Selective Activation of Human Dendritic Cells by OM-85 through a NF-kB and MAPK Dependent Pathway (original) (raw)
Figure 1
Activation of the NF-kB and MAPK pathways by OM-85 in MoDC.
A) Immature human MoDC were stimulated with 100 µg/ml OM-85 for 30, 60, 90 and 120 minutes. 100 ng/ml LPS was used as a positive control. After cell lysis and protein fractionation, cytoplasmic (Cyto) and nuclear (Nuclei) extracts were blotted against NF-kB p65 and IkBα. β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of eight. B) EMSA (upper panel) and supershift (lower panel) showing the induction of NFkBp65-DNA binding activity by OM-85 in human moDC stimulated as in A). Signal specificity was assessed by competing each sample with a 125-fold excess unlabeled probe (lanes 2,4,6,8,10 upper panel). The image depicts results obtained in one representative donor out of four. C) OM-85 induces the production of luciferase in THP1 cells bearing a NF-kB-reporter plasmid (NF-kB pGL4, striped histograms). THP1 cells were stimulated with 1 µg/ml LPS and 1000 µg/ml OM-85. As expected, THP1 untransfected cells (untransfected, empty histograms) did not produce luciferase in response to stimulation. Similar results were obtained when cells were transfected with the pGL4 empty backbone (pGL4, black histograms). Results are expressed as mean+/−SD of three independent experiments. *P value<0.01 by Dunnett's Multiple Comparison Test. D) OM-85 activates the MAPK pathway. Cell extracts prepared as in A) were blotted with antibodies specific for phophorilated ERK1/2 (Cyto, upper panel) and total ATF2 and c-Jun (Nuclei, lower panel). β-actin and Lamin A/C represent loading controls for cytoplasmic and nuclear proteins respectively. The image depicts results obtained in one representative donor out of three. E) OM-85 induces NF-kB- and MAPK-dependent gene transcription. Immature MoDC were stimulated with 100 µg/ml OM-85 (open circles) and 100 ng/ml LPS (black circles) for 2, 4, 8 and 24 hours. After RNA extraction, reverse transcription and DNAse I digestion, samples were amplified by Q-PCR using gene-specific primers. Results represent means+/−SE of three independent donors and are expressed as fold induction (FI) over unstimulated samples (0).