IMQ induces cytosolic and mitochondrial Ca²+ flux, leading to mitochondrial dysfunction and inflammasome activation in DCs.

a, WT and MyD88-deficient BMDCs were pre-stained with Fluo-8, followed by IMQ stimulation. Ca2+ influx was monitored by fluorescent microscopy. Left panel shows representative Fluo-8 fluorescent intensity images. Right panel shows violin plots (n = 20). b, BMDCs were pre-stained with Rhod-2-AM reagent, followed by LPS stimulation. Mitochondrial Ca2+ was analyzed by flow cytometry (n = 4). Left panel shows a representative flow cytometry image. c, BMDCs were pretreated with 2-APB, followed by IMQ stimulation. Intracellular ROS was analyzed by flow cytometry (n = 3). d, e, f, BMDCs were pretreated with LPS, followed by 2-APB treatment, and stimulated with IMQ. IL-1β concentrations in cell culture supernatant were measured by ELISA (d) (n = 3). Cells were stained with PI and Annexin V, and cell death was analyzed by flow cytometry (e) (n = 3). Cell lysates were analyzed by immunoblotting for cleaved caspase-1 (p20) (f). Data are representative immunoblot analysis images of two independent experiments. g, BMDCs were stimulated with IMQ or RSQ, stained with JC-1 reagent, and mitochondrial damage was analyzed by flow cytometry (n = 3). Left panel shows representative flow cytometry images. h, BMDCs were pre-stained with MitoSOX red reagent, stimulated with IMQ or RSQ, and mitochondrial ROS was analyzed by flow cytometry (n = 4). Left panel shows representative flow cytometry images. i, BMDCs were pretreated with LPS, followed by treatment with the indicated anti-oxidants, stimulated with IMQ, and IL-1β concentrations in cell culture supernatants were measured by ELISA (n = 3). j, BMDCs were pretreated with LPS, followed by treatment with the indicated anti-oxidants, stimulated with IMQ, stained with PI and Annexin V, and cell death was analyzed by flow cytometry (n = 3). k, BMDCs were pretreated with mitoquinone (MitoQ), followed by IMQ stimulation, and IL-1β concentrations in cell culture supernatants were measured by ELISA (n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (c-e and g-k) or Student’s t-test (a and b). (*: p > 0.05, ***: p > 0.001).

IMQ promotes UPR and facilitates Il23a expression in DCs.

a, BMDCs were pretreated with LPS, followed by IMQ stimulation, and VDAC1-IP3R1 interactions were analyzed by PLA. Left panel shows representative PLA images (n = 200). b, WT and MyD88-deficient BMDCs were stimulated with IMQ or DTT as a positive control, and the expression of short form Xbp1 (Xbp1s) mRNA was measured by qPCR (n = 3). c, BMDCs were stimulated with IMQ or DTT as a positive control, and cell lysates were analyzed by immunoblotting for the phosphorylated form of PERK and eIF2α. Data are representative images of two independent experiments. d, WT and MyD88-deficient BMDCs were stimulated with IMQ, and the expressions of Dnajb9, Ddit3, and Il23a mRNA were measured by qPCR (n = 3). e, BMDCs were pretreated with IRE1α ribonuclease inhibitor 4μ8c, followed by IMQ or RSQ stimulation, and the expression of Il23a mRNA was measured by qPCR (n = 3). f, HEK293T cells were transfected with an Il23a promoter reporter plasmid together with the indicated expression plasmids, and luciferase activity was measured (n = 3). g, BMDCs were stimulated with RSQ and thapsigargin, and the expressions of Il23a, Il6, and Xbp1s mRNA were measured by qPCR (n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (b and d-g) or Student’s t-test (a). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

IMQ drives psoriasis-associated gene expression in keratinocytes via the IRE1α-XBP1s axis of the UPR.

a, Mouse keratinocytes were stimulated with IMQ, and the expressions of Xbp1s, Hspa5, and Ddit3 mRNA were measured by qPCR (n = 3). b, Mouse keratinocytes were pre-stained with Fluo-8, followed by IMQ stimulation. Ca2+ influx was monitored by fluorescent microscopy (n = 40). c, Mouse keratinocytes were stimulated with IMQ, and cell lysates were analyzed by immunoblotting for the phosphorylated form of eIF2α and PERK. Data are representative images of two independent experiments. d, Mouse keratinocytes were stimulated with IMQ, and the gene expression profile was analyzed by RNA-seq. Data are shown as a volcano plot. Red and blue plots represent significantly upregulated and downregulated genes (p < 0.05, |log2FoldChange| > 1), respectively. e, Upregulated genes in (d) were compared with those in skin from human psoriasis patients. Data are shown as a Venn diagram. f, Upregulated genes in (d) were subjected to GO analysis (BP2). Data shows the top 10 (p-value) enriched biological processes. g, Data shows a heat map of genes upregulated in (d). h, Mouse keratinocytes were pretreated with 4μ8c, followed by IMQ stimulation, and the expressions of Xbp1s, Ccl20, Nr4a3, and Defb14 mRNA were measured by qPCR (n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (h) or Student’s t-test (a and b). (***: p > 0.001).

UPRs exacerbate IMQ-induced dermatitis, and Ca2+ flux inhibition ameliorates it.

a, b, IMQ cream was applied to the ear lobes of mice. Total RNA was extracted from the ears and the expressions of Xbp1s, Hspa5, and Hsp90b1 mRNA were measured by qPCR (n = 4) (a). DNA was extracted from the ears and serum, and the amount of mtDNA was measured by qPCR (n = 4) (b). c, d, e, f, IMQ cream and tunicamycin, an ER stress inducer, were applied daily to the ear lobes of mice. Ear thickness was measured daily (n = 3) (c). Mouse ears were sampled and subjected to histological analysis (H&E staining) (d). The epidermal thickness in (d) was measured (n = 10) (e). Total RNA was harvested from the ears, and the expression of Xbp1s, S100a9, Il17a, Cxcl1, and Defb14 mRNA was measured by qPCR (n = 3) (f). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (f) or Student’s t-test (a-c and e). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

Ca2+ flux inhibition ameliorates dermatitis.

a, b, c, d, IMQ cream and 2-APB were applied daily to the ear lobes of mice. Ear thickness was measured daily (n = 5) (a). Mouse ears were sampled and subjected to histological analysis (H&E staining). Data are shown as representative histological analysis images (b). The epidermal thickness in (b) was measured (n = 10) (c). Total RNA was harvested from the ear and the expression of S100a9, Il17a, Xbp1s, and Defb14 mRNA was measured by qPCR (n = 5) (d). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated using one-way ANOVA with Tukey’s test. (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

Mitochondrial DNA and mBD14 complex activate pDC via TLR9.

a, BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA was harvested from cytosolic and mitochondrial fractions, and the amount of mitochondrial DNA and nuclear DNA (nDNA) were measured by qPCR (n = 3). b, BMDCs were pretreated with LPS and 2-APB, followed by IMQ stimulation. DNA was harvested from cytosolic fraction, and the amount of mitochondrial DNA was measured by qPCR (n = 3). c, BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA was harvested from cytosolic fraction, and oxidized DNA was detected by dot blot analysis. Data are representative images of two independent experiments. d, BMDCs were stimulated with IMQ, and mitochondria-enriched fractions were collected from the cell culture supernatants and lysed. Western blot analysis was performed using an anti-TOM20 antibody to visualize mitochondrial content in the extracellular fraction. Data are representative images of two independent experiments. e, WT and NLRP3-deficient BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA in cell culture supernatants was harvested and the amount of mitochondrial DNA was measured by qPCR (n = 3). f, Bone marrow-derived pDCs were stimulated with DNA isolated from the cytosolic fraction of BMDCs stimulated with IMQ, and mBD14. Total RNA was extracted from the pDCs and the expression of Il6 and Tnf mRNA was measured by qPCR (n = 4). g, BMDCs were stimulated with synthesized mtDNA or oxidized mtDNA with or without recombinant mBD14. The expression of Tnf and Il6 mRNA was measured by qPCR (n = 3) (upper panel). The concentration of TNF-α and IL-6 in cell culture supernatants was measured by ELISA (n = 3) (bottom panel). h, WT and TLR9-deficient bone marrow-derived pDCs were stimulated with mtDNA or oxidized mtDNA with or without mBD14. The expression of Il6 mRNA was measured by qPCR (n = 3). i, BMDCs were stimulated with oxidized double stranded-DNA synthesized from mitochondrial or genomic DNA. The expression of Il6 mRNA was measured by qPCR (n = 3). j, HEK293T cells were transfected with FLAG-mBD14 or FLAG-hBD3 expression plasmids. Cell lysates were subjected to DNA immunoprecipitation with anti-FLAG antibody and immunoprecipitants were analyzed by qPCR (n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (e-j) or Student’s t-test (a and b). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

IMQ interacts with Gelsolin and Gelsolin deficiency increases UPRs.

a, Schematic representation of the method to identify IMQ-binding proteins. b, IMQ-conjugated or control magnetic beads were incubated with whole cell lysate prepared from Gelsolin-overexpressed HEK293T cells. After pull-down, proteins were eluted from each bead, and subjected to western blot analysis for FLAG-Gelsolin. Data are representative images of two independent experiments. c, FLAG-Gelsolin was transiently expressed in MEFs and cells were stimulated with IMQ. Gelsolin was visualized in green, Calnexin (ER) and TOM20 (mitochondria) were stained in red, and nuclei were counterstained with Hoechst. Data are representative images of two independent experiments. Scale bars represent 10 μm. d, WT and Gelsolin (Gsn)-deficient MEFs were lysed, and cell lysates were analyzed by western blot analysis for Gelsolin expression. Data are representative images of two independent experiments. e, f, WT (Control) and Gsn-deficient MEFs were stimulated with IMQ, and Xbp1s (e), Ddit3 (f) and Hspa5 (f) expression was measured by qPCR analysis (e: n = 4, f: n = 3) g, WT and Gsn-deficient MEFs were pre-stained with MitoSOX red reagent, stimulated with IMQ, and mitochondrial ROS was analyzed by flow cytometry (n = 4). h, WT and Gsn-deficient MEFs were pretreated with LPS, followed by IMQ stimulation, and VDAC1-IP3R1 interactions were analyzed by PLA. (n = 10). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P-values were calculated by one-way ANOVA with Tukey’s test (e and f) or Student’s t-test (g and h). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

Human and mouse psoriatic lesions exhibit reduced Gelsolin and enhanced ER stress.

a, b, Datasets (GSE289485 and GSE117405) were re-analyzed, and each gene expression was compared using normalized read counts. (mouse [n = 6], human: non-lesionl [n = 9], lesional [n = 8]) c, Pearson correlation analysis between Gelsolin and Hspa5 in (b). d, Schematic representation of this study. IMQ induces ER-mitochondria contact and calcium overload in dendritic cells, triggering UPR activation and mitochondrial dysfunction. Released mtDNA, together with mBD14, activates plasmacytoid DCs, amplifying inflammation. Gelsolin binds IMQ and attenuates these stress responses, with its downregulation in psoriatic lesions correlating with enhanced UPR signaling.