FIGURE 1.

Induction of KLF4 expression in human blood-derived PMNs by S. pneumoniae requires LytA-dependent bacterial autolysis. PMNs isolated from human blood were stimulated with D39, D39Δcps, or R6× pneumococci with different MOIs (1, 10, or 100) for 3 or 6 h (A–D), with 0.05 ng/μL MALP-2 (M-2), 10 ng/μL CpG, or 0.1 ng/μL LPS for 6 h (B), or with R6×, R6×ΔlytA (MOI 1), or 5 μg/mL R6× DNA alone or in combination with R6×ΔlytA (MOI 1) (E). Cell lysates were collected after stimulation and analyzed for KLF4 expression using Western blotting. β-Actin confirmed equal protein loading. The densitometries of the KLF4 and β-actin bands were quantified using an Odyssey 2.0 infrared imaging system. The ratios of the KLF4 and β-actin densitometries were calculated and shown as the fold change of induction to unstimulated PMNs (control, C). Quantifications show the mean with standard deviation of at least three (A–D) or four (E) independent experiments. Statistics: Two-way ANOVA with Bonferroni post hoc test (A). Kruskal–Wallis test with Dunn multiple-comparisons test (B–E). *p < 0.05; ***p < 0.001. Each experiment was performed with PMNs isolated from the buffy coat of a different donor.