Macrophage response to pneumococcus

Whole blood was obtained from healthy volunteers. Ethical approval was granted by South Sheffield Regional Ethics committee (07/Q2305/7). Peripheral blood mononuclear cells were separated by differential centrifugation using a Ficoll-Paque gradient and differentiated into monocyte derived macrophages (MDM) for 14 d as previously described in 24 well plates (Corning) (26 (link)). Bacteria were washed in PBS and re-suspended in RPMI 1640 supplemented with 10% pooled human immune serum (from previously vaccinated volunteers with demonstrable antibody levels to serotype 2 pneumococci) (27 (link)). MDM were challenged with either opsonised S. pneumoniae, Δply or PBS, at a MOI of 10, rested on ice for 1 h and incubated at 37°C in 5% CO₂ for a further 3 h (28 (link)). For certain experiments cells were treated with 3 μM vorinostat (SAHA, Sigma) or 0.5% DMSO (vehicle control) for 30 min prior to bacterial challenge and vorinostat reintroduced after bacterial challenge.