Molecular Medicine Israel

A druggable copper-signalling pathway that drives inflammation


Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2,3,4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.Main

Inflammation is a complex physiological process that enables clearance of pathogens and repair of damaged tissues. However, uncontrolled inflammation driven by macrophages and other immune cells can result in tissue injury and organ failure. Effective drugs against severe forms of inflammation are scarce5,6, and there is a need for therapeutic innovation7.The plasma membrane glycoprotein CD44 is the main cell surface receptor of hyaluronates8,9,10. It has been associated with biological programmes11 that involve cells capable of acquiring distinct phenotypes independently of genetic alterations, which is commonly defined as cell plasticity12,13. For instance, inflammatory macrophages are marked by increased expression of CD44 and its functional implication in this context has been demonstrated14,15. However, the mechanisms by which CD44 and hyaluronates influence cell biology remain elusive14,16,17,18. The recent discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in cancer cells links membrane biology to the epigenetic regulation of cell plasticity, where increased iron uptake promotes the activity of α-ketoglutarate (αKG)-dependent demethylases involved in the regulation of gene expression19. Hyaluronates have been shown to induce the expression of pro-inflammatory cytokines in alveolar macrophages (AMs)20, and macrophage activation relies on complex regulatory mechanisms occurring at the chromatin level21,22,23. This body of work raises the question of whether a general mechanism involving CD44-mediated metal uptake regulates macrophage plasticity and inflammation.Here we show that macrophage activation is characterized by an increase of mitochondrial copper(II), which occurs as a result of CD44 upregulation. Mitochondrial copper(II) catalyses NAD(H) redox cycling, thereby promoting metabolic changes and ensuing epigenetic alterations that lead to an inflammatory state. We developed a metformin dimer that inactivates mitochondrial copper(II). This drug induces metabolic and epigenetic shifts that oppose macrophage activation and dampen inflammation in vivo.

CD44 mediates cellular uptake of copper

To study the role of metals in immune cell activation, we generated inflammatory macrophages using human primary monocytes isolated from blood (Fig. 1a). Activated monocyte-derived macrophages (aMDMs) were characterized by the upregulation of CD44, CD86 and CD80, together with a distinct cell morphology (Fig. 1b and Extended Data Fig. 1a–c).

Using inductively coupled plasma mass spectrometry (ICP-MS), we detected higher levels of cellular copper, iron, manganese and calcium in aMDMs compared with non-activated MDMs (naMDMs) (Fig. 1c and Extended Data Fig. 1d). In contrast to other metal transporters, knocking down CD44 antagonized metal uptake (Fig. 1d and Extended Data Fig. 1e,f) and, unlike CD44, levels of these other metal transporters did not increase upon macrophage activation (Fig. 1e). Of note, levels of these transporters remained unchanged under CD44-knockdown conditions (Extended Data Fig. 1g). Treating MDMs with an anti-CD44 antibody24 antagonized metal uptake upon activation (Fig. 1f and Extended Data Fig. 2a). Conversely, supplementing cells with hyaluronate upon activation increased metal uptake, whereas addition of a permethylated hyaluronate25, which is less prone to metal binding, had no effect (Fig. 1g and Extended Data Fig. 2b). Inflammatory macrophages were also characterized by the upregulation of hyaluronate synthases (HAS) and the downregulation of the copper export proteins ATP7A and ATP7B (Extended Data Fig. 2c). Nuclear magnetic resonance revealed that hyaluronate interacts with copper(II) and that this interaction can be reversed by lowering the pH (Fig. 1h). Fluorescence microscopy showed that labelled hyaluronate colocalized with a lysosomal copper(II) probe26 in aMDMs (Fig. 1i). Cotreatment with hyaluronidase—which degrades hyaluronates—or knocking down CD44 reduced lysosomal copper(II) staining (Fig. 1i and Extended Data Fig. 2d). In aMDMs, the copper transporter CTR2 colocalized with the endolysosomal marker LAMP2, and CTR2 knockdown led to increased lysosomal copper(II) staining (Extended Data Fig. 2e,f). Collectively, these data indicate that in aMDMs, CD44 mediates the endocytosis of specific metals bound to hyaluronate, including copper.

Mitochondrial Cu(II) regulates cell plasticity

We evaluated the capacity of copper(I) and copper(II) chelators, including ammonium tetrathiomolybdate (ATTM), D-penicillamine (D-Pen), EDTA and trientine to interfere with macrophage activation. We also studied metformin, a biguanide used for the treatment of type-2 diabetes, because it can form a bimolecular complex27 with copper(II). Metformin partially antagonized CD86 upregulation, albeit at high concentrations, in contrast to the marginal effects of other copper-targeting molecules (Fig. 2a and Extended Data Fig. 3a).

To reduce the entropic cost inherent to the formation of bimolecular Cu(Met)2 complexes27,28, we tethered two biguanides with methylene-containing linkers to produce the lipophilic copper clamps LCC-12 and LCC-4,4 (Fig. 2b), which contain 12 and 4 linking methylene groups, respectively. LCC-4,4 displays distal butyl substituents to exhibit a lipophilicity similar to that of LCC-12. We compared simulated structures of copper(II) complexes with the lowest energies using molecular dynamics and discrete Fourier transform with a Cu(Met)2 complex using the crystal structure of the latter as benchmark28 (Extended Data Fig. 3b). Cu–LCC-12 adopted a geometry similar to that of Cu(Met)2, whereas Cu–LCC-4,4 lacked bonding angle symmetry and exhibited imine–copper bonds out of plane. The calculated free energy of Cu–LCC-4,4 was 16.6 kcal mol−1 higher than that of Cu–LCC-12, suggesting that Cu–LCC-4,4 is a less stable copper(II) complex. High-resolution mass spectrometry (HRMS) confirmed the formation of monometallic copper biguanide complexes, with Cu–LCC-12 being the most stable (Fig. 2c and Extended Data Fig. 3c). LCC-12 did not form stable complexes with other divalent metal ions (Extended Data Fig. 3d). A reduction in the UV absorbance of LCC-12 upon addition of copper(II) chloride indicated complex formation at low micromolar concentrations. This was confirmed by the appearance of coloured solutions characteristic of metal complexes (Extended Data Fig. 3e,f). Notably, even at a 1,000-fold lower dose, LCC-12 antagonized the induction of CD86 and CD80 in aMDMs more potently than metformin (Fig. 2d). The effect of LCC-4,4 used at 10 μM was moderate, consistent with the reduced capacity of this analogue to form a complex with copper(II). As reported for metformin29, LCC-12 induced AMPK phosphorylation, albeit at a much lower concentration, suggesting that phenotypes induced by metformin are linked to copper(II) targeting (Extended Data Fig. 3g).

Next, we evaluated the effect of LCC-12 on other cell types that can upregulate CD44 upon exposure to specific biochemical stimuli. LCC-12 interfered with the activation of dend

ritic cells and T lymphocytes and the expression of several cell surface molecules on alternatively activated macrophages (Extended Data Fig. 4a,b). By contrast, LCC-12 did not interfere with the activation of neutrophils, a process that is not marked by CD44 upregulation. Copper signalling has previously been linked to cancer progression30,31,32. Human non-small cell lung carcinoma cells and mouse pancreatic adenocarcinoma cells undergoing epithelial–mesenchymal transition (EMT)—a cell biology programme that can promote the acquisition of the persister cancer cell state and metastasis12,33—were characterized by CD44 upregulation and increased cellular copper. Consistently, LCC-12 interfered with EMT, as shown by the levels of the epithelial marker E-cadherin, mesenchymal markers vimentin and fibronectin, the EMT transcription factors Slug and Twist as well as the levels of pro-metastatic protein CD109 (Extended Data Fig. 4c,d). These data support a general mechanism involving copper that regulates cell plasticity.

Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging of aMDMs revealed a subcellular localization of the isotopologue 15N,13C-LCC-12 that overlapped with the signals of 197Au-labelled cytochrome c, suggesting that LCC-12 targets mitochondria (Extended Data Fig. 5a,b). Fluorescent in-cell labelling of the biologically active but-1-yne-containing analogue LCC-12,4 using click chemistry34 gave rise to a cytoplasmic staining pattern that colocalized with cytochrome c (Fig. 2e,f). The mitochondrial staining of LCC-12,4 was reduced upon cotreatment with carbonyl cyanide chlorophenylhydrazone (CCCP), a small molecule that dissipates the inner mitochondrial proton gradient, indicating that LCC-12 accumulation in mitochondria is driven by its protonation state (Fig. 2g). Labelling alkyne-containing small molecules in cells requires a copper(I) catalyst generated in situ from added copper(II) and ascorbate34,35,36,37. We investigated whether the mitochondrial copper(II) content in aMDMs would allow in-cell labelling without the need to experimentally add a copper catalyst. Fluorescent labelling of LCC-12,4 used at a concentration of 100 nM, which is lower than the biologically active dose of LCC-12, occurred in aMDMs in the absence of added copper(II) and a strong staining was observed only in aMDMs when ascorbate was used for labelling (Fig. 2h,i). Furthermore, the fluorescence intensity of labelled LCC-12,4 was reduced when a 100-fold molar excess of LCC-12 was used as a competitor (Extended Data Fig. 5c). These data support the existence of a druggable pool of chemically reactive copper(II) in mitochondria. Consistent with this, levels of copper increased in mitochondria upon macrophage activation together with those of manganese (Fig. 2j and Extended Data Fig. 5d,e), whereas levels of copper in the endoplasmic reticulum and nucleus remained unaltered (Extended Data Fig. 5f,g). Notably, aMDMs were characterized by an increase of nuclear iron, hinting at an increased activity of αKG-dependent demethylases as previously shown in cancer cells undergoing EMT19 (Extended Data Fig. 5f). LCC-12 treatment did not alter the total cellular and mitochondrial copper content of aMDMs, indicating that LCC-12 does not act as a cuprophore38 (Extended Data Fig. 5h,i). By contrast, LCC-12 reduced the fluorescence of a mitochondrial copper(II) probe39 in aMDMs, supporting direct copper binding in mitochondria (Extended Data Fig. 5j). Notably, the mitochondrial metal transporters SLC25A3 and SLC25A37 were upregulated in aMDMs (Extended Data Fig. 5k). Knocking down the expression of these transporters or CD44 did not reduce labelled LCC-12,4 fluorescence (Extended Data Fig. 5l–n), whereas knocking down CD44 led to marked reduction of mitochondrial copper (Fig. 2k). This indicates that, unlike the proton gradient, mitochondrial copper does not drive mitochondrial accumulation of biguanides. As a control, labelling an alkyne-containing derivative of the copper(II) chelator trientine, which did not exhibit a potent effect against macrophage activation, revealed nuclear accumulation, providing a rationale for the lack of biological activity of this and potentially other copper-targeting drugs in this context (Extended Data Fig. 5o,p).

Cu(II) regulates NAD(H) redox cycling

Higher mitochondrial levels of manganese in aMDMs pointed to a functional role of the superoxide dismutase 2 (SOD2) in the context of macrophage activation. The amount of SOD2 protein increased in mitochondria upon activation, whereas the amount of catalase decreased (Fig. 3a,b). Mitochondrial hydrogen peroxide, a product of superoxide dismutase and substrate of catalase, increased accordingly (Fig. 3c,d). In cell-free systems, copper(II) can catalyse the reduction of hydrogen peroxide by various organic substrates40,41. In the presence of copper(II), NADH reacted with hydrogen peroxide to yield NAD+, whereas the absence of copper(II) yielded a complex mixture of oxidation products (Fig. 3e and Extended Data Fig. 6a). Consistently, copper(II) favoured the conversion of 1-methyl-1,4-dihydronicotinamide (MDHNA), a structurally less complex surrogate of NADH, into 1-methylnicotinamide (MNA+), whereas a product of epoxidation was formed preferentially in the absence of copper (Extended Data Fig. 6b,c). Thus, copper(II) redirects the reactivity of hydrogen peroxide towards NADH. Under reaction conditions similar to those found in mitochondria, NADH was rapidly consumed to yield NAD+ in the presence of copper(II) (Fig. 3f and Extended Data Fig. 6d). This reaction was inhibited by LCC-12, whereas the effects of LCC-4,4 and metformin were marginal (Fig. 3f). Molecular modelling supported a reaction mechanism in which copper(II) activates hydrogen peroxide, facilitating its reduction through the transfer of a hydride from NADH (Extended Data Fig. 6e). Copper(II) acts as a catalyst that lowers the energy of the transition state with a geometry favouring this reaction. Molecular modelling also supported the inactivation of this reaction by biguanides through direct copper(II) binding (Extended Data Fig. 6f).

Mitochondrial NADH levels were higher and NAD+ levels were lower in aMDMs compared with naMDMs, suggesting an enhanced activity of mitochondrial enzymes reliant on NAD+ (Fig. 3g and Supplementary Table 1). Treating MDMs with LCC-12 during activation led to a reduction of NADH and NAD+ (Fig. 3g and Supplementary Table 1). This suggests that copper(II) catalyses the reduction of hydrogen peroxide by NADH to produce NAD+ and that biguanides can interfere with this redox cycling, leading instead to other oxidation by-products (Fig. 3e). NADH and copper were found in mitochondria of aMDMs at an estimated substrate:catalyst ratio of 2:1, which is even more favourable for this reaction to take place than the 20:1 ratio used in the cell-free system (Extended Data Fig. 6g). Macrophage activation was accompanied by altered levels of several metabolites whose production depends on NAD(H) (Fig. 3h and Supplementary Table 2). LCC-12-induced metabolic reprogramming of aMDMs was marked by a reduction of αKG and acetyl-coenzyme A (acetyl-CoA) (Fig. 3i). LCC-12 also caused a reduction of extracellular lactate and accumulation of glyceraldehyde 3-phosphate in aMDMs consistent with the reduced activity of NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase (Extended Data Fig. 6h,i). Collectively, these data support the central role of mitochondrial copper(II) in the maintenance of a pool of NAD+ that regulates the metabolic state of inflammatory macrophages.

Mitochondrial Cu(II) regulates transcription

Transcription is co-regulated by chromatin-modifying enzymes, whose expression levels and recruitment at specific genomic loci shape gene expression. The turnover of specific enzymes such as iron-dependent demethylases and acetyltransferases relies on αKG and acetyl-CoA42. The finding that LCC-12 interfered with the production of these metabolites and opposed macrophage activation pointed to epigenetic alterations that affect the expression of inflammatory genes. We analysed the transcriptomes of aMDMs versus those of naMDMs by RNA sequencing (RNA-seq) (Supplementary Table 3) and compared them to transcriptomics data obtained from bronchoalveolar macrophages of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)43 and from human macrophages exposed in vitro to Salmonella typhimurium44Leishmania major45 or Aspergillus fumigatus46 (Supplementary Table 4). Gene ontology (GO) analysis revealed three groups of GO terms comprising upregulated genes, belonging to inflammation, metabolism and chromatin (Fig. 4a). Notably, the GO terms of these genes included endosomal transport, cellular response to copper ion, response to hydrogen peroxide and positive regulation of mitochondrion organization. Similar signatures were obtained for macrophages exposed to distinct pathogens (Extended Data Fig. 7a,b and Supplementary Table 5), as defined by GO terms and increased RNA amounts for genes involved in inflammation (Fig. 4b and Extended Data Fig. 7c). aMDMs exhibited upregulated genes encoding CD44, sorting nexin 9 (SNX9), a regulator of CD44 endocytosis, and metallothioneins (MT2A and MT1X) involved in copper transport and storage, whereas expression levels of ATP7A and ATP7B were downregulated (Supplementary Table 3). Genes involved in chromatin and histone modifications were upregulated in aMDMs and similar genes encoding iron-dependent demethylases and acetyltransferases were upregulated in bronchoalveolar macrophages from individuals infected with SARS-CoV-2, as well as in macrophages exposed to other pathogens (Fig. 4c and Extended Data Fig. 7d). These data indicate that distinct classes of pathogens trigger similar epigenetic alterations47, leading to the inflammatory cell state. In aMDMs, variations in protein levels including increases in iron-dependent demethylases and acetyltransferases were consistent with the RNA-seq data (Extended Data Fig. 8a,b and Supplementary Table 6). Changes in levels of specific demethylases and acetyltransferases were associated with alterations of their targeted marks (Extended Data Fig. 8c,d). Chromatin immunoprecipitation sequencing (ChIP–seq) revealed a global increase of the permissive acetyl marks H3K27ac, H3K14ac and H3K9ac together with a reduction of repressive methyl marks H3K27me3 and H3K9me2 at inflammatory gene loci, with consistent effects on the transcriptional profile of aMDMs (Fig. 4d, Extended Data Fig. 8e,f and Supplementary Table 7). LCC-12 treatment induced a downregulation of genes related to NAD(H) and αKG metabolism, regulation of chromatin and inflammation (Fig. 4e and Supplementary Table 8). Inactivating mitochondrial copper(II) also promoted the downregulation of inflammatory genes at the RNA and protein levels (Fig. 4f, Extended Data Fig. 9a,b and Supplementary Table 3), reflecting a complex epigenetic reprogramming toward a distinct cell state (Extended Data Fig. 9c). LCC-12 treatment reduced H3K27ac, H3K14ac and H3K9ac and increased H3K27me3 and H3K9me2 levels (Extended Data Fig. 9d), which was associated with the downregulation of targeted inflammatory genes (Fig. 4g, Extended Data Fig. 9e,f and Supplementary Table 7). Thus, the LCC-12-induced decreases in αKG and acetyl-CoA were associated with a reduced activity of iron-dependent demethylases and acetyltransferases, respectively. Notably, knocking down expression of SOD2 or the mitochondrial copper transporter SLC25A3 reduced the inflammatory signature of macrophages (Extended Data Fig. 9g,h). Similarly, knocking out CD44 antagonized epigenetic programming of inflammation in aMDMs without adversely affecting the expression of other metal transporters (Fig. 4h, Extended Data Fig. 9i–k and Supplementary Table 9). Together, these data indicate that hydrogen peroxide is a driver of cell plasticity and that mitochondrial copper(II) controls the availability of essential metabolic intermediates required for the activity of chromatin-modifying enzymes, which enables rapid transcriptional changes underlying the acquisition of distinct cell states…

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