Figure 4
Bacterial killing by MSC mediated in part by the antimicrobial peptide Cramp. The ability of MSC to kill S. aureus was assessed by co-culture of bacteria directly with MSC (A) or with MSC CM (B). MSC (5 × 105 cells/well) were co-cultured with bacteria (MOI = 2) in triplicate wells of 24-well plates for 3 h, then bacteria were collected from supernatants resuspended by gentle pipetting, and CFU were determined by serial dilution and manual counting. Bacteria were incubated directly in MSC CM for 3 h in (B). In (C) synergistic killing of S. aureus by MSC CM and cefazolin. Cefazolin was added at a dose of 50 ng/ml and bacteria added at 1 × 106 per well to 1 ml of MSC-CM or media alone in a 24 well plate and incubated for 3 hours at 37 °C, *denotes p < 0.05 as assessed by ANOVA and Tukey multiple means post-test. Synergy assessed via two way ANOVA for detection of significant interaction, as described previously46. Immunocytology was used to assess intracellular expression of the antimicrobial peptide Cramp by MSC in (D), as described in Methods. Cells were immunostained with an irrelevant antibody (isotype panel insert) or with an anti-Cramp antibody (D) and cells were evaluated by fluorescence microscopy. Synergistic killing between antimicrobial peptide LL-37 and beta lactam antibiotic demonstrated in (E). Human LL-37 at 30 ug/ml was incubated with 50 ng/ml Cefazolin for 3 hours with 1 × 106 CFU/ml of S. aureus. In (F), MSC were co-cultured with bacteria and the effects of Cramp neutralization on bacterial killing were assessed Synergistic killing of bacteria was demonstrated between MSC and cefazolin (C) and between LL-37 and cefazolin (E). *Denotes p < 0.05 as assessed by ANOVA and Tukey multiple means post-test. Similar results were obtained in two additional experiments.
