Figure 1.
Genz-529648 reduces P. aeruginosa stimulated IL-8 mRNA expression and protein release.
Panels A and B. Genz-529648 reduces P. aeruginosa stimulated IL-8 mRNA expression. IB3-1 (A) and CuFi-1 (B) cells were treated with a range of doses of Genz-529648 (1–100 nM) or solvent alone for 1 hour and then infected with PAO1 for 4 hours at 37°C. The inflammatory response was evaluated by studying the expression of IL-8 mRNA, which was measured by Real-time qPCR and obtained by comparing the ratio IL-8 and the housekeeping gene GAPDH between non-infected and infected cells. The results are expressed as the % of untreated cells and represent the mean ± standard error of the mean of 4 independent experiments in duplicate. Comparisons between groups were made by using Student’s t tests. Panels C and D. Genz-529648 reduces the P. aeruginosa induced IL-8 secretion. IB3-1 (C) and CuFi-1(D) cells were treated with Genz-529648 (100 nM) for 1 hour prior to infection with heat killed PAO1 for 24 hours. Data reported are the mean ± standard error of the mean of 4 independent experiments in duplicate. Comparisons between groups were made by using Student’s t tests.
Table 1.
Inhibition of P. aeruginosa stimulated IL-8 mRNA expression by alkylated iminosugars in IB3-1 and CuFi-1 cells.
Figure 2.
Infection with PAO1 increases β-glucosidase activity in IB3-1 and CuFi-1 cells.
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 t tests.
Figure 3.
Miglustat and Genz-529648 inhibit GBA2 activity in IB3-1 and CuFi-1 cells infected by P. aeruginosa.
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 figure 2. 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 t tests.
Figure 4.
Transfection with GBA2 siRNA reduces the expression of GBA2 in CF bronchial cells.
IB3-1 (A), CuFi-1 (B) or CF primary bronchial cells (C) were transfected with GBA2 siRNA or scrambled oligonucleotides for 24 h. GBA2 mRNA expression was measured by Real-time qPCR and obtained by comparing the ratio GBA2 and the housekeeping gene GAPDH between scrambled or siRNA treated cells. The data reported on the y-axis are relative to scrambled-treated cells and represent the mean ± SE of five (IB3-1, panel A), eight (CuFi-1, panel B) and four (CF primary bronchial, panel C) independent experiments performed in duplicate. Comparisons between groups were made by using Student’s t tests.
Figure 5.
Reduction of IL-8 is associated with a relevant decrease of GBA2 expression in CF bronchial cells.
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 figure 1. 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 t tests.
Figure 6.
GBA2 silencing reduces the IL-8 protein release in CuFi-1 cells.
CuFi-1 cells were transfected with GBA2 siRNA or scrambled oligonucleotides and then infected with PAO1 as indicated in figure 5. 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 t tests.
Figure 7.
GBA2 silencing reduces GBA2 activity in CuFi-1 cells.
CuFi-1 cells were transfected with GBA2 siRNA or scrambled oligonucleotides as indicated in figure 5. Eighteen or 42 hours after transfection, the cells were scraped and treated as indicated in figure 2. 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 t tests.
Figure 8.
Infection with PAO1 increases whole cell ceramides in CF bronchial epithelial cells.
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 figure 3. 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 t tests.
Figure 9.
Treatment with Genz-529648 reduces the ceramide content in IB3-1 cells infected with PAO1.
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 figure 3. 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 t tests (B).
Figure 10.
Metabolic pathways involved in ceramide formation.
Schematic representation of the primary metabolic pathways involved in ceramide production. Ceramide can be produced by the de novo 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 de novo 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).