Lung accumulation of Th 17 cells but not T regs is limited in the absence of MyD88 signalling.

<p>Lymphocyte numbers in the bronchoalveolar lavage (BAL) fluid (<b>A</b>) and CD4+ T lymphocyte numbers in lung suspensions (<b>B</b>) obtained from wild-type (WT) and MyD88-knockout (MyD88-KO) mice 60 days after silica (SiO₂) or saline solution (NaCl) treatment. Expression of Foxp3 (<b>C</b> and <b>D</b>), IL-17A (<b>E</b>) and RORγ (<b>F</b>) analyzed by RTqPCR in lungs (<b>C</b>) and in lung CD4+ T lymphocytes (<b>D–F</b>) obtained from WT and MyD88-KO mice 60 days after silica or saline treatment. Expression of Foxp3 and IL-17A was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t-test. ns indicates no statistically significant difference and * = p<0.05 and ** = p<0.01 indicates statistically significant difference between values measured in silica-treated WT mice and silica-treated KO mice.</p

Lung inflammation but not collagen accumulation is reduced in MyD88-KO mice treated with silica particles.

<p>Neutrophil (<b>A</b>) and macrophage (<b>B</b>) numbers in the bronchoalveolar lavage (BAL) fluid of wild-type (WT) and MyD88-knockout (MyD88-KO) mice treated with silica (SiO₂, 2.5 mg/mouse) or saline solution (NaCl) and sacrificed at day 60. Quantification of IL-1β (<b>C</b>) by ELISA in the BAL fluid of WT and MyD88-KO mice treated with SiO₂ or NaCl at day 60. Lung fibrosis assessed by OH-proline contents (<b>D</b>) in WT and MyD88-KO mice after silica or saline instillation (d60). Bars represent means ± SEM (n = 4–6). These results were treated statistically by a t- test. ns indicates no statistically significant difference and ** = p<0.01 indicate statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Robust fibrotic response in the absence of innate immune receptors or ligands.

<p>Hydroxyproline (OH-proline) content assessed in lung homogenates of mice 60 days after saline (NaCl) or silica (SiO₂) instillation in wild-type (WT), IL-1R- (<b>A</b>), ASC- (<b>B</b>), NALP3- (<b>C</b>), IL-1β- (<b>D</b>), TRIF- (<b>E</b>), TLR2/4- (<b>F</b>), TLR3- (<b>G</b>), γδTCR- (<b>H</b>), IL-23p19- (<b>I</b>) knockout (KO) mice. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t- test. ns indicates no statistically significant difference and ** = p<0.01 indicates statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Lung expression of TGF-β1, IL-10 and PDGF-B is not reduced in MyD88-KO mice treated with silica particles.

<p>Lung expression of TGF-β1 (<b>A</b>), IL-10 (<b>B</b>) and PDGF-BB (<b>C</b>) analyzed by RTqPCR in tissue or by ELISA in BAL of wild-type (WT) and MyD88-knockout (MyD88-KO) mice 60 days after silica (SiO₂) or saline (NaCl) treatment. Expression of PDGF B (<b>D</b>) analyzed by RTqPCR in lung CD4+ T lymphocytes purified from NaCl- or SiO₂-treated WT and MyD88-KO mice at day 60. Expression of TGF-β1, IL-10 and PDGF-B was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a t- test and no statistically significant difference (ns) between values measured in silica-treated WT mice and silica-treated KO mice was noted.</p

MyD88-KO mice treated with silica particles developed limited granuloma and neutrophil infiltration but marked fibrosis and lymphocyte accumulation.

<p>5-µm sections of paraffin-embedded lung tissue of wild-type (WT) and MyD88- knockout (MyD88-KO) mice treated with silica (SiO₂, day 60) were stained with hematoxylin and eosin (panels <b>A–B</b> and <b>I–J</b>; neutrophils are indicated by black arrows and lymphocyte aggregates by an asterisk), with Masson's trichrome (panels <b>C–F</b>) or with silver (panels <b>G–H</b>). Sections are representative of 3–4 mice examined. Magnification 50X (panels A and B), 100X (panels <b>C–H</b>) and 400X (panels I–J). Histological quantification of fibrotic nodule, diffuse fibrosis and lymphoid aggregate numbers (<b>K</b>). These results were treated statistically by a t- test. * = p<0.05, ** = p<0.01 indicate statistically significant difference between values measured in silica-treated WT and silica-treated KO mice.</p

Pulmonary accumulation of T regs and Th17 lymphocytes is associated with the development of silica-induced lung fibrosis.

<p>Number of CD4+ (<b>A</b>) and CD8+ (<b>B</b>) analyzed by flow cytometry in lung cell suspensions obtained from wild-type mice after silica (SiO₂, d3 to d60) or saline (d0) instillation. Expression of Foxp3 (<b>C</b>) and IL-17A (<b>D</b>) analyzed by RTqPCR in the lungs obtained from wild-type mice after silica (d3 to d60) or saline (d0) treatment. Expression of Foxp3 and IL-17A was normalized on that of 18S RNA. Bars represent means ± SEM (n = 3–5). These results were treated statistically by a Student Neuman-Keul's test. * = p<0.05, ** = p<0.01, *** = p<0.001 indicate statistically significant difference between values measured in saline-treated mice and silica-treated mice.</p

Lung histological responses to bleomycin are attenuated by vardenafil.

<p>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.</p

Exaggerated proliferation and myofibroblast differentiation of CF fibroblasts.

<p>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 of³H-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. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p

Vardenafil prevents overresponsive proinflammatory status in CF fibroblasts.

<p>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. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p

Exaggerated CF lung responses to bleomycin are attenuated by vardenafil.

<p>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. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p