutrophils stimulated with 1% unconditioned medium or 1% BCM of S. aureus (LS1, LS1∆hla or LS1∆agr/sarA), or 100 µg/mL α-hemolysin for 10 min at 37 °C. Microscopic images are given as overlay of 5-LOX (red) and FLAP (green) staining and are representative for three independent experiments. e LM formation of human neutrophils was assessed after pre-incubation with 300 nM MK886, 3 µM zileuton, or vehicle (0.1% DMSO) prior to stimulation with 1% BCM of S. aureus (LS1) for 10 min at 37 °C. Data are expressed in percentage of vehicle control as mean ± SEM of three different donors. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. vehicle control using two-tailed Student’s t test. f LM formation of neutrophils shown in e as percentage of vehicle control. g Neutrophils were treated with 1% BCM of S. aureus (LS1, LS1∆hla, and LS1∆agr/sarA) or unconditioned medium (vehicle) for 10 min at 37 °C followed by automatic cell counting including trypan blue staining. Viability is presented as percentage of viable cells vs. total cell number. h Lactate dehydrogenase (LDH) release of neutrophils was measured upon stimulation with 1% BCM of S. aureus (LS1, LS1∆hla, LS1∆agr/sarA) or 1% Triton X-100 for 10 min at 37 °C. Values are presented as percentage of Triton X-100 treatment and as mean ± SEM, n = 3. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 LS1 vs. vehicle control or *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the corresponding LS1 wild-type strain using one-way ANOVA with Tukey multiple comparison post-hoc test