Constitutive Activation of Receptor Tyrosine Kinases in IPF Cells is Necessary for Differentiation into Myofibroblasts.

<p>Baseline levels of p-tyrosine proteins were assessed by western blotting in early and late passage control (n = 4) and IPF (n = 7) cells. Representative immune-blots are shown in panel A. Then, to address whether p-tyrosine signalling was inducible upon exogenous oxidative stress early passage control fibroblasts (n = 3) were treated for 15 min with increasing concentration of H₂O₂. Pre-treatment of cells with the ROS scavenger NAC (10 mM for 1 h) was also analysed. Modulation of p-tyrosine levels are shown in a representative immune-blot in panel B (left). Conversely, representative changes of p-tyrosine levels in early passage IPF cells (n = 3) treated with NAC (10 mM for 1 h) are shown on the right. To investigate whether myofibroblast differentiation is dependent upon tyrosine kinase signalling, changes of α-SMA expression were assessed by means of western blotting in early passage control (n = 3) and IPF (n = 5) fibroblasts treated for 30 min with the RTKs inhibitors AG1296 (anti-PDGF-R, 2 µM) and AG1478 (anti-EGF-R, 2 µM). Representative immune-blots of α-SMA/tubulin and relative statistics are shown in panel C. *p<0.001 versus untreated IPF; **p<0.01 versus untreated IPF. Finally, to investigate whether RTKs inhibition abrogates peroxide-induced α-SMA expression early passage control cells (n = 3) were pre-treated for 30 min with the RTKs inhibitors AG1296 (2 µM) and AG1478 (2 µM) and then exposed for 2 h to H₂O₂ (200 µM). Panel D shows representative immune-blots of α-SMA/tubulin and relative statistics. Results are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.05 versus basal; **p<0.001 versus H₂O₂ treated cells.</p

Negative Modulation of the Pro-Fibrotic Phenotype by ROS Scavenging.

<p>Control and IPF fibroblasts were treated for 48 h with the ROS scavenger NAC (5 mM). Panels A and B show representative immune-blots for α-SMA/tubulin and relative statistics in early passage control (n = 3) and IPF (n = 3) cells, respectively. Panel C illustrates changes of type-I collagen expression in early passage IPF fibroblasts. Changes of α-SMA and type-I collagen expression in NAC-treated late passage IPF fibroblasts (n = 3) are shown in panels D and E, respectively. Data are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.05 versus basal.</p

Chronic Oxidative Stress Induces a Pro-Fibrotic Phenotype.

<p>Changes of α-SMA expression were investigated by means of western blot in early control fibroblasts (n = 3) upon exogenous administration of hydrogen peroxide. Panel A shows changes of α-SMA expression upon cell treatment with increasing concentrations of H₂O₂ for 48 h. Conversely, panel B shows changes of α-SMA by treating cells with a fixed concentration of H₂O₂ (200 µM) at different time points. A representative immune-blot of α-SMA/tubulin is also shown. Panel C shows type-I collagen expression, assessed by real time PCR, in cells incubated for 48 h in the presence of H₂O₂ (200 µM). Expression of α-SMA in H₂O₂ (200 µM)-treated early passage IPF fibroblasts (n = 3) over time is shown in panel D. All data are expressed as mean fold change ± SD and are representative of three independent experiments. *p<0.001 versus basal; **p<0.05 versus basal.</p

IPF Cells Produce High Levels of ROS.

<p>The baseline ability of control (n = 4) and IPF (n = 7) cells to generate ROS was measured at different culture passages with different methods. The intracellular content of ROS was first measured fluorimetrically in DCHF-DA loaded cells at early passages. Panel A shows levels of DCF fluorescence in early (full bars) and late (dashed bars) passage control and IPF cells. *p<0.05 versus control cells. Representative flow cytometry dot plots of the distribution of DCF fluorescence in control and IPF fibroblasts at different culture passages are shown in panel B. Panel C shows O₂^- production, estimated by means of superoxide dismutase-inhibitable cytocrome c reduction, respectively in early (full bars) and late (dashed bars) passage control and IPF fibroblasts. All data are reported as mean value ± SD and are representative of three independent experiments. *p<0.01 versus early passage control cells; **p<0.05 versus early passage control cells; ***p<0.001 versus early passage IPF cells.</p

ROS Generation in IPF Fibroblasts Occurs Through the Activation of a Membrane NADPH Oxidase-Like System.

<p>Basal levels of ERK phosphorylation were assessed in control (n = 4) and IPF (n = 7) fibroblasts at different culture passages. Further, to determine whether a functional NADPH oxidase complex was involved in the generation of ROS, changes of p-ERK expression were analysed in response to treatment with DPI (20 µM), a flovoprotein inhibitor. Modulation of p-ERK levels was analysed upon 30 min of cell treatment. Representative immune-blots of p-ERK/tubulin and relative statistics are respectively shown in panels A and B. *p<0.001 versus basal control cells; **p<0.001 versus basal IPF cells. To investigate whether ERK signalling was inducible upon oxidative stress, early passage control (n = 3) and IPF fibroblasts (n = 3) were treated with increasing concentrations of H₂O₂ for 30 min. Pre-treatment of cells with the ROS scavenger NAC (10 mM for 1 h) was also analysed. Representative immune-blots and relative statistics of three independent experiments are reported in panels C (*p<0.01 versus basal; **p<0.01 versus 1 mM H₂O₂ treated cells) and D (*p<0.05 versus basal; **p<0.01 versus basal). To address whether ERK activation is necessary for α-SMA expression, modulation of peroxide-induced α-SMA (H₂O₂ 200 µM for 2 h) was analysed in early passage control cells (n = 3) pre-treated with UO126 (10 mM for 30 min). Similarly, α-SMA expression was evaluated in early passage IPF fibroblasts (n = 3) in response to UO126. Panel E shows representative immune-blots of α-SMA/tubulin and relative statistics. All data are expressed as mean fold change ± SD. *p<0.05 versus H₂O₂ treated cells; **p<0.05 versus basal.</p

IL-17A+ and IL-17F+ cell frequency positively correlates with disease duration.

<p>IL-17A+ and IL-17F+ cells were identified by immunohistochemistry. Each symbol represents a single individual. Significance was assessed by Pearson's correlation test. mo  =  months.</p

Co-expression of IL-17A with IL-17C or IL-17E.

<p>Indirect immunofluorescence analysis was used to assess the expression of IL-17A (green), in combination with IL-17C (<b>A</b>) or IL-17E (<b>B</b>) or IL-17F (<b>C</b>) (red), and DAPI staining for nuclei (blue) in the dermis of one biopsy of 3 assessed (1 HD, 1 SSc and 1 morphea). Original magnification 40X.</p

Clinical characteristics of the study populations.

<p>ACA: anti-centromere antibody; ANA: anti-nuclear antibody; ATA: anti-topoisomerase-1 antibody; DU: digital ulcer; GERD: gastro-esophageal reflux disease; ILD: interstitial lung disease; MRRS: Modified Rodnan Skin Score; n/a not available.</p

IL-17F and IL-17E, but not IL-17C, enhance MCP-1 and MMP-1 production by normal and SSc fibroblasts.

<p>Dermal fibroblasts were cultured in triplicate in the presence of increasing amounts (3, 30, 300, 600 ng/ml) of IL-17C, IL-17E or IL-17F for 48 h. Box-plot show the levels of MCP-1, IL-6, IL-8, MMP-1 and type I collagen assessed in fibroblast culture supernatants from 4 HD and 4 SSc. The box represents values between 25^th and 75^th percentile with a line at the median (50^th percentile). The whiskers extend above and below the box to show the highest and the lowest values. IL-17A (30 ng/ml), TNF (A, 1 ng/ml) or TGF-β (B, 10 ng/ml) were used as controls. Significance versus nil condition was assessed by one-sample t-test.</p

Frequency of IL-17A+ and IL-17F + cells in SSc and morphea.

<p><b>A.</b> Immunohistochemical analysis of IL-17A and IL-17F in healthy (HD), lesional SSc and morphea skin. Arrows indicate IL-17A (upper panels) or IL-17F (lower panels) positive cells. Results are representative of 14 SSc, 5 Morphea and 8 HD individuals. Original magnification 20X, scale bar 100 µm. Insets, 2X. <b>B.</b> Frequency of IL-17A+ and IL-17F+ cells expressed as percentage of total cells. Each symbol represents a distinct individual and the line depicts the mean. Empty and full dots refer to limited and diffuse SSc, respectively. Significance was assessed by Mann-Whitney test.</p