Stiffness induces stress-associated TF s and inhibits ΙFN-I induction in pDCs via the modulation of the glycolysis pathways. (A) Purified pDCs from HDs (n = 2–4) were cultured at different stiffness (low stiffness: 0.2 kPa and high stiffness: 50 kPa) in the presence of a TLR9 agonist (C274 at 0.075 μM) for 5 h and analyzed by RNA-seq. All modulated pathways were identified using QIAGEN IPA. (B) Purified pDCs from HDs (n = 7; three independent experiments) were cultured at a stiffness of 0, 0.2, 2, and 50 kPa, and gene expression level of XBP1 splicing was analyzed by Q-PCR. (C) Purified pDCs from HDs were cultured at a stiffness of 0, 0.2, and 50 kPa, followed by incubation with the TLR9 agonist (C274 at 0.075 μM). The gene expression level of XBP1 splicing was analyzed by Q-PCR. (D) pDCs (n = 4; three independent experiments) were cultured at a stiffness of 0 or 50 kPa alone or with the NRF2 activator (KI696-5 μM) for 5 h. NRF2 protein levels were assessed by western blot at a 5 h time point. ImageJ was used to quantify the intensity of the band in the western blots and changes in the ratio between NRF2 and β-actin levels as compared to medium alone (normalized as 1) are indicated. (E and F) Purified pDCs from HDs (n = 3–4; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696 at 5 μM) for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 15 h. (E) IL6 secretion was analyzed by ELISA. (F) Cell viability was assessed by flow cytometry after 6 h of culture with the TLR9 agonist alone or with KI696. (G–K) Purified pDCs from HDs (n = 4–8; four independent experiments) were cultured in media alone or with the NRF2 activator (4-octyl itaconate [4-OI]-100 μM) for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM). (G) Cell viability was assessed by flow cytometry. (H and I) After 5 h of culture, gene expression levels of (H) HMOX1 and (I) IFNA2 were analyzed by Q-PCR. (J and K) After 15 h of culture, (J) IFNα and (K) IL6 secretion were quantified by ELISA. (L) Purified pDCs from HDs (n = 2–4) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by the TLR9 agonist (C274 at 0.075 μM) for 5 h and analyzed by RNA-seq. All modulated pathways were identified using QIAGEN IPA. Upregulated and downregulated pathways are indicated in red and blue, respectively, and the size represented the significance of the pathways. (M) Graphical representation of the interconnection of glycolysis with PPP. (N) Purified pDCs from HDs (n = 4; three independent experiments) were cultured either with media alone or with KI696 for 4 h. Intracellular ATP was quantified according to the manufacturer’s protocol and normalized to medium. (O) Purified pDCs from HDs (n = 6; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696) alone or with sodium pyruvate (10 mM) for 1 h, followed by incubation with the TLR9 agonist for 5 h and gene expression level of IL6 was analyzed by Q-PCR. (P) Purified pDCs from HDs (n = 2–3; two to three independent experiments) were transfected with CRISPR-Cas9 complex targeting either G6PD or TKT on exon 2 and exon 3 respectively using Lipofectamine CRISPRMAX reagent and cultured with IL3 (20 ng/ml) for 72 h. Gene expression level of TKT and G6PD was analyzed by Q-PCR. 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.