Fig. 7
The conditioned medium from astrocytes pretreated with Hp OMVs promotes secretion of IFNγ and CAD cell death. a Representation of the different treatments: (1) untreated astrocytes, or treated with or without TNF or OMVs, in the presence or absence of NF-κB inhibitors; (2) after treatment, cells were washed and the medium was changed to SFM; (3) the collected astrocyte-conditioned medium (ACM) was added to differentiated CAD cells to evaluate cell viability and DEVDase activity (caspase 3); (4) collected ACM was used to quantify TNF, IL-6, IL-1β, and IFNγ using the MILLIPLEX RCYTMAG-65 K kit. b Dot plots of CAD cells using flow cytometry. CAD cells were harvested 24 h after treatment with ACM from astrocytes (ACM Control), etoposide (10 µM), or ACM obtained after treatment with TNF or OMVs for 24 h. Cells (105) were incubated with AV conjugated to Alexa Fluor 488 and propidium iodide (PI). Dead cells are shown in the upper left quadrant (AV-negative but PI-positive, Q1); late or necrotic apoptotic cells, in the upper right quadrant (AV-positive and PI-positive, Q2); early apoptotic cells, in the lower right quadrant (AV-positive but PI-negative, Q3); and viable cells in the lower left quadrant (Q4). c The graph shows the percentage of the total dead cell populations (Q1 + Q2 + Q3) of differentiated CAD cells after the different treatments (etoposide or ACM ± IMD 0354 inhibitor pretreatment). Values in the graph are the means ± S.E.M.; n = 4; ns, non-significant, *p < 0.05, **p < 0.01, compared to ACM-control; and &p < 0.05, compared to ACM-OMVs. d The graph shows the fold change of the DEVDase activity of CAD cells treated with 10 µM etoposide as a positive inducer of apoptosis, or of CAD cells treated with ACM from TNF-, or OMV-treated astrocytes ± 1 µM IMD 0354 inhibitor, compared to the ACM control. The results are expressed as the means ± S.E.M.; n = 3; ns, non-significant, *p < 0.05 and **p < 0.01, compared to the AMC-control; and ##p < 0.01, compared to AMC-TNF. e–h Quantification of the cytokines TNF (e), IL-6 (f), IL-1β (g) and IFNγ (h). Values in the graph are expressed as the means ± S.E.M; n = 3–4. **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared to the ACM-control; ####p < 0.0001, compared to ACM-OMVs; †††p < 0.001 and ††††p < 0.0001, compared to ACM-TNF