Extracellular GSH compromises the interaction of <i>B. cenocepacia</i> with mucociliary-differentiated CF bronchial epithelial cells. Panel A.

<p>MIP (Maximum Intensity Projection) from confocal system acquisition (Olympus IX 81 inverted microscope, software FV 1000) of monolayers infected with <i>B. cenocepacia</i> LMG 16656 for 3 hours, in absence (<b>upper</b>) or in presence (<b>bottom</b>) of 10 mM extracellular GSH. Cells were washed, fixed and permeabilized as described in Materials and Methods. Bacteria (red) and zona occludens (green) were detected using specific antibodies (R418 and anti-ZO1, respectively). Bar = 20 µm. <b>Panel B.</b> Invasion of mucociliary-differentiated CF bronchial epithelial cells by <i>B. cenocepacia</i> LMG 16656 in absence or in presence of GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayers. The reported values represent the mean ± SD obtained by measuring LMG 16656 invasion ability from six individual cultures. (*p = 0.01; #p = 0.002; **p<0.05).</p

Extracellular GSH decreases <i>B. cenocepacia</i> invasion. Panel A.

<p>Invasion of 9HTEo- and CFTE29o- by <i>B. cenocepacia</i> LMG 16656 was assayed in presence of 0, 0.1, 1 and 10 mM GSH. The % of invasion indicates the ratio between the number of intracellular bacteria recovered from infected cells with respect to the bacteria added to the cell monolayer. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion in three independent experiments. Asterisks denote statistically significant results (* p<0.05; ** p<0.01 and *** p<0.0001, respectively). White bar: untreated cells; grey bar: cells treated with 0.1 mM GSH; dotted bar: cells treated with 1 mM GSH; black bar: cells treated with 10 mM GSH. <b>Panel B.</b> 9HTEo- and CFTE29o- were pretreated with 0, 0.1, 1 and 10 mM GSH for 3 hours at 37°C, washed to remove GSH and then used in the infection assay. Results, which are expressed as the mean ± SD of intracellular bacteria isolated from cells pretreated with GSH with respect to untreated cells, are the average of three independent experiments (**p<0.01; ***p<0.0001). Bar colors are as in panel A. <b>Panel C.</b> Invasion of C38 and IB3-1 by <i>B. cenocepacia</i> LMG 16656 in presence of 0 and 10 mM GSH. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion ability in three independent experiments (*** p<0.0001). White bars: control cells; black bars: cells treated with 10 mM GSH. <b>Panel D.</b> Invasion of 9HTEo-, CFTE29o-, C38 and IB3-1 by <i>B. cenocepacia</i> 6L in presence of 0 and 10 mM GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD obtained by measuring 6L invasion ability in three independent assays (** p<0.01). White bars: control cells; black bars: cells treated with 10 mM GSH.</p

Extracellular GSH modifies <i>B. cenocepacia</i> LMG 16656 adhesion, but not intracellular replication. Panel A.

<p>Effect of extracellular GSH on <i>B. cenocepacia</i> LMG 16656 adhesion to 9HTEo- and CFTE29o- cells. Bars indicate the % of the bacteria adhering to cells with respect to the bacteria added to the cell monolayer. Results are the average ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (*p<0.05; ** p<0.01). <b>Panel B.</b> Effect of 10 mM GSH on the total number (adherent + intracellular bacteria) of <i>B. cenocepacia</i> LMG 16656 recovered after 3 hours of 9HTEo- epithelial cell infection. Results are shown as % of total bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (***p<0.0001). <b>Panel C. </b><i>B. cenocepacia</i> LMG 16656 replication within 9HTEo- cells. Fold replication values were determined by dividing the intracellular bacterial load at 1, 2.5 and 4 h post-infection by that determined after 30 minutes of infection. Results are the mean ± SD of three independent experiments.</p

Effect of extracellular GSH on pro-inflammatory cytokines expression stimulated by <i>B. cenocepacia</i> LMG 16656.

<p>9HTEo-, CFTE29o- cells were incubated in presence or absence of 10 mM extracellular GSH and infected with <i>B. cenocepacia</i> LMG 16656 (10 CFU/cell) in presence of 0 and 10 mM GSH for 3 hours. The expression of IL-8, TNF-α and IL-1β was analyzed by RT-PCR. Data are from a typical experiment out of three giving qualitatively similar results. Each data point is the average of three independent measures on each sample.</p

Cytofluorimetric analysis of surface thiols.

<p>After an incubation in presence or absence of 10 mM extracellular GSH, cells were treated with 10 µM Alexa fluor C₅-maleimide to label surface free thiols and then analyzed by FACScalibur system, as described in Materials and Methods. The histograms are from a typical experiment out of three giving essentially identical results.</p