A druggable copper-signalling pathway that drives inflammation

Abstract

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 diseases^2,3,4. 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 scarce^5,6, and there is a need for therapeutic innovation⁷.

The plasma membrane glycoprotein CD44 is the main cell surface receptor of hyaluronates^8,9,10. 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 plasticity^12,13. For instance, inflammatory macrophages are marked by increased expression of CD44 and its functional implication in this context has been demonstrated^14,15. However, the mechanisms by which CD44 and hyaluronates influence cell biology remain elusive^14,16,17,18. 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 level^21,22,23. 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).