Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis

Abstract

Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

Figure 1. Exaggerated CF lung responses to bleomycin are attenuated by vardenafil.

a) Soluble collagen content in homogenized unlavaged lungs; b) lymphocyte counts, c) CCL-2, d) IL-6, and e) TGF-β1 in bronchoalveolar lavage (BAL); and f) TIMP-1 in homogenized unlavaged lungs from CF mice homozygous for the F508del mutation and from wild-type (WT) mice 10 days after deposition into the lungs by pharyngeal aspiration of a single dose of 0.015 U bleomycin (Bleo). In case of simultaneous treatment with bleomycin and vardenafil, animals were given a first intraperitoneal injection of 0.14 mg/kg vardenafil (Vard) on the day before the bleomycin dose and every day thereafter until the day before sampling. Values are means ± SEM of 5 animals per group from a representative experiment selected from a series of 3 experiments with similar results. *: P<0.05; **: P<0.01; *** P<0.001 for comparison of mean values.

Figure 2. Lung histological responses to bleomycin are attenuated by vardenafil.

Lung histological sections of wild-type (a,b,e,f,i,j) and CF mice homozygous for the F508del mutation (c,d,g,h,k,l) 10 days after treatment with saline (NaCl; a–d), bleomycin (Bleo; e–h) or bleomycin and vardenafil (Bleo+Vard; i–l) were stained with hematoxylin and eosin (a,c,e,g,i,k); impregnated with silver (b,d,f,h,j,l) or stained with Masson’s trichrome (inserts). Bars, 100 µm in panels a,c,e,g,i,k; 20 µm in panels b,d,f,h,j,l; and 40 µm in inserts. Representative micrographs from 5 mice per group.

Figure 3. Mouse fibroblasts express CFTR protein.

CFTR expression in mouse nasal epithelial cells in primary cultures, fibroblasts (3T3 cell line and lung cells at passages #2 and #3) and macrophages (J774 cell line and alveolar and peritoneal cells in primary cultures). a) Immunoblots of precipitates for IgG blank and lung fibroblasts from Cftr knockout mice (KO), wild-type (WT) and CF mice homozygous for the F508del mutation. Immunoprecipitates (IP) using 4×10⁶ fibroblasts grown in Petri dishes were lysed in a buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) supplemented with Complete PIC (Roche) and incubated with mouse anti-CFTR antibody clone 24-1 coupled with G protein-conjugated magnetic Dynabeads. Data selected from at least 4 experiments with similar results. As expected for mouse CFTR, a band was recognized at 160 kDa but not detected when IPs were performed with non-immune IgG. b) Immunofluorescence labelling for CFTR (green) in fibroblasts purified from Cftr knockout (KO) mice, wild-type mice with (WT) or without CFTR antibody (w/o Ab), and CF mice homozygous for the F508del mutation. Fibroblasts grown on collagen-coated cover glasses were fixed with acetone and permeabilized with 0.25% Triton X-100. Detection was obtained using anti-mouse AlexaFluor 488 secondary antibody after overnight incubation with anti-CFTR antibody clone 24-1. Nuclei stained with DAPI blue. Bars: 20 µm. Data selected from at least 3 experiments with similar results. c) Total CFTR mRNA expression, using 18S rRNA as a reference. Data expressed as means ± SEM of 3–8 samples per group.

Figure 4. Exaggerated proliferation and myofibroblast differentiation of CF fibroblasts.

Cell proliferation and myofibroblast differentiation in cultured lung (A–D) and skin (E–H) fibroblasts at the second passage purified from CF mice homozygous for the F508del mutation and from wild-type (WT) mice. A,e) Uptake of ³H-thymidine (1 µCi/well) assessed in cultured cells seeded at 30×10³ cells/well, in the absence of any added growth factor to culture media or in the presence of 1 to 100 ng/ml human rPDGF-BB for 1 h. After 48 h, adherent cells were trypsinized before ³H-thymidine counting. Data expressed as counts per minute (cpm). b,f) Cell growth analysis assessed by daily counting, in a Neubauer chamber, of trypsinized cells cultured in the absence of any added growth factor to culture media. c,g) α-SMA mRNA expression, using 18S RNA as a reference, assessed in the absence of any added growth factor to culture media or in the presence of 1 to 100 ng/ml human rTGF-β1 for 24 h. d) α-SMA mRNA expression assessed in the presence of 0.1 to 50 µM vardenafil (Vard) for 6 h. h) Micrographs of fibroblast cultures under stimulation with 10 ng/ml human rTGF-β1. Arrows identify formation of cellular aggregates. Bars: 100 µm. Values are means ± SEM of 3 multi(96)well cultures per group from a representative experiment selected from at least 3 experiments with similar results. *: P<0.05; **: P<0.01; *** P<0.001 for comparison of mean values.

Figure 5. Vardenafil prevents overresponsive proinflammatory status in CF fibroblasts.

a,b–g) mRNA and c) protein expression of proinflammatory cytokines in response to 0.1 mg/ml LPS in lung (a,b,e–g) and skin (c,d) cultured fibroblasts at the second passage purified from CF mice homozygous for the F508del mutation and from wild-type (WT) mice. At the mRNA level, markers were assessed 3 h after LPS stimulation. At the protein level, CCL-2 (c) was assessed 24 h after LPS stimulation. Vardenafil (Vard; 0.1 µM) was used for TNF-α (e), IL-1β (f) and IL-6 (g) mRNA expression studies. 18S RNA used as a reference gene. Values are means ± SEM of 3 multi(96)well cultures per group from a representative experiment selected from at least 3 experiments with similar results. *: P<0.05; **: P<0.01; *** P<0.001 for comparison of mean values.