key: cord-0738966-yxe19kwe
authors: Solier, Stéphanie; Müller, Sebastian; Cañeque, Tatiana; Versini, Antoine; Baron, Leeroy; Gestraud, Pierre; Servant, Nicolas; Emam, Laila; Mansart, Arnaud; Pantoș, G. Dan; Gandon, Vincent; Sencio, Valentin; Robil, Cyril; Trottein, François; Bègue, Anne-Laure; Salmon, Hélène; Durand, Sylvère; Wu, Ting-Di; Manel, Nicolas; Puisieux, Alain; Dawson, Mark A.; Watson, Sarah; Kroemer, Guido; Annane, Djillali; Rodriguez, Raphaël
title: Discovery of a druggable copper-signaling pathway that drives cell plasticity and inflammation
date: 2022-03-29
journal: bioRxiv
DOI: 10.1101/2022.03.29.486253
sha: 94e13007eff09a53ea7c6e23582401b7432ddb34
doc_id: 738966
cord_uid: yxe19kwe

Inflammation is a complex physiological process triggered in response to harmful stimuli. It involves specialized cells of the immune system able to clear sources of cell injury and damaged tissues to promote repair. Excessive inflammation can occur as a result of infections and is a hallmark of several diseases. The molecular basis underlying inflammatory responses are not fully understood. Here, we show that the cell surface marker CD44, which characterizes activated immune cells, acts as a metal transporter that promotes copper uptake. We identified a chemically reactive pool of copper(II) in mitochondria of inflammatory macrophages that catalyzes 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 a rationally-designed dimer of metformin triggers distinct metabolic and epigenetic states that oppose macrophage activation. This drug reduces inflammation in mouse models of bacterial and viral (SARS-CoV-2) infections, improves well-being and increases survival. Identifying mechanisms that regulate the plasticity of immune cells provides the means to develop next-generation medicine. Our work illuminates the central role of copper as a regulator of cell plasticity and unveils a new therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Inflammation is a complex physiological process triggered in response to harmful stimuli. Inflammation can occur as a result of infection. It is a complex biological process regulated by 55 pro-inflammatory and anti-inflammatory mediators that orchestrate a balanced response to enable clearance of pathogens and tissue repair 1 . When this balance is lost, excessive inflammation driven by macrophages and other immune cells results in tissue injury and organ failure 2,3 . Sepsis, a deregulated host-response to infection, places a major burden on healthcare systems with approximately 50 million cases per year and 11 million deaths, which represent 1 60 out of 5 of global mortalities 4 . Effective drugs against severe forms of inflammation are scarce 5, 6 , calling for therapeutic innovation.

Macrophages are immune cells that mediate tissue repair and host defense against pathogens, in particular through the production of growth factors and cytokines. An excessive response of macrophages to infection can be damaging [7] [8] [9] . Inflammatory macrophages are characterized by 65 elevated expression of CD44, a cell surface marker involved in inflammation 10 . CD44-deficient mice have a reduced capacity to resolve lung inflammation and accumulate hyaluronic acids (HA) in lungs, while reconstituted CD44-positive alveolar macrophages reverse the inflammatory phenotype 11 . The molecular bases underlying the role of CD44 in inflammation remain elusive. 70 HA are negatively charged biopolymers that interact with positively charged metal ions. The recent finding that CD44 mediates endocytosis of iron-bound HA in cancer cells provides a new paradigm that connects membrane biology to the epigenetic regulation of cell plasticity 12 .

In particular, increased iron uptake promotes the activity of a-ketoglutarate (aKG)-dependent demethylases involved in the regulation of gene expression. HA have also been shown to induce 75 the expression of pro-inflammatory cytokines in alveolar macrophages 13 , and macrophage activation relies on complex regulatory mechanisms occurring at the chromatin level [14] [15] [16] [17] . This body of work raises the question whether a mechanism involving CD44-mediated metal uptake regulates macrophage plasticity.

Here, we show that macrophage activation is characterized by an increase of mitochondrial 80 copper(II), which is mediated by CD44. We discovered that mitochondrial copper(II) catalyzes NAD(H) redox cycling, thereby promoting epigenetic alterations that lead to the inflammatory state. We have developed a small molecule dimer of metformin termed LCC-12 that selectively targets mitochondrial copper(II). This drug induces metabolic and epigenetic shifts that oppose macrophage activation and dampen inflammation in vivo.

To study the role of metals in immune cell activation, we produced inflammatory macrophages 90 from primary monocytes freshly isolated from blood of human donors (Fig. 1a) . These cells were differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF), then treated with lipopolysaccharide (LPS) and interferon gamma (IFNg) to produce activated monocyte-derived macrophages (aMDM) (Fig. 1a) . The inflammatory state of aMDM was characterized by the upregulation of the cell surface markers CD80 and CD86, as well as a 95 distinct cell morphology (Extended Data Fig. 1a-c) . Consistent with the literature 10 , aMDM were also characterized by increased levels of CD44 (Fig. 1b) .

Given that CD44 mediates endocytosis of iron in cancer cells 12 , we investigated whether inflammatory macrophages similarly exploit such a mechanism. Using inductively coupled plasma mass spectrometry (ICP-MS), we observed higher levels of cellular copper, iron, 100 manganese and calcium in aMDM compared to non-activated MDM (naMDM) ( Fig. 1c and Extended Data Fig. 1d ). In contrast, the cellular content of other metals studied, such as magnesium, cobalt, nickel and zinc, was not significantly altered (Extended Data Fig. 1d) .

Interestingly, levels of the copper transporter Ctr1 or the iron transporter TfR1 remained mostly unchanged during macrophage activation (Fig. 1d, e) . Knocking down CD44 by means of RNA 105 silencing or treating aMDM with the therapeutic anti-CD44 antibody RG7356, which interferes with HA binding to CD44 18 , reduced copper and iron uptake (Fig. 1f, g Fig. 1e, f) . Conversely, supplementing cells with HA during activation further increased intracellular levels of these metals ( Fig. 1h and Extended Data Fig. 1g) . These data indicate that CD44 and HA promote the uptake of copper and iron in aMDM. To substantiate the role 110 of CD44 in mediating copper uptake, we investigated the propensity of HA to form complexes with copper using a HA tetrasaccharide, the proton signals of which can be resolved by nuclear magnetic resonance (NMR) spectroscopy. Adding copper(II) to HA in water led to a line broadening of the proton signals of HA (Fig. 1i) . Acidification of the media to protonate the carboxylate of HA and disrupt copper binding restored the signals of unbound HA, showing 115 that the negatively-charged polysaccharides can dynamically interact with copper(II) under physiologically relevant conditions. Next, we confirmed CD44-dependent copper endocytosis using a fluorescent probe designed to detect copper in lysosomes. Specifically, we used Lys-Cu, whose fluorescence selectively increases upon copper(II) binding 19 . Fluorescence microscopy revealed that a 120 fluorescently labeled HA colocalized with this probe in aMDM (Fig. 1j) .

Following this, we set out to explore copper levels in a model of inflammation representative of our experimental setup. To this end, we treated mice with LPS intraperitoneally and isolated small peritoneal macrophages (SPM). SPM from mice treated with LPS exhibited higher CD44 levels together with higher copper content compared to 125 healthy mice ( Fig. 1k-m) . Collectively, these data indicate that inflammatory macrophages internalize copper through CD44-mediated HA uptake, raising the hypothesis that copper plays a functional role in macrophage activation. 130 To explore functional roles of copper signaling in the context of inflammation, we evaluated the capacity of the copper chelators D-penicillamine (D-Pen) and ammonium tetrathiomolybdate (ATTM) 20 , to interfere with macrophage activation. In this focused screen we also included metformin (Met), an FDA-approved biguanide used for the treatment of type-2 diabetes, because it forms complexes with copper(II) in cell-free settings 21 . Met partially 135 antagonized CD86 upregulation, albeit at high concentrations (e.g. 10 mM), contrasting the marginal effects of other clinically approved copper-targeting drugs (Fig. 2a) .

Met can form a stable bimolecular complex with copper(II) 21, 22 . To alleviate the entropic cost inherent to the formation of supramolecular complexes and thus, to increase the copper binding capacity of biguanides, we synthesized a series of dimeric small molecules, where two 140 biguanide moieties were tethered with a methylene-containing linker (Fig. 2b) . The design of such 'lipophilic copper clamps' (LCCs) was computationally guided and consisted in varying the number of methylene groups separating two biguanide units to ensure optimal geometry for copper(II) binding. Thus, we synthesized the dimers of biguanides LCC-12 and LCC-4,4

harboring twelve and four methylene-containing linkers, respectively. Importantly, LCC-4,4 145 was equipped with two distal butyl substituents such that both LCC-12 and LCC-4,4 have identical molecular formulae and exhibit a comparable lipophilicity. We used molecular dynamics simulation to predict the structure of copper(II) complexes with the lowest energies, comparing the geometry of each copper complex with that of an experimentally produced Cu(Met)2 complex 22 . This took into account bonding angles around the metal center and the 150 planarity of the system, given that copper(II) complexes can adopt square planar geometries.

The modeled geometry of Cu(Met)2 resembled that of the crystal structure of a Cu(Met)2 complex 22 , validating our theoretical methodology (Fig. 2c) . Cu-LCC-12 adopted a similar geometry according to bonding angles around copper and the planar geometry of each of the two biguanides bound to the metal ion. These two biguanides occupied distinct planes, whereas 155 Cu(Met)2 was planar overall. In contrast, Cu-LCC-4,4 lacked bonding angle symmetry and exhibited imine-copper bonds out of plane, indicating a geometrically constrained structure ( Fig. 2c) . Accordingly, the calculated free energy of Cu-LCC-4,4 was 16.6 kcal/mol higher compared to that of Cu-LCC-12, predictive of a less stable copper(II) complex.

High-resolution mass spectrometry (HRMS) confirmed the formation of monometallic 160 copper complexes with Met, LCC-12 and LCC-4,4 ( Fig. 2d) . Cu-LCC-12 was more stable than When added to aMDM, LCC-12 antagonized CD80 and CD86 induction more potently than Met, even at a 1000-fold lower dose (e.g. 10 µM LCC-12 versus 10 mM Met) (Fig. 2e) . In contrast, the effect of LCC-4,4 used at 10 µM was moderate, consistent with the reduced capacity of this analog to interact with copper(II). These data support the idea that copper(II) is 175 a mechanistic target of biguanides.

Next, we evaluated the effect of LCC-12 in other cell types expressing CD44. Interestingly, expression of activation markers by human CD4 + or CD8 + T lymphocytes, dendritic cells and monocytes, which upregulate CD44 levels upon appropriate stimulation, was reduced upon LCC-12 treatment (Extended Data Fig. 2d-h) . In contrast, the activation of neutrophils, which did not upregulate CD44, was not affected by LCC-12. Furthermore, in mouse pancreatic adenocarcinoma cells, TGF-b-induced epithelial-mesenchymal plasticity (EMP) was characterized by CD44 upregulation and increased copper levels. In this system, LCC-12 interfered with EMP (Extended Data Fig. 2i) . Altogether, these data illustrate the general nature of this copper signaling pathway as a regulator of cell plasticity. 185 

To gain further insights into the mechanism of action (MoA) of LCC-12, we employed nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, which qualitatively assesses the cellular distribution of specific isotopes. The subcellular localization of the isotopologue 190 15 N-13 C-LCC-12 overlapped with an 197 Au-labeled antibody specific for cytochrome c (Cyt c), suggesting that LCC-12 targets mitochondria in aMDM ( Fig. 3a, b) . To confirm this finding, we developed the biologically active alkyne-containing LCC-12,3 that can be fluorescently labeled in cells by means of click chemistry. This methodology has previously been used to inform on the subcellular localization and putative sites of action of small molecules 23 . In 195 aMDM, the labeled small molecule was detected as cytoplasmic puncta that colocalized with Cyt c, confirming accumulation of LCC-12 in mitochondria (Fig. 3c, d) . The mitochondrial labeling of LCC-12,3 was reduced upon cotreatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a protonophore that dissipates the inner mitochondrial proton gradient (Fig.   3e ). This indicated that LCC-12 accumulation in mitochondria is driven by its protonation state. 200 Labeling small molecules in cells by means of click chemistry requires a copper(I) catalyst generated in situ from added copper(II) and ascorbate (Asc) as a reducing agent. Given that our study converged towards mitochondrial copper(II) as a mechanistic target of LCC-12, we investigated whether the natural abundance of mitochondrial copper in aMDM would allow for click labeling without the need to experimentally add a metal catalyst. We found that 205 fluorescent labeling of LCC-12,3 used at a concentration of 100 nM, which is 100-fold lower than the biologically active dose of LCC-12, occurred in aMDM in absence of exogenous copper, leading to a fluorescent signal that colocalized with mitotracker. This staining pattern was observed only when MDM were activated (Fig. 3f) , and in the presence of exogenous Asc ( Fig. 3g) . Furthermore, the fluorescence intensity of labeled LCC-12,3 was substantially 210 reduced when a 100-fold molar excess of LCC-12 was used as a non-clickable competitor (Extended Data Fig. 3a) . ICP-MS measurement using isolated mitochondria revealed that copper levels were higher in aMDM compared to naMDM (Fig. 3h) . Interestingly, levels of manganese were also increased in mitochondria of aMDM, whereas the contents of other metals studied was not significantly altered (Extended Data Fig. 3b, c) . Taken together, these data 215 support the existence of a chemically reactive pool of copper(II) in mitochondria of aMDM.

We next set out to identify mitochondrial processes reliant on copper(II) that contribute to the inflammatory phenotype. In mitochondria, copper is essential for the function of cytochrome c 220 oxidase (also termed Complex IV), a component of the electron transport chain (ETC) that catalyzes the reduction of molecular oxygen. Although the regulation of cellular copper homeostasis is not fully understood, it has previously been argued that copper is tightly bound to proteins and mostly handled as a copper(I) species 24 . However, our data suggested that copper(II) plays a critical role in inflammatory macrophages. 225 Higher mitochondrial levels of manganese in aMDM pointed to a functional role of the superoxide dismutase 2 (SOD2). In line with this, SOD2 protein levels increased in mitochondria upon activation (Fig. 4a, b) , together with levels of mitochondrial hydrogen peroxide, a product of superoxide dismutation catalyzed by SOD2 (Fig. 4c, d) . In cell-free systems, copper(II) can catalyze the reduction of hydrogen peroxide using various organic 230 substrates as sources of electrons 25, 26 . Mass spectrometry indicated that in the presence of copper(II), hydrogen peroxide reacted with NADH to yield NAD + (Fig. 4e) . However, in absence of copper, hydrogen peroxide reacted with NADH to yield a complex mixture of products including an epoxide whose structure was supported by spectral data (Fig. 4e) .

Molecular modeling also supported a plausible reaction of epoxidation. In agreement with this, 235 copper(II) favored the conversion of 1-methyl-1,4-dihydronicotinamide (MDHNA), which was used as a structurally less complex surrogate of NADH, into 1-methylnicotinamide (MeNAD + ), whereas a product of epoxidation formed preferentially in absence of copper (Extended Data Fig. 4b) . Thus, copper reprograms the reactivity of hydrogen peroxide towards NADH, protecting the latter from oxidative degradation. To mimic the conditions found in the mitochondrial matrix, we next performed this reaction at 37 °C and pH 8. The rate of the reaction was evaluated by monitoring the concentration of NADH over time. When the reaction was performed in the presence of copper(II) and imidazole as 245 stabilizing copper ligand, NADH was rapidly consumed to yield NAD + (Fig. 4f) , as confirmed by mass spectrometry and NMR spectroscopy (Extended Data Fig. 4c) . This reaction was inhibited by LCC-12 in a dose-dependent manner, whereas the effect of LCC-4,4 and Met was marginal (Fig. 4f) . Taken together, LCC-12 can effectively compete against hydrogen peroxide for copper(II) binding, thereby inhibiting this copper(II)-catalyzed reaction. Using MDHNA as 250 a model substrate, molecular modeling supported a reaction mechanism involving the formation of a copper(II)-hydrogen peroxide complex, in which copper activates hydrogen peroxide facilitating the transfer of a hydride from NADH onto hydrogen peroxide (Extended Data Fig.   4d ). Copper(II) can act as a metal catalyst that lowers the energy of the transition state (TS) with a geometry that favors this reaction. Consistent with our findings, molecular modeling also 255 supported that, by forming a complex with copper(II), biguanides can interfere with the binding and activation of hydrogen peroxide (Extended Data Fig. 4e) .

The higher abundance of copper(II) and hydrogen peroxide in mitochondria from aMDM compared to naMDM prompted us to investigate the biological relevance of this reaction in inflammatory macrophages. To this end, we quantified mitochondrial NADH and NAD + in 260 aMDM by mass spectrometry-based metabolomics. Mitochondrial NADH levels were higher, whereas NAD + levels were lower in aMDM compared to naMDM, suggesting an enhanced activity of mitochondrial enzymes reliant on NAD + (Fig. 4g and Supplementary Table 1 ). In agreement with data obtained from our cell-free system, treating aMDM with LCC-12 during activation led to a decrease of both mitochondrial NAD + and NADH ( Fig. 4g and   265 Supplementary Table 1) . This is consistent with the idea that copper(II) catalyzes the reduction of hydrogen peroxide by NADH to produce NAD + in cells, and that biguanides can interfere with this redox cycling, leading instead to other oxidation by-products. Notably, NADH and copper were found in mitochondria of aMDM at an estimated substrate/catalyst ratio of 2:1, which is even more favorable for this reaction to take place than the 20:1 ratio used 270 in the cell-free system (Fig. 4h) . This substantiates the existence of a copper(II)-catalyzed reduction of hydrogen peroxide by NADH in mitochondria.

Quantitative metabolomics analysis of total cellular extracts indicated that macrophage activation was accompanied by altered levels of several metabolites whose production depend on NAD(H) (Fig. 4i and Supplementary Table 2) . In particular, LCC-12-induced metabolic 275 reprogramming of aMDM was marked by a reduction of aKG and acetyl-coenzyme A (acetyl-CoA) (Fig. 4j) . Collectively, these data support the central role of mitochondrial copper(II), which maintains a pool of NAD + and regulates the metabolic state of inflammatory macrophages.

Transcriptional programs underlying macrophage activation involve alterations of the epigenetic landscape [14] [15] [16] [17] 27 . aKG and acetyl-CoA are key metabolites required to regulate 285 epigenetic plasticity 28 . These two metabolites are co-substrates of iron-dependent demethylases and acetyl transferases (ATs), which can target histones and nucleobases. Our findings that LCC-12 interfered with the production of these metabolites and antagonized macrophage activation, pointed to epigenetic alterations that affect the expression of inflammatory genes.

Comparing transcriptional changes triggered by various pathogens can reveal general 290 regulatory mechanisms of macrophage activation. To explore this, we analyzed the transcriptome of aMDM compared to naMDM by RNA-seq (Supplementary Table 3) , and compared this to transcriptomics data obtained from bronchoalveolar macrophages of patients infected with SARS-CoV-2 29 and from human macrophages exposed in vitro to Salmonella typhimurium 30 , Leishmania major 31 or Aspergillus fumigatus 32 (Supplementary Table 4) . 295 Gene ontology (GO) analysis revealed three main GO term groups, which belonged to inflammation, metabolism and chromatin. Importantly, GO terms of upregulated genes in aMDM included endosomal transport, cellular response to copper ion, response to hydrogen peroxide and positive regulation of mitochondrion organization (Fig. 5a) . Furthermore, we observed striking similarities between the transcriptomics datasets (Extended Data Fig. 5a , b 300 and Supplementary Table 5 ). In particular, we found increased RNA levels of genes encoding inflammatory cytokines such as IL-6, IL-1b and TNFa, as well as genes encoding proteins involved in the JAK/STAT signaling pathway, the inflammasome and Toll-Like Receptors (TLRs) (Fig. 5b and Extended Data Fig. 5c ). In addition, aMDM upregulated genes that encode sorting nexin 9 (SNX9), a regulator of CD44 endocytosis 33 , the lysosomal copper 305 transporter 2 (CTR2) and metallothioneins (MT2A, MT1X), which are involved in copper transport and storage (Supplementary Table 3 ). In contrast, the gene coding for COX11, a chaperone involved in copper incorporation into cytochrome c oxidase was downregulated.

Next, we explored epigenetic gene signatures and found that genes involved in chromatin and histone modifications were upregulated in aMDM (Fig. 5a, c, d) . A similar subset of genes 310 encoding iron-dependent demethylases and ATs was upregulated in bronchoalveolar macrophages of patients infected by SARS-CoV-2 and in macrophages exposed to other pathogens (Extended Data Fig. 5d) . These data indicate that macrophage activation relies on similar epigenetic reprogramming irrespective of the pathogens involved 34 and is a key element of macrophage activation. In aMDM, we observed a reduction of H3K9me2, H3K27me3 and 315 H3K36me2, which are substrates of iron-dependent demethylases (Fig. 5e) . We also found an increase of H3K9ac, H3K14ac and H3K27ac, which are products of ATs. It is noteworthy that the higher iron load detected in aMDM is consistent with an increased demethylase activity, which contributes to establishing the inflammatory phenotype. Depletion of repressive marks (e.g. H3K9me2, H3K27me3) and increase of marks associated with active transcription (e.g. 320 H3K9ac, H3K14ac, H3K27ac) provide a mechanistic rationale underlying the expression of inflammatory genes in macrophages.

LCC-12 treatment led to an increase of methylation and a reduction of acetylation of specific histone residues including H3K9 and H3K27 (Fig. 5f, g) . Thus, LCC-12-induced decrease of aKG and acetyl-CoA reduced the activity of iron-dependent demethylases and ATs, 325 respectively. Accordingly, LCC-12 altered the gene expression signature of aMDM (Supplementary Table 3) , reflecting a complex epigenetic reprogramming toward a distinct cell state (Fig. 5h) . Notably, targeting mitochondrial copper(II) downregulated genes related to NAD(H) and aKG metabolism, regulation of chromatin and inflammation ( Fig. 5i and Supplementary Table 6 ). In aMDM, LCC-12 induced a downregulation of genes encoding 330 IL6, STAT1, JAK2, components of the inflammasome and TLRs among others (Fig. 5j) .

Consistently, secretion of pro-inflammatory cytokines was reduced (Fig. 5k) . Altogether, these data advocate for a biological mechanism whereby inflammatory macrophages increase mitochondrial copper(II) levels to replenish the pool of NAD + . This process is involved in the production of aKG and acetyl-CoA, metabolites that are required for the epigenetic regulation 335 of inflammatory gene expression.

Next, we investigated the biological effect of LCC-12 in mouse models of bacterial infection, where inflammatory macrophages play a central role 2, 7 . At a dose of 3 mg/kg by intraperitoneal 340 (IP) injection, LCC-12 did not provoke any adverse clinical signs. In contrast, at 10 mg/kg, signs of piloerection that vanished overtime were observed in 60% of the cohort, whereas additional signs of ptosis, reduced activity and death occurred in 40% of the cohort. We measured a maximal bioavailability of 61% fifteen minutes after intraperitoneal injection. LCC-12 was stable in plasma and exhibited moderate binding to plasma proteins. 345 First, we evaluated the effect of LCC-12 at 0.3 mg/kg/day, a dose at least 10 times lower than the maximum tolerated dose (MTD), in two well-established models of sepsis, namely (i) endotoxemia induced by LPS and (ii) cecal ligation and puncture (CLP). The former was chosen to reflect our mechanistic model of macrophage activation, whereas the latter is representative of the pathophysiology of subacute polymicrobial abdominal sepsis occurring in humans 35 .

Like SPM from mice treated with LPS (Fig. 1m) , peritoneal tissues exhibited higher levels of copper after local injection of LPS (Fig. 6a) . Treatment with LCC-12 fully protected animals from LPS-induced death and prevented the reduction of body temperature (Fig. 6b, c) . It is noteworthy that LCC-12 performed better than high doses of the anti-inflammatory glucocorticoid dexamethasone (DEX), which is used for the clinical management of acute 355 inflammation and severe forms of COVID-19 5 . Importantly, LCC-12 treatment decreased the inflammatory state of SPM in vivo (Fig. 6d) . Consistently, in a model of CLP-induced lethal sepsis, LCC-12 delayed death and increased the survival rate of mice, comparing favorably to DEX (Fig. 6e) .

Next, we evaluated the effect of LCC-12 in a model of viral infection. K18-hACE2 360 transgenic mice infected with SARS-CoV-2 displayed increased levels of CD44 in lung tissues ( Fig. 6f) . Similarly, lung tissues were characterized by alterations of genes related to mitochondrial metabolism, regulation of chromatin and inflammation (Extended Data Fig. 6a and Supplementary Table 7 ). In particular, viral infection promoted the expression of inflammatory genes and some genes encoding ATs and iron-dependent demethylases 365 (Extended Data Fig. 6b, c and Supplementary Table 8) . Treatment with LCC-12 administered by inhalation perturbed the expression of genes involved in the regulation of chromatin and inflammation ( Fig. 6g and Supplementary Table 9) , with a reduced expression of inflammatory genes (Fig. 6h, Extended Data Fig. 6d and Supplementary Table 8) . Taken together, these data indicate that targeting mitochondrial copper(II) interferes with acquisition 370 of the inflammatory state in vivo and confers therapeutic benefits.

We have uncovered a copper-signaling pathway that regulates the plasticity of macrophages toward an inflammatory state. We report that the pro-inflammatory cell surface marker CD44 375 mediates copper uptake to promote macrophage activation. We provide evidence for the existence of a labile pool of copper(II) in mitochondria that characterizes the inflammatory cell state and describe a previously uncharted chemical reaction that takes place in this organelle.

We found that NAD + maintenance enables epigenetic programming to unlock the expression of inflammatory genes. Transcriptomics data of macrophages exposed to distinct classes of 380 pathogens, including bacteria and viruses, illustrate the general nature of this mechanism.

Macrophages play multiple roles in health and disease. For instance, inflammation is part of the etiology of a plethora of diseases and biological processes such as cancer, aging and obesity 36 .

Thus, unraveling and manipulating mechanisms underpinning cell activation and plasticity is crucial for the design of novel therapeutic strategies. We show that mitochondrial copper(II) is 385 druggable using a rationally-designed small molecule, which leads to increased survival and improved well-being in murine models of sepsis.

The cell surface marker CD44 has previously been linked to inflammation, immune response and cancer progression. Here, we show that CD44 mediates cellular uptake of copper, which regulates cell plasticity in distinct cell types. Thus, CD44 can be more generally defined as a 390 regulator of cell plasticity. Copper has previously been shown to play a role in immune defense and cancer 37, 38 . For example, copper has been proposed to promote the production of reactive oxygen species as a defense mechanism against bacteria 39 . Furthermore, targeting copper trafficking was reported to attenuate cancer cell proliferation 40 .

Our data indicate that the mitochondrial copper(II) content increases during macrophage 395 activation and that this pool can be targeted with small molecules. Furthermore, inflammatory macrophages exhibit increased levels of hydrogen peroxide in mitochondria. While this substrate is often described as a potentially toxic by-product of respiration, our data suggest a distinct role. Macrophages upregulate the production of hydrogen peroxide to replenish the pool of NAD + enzymatic cofactor from NADH, which may be energetically more advantageous for 400 cells compared to de novo biosynthesis. In this context, hydrogen peroxide is a functional metabolite. Our data support the existence of a chemical reaction in which copper(II) activates hydrogen peroxide in mitochondria, enabling its reduction by NADH through a hydride transfer to yield NAD + . Conversely, in absence of copper, the reaction of NADH and hydrogen peroxide yields by-products of oxidation. Thus, in inflammatory macrophages, copper(II) protects 405 NADH from degradation, while maintaining NAD(H) redox cycling. This is consistent with the reactivity of enamines (e.g. NADH, MDHNA) towards dimethyldioxirane, an organic equivalent of hydrogen peroxide that can promote the production of highly reactive epoxides prone to solvolysis and alkylation 41 . Interestingly, epoxide hydrolases have been detected in the mitochondria of mammalian cells to prevent accumulation of these chemically reactive species 410 that can potentially lead to alkylation by-products 42 .

Manganese is exploited by SOD2 for the dismutation of superoxide 43 , and iron serves as a metal catalyst of demethylases consuming molecular oxygen 44 . This raises the question of whether this copper-catalyzed reaction is similarly assisted by an enzyme. The experimental setup we employed indicates that this reaction can be performed in an enzyme-free manner. 415 Imidazole, a functional group of histidine (His) residues found in mitochondrial proteins, enhances the rate of this reaction through binding to copper(II), arguing in favor of a putative enzyme-mediated process. A plausible hypothesis points to complex I of the ETC, which contains a NADH binding site and imidazole-containing His residues in proximity of flavin mononucleotide (FMN) 45 , being involved in the reduction of hydrogen peroxide by NADH. 420 There, copper(II) bound to His may activate hydrogen peroxide with a geometry poised for hydride transfer. This is further supported by the observation that Met and a copper(II)thiosemicarbazone complex inhibit complex I of the ETC 46,47 . Complex I may accommodate other substrates than FMN, promoting reduction of hydrogen peroxide.

Metals are ubiquitous in cell biology, serving multiple functions. While other processes may 425 be at play, our data illuminate a central role of copper in the activation of macrophages by promoting the expression of inflammatory genes. This involves NAD + -dependent production of aKG and acetyl-CoA, two metabolites required for the regulation of epigenetic plasticity.

These metabolites enable demethylation and acetylation of chromatin marks to unlock the expression of inflammatory genes. We identified a series of iron-dependent demethylases and 430 acetyl transferases reliant on these co-substrates that were upregulated in aMDM and macrophages exposed to various pathogens, suggesting a common epigenetic mechanism underlying macrophage activation.

We designed a dimer of biguanides able to form a near-square planar copper(II) complex.

Upon binding to copper(II), LCC-12 prevents activation of hydrogen peroxide, which reduces 435 levels of NAD(H) and interferes with aKG and acetyl-CoA biosynthesis. This in turn impairs the enzymatic activity of specific demethylases and ATs, causing increased histone methylation and a reduced expression of inflammatory genes. LCC-12 exhibits therapeutic effects in several models of acute inflammation, illustrating the pathophysiological relevance of this copper(II)triggered molecular chain of events. Acute inflammation is therefore reminiscent of a metabolic 440 disease that can be rebalanced by controlling cell plasticity through the targeting of mitochondrial copper(II) (Extended Data Fig. 7) .

A remarkable feature of LCC-12 is to selectively target mitochondrial copper(II), a unique property that may not be achieved by genetic manipulation or other clinically approved small molecules. Regardless, it appears that the metformin-inspired mitochondrial copper(II) 445 interactor LCC-12 may serve as a lead compound for developing anti-inflammatory agents based on this MoA. Due to its broad anti-inflammatory action, metformin exhibits positive effects on human health and is actually being studied as an anti-aging drug in a large NIHsponsored human clinical trial 48 

Hallmarks of health

Septic shock

Cytokine release syndrome in severe COVID-19

Global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease Study

Dexamethasone in Hospitalized Patients with Covid-19

Hydrocortisone plus Fludrocortisone for Adults with Septic Shock

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages

Macrophage biology in development, 655 homeostasis and disease

Macrophage plasticity and polarization: in vivo veritas

A crucial role for CD44 in inflammation

Resolution of lung inflammation by CD44

CD44 regulates epigenetic plasticity by mediating iron endocytosis

Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity

Trained immunity: A program of innate immune memory in health and disease

The Emerging Role of Epigenetics in Inflammation and Immunometabolism

First-in-human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors

A dual-emission fluorescenceenhanced probe for imaging copper(ii) ions in lysosomes

Transition metal chelators, pro-chelators, and ionophores as small molecule cancer chemotherapeutic agents

Zur Konstitution der Schwermetall-Komplexverbindungen des Biguanids

Bis(1,1-dimethylbiguanido)copper(II) octahydrate

Visualizing biologically active small molecules in cells using click chemistry

Iron and copper in mitochondrial diseases

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Activation of Hydrogen Peroxide for Oxidation by Copper(II) Complexes

The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory

The evolving metabolic landscape of chromatin biology and epigenetics

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases

Phylogenetic perspectives in innate immunity

Animal models of sepsis: setting the stage

The spectrum of inflammatory responses

Copper signaling in the brain and beyond

Connecting copper and cancer: from transition metal signalling to metalloplasia

A role for the ATP7A coppertransporting ATPase in macrophage bactericidal activity

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

Epoxidation of Enamines by Dimethyldioxirane: Formation of 1,4-Dioxanes by Enamine Epoxide Dimerization

Epoxide hydrolase activity in the mitochondrial fraction of mouse liver

Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria

Role of TET enzymes in DNA methylation, 735 development, and cancer

Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I

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