Fig 2: Co-localisation of human CD1c+ DC with ferroptotic PTEC in fibrotic kidney tissue.Representative serial section immunohistochemical (IHC) staining of control/non-fibrotic (left panel) and fibrotic (middle panel) kidney tissue probed for (A) aquaporin-1, (B) GPX4, (C) 4-HNE and (D) CD1c. Regions of CD1c+ DC co-localisation with aqupaporin-1+ PTEC displaying evidence of ferroptotic cell death (↓ GPX4 and ↑ 4-HNE) are highlighted with black arrows. Scale bars represent 60 µm. Quantitative analysis (positive pixel intensity/µm2 total area) of IHC staining in control and fibrotic tissue is presented (right panels). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from four randomly selected areas for each tissue sample. *P < 0.05, ***P < 0.001, Welch’s t test.

Fig 3: Significantly elevated tubulointerstitial CD1c+ DC with ASC specks in fibrotic kidney tissue.A, B Immunofluorescent labelling of frozen sections from control/non-fibrotic (A) and fibrotic kidney tissue (B) stained for ASC (green), CD1c (red), aquaporin-1 (AQP-1) (white) and DAPI (blue). Scale bars represent 50 µm for small frames (left panels) and 20 µm for large frames (right panels). ASC-speck-positive CD1c+ DC are highlighted with white arrows. C Quantification (mean cells/mm2) of ASC-speck-negative CD1c+ DC (white bars) and ASC-speck-positive CD1c+ DC (grey bars) in control/non-fibrotic kidney tissue (n = 4) and fibrotic kidney tissue (n = 4). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from five randomly selected areas for each tissue sample. *P < 0.05, **P < 0.01, Welch’s t test.

Fig 4: Hypoxic PTEC trigger CD1c+ DC-derived IL-1β/IL-18 via activation of the NLRP3 inflammasome.A NLRP3 mRNA expression relative to housekeeping gene β-2-microglobulin (B2M) in flow cytometry sorted CD1c+ DC freshly isolated (0 h) or following 24-h co-culture (DC & PTEC) with pre-conditioned normoxic (white bar) or hypoxic (grey bar) PTEC in the presence of poly I:C. DC alone cultures are included as controls. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. *P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. B Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c+ DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody); n = 6. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. C, D Fold changes (relative to NIL inhibitor) in secreted IL-1β (C) and IL-18 (D) protein levels (measured by LEGENDplex™ assay) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c+ DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 6. *P < 0.05; **P < 0.01; ****P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test.