CXCL4 counters the NRF2-driven inhibition of pDCs and NRF2 cannot prevent IFN-I in patients with SSc. (A–D) sc-RNA-seq of SSc patients from the published paper (Gur et al., 2022) were reanalyzed. (A) Expression of genes of the Hippo pathway in pDCs from the blood or the skin of 56 healthy donors and 97 SSc patients. (B) Differentially regulated transcriptional factors in skin-infiltrating pDCs as analyzed by QIAGEN IPA. Upregulated and downregulated genes are identified in red and blue, respectively. (C) Expression level of NFE2L2 (low versus high) in pDCs from the blood or the skin of SSc patients. (D) Expression of ISGs in pDCs with high or low expression of NFE2L2 from the skin of SSc patients. (E) Purified pDCs from HDs (n = 5; four independent experiments) were cultured at a stiffness of 0 or 50 kPa for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) alone or with CXCL4 (0.3, 1, and 3 μg/ml) for 15 h. IFNα secretion was analyzed by ELISA. (F) Purified pDCs from HDs (n = 5; four independent experiments) were cultured in media alone or with the NRF2 activator (KI696) for 1 h, followed by incubation with the TLR9 agonist (C274 at 0.075 μM) alone or with CXCL4 (3 μg/ml) for 15 h. IFNα secretion was analyzed by ELISA. (G) 8–10-wk-old C57BL/6 mice (n = 5–8 mice per group; four independent experiments) were either shaved only or tape stripped (TAPE) and injected i.p. with either vehicle or KI696 along with intradermal injection of murine CXCL4 (2 µg per mice). Blood and skin biopsies were collected on day 1 after tape stripping. Gene expression levels of IRF7, MX1, ISG20, and ISG15 were analyzed by Q-PCR. Individual donors or mice are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann–Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001.