Reduction of IL-8 is associated with a relevant decrease of GBA2 expression in CF bronchial cells.

<p>IB3-1 (A), CuFi-1 (B) or CF primary bronchial cells (C) were transfected with GBA2 siRNA or scrambled oligonucleotides for 24 h and then infected with PAO1 (10–50 CFU/cell). IL-8 mRNA expression was measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g001" target="_blank">figure 1</a>. The data reported on the y-axis are relative to scrambled-treated cells (A, B and C) or scrambled-treated uninfected cells (D, E and F) and represent the mean ± SE of five (IB3-1, panels A and D), eight (CuFi-1, panels B and E) and four (CF primary bronchial, panels C and F) independent experiments performed in duplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

Miglustat and Genz-529648 inhibit GBA2 activity in IB3-1 and CuFi-1 cells infected by <i>P. aeruginosa</i>.

<p>IB3-1 and CuFi-1 cells were treated with [2 µM] miglustat, [10 nM] Genz-529648 or solvent alone for 1 hour prior to infection with heat-killed PAO1 for 4 hours. Total β–glucosidase (A), GBA1 (B) and GBA2 (C) activities were measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g002" target="_blank">figure 2</a>. The data reported are the mean ± standard error of the mean of 3 (IB3-1) or 2 (CuFi-1) independent experiments in triplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

GBA2 silencing reduces the IL-8 protein release in CuFi-1 cells.

<p>CuFi-1 cells were transfected with GBA2 siRNA or scrambled oligonucleotides and then infected with PAO1 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g005" target="_blank">figure 5</a>. The supernatants were collected at the end of infection, and IL-8 protein release was measured as detailed in the “Methods” section. The data reported are the mean ± SE of eight independent experiments performed in duplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

Inhibition of <i>P. aeruginosa</i> stimulated IL-8 mRNA expression by alkylated iminosugars in IB3-1 and CuFi-1 cells.

<p>Inhibition of <i>P. aeruginosa</i> stimulated IL-8 mRNA expression by alkylated iminosugars in IB3-1 and CuFi-1 cells.</p

Treatment with Genz-529648 reduces the ceramide content in IB3-1 cells infected with PAO1.

<p>IB3-1 cells subjected to the SL metabolic labeling with (1-³H)sphingosine were treated with [10 µM] amitriptyline alone, in combination with [10 nM] Genz-529648, or with solvent alone and infected with PAO1 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g003" target="_blank">figure 3</a>. After lipid extraction, (³H)ceramide was separated from the other radioactive SLs by HPTLC, as detailed in the online supplement, and detected by digital autoradiography (total lipid extracts amounts corresponding to 4 µg of cellular proteins were applied on a 4-mm line. Time of acquisition: 48 hours). The digital autoradiography represents data obtained in three different experiments (A). The ceramide content was quantified by specific β-Vision software, and the data reported are the mean ± SE of three independent experiments. Comparisons between groups were made by using Student’s <i>t</i> tests (B).</p

Metabolic pathways involved in ceramide formation.

<p>Schematic representation of the primary metabolic pathways involved in ceramide production. Ceramide can be produced by the <i>de novo</i> biosynthesis, the hydrolysis of sphingomyelin (SM) by the action of sphingomyelinases and the catabolism of glycosphingolipids (GSL). In particular, it has been observed that in CF bronchial epithelial cells, the use of inhibitors of these pathways resulted in a reduction of ceramide. Myriocin acts on the first step of the <i>de novo</i> biosynthesis through the inhibition of the Serine-palmitoyl transferase (SPT); amitriptyline inhibits the acid SMase (ASM) responsible for SM catabolism; and miglustat, NB-DGJ and Genz-529648 are inhibitors of the β-glucosidases GBA1 and GBA2, which are involved in the hydrolysis of the glucosylceramide (GlcCer).</p

GBA2-Encoded β-Glucosidase Activity Is Involved in the Inflammatory Response to <i>Pseudomonas aeruginosa</i>

<div><p>Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by <i>P. aeruginosa</i> and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by <i>P. aeruginosa</i>. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely <i>N</i>-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by <i>P. aeruginosa</i>. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.</p></div

Infection with PAO1 increases β-glucosidase activity in IB3-1 and CuFi-1 cells.

<p>IB3-1 and CuFi-1 cells were infected with heat-killed PAO1 for 4 hours. The cells were then scraped and centrifuged; the cellular pellets were resuspended in water containing protease inhibitors and sonicated. Similar amounts of cellular proteins were used to perform the enzymatic assays to detect the activities of total β-glucosidase (A), GBA1 (B) and GBA2 (C), as reported in the Methods section. The data reported are the mean ± standard error of the mean of 4 (IB3-1) or 3 (CuFi-1) independent experiments in triplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

GBA2 silencing reduces GBA2 activity in CuFi-1 cells.

<p>CuFi-1 cells were transfected with GBA2 siRNA or scrambled oligonucleotides as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g005" target="_blank">figure 5</a>. Eighteen or 42 hours after transfection, the cells were scraped and treated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g002" target="_blank">figure 2</a>. Total β-glucosidase (B) and GBA2 (A) activities were measured as reported in the Methods section. The data reported are the mean ± standard error of the mean of 2 independent experiments in triplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

Infection with PAO1 increases whole cell ceramides in CF bronchial epithelial cells.

<p>IB3-1 (A) and CuFi-1 (B) cells were treated with [2 µM] miglustat, [10 nM] Genz-529648 or solvent alone and infected with PAO1 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g003" target="_blank">figure 3</a>. After infection, whole cell ceramides were analyzed by LC-MS and LC-MS/MS methods as described in the online supplement. The data reported are the mean ± SE of three independent experiments performed with both cell lines. Comparisons between groups were made by using Student’s <i>t</i> tests.</p