Increasing stiffness inhibits IFN-I response in pDCs via integrin-β1. (A–D) Purified pDCs from HDs (n = 4) were cultured either at 0.2 or 50 kPa for 5 h and analyzed by RNA-seq. (A) PCA plot of RNA-seq analysis. (B) Heatmap of all differentially regulated genes by stiffness, followed by pathways analysis using hallmark genes analysis for cluster 1. (C) Canonical functional activation bar chart as identified by QIAGEN IPA, showing the top 11 that were differentially regulated by stiffness in human pDCs. (D) Volcano plot comparing gene expression in pDCs at 0.2 or 50 kPa. Colors on the graph indicate differentially expressed genes, and the genes involved in cell migration are indicated. (E and F) Purified pDCs from HDs (n = 5–10; three to five independent experiments) were cultured with media alone or at different stiffness (as indicated) alone or either with the TLR9 agonist (C274 at 0.075 μM) or the TLR7 agonist (influenza virus, VR-95 at 0.5 pfu/cell). Supernatants were collected after 15 h and IFN-α secretion quantified by ELISA. (G) Purified pDCs from HDs (n = 9; five independent experiments) were first incubated in media alone or with a neutralizing antibody against integrin-β1 (20 μg/ml) for 1 h, then cultured at 50 kPa stiffness alone or with a TLR9 agonist (C274 at 0.075 μM). Supernatants were collected after 15 h and IFN-α secretion quantified by ELISA. (H) Purified pDCs from HDs (n = 4; three independent experiments) were cultured in media alone or with an integrin-β1 agonist (pyrintegrin: 0.5 or 1 μM) for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 5 h. RNA was collected and analyzed for IFNA2 expression. Individual donors are indicated; all results are represented as mean ± SEM, and statistical significance was evaluated using Mann–Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.