Fig 2: CD44 mediates the uptake of metals in inflammatory macrophages.a, Flow cytometry of cell surface markers in MDM. Monocytes treated as indicated to obtain pro-inflammatory or anti-inflammatory states. b, Flow cytometry of cell surface markers in MDM. Data representative of n = 13 donors. c, Bright field microscopy images of MDM. Scale bar, 20 μm. Morphological observations representative of n = 128 donors. d, ICP-MS of cellular metals in MDM (n = 9 donors). e, ICP-MS of cellular metals in aMDM under knockdown conditions of indicated genes (n = 6 donors). f, Western blots of cellular metal transporters in aMDM under knockdown conditions of indicated genes (n = 4 donors). g, Western blots of cellular metal transporters in aMDM under CD44 knockdown conditions (n = 6 donors). For d, f and g two-sided Mann-Whitney test. For e, Kruskal-Wallis test with Dunn’s post-test. Box plots: boxes represent interquartile range and median and whiskers indicate the minimum and maximum values. Each colored dot represents a distinct donor for a given panel. Source data

Fig 3: Development of a small molecule inactivator of mitochondrial copper(ii).a, Flow cytometry of cell surface markers in MDMs treated with metformin (Met) (n = 8 donors). b, Molecular structures of metformin, LCC-12 and LCC-4,4. c, HRMS of a Cu–LCC-12 complex. d, Flow cytometry of cell surface markers in MDMs treated with LCC-12 or LCC-4,4 (n = 9 donors). e, Experimental procedure of in-cell labelling of LCC-12,4. f, Fluorescence microscopy of labelled LCC-12,4 in aMDMs (n = 6 donors). At least 50 cells were quantified per donor. Cyt c, cytochrome c. g, Fluorescence microscopy of labelled LCC-12,4 in aMDMs treated with CCCP. h, Fluorescence microscopy of labelled LCC-12,4 in MDMs. In-cell labelling is performed with ascorbate and without added copper(ii). i, Fluorescence microscopy of labelled LCC-12,4 in aMDMs. In-cell labelling is performed in the presence or absence of ascorbate (asc) and without added copper(ii). j, ICP-MS of mitochondrial copper in MDMs (n = 6 donors). k, ICP-MS of mitochondrial copper in aMDMs under CD44-knockdown conditions (n = 6 donors). g–i, Two-sided unpaired t-test, representative of n = 3 donors. Data are mean ± s.d. a,d, Kruskal–Wallis test with Dunn’s post test. j,k, Two-sided Mann–Whitney test. In graphs, each coloured dot represents an individual donor for a given panel. Scale bars, 10 µm.Source data

Fig 4: CD44 mediates copper uptake.a, Experimental setup used to generate inflammatory monocyte-derived macrophages (MDMs). b, Flow cytometry of CD44 in MDMs. Data are representative of n = 13 donors. AU, arbitrary units. c, ICP-MS of cellular copper in MDMs (n = 9 donors). d, ICP-MS of cellular copper in aMDMs with short interfering RNA (siRNA) knockdown of indicated receptors and transporters (n = 6 donors). Copper transporter 1 (CTR1) is encoded by SLC31A1, CTR2 is encoded by SLC31A2, transferrin receptor 1 (TFR1) is encoded by TFRC, and divalent metal transporter 1 (DMT1) is encoded by SLC11A2. siCtrl, control siRNA. e, Representative western blots of metal transporters in MDMs (n = 7 donors). FC, fold change. f, ICP-MS of cellular copper in MDMs treated with anti-CD44 antibody RG7356 during activation (n = 7 donors). g, ICP-MS of cellular copper in MDMs treated with hyaluronate (0.6–1 MDa) (HA) or permethylated hyaluronate (meth–HA) during activation (n = 6 donors). h, Molecular structure of hyaluronate tetrasaccharide (top) and 1H NMR spectra (bottom) of copper–hyaluronate complexation experiment, recorded at 310 K in D2O. i, Fluorescence microscopy of a lysosomal copper(ii) probe (Lys-Cu) and FITC–hyaluronate in aMDMs treated with hyaluronidase (HD). At least 30 cells were quantified per donor (n = 6 donors). Scale bar, 10 μm. Rel., relative. c,e,f,i, Two-sided Mann–Whitney test. d,g, Kruskal–Wallis test with Dunn’s post test. In all box plots in the main figures, boxes represent the interquartile range, centre lines represent medians and whiskers indicate the minimum and maximum values. In graphs, each coloured dot represents an individual donor for a given panel.Source data

Fig 5: Mitochondrial copper(ii) regulates the epigenetic states and transcriptional programmes of inflammatory macrophages.a, GO term analysis of upregulated genes in aMDMs (n = 10 donors). Adj. P, adjusted P value. b, RNA-seq analysis of MDMs. Macrophage inflammatory signature genes are highlighted. The dashed line indicates an adjusted P value of 0.05 (n = 10 donors). c, RNA-seq analysis of MDMs. Iron-dependent demethylase and acetyltransferase signature genes are highlighted. The dashed line indicates an adjusted P value of 0.05 (n = 10 donors). d, Correlation for a representative donor of ChIP–seq reads count of histone marks in genes against RNA-seq of gene transcripts in MDMs (n = 10 donors). e, GO term analysis of genes in aMDMs (n = 10 donors) whose expression levels are downregulated upon treatment with LCC-12 (n = 5 donors). f, RNA-seq analysis of aMDMs (n = 10 donors) and MDMs treated with LCC-12 during activation (n = 5 donors). Macrophage inflammatory signature genes are highlighted. The dashed line indicates an adjusted P value of 0.05. g, Correlation for a representative donor of ChIP–seq reads count of histone marks in genes against RNA-seq of gene transcripts in aMDMs (n = 10 donors) and MDMs treated with LCC-12 during activation (n = 5 donors). h, RNA-seq analysis of CD44-knockout (KO) and wild-type (WT) aMDMs. Representative of n = 4 donors. Gating strategy is shown in the Supplementary Information. Macrophage inflammatory signature genes are highlighted. The dashed line indicates an adjusted P value of 0.05. In a–c,e,f, Differential gene expression was assessed with the limma/voom framework. GO enrichment was assessed with the enrichGO method from clusterProfiler. P values were corrected for multiple testing with the Benjamini–Hochberg procedure.