Fig 1: Effect of CXCL9(74–103) on TGF‐β1 and PDGF. (a) Competition of recombinant human TGF‐β1 with CXCL9(1–78) or CXCL9(74–103) for binding to heparan sulphate‐coated plates was tested. Results are expressed as the percentage inhibition of TGF‐β1 binding to heparan sulphate and shown as mean ± SEM (n = 2). (b) The xCELLigence Real‐Time Cell Analyzer was used to measure fibroblast migration. PDGF‐β (30 ng mL−1) or medium (control) was added to the wells of the lower chamber, and medium with or without CXCL9(74–103) (0.3 or 3 µm) was added with the fibroblasts in the upper chamber. In each experiment, all conditions were tested at least in duplicate. Data represent mean ± SEM (n = 3). *P < 0.05, ***P < 0.001 versus PDGF. (c) To determine the effect of CXCL9(74–103) on TGF‐β1‐induced CTGF production, fibroblasts were seeded in six‐well plates and either left untreated or stimulated with TGF‐β1 in the presence or absence of 100 nm of CXCL9(74–103). After 48 h, an enzyme‐linked immunosorbent sandwich assay (ELISA) was used to measure CTGF production (n = 5; conditions were tested in duplicate in each experiment). *P < 0.05.
Fig 2: IL-31 induced SSc-like phenotype in DFs.a, b DFs from dcSSc patients and healthy controls were cultured for 24 h with rhIL-31 (0, 10, 25, 50, or 100 ng/ml) to examine Col1a1 and Col1a2 mRNA expression (a) and type I collagen production (b) by real-time PCR and ELISA, respectively. Exact p values (Ctrl + IL-31 10 ng/ml vs. Ctrl, Ctrl + IL-31 25 ng/ml vs. Ctrl, Ctrl + IL-31 50 ng/ml vs. Ctrl, Ctrl + IL-31 100 ng/ml vs. Ctrl, SSc + IL-31 10 ng/ml vs. SSc, SSc + IL-31 25 ng/ml vs. SSc, SSc + IL-31 50 ng/ml vs. SSc, SSc + IL-31 100 ng/ml vs. SSc) = 0.690, 0.095, 0.056, 0.008, 0.032, 0.016, 0.008, 0.008 (Col1a1); 0.421, 0.421, 0.222, 0.016, 0.008, 0.008, 0.008, 0.008 (Col1a2); 0.508, 0.127, 0.841, 0.008, 0.008, 0.008, 0.008, 0.008 (Type I collagen). Relative fold differences (Ctrl + IL-31 10 ng/ml, Ctrl + IL-31 25 ng/ml, Ctrl + IL-31 50 ng/ml, Ctrl + IL-31 100 ng/ml, SSc + IL-31 10 ng/ml, SSc + IL-31 25 ng/ml, SSc + IL-31 50 ng/ml, SSc + IL-31 100 ng/ml) = 1.10, 1.16, 1.19, 1.53, 1.47, 2.10, 2.24, 2.71, 2.86 (Col1a1); 1.11, 1.10, 1.31, 1.54, 1.83, 2.35, 2.54, 2.75, 3.25 (Col1a2). c Protein levels of TGF-ß1, CTGF, MMP-1, MMP-3, and MMP-9 were assessed by ELISA in the supernatants of dcSSc DFs treated with or without rhIL-31 (50 ng/ml). Exact p values = 0.008 (TGF-ß1), 0.587 (CTGF), 0.008 (MMP-1), 0.016 (MMP-3), 0.024 (MMP-9). d Representative western blot analysis of a-SMA in dcSSc DFs treated with or without rhIL-31 (50 ng/ml; left). Quantification of protein expression normalized to ß-actin (right). Exact p value = 0.008. e Relative mRNA expression level of Acta2 was evaluated by real-time PCR in dcSSc DFs treated with or without rhIL-31 (50 ng/ml). Exact p value = 0.008. Relative fold difference = 2.85. f BrdU incorporation was quantified by ELISA in dcSSc DFs treated with or without rhIL-31 (50 ng/ml). The absorbance at 450 nm was measured. Exact p value = 0.008. g DFs from dcSSc patients were treated with or without rhIL-31 (50 ng/ml) and analyzed for apoptosis by flow cytometry. Representative dot plots were shown (left). Annexin-V+, 7-AAD- cells were considered early apoptotic cells, and Annexin-V+, 7-AAD+ cells were considered late apoptotic cells, respectively (right). Exact p values (IL-31 vs. media) = 0.008 (early apoptosis); 0.008 (late apoptosis). h DFs from dcSSc patients and healthy controls were pretreated with mitomycin C, scratched to make a wound, and incubated for 24 h with rhIL-31 (50 ng/ml) or media alone. Representative microscopic images were shown (left, scale bar=500 µm). Red lines show the borders of the wounds. Wound closure was expressed as the percentage of wound reduction from the original wound (right). Exact p values (Ctrl + media vs. Ctrl + IL-31, Ctrl + media vs. SSc + media, Ctrl + media vs. SSc + IL-31, Ctrl + IL-31 vs. SSc + media, Ctrl + IL-31 vs. SSc + IL-31, SSc + media vs. SSc + IL-31) = 0.005, 0.0000001, 0.00000000006, 0.00008, 0.000000004, 0.00002. i, j DFs obtained from dcSSc patients were cultured with rhIL-31 (50 ng/ml) or media alone for 24 h, and expression levels of IL-6, IL-33, and CCL2 were measured by real-time PCR (i) and ELISA (j). Exact p values = 0.008 (Il6); 0.032 (Il33); 0.008 (Ccl2); 0.008 (IL-6); 0.032 (IL-33); 0.008 (CCL2). Relative fold differences = 1.99 (Il6); 1.37 (Il33); 2.39 (Ccl2). n = 5. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. unstimulated Ctrl or SSc fibroblasts (a, b) or media (c–g, i, and j). Significance was determined by two-tailed Mann-Whitney U test (a–g, i, and j) and one-way analysis of variance followed by Tukey’s post hoc comparison test (h). Ctrl, healthy controls; OD, optical density. Source data are provided as a Source Data file.
Fig 3: IL-31 up-regulated Th2- and fibrosis-related cytokines in BLM-SSc mice.a Relative mRNA expression levels of Il4, Il6, Il10, Il17a, Tnf, Tgfb1, and Ifng in the skin and lungs were evaluated by real-time PCR. Exact p values (PBS-Ctrl + sham vs. PBS-Ctrl + IL-31, PBS-Ctrl + sham vs. BLM-SSc + sham, PBS-Ctrl + sham vs. BLM-SSc + IL-31, PBS-Ctrl + IL-31 vs. BLM-SSc + sham, PBS-Ctrl + IL-31 vs. BLM-SSc + IL-31, BLM-SSc + sham vs. BLM-SSc + IL-31) = 0.866, 0.039, 0.00006, 0.162, 0.0003, 0.021 (Il4, skin); 0.403, 0.00000000006, 0.0000000000002, 0.0000000003, 0.0000000000004, 0.000001 (Il6, skin); 0.034, 0.004, 0.000003, 0.699, 0.0007, 0.007 (Il10, skin); 0.926, 0.000007, 0.0000005, 0.00002, 0.000001, 0.344 (Il17a, skin); 0.977, 0.012, 0.063, 0.005, 0.029, 0.832 (Tnf, skin); 0.572, 0.0001, 0.00000001, 0.001, 0.00000008, 0.0001 (Tgfb1, skin); 0.428, 0.00007, 0.002, 0.001, 0.040, 0.361 (Ifng, skin); 0.00008, 0.003, 0.0000002, 0.286, 0.008, 0.0002 (Il4, lung); 0.023, 0.00001, 0.0000001, 0.007, 0.00002, 0.038 (Il6, lung); 0.002, 0.227, 0.00003, 0.085, 0.231, 0.002 (Il10, lung); 0.192, 0.000008, 0.000001, 0.0004, 0.00005, 0.686 (Il17a, lung); 0.641, 0.013, 0.041, 0.127, 0.321, 0.937 (Tnf, lung); 0.967, 0.0002, 0.0000005, 0.0005, 0.000001, 0.014 (Tgfb1, lung); 0.973, 0.00003, 0.0001, 0.00007, 0.0003, 0.868 (Ifng, lung). Relative fold differences (PBS-Ctrl + IL-31, BLM-SSc + sham, BLM-SSc + IL-31) = 1.55, 3.12, 5.45 (Il4, skin); 2.14, 13.09, 19.11 (Il6, skin); 2.59, 3.15, 5.16 (Il10, skin); 1.22, 3.63, 4.24 (Il17a, skin); 0.89, 1.97, 1.74 (Tnf, skin); 1.34, 2.57, 4.12 (Tgfb1, skin); 1.68, 3.71, 2.98 (Ifng, skin); 3.59, 2.81, 5.20 (Il4, lung); 5.56, 10.92, 15.12 (Il6, lung); 2.29, 1.56, 2.85 (Il10, lung); 2.35, 5.74, 6.45 (Il17a, lung); 1.27, 1.81, 1.68 (Tnf, lung); 1.16, 2.98, 4.22 (Tgfb1, lung); 1.22, 4.34, 3.95 (Ifng, lung). b Protein levels of TGF-ß1 were evaluated by ELISA. Exact p values (PBS-Ctrl + sham vs. PBS-Ctrl + IL-31, PBS-Ctrl + sham vs. BLM-SSc + sham, PBS-Ctrl + sham vs. BLM-SSc + IL-31, PBS-Ctrl + IL-31 vs. BLM-SSc + sham, PBS-Ctrl + IL-31 vs. BLM-SSc + IL-31, BLM-SSc + sham vs. BLM-SSc + IL-31) = 0.924, 0.00006, 0.0000003, 0.0002, 0.0000007, 0.022 (skin); 0.805, 0.00003, 0.0000004, 0.0002, 0.000002, 0.076 (lung). c, d Relative mRNA levels and protein levels of Ctgf were evaluated by real-time PCR (c) and ELISA (d), respectively. Exact p values (PBS-Ctrl + sham vs. PBS-Ctrl + IL-31, PBS-Ctrl + sham vs. BLM-SSc + sham, PBS-Ctrl + sham vs. BLM-SSc + IL-31, PBS-Ctrl + IL-31 vs. BLM-SSc + sham, PBS-Ctrl + IL-31 vs. BLM-SSc + IL-31, BLM-SSc + sham vs. BLM-SSc + IL-31) = 0.912, 0.000000003, 0.0000000005, 0.000000007, 0.000000001, 0.391 (Ctgf, skin); 0.967, 0.00000007, 0.000000009, 0.0000001, 0.00000002, 0.393 (Ctgf, lung); 0.972, 0.00006, 0.00002, 0.00003, 0.00001, 0.941 (CTGF, skin); 0.933, 0.005, 0.006, 0.017, 0.019, 0.999 (CTGF, lung). Relative fold difference (PBS-Ctrl + IL-31, BLM-SSc + sham, BLM-SSc + IL-31) = 1.14, 3.79, 4.13 (skin); 1.12, 3.86, 4.30 (lung). e Relative mRNA expression levels of Mmp3, Mmp9, Mmp13, Timp1, Timp2, and Timp3 were assessed in the lungs of BLM-SSc mice by real-time PCR. Exact p values = 0.008 (Mmp3); 0.016 (Mmp9); 0.008 (Mmp13); 0.008 (Timp1); 0.008 (Timp2); 0.008 (Timp3). Relative fold differences = 0.79 (Mmp3); 0.72 (Mmp9); 0.68 (Mmp13); 2.06 (Timp1); 1.42 (Timp2); 1.26 (Timp3). n = 5. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using one-way analysis of variance followed by Tukey’s post hoc comparison test (a-d) and two-tailed Mann–Whitney U test (e). The results shown are representative of three independent experiments with similar results. PBS-Ctrl, PBS-treated control. Source data are provided as a Source Data file.
Fig 4: Summary of proposed mechanism for OPG in senescence and fibrosis. In response to stiffness, Primary human lung fibroblasts show increased proliferation, higher deposition of ECM proteins and higher secretion of SASP and fibrosis related cytokines. OPG is proposed to act as a regulatory mechanism between senescence and fibrosis by binding to RANKL and CTGF, and potentially inhibiting their function.
Fig 5: Stiffness induced a secretory phenotype in Primary human lung fibroblasts cultured on stiff matrices. (A,B) DCN gene expression and secreted protein were measured after 7 days of culture on soft (3.94 ± 0.87 kPa) and stiff (11.34 ± 3.82 kPa) hydrogels. (C–H) Levels of secreted factors RANKL, OPG, CTGF and TGF-β1 including TGF-β1 and CTGF gene expression after 7 days of culture on soft and stiff hydrogels. I) Correlation was calculated between OPG, RANKL and all other secreted factors. Panel J shows a fluorescent staining for DCN, DAPI was used for identification of cell nuclei. Illustrated photographs are representative images of in total 7 unique donors. Gene expression data were normalised against 18S and expressed as 2−ΔCT x 106. Secreted matrix proteins and cytokines were normalised to cell counts and expressed as pg/ml or ng/ml 105 cells. Differences between soft and stiff hydrogels were analysed by a Wilcoxon matched pairs signed rank test. Correlation was calculated using Pearson correlation coefficient. (n = 6) Scale bar = 50 µm. Each donor has a different unique colour (blue, red, yellow, green, black, and grey).
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