Peripheral blood mononuclear cells undergo apoptosis following challenge with Streptococcus pneumoniae.

<p>A) The percentage of Annexin V⁺ peripheral blood lymphocytes (PBL) identified by forward (FSC) and side scatter characteristics (SSC) 4 and 24 h after either mock-infection (multiplicity of infection (MOI) = 0) of peripheral blood mononuclear cells (PBMC) or challenge with D39 <i>Streptococcus pneumoniae</i> (MOI = 10), n = 6. B) Loss of inner mitochondrial transmembrane potential (Δψ_m) was detected with the cationic dye JC-1 in PBL 16 h post challenge of PBMC (MOI = 0–10). Representative histograms demonstrate the gain of green and loss of red fluorescence following mock infection (tinted histogram, MOI = 0) or pneumococcal challenge (filled histogram, MOI = 10), the graph depicts the percent loss of red fluorescence indicative of loss of Δψ_m, n = 6. C) Caspase 3 activation in PBMC 16 h post challenge (MOI = 0 or 10), recorded as caspase 3 relative luminescence units (Caspase 3 RLU), n = 3. D) Dot plots of the FSC and SSC profile of PBMC demonstrating a loss in FSC 16 h post challenge (MOI = 0 or 10), representative of three individual experiments. E) Representative images of PBMC 24 h following challenge (MOI = 0 or 10) and stained with DAPI and terminal UTP nick-end labeling (TUNEL). Results are typical of three independent experiments. F) A representative image showing Annexin V and TOPRO-3 staining is shown at MOI = 0 and 10 and the mean percentage Annexin V⁺ PBL gated by FSC and SSC after challenge of plastic purified PBMC (MOI = 0–50) for 6 h, n = 3. G) Representative histograms and graph of mean percentage CD3⁺ T-cells with accumulation of hypodiploid DNA (Sub G0/1) following challenge (MOI = 0 or 50) for 6 h. T-cells were identified by labeling with FITC conjugated anti-CD3, n = 4. H) The percentage Sub G0/1 CD3⁺ T-cells from PBMC pre-treated with either vehicle control (DMSO) or the pan-caspase inhibitor Z-VAD-FMK (10 µM) and mock-infected (D39−) or challenged with <i>S. pneumoniae</i> (D39+, MOI = 10) for 16 h, n = 3. Statistical analyses by ANOVA or t-test; * p<0.05, ** p<0.01, *** p<0.001.