Figure 3.

Group A Streptococcus (GAS) infection increases caspase expression in human neutrophils and induces proinflammatory cytokine IL-1β and TNF-α release. (A) Human neutrophil (PMN) lysates were prepared at 30, 60 and 180 min during GAS infection and compared to uninfected neutrophils (0, 30, 60, 180 min) via immunoblotting of caspase-3, caspase-1, caspase-4 and GAPDH. Images shown are from a single donor and are representative of triplicate experiments using different donors. Immunoblot bands were quantified (ImageJ) and normalised over total protein ( Figure S2 ). SE=short exposure, LE=long exposure. (B) Inflammatory caspase activation in human neutrophils due to GAS infection was confirmed via flow cytometry using FLICA (FAM-YVAD-FMK) (n=3 donors). Flow cytometry gating strategy used singlets/PMNs ( Figures S3A, B ) and FAM-FLICA fluorescence ( Figure S3C ). The release of cytokines from neutrophils during GAS infection was measured using the LEGENDplex™ human inflammation cytometric bead assay over 360 min. Neutrophils differentially released (C) IL-1β, (D) TNF-α, (E) IL-8 and (F) IL-18 in response to GAS infection (duplicate measurements, n=6 donors). (B–F) Results are the pooled means ± SD. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.