Figure 2.
Skin stiffness inhibits ΙFN-I induction via NRF2 activation. (A–D) Purified pDCs from HDs (n = 2–4) were cultured at different stiffnesses (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. (A) PCA plot of RNA-seq analysis. (B) Volcano plot showing type I IFN genes. (C) All differential regulated genes were analyzed for transcriptional factors that are either activated or inhibited by stiffness in TLR9-activated pDCs and were identified using QIAGEN IPA. Upregulated and downregulated TFs are indicated in red and blue, respectively. (D) Heatmaps of the genes that are transcriptionally regulated by the transcriptional factor NFE2L2 (NRF2) in Fig. 2 C. (E) Purified pDCs from HDs (n = 5; three independent experiments) were cultured either in media alone or with the NRF2 activator (KI696-5 μM) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. HMOX1 (marker gene induced by NRF2 activation) gene expression was analyzed by Q-PCR. (F–H) pDCs (n = 4; three to four independent experiments) were cultured at a stiffness of 0 or 50 kPa alone or with the NRF2 activator (KI696-5 μM) for 5 h. (F and G) NRF2 protein levels were assessed by flow cytometry. (H) NRF2 protein levels were assessed by western blot at a similar time point. (I) Purified pDCs from HDs (n = 3; three independent experiments) were cultured at a stiffness of 0 or 50 kPa for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. HMOX1 gene expression was analyzed by Q-PCR. (J and K) Purified pDCs from HDs (n = 5; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696-5 μM) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM). (J) Supernatant were collected after 15 h and IFNα secretion was analyzed by ELISA. (K) RNA was collected after 5 h, and IFNA2 gene expression was analyzed by Q-PCR. (L) Purified pDCs from HDs (n = 5; three independent experiments) were first cultured for 1 h either in media alone or with the NRF2 inhibitor (ML385-2 μM), then cultured at a stiffness of 0 or 50 kPa for another 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 5 h. IFNA2 gene expression 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. Source data are available for this figure: SourceData F2. Refer to the image caption for details.

Skin stiffness inhibits ΙFN-I induction via NRF2 activation. (A–D) Purified pDCs from HDs (n = 2–4) were cultured at different stiffnesses (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. (A) PCA plot of RNA-seq analysis. (B) Volcano plot showing type I IFN genes. (C) All differential regulated genes were analyzed for transcriptional factors that are either activated or inhibited by stiffness in TLR9-activated pDCs and were identified using QIAGEN IPA. Upregulated and downregulated TFs are indicated in red and blue, respectively. (D) Heatmaps of the genes that are transcriptionally regulated by the transcriptional factor NFE2L2 (NRF2) in Fig. 2 C. (E) Purified pDCs from HDs (n = 5; three independent experiments) were cultured either in media alone or with the NRF2 activator (KI696-5 μM) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. HMOX1 (marker gene induced by NRF2 activation) gene expression was analyzed by Q-PCR. (F–H) pDCs (n = 4; three to four independent experiments) were cultured at a stiffness of 0 or 50 kPa alone or with the NRF2 activator (KI696-5 μM) for 5 h. (F and G) NRF2 protein levels were assessed by flow cytometry. (H) NRF2 protein levels were assessed by western blot at a similar time point. (I) Purified pDCs from HDs (n = 3; three independent experiments) were cultured at a stiffness of 0 or 50 kPa for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM) for 5 h. HMOX1 gene expression was analyzed by Q-PCR. (J and K) Purified pDCs from HDs (n = 5; three independent experiments) were cultured in media alone or with the NRF2 activator (KI696-5 μM) for 1 h, followed by incubation with the TLR9 agonist (C274-0.075 μM). (J) Supernatant were collected after 15 h and IFNα secretion was analyzed by ELISA. (K) RNA was collected after 5 h, and IFNA2 gene expression was analyzed by Q-PCR. (L) Purified pDCs from HDs (n = 5; three independent experiments) were first cultured for 1 h either in media alone or with the NRF2 inhibitor (ML385-2 μM), then cultured at a stiffness of 0 or 50 kPa for another 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) for 5 h. IFNA2 gene expression 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. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal