Résumé de thèse

La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique caractérisée par des atteintes articulaires. Les synoviocytes, cellules qui tapissent la membrane synoviale, deviennent réfractaires à l'apoptose et, en produisant la cytokine inflammatoire Interleukin 6 (IL-6), participent à la chronicité de l'inflammation qui est à l'origine de la perte osseuse dans la PR.

Le zinc (Zn) et le cadmium (Cd) partagent leurs propriétés physico-chimiques et leurs mécanismes de transport cellulaire. Le Zn régule des fonctions du système immunitaire, contrairement au Cd avec des propriétés sur le système immunitaire peu décrites dans la littérature.

Notre travail vise à identifier les mécanismes impliqués dans la modification de l'homéostasie des métaux dans les synoviocytes par l'inflammation et les conséquences de cette altération.

L'inflammation induit l'accumulation des métaux dans les synoviocytes en augmentant l'expression de l'importeur ZIP-8. En revanche, l'expression de l'exporteur ZnT1 et des metallothionéines (MTs, régulateurs de l'homéostasie des métaux) est dépendante de la présence des métaux. L'affinité Cd-MTs permet une accumulation irréversible du Cd dans les cellules qui réduit leur prolifération et la production d'IL-6.

Les effets antiprolifératif et anti-inflammatoire du

Résumé substantiel:

La polyarthrite rhumatoïde (PR), une maladie inflammatoire chronique, est le plus fréquent des rhumatismes inflammatoires. La PR est une maladie systémique, caractérisée par des atteintes articulaires liées à une hyper-prolifération locale des synoviocytes, cellules fibroblastiques qui tapissent la membrane synoviale. Les synoviocytes deviennent réfractaires à l'apoptose et participent au développement et à la chronicité de l'inflammation en produisant notamment de l'interleukin 6 (IL-6).

La réaction inflammatoire chronique dans les articulations est à l'origine d'une importante perte osseuse entraînant une perte de mobilité des patients et, par conséquent, une baisse de la qualité de vie.

Le zinc (Zn) et le cadmium (Cd) appartiennent au groupe 12 des métaux divalents et partagent de nombreuses caractéristiques, notamment les propriétés physico-chimiques et leurs mécanismes de transport cellulaire. Le Zn est un important régulateur des fonctions du système immunitaire et l'inflammation chronique ainsi que le développement de plusieurs maladies chroniques ont été associé à une carence de Zn. Contrairement au Zn, le rôle du Cd dans la régulation du système immunitaire est peu décrit dans la littérature.

Notre travail vise à identifier les mécanismes impliqués dans la modification de l'homéostasie des métaux dans les synoviocytes en conditions inflammatoires. Les conséquences de l'altération de l'homéostasie des métaux par l'inflammation ont été également étudiées.

Les synoviocytes de patients atteints de PR, utilisés comme modèle cellulaire d'inflammation chronique, ont été d'abord exposés aux cytokines proinflammatoires IL-17 et TNF-α, afin de recréer un environnement inflammatoire in vitro. Puis, Zn et au Cd ont été ajouté, seuls ou associés. Après avoir étudié les mécanismes de régulations et les effets du Zn et du Cd sur les synoviocytes in vitro, nous avons testé leurs effets in vivo dans un modèle d'arthrite chez le rat.

Les résultats obtenus montrent que la combinaison IL-17/TNF-α induit l'accumulation des métaux dans les synoviocytes en augmentant l'expression de l'importeur transmembranaire ZIP-8, qui néanmoins n'est pas sensible à la concentration des métaux dans le milieu de culture. En revanche, l'expression de l'exporteur ZnT1 et des metallothionéines (MTs, régulateurs de l'homéostasie des métaux) est dépendante de la présence des métaux. Les résultats obtenus nous ont permis de développer un modèle mathématique représentant l'effet des cytokines sur l'homéostasie des métaux dans les synoviocytes, qui pourra être appliqué à d'autres types cellulaires.

Nous avons montré que le Cd induit une expression plus élevée des MTs que le

Zn, dû à une plus grande affinité des MTs pour le Cd. La présence de cytokines inflammatoires renforce encore cet effet et permet l'accumulation irréversible du Cd dans la cellule. Pour cette raison, lorsque le Cd est ajouté dans le milieu de culture, la prolifération des cellules et la production de la cytokine pro-inflammatoire IL-6 est réduite. Au contraire, lorsque le Zn est ajouté, la viabilité des synoviocytes et l'inflammation qui en dépend augmente.

Les effets antiprolifératif et anti-inflammatoire du Cd ont été testés dans le modèle d'arthrite induite par adjuvant chez le rat. Dans ce cas, le Cd est utilisé comme traitement local par injection intra-articulaire afin de réduire l'inflammation et la destruction de l'articulation qui en découle. Les résultats montrent qu'une seule injection de Cd à faible dose prévient la perte osseuse dans les articulations et réduit les scores cliniques d'arthrite.

L'effet antiprolifératif et anti-inflammatoire du Cd, pourrait représenter une nouvelle approche thérapeutique locale pour le traitement de la PR mais aussi pour d'autres pathologies caractérisées par une hyperplasie et une inflammation localisées. Cette approche est d'ailleurs déjà utilisée dans certaines thérapies anticancéreuses, par l'utilisation de la toxicité intrinsèque de certains nanomatériaux.

Thesis summary

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Synoviocytes, cells forming the inner layer the synovium, become refractory to apoptosis and participate in the chronicity of inflammation through the production of IL-6. The perpetuation of inflammation causes an important induction of bone loss in joints. Zinc (Zn) and Cadmium (Cd) share many physico-chemical properties and cell transport mechanism. Zn is known as a regulator of the normal function of the immune system, while Cd properties on the immune system are not well defined.

Our aim was to provide information on the metal homeostasis mechanisms in synoviocytes during chronic inflammation and on the consequences of metal homeostasis changes. After studying the differential effect of Zn and Cd at the cellular level, we could provide an innovative tool to control synoviocyte contribution to rheumatoid arthritis, which was tested on an in vivo model of arthritis.

Results show that IL-17/TNF-α combination drives the accumulation of metals inside synoviocytes through the enhancing of ZIP-8 importer expression and regardless of the concentration of metals in the culture medium. In contrast, the expression of the metal exporter ZnT1 and of the homeostasis regulators metallothioneins (MTs) was primarily dependent on metal levels. In this study, we focused principally on three pro-inflammatory cytokines:

Addition of

interleukin-17 (IL-17), interleukin-1 (IL-1), and TNF-α. These three cytokines are at the beginning of the inflammatory cascade as represented in figure 1 and can induce the secretion of other pro-inflammatory cytokines such as IL-6, IL-8 or CCL-20 from the cells of the tissue on which they act. This promotes the loop of inflammation, by the enhanced production of IL-17, IL-1 and TNF-α (figure 1).

IL-1β is produced by activated macrophages and monocytes. It is involved in the generation of systemic and local responses to infection, injury, and other

immunological challenges and it is the primary cause of chronic and acute inflammation (4). IL-1β is produced as an inactive cytoplasmic precursor (proIL-1β) that must be cleaved (at Asp116) to generate the mature active form. The IL-1βconverting enzyme (ICE), better known as caspase-1 ( 5) is required for its cleavage.

TNF-α is mostly produced by activated macrophages but it is also expressed by neutrophils, NK-cells, endothelial cells and activated lymphocytes. The cytokine elicits biological effects by two specific receptors: TNFR1 (CD120a) -present on all kinds of cells, and TNFR2 (CD120b) -more restricted, with the highest expression on Treg lymphocytes [START_REF] Sedger | TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants -past, present and future[END_REF].

Both IL-17 and IL-1β can act synergistically with TNF-α at the site of inflammation mobilizing and activating leukocytes, inducing B and T cell proliferation and natural killer cell cytotoxicity. The synergy between IL-17 and TNF-α was previously studied in our laboratory, in the context of rheumatoid arthritis (RA) and other chronic inflammatory diseases [START_REF] Miossec | Interleukin-17 in rheumatoid arthritis: If T cells were to contribute to inflammation and destruction through synergy[END_REF][START_REF] Hot | Combination of IL-17 and TNFα induces a proinflammatory, pro-coagulant and pro-thrombotic phenotype in human endothelial cells[END_REF]. It was previously described that IL-17 primes the cells at the site of inflammation to respond to other cytokines, as TNF-α, by increasing the expression of their receptor, in this case TNFR2 [START_REF] Zrioual | Genome-Wide Comparison between IL-17A-and IL-17F-Induced Effects in Human Rheumatoid Arthritis Synoviocytes[END_REF]. The synergism of IL-1β and TNF-α is also a commonly reported phenomenon, which induces the expression of endothelial adhesion molecules. These molecules are essential for the adhesion of leukocytes to endothelial surface prior to migration into the tissues [START_REF] Dinarello | Proinflammatory cytokines[END_REF].

These synergies between IL-17, TNF and IL-1β induce in loop the secretion of other cytokines, notably IL-8, IL-6 and CCL-20 from the cells at the site of inflammation. The production of the latter is considered as a marker of response to the first three and moreover can induce in loop their production (Fig. 1).

IL-8 is produced by macrophages and other cells (as epithelial cells). It binds CXCR1 and CXCR2 receptors and functions as a chemokine, activating neutrophils to degranulate and causing tissue damage at the site of inflammation. IL-8 can also be a potent promoter of angiogenesis.

IL-6 demonstrates contradictory activities: this cytokine is mainly recognized as a pro-inflammatory molecule, but under some circumstances, e.g. disease context, it can exhibit anti-inflammatory properties. IL-6 plays a pivotal role during the transition from innate to acquired immunity [START_REF] Jones | Directing transition from innate to acquired immunity: defining a role for IL-6[END_REF], leading to the switch from neutrophils to monocyte recruitment [START_REF] Chomarat | IL-6 switches the differentiation of monocytes from dendritic cells to macrophages[END_REF], by up-regulating M-CSF receptor expression and by programming Th17 differentiation through IL-21 and IL-23 pathways [START_REF] Zhou | IL-6 programs TH-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways[END_REF]. Simultaneously, IL-6 can enhance the production of antiinflammatory IL-1Ra and suppresses pro-inflammatory cytokines such as IL-1, TNFα and IL-12 in other disease contexts [START_REF] Scheller | The pro-and antiinflammatory properties of the cytokine interleukin-6[END_REF]. IL-6 receptor has a heterodimeric structure and it is composed of gp130 and IL-6Rα subunits. Soluble form of IL-6Rα (sIL-6Rα) and gp130 (sgp130) are abundantly present in body fluids. The complex effect of IL-6 may relate to the fact that the final cell response to IL-6 depends upon the ratio between the membrane-bound IL-6Rα and gp130 on one side, and the availability of IL-6, sIL-6Rα and sgp130 on the other [START_REF] Chizzolini | Cytokines in chronic rheumatic diseases: is everything lack of homeostatic balance?[END_REF].

Chemokine (C-C motif) ligand 20 (CCL-20

) is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes, preferentially Th17 [START_REF] Hirota | Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model[END_REF][START_REF] Alcaide | Difference in Th1 and Th17 Lymphocyte Adhesion to Endothelium[END_REF], and weakly attracts neutrophils through the receptor CCR6 at the site of inflammation. It is produced by immune cells but also cells at the site of inflammation and it can be upregulated by IL-1β, TNF-α and IFN-γ and downregulated by IL-10 (17).

Chronic inflammation

This series of biochemical events propagate and mature the inflammatory response. Prolonged, non-resolving inflammation is known as chronic inflammation and leads to a progressive shift in the type of cells present at the site of inflammation: from mainly neutrophils during acute inflammation to a lymphocyte-, plasma cell-and macrophage-enriched infiltration. The development of chronic inflammation is due to the persistent non-resolvable acute inflammation: nondegradable pathogens, viral infection, persistent foreign bodies, or autoimmune reactions can last for months or even years.

Pro-inflammatory cytokines as IL-6, IL-8, IL-1β and IL-17 continue to be produced in loop, causing a simultaneous destruction and healing of the tissue due to the inflammatory process, in turn leading to fibrosis, angiogenesis and eventually necrosis. Other factors involved in the persistence of inflammation can be reactive oxygen species (ROS) and some proteolytic enzymes [START_REF] Bryniarski | Modulation of macrophage activity by proteolytic enzymes. Differential regulation of IL-6 and reactive oxygen intermediates (ROIs) synthesis as a possible homeostatic mechanism in the control of inflammation[END_REF]. The changes involved in the process of inflammation are represented in the figure 2.

Chronic inflammatory autoimmune diseases

Inappropriate inflammatory response, when there are no foreign substances to fight off, leads to autoimmunity; an autoimmune disease is described as a selfperpetuating immune idiopathic reaction that results in tissue damage and inflammation.

Autoimmune diseases can be classified as systemic or local, depending on the extension of inflammation and on the presence or absence, respectively, of autoantibodies. In the first group are included diseases as rheumatoid arthritis (RA), dermatomyositis, scleroderma and systemic lupus erythematous (SLE); diseases as psoriasis (dermatologic), multiple sclerosis (MS, neurologic), or inflammatory bowel disease (IBD, gastrointestinal) are part of the second group. IL-17, alone or in association with TNF-α have been described as important for the pathogenesis and development of numerous of these diseases [START_REF] Chizzolini | Cytokines in chronic rheumatic diseases: is everything lack of homeostatic balance?[END_REF].

RA, being the expertise of our research group, was used as the model of chronic inflammatory disease in this work. Moreover, RA is a systemic disease, but its manifestation is primarily local: the articulation is destroyed by the perpetuation of inflammation, which causes tissue destruction with issues in self-reparation, tissue infiltration by immune system cells and neo-angiogenesis [START_REF] Firestein | Evolving concepts of rheumatoid arthritis[END_REF].

Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease of unknown etiology. The overall world prevalence of RA is approximately 1% to 2%.

The physiopathology of RA is still not clear but involves a complex interplay of different causes: genetic, hormonal and environmental. Genetic factors represent a high risk in developing RA, with an estimated heritability of 60%. There are more than 30 known polymorphisms associated with RA, notably on the gene PTPN22 [START_REF] Begovich | A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis[END_REF]. The most significant genetic risk factors for RA are variations in human leukocyte antigen (HLA) genes, especially the HLA-DRB1 gene. Several alleles of this gene (HLA-DRB1*0401, *0404, *0101) contain a common amino acid motif (QKRAA), called shared epitope, which confer an increased susceptibility to the disease [START_REF] Holoshitz | The Rheumatoid Arthritis HLA-DRB1 Shared Epitope[END_REF].

The frequency of RA is three times higher in woman, indicating an implication of sexual hormones in triggering the pathology, but this tendency is decreasing with age. Estrogens are implicated as enhancers of humoral immunity, while androgens and progesterone are natural immune suppressors [START_REF] Cutolo | Sex hormone adjuvant therapy in rheumatoid arthritis[END_REF]. Coherently, several studies demonstrated a relative low androgen levels in RA patients [START_REF] Gibofsky | Overview of epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis[END_REF].

Between the environmental factors, infectious agents (e.g., Epstein-Barr virus, cyto-megalovirus, proteus species, and Escherichia coli) and their products (e.g., heat-shock proteins) have long been linked with rheumatoid arthritis and, although unifying mechanisms remain elusive, some form of molecular mimicry is postulated [START_REF] Mcinnes | The pathogenesis of rheumatoid arthritis[END_REF]. The gastrointestinal microbiota has also been implicated in triggering autoantibody production, depending on the present bacteria [START_REF]Overview of Epidemiology, Pathophysiology, and Diagnosis of Rheumatoid Arthritis[END_REF]. The idea that intestinal micro-organisms are associated with the development of RA comes from the evidence that the gut microbiota helps to maintain the homeostasis balance of intestinal immunity and regulates the immune response. Moreover, associations between the presence of periodontal disease and RA have been described in literature [START_REF] Marotte | The association between periodontal disease and joint destruction in rheumatoid arthritis extends the link between the HLA-DR shared epitope and severity of bone destruction[END_REF][START_REF] Mercado | Is there a relationship between rheumatoid arthritis and periodontal disease?[END_REF]. Most recently, Porphyromonas gingivalis, a periodontopathic bacterium, has been recognized as a possible link between periodontitis, peptide citrullination, autoantibody formation and joint inflammation [START_REF] Arvikar | Clinical correlations with Porphyromonas gingivalis antibody responses in patients with early rheumatoid arthritis[END_REF][START_REF] Rinaudo-Gaujous | A6.7 Evaluation of porphyromonas gingivalis serology in rheumatic and non-rheumatic inflammatory disease[END_REF].

Smoking and other forms of bronchial stress (e.g., exposure to silica) increase the risk of rheumatoid arthritis among people with susceptibility HLA-DR alleles.

The association between toxic metals exposure via cigarette smoking and rheumatoid arthritis incidence was also evaluated as an independent risk factor, concluding in a possible synergistic effect with other risk factors for RA development [START_REF] Hutchinson | Cadmium, one of the villains behind the curtain: has exposure to cadmium helped to pull the strings of seropositive rheumatoid arthritis pathogenesis all along?[END_REF]. Exposure to heavy metal also occurs with water and food assimilation, or occupational work and could represent an environmental risk factor for RA.

Systemic inflammation in RA

RA is a systemic inflammatory disease characterized by heterogeneity in features and localization that can involve tissues and organs other than synovial joints. For that reason the severity and prognosis of RA can also vary significantly among individuals.

Extra-articular manifestations of RA are well described (skin, ocular, cardiovascular, pulmonary, etc.) and they are the main cause of co-morbidity and premature mortality (50-60% due to cardiovascular lesions) [START_REF] Cojocaru | Extra-articular Manifestations in Rheumatoid Arthritis[END_REF].

With the discovery of the rheumatoid factor (RF) in the '50s, it was highlighted that infections, or other factors, may trigger the induction of systemic inflammation.

RF is a high-affinity auto-antibody against the Fc portion of immunoglobulin and may form with it immune complexes, which contribute to the disease. RF has long served as a diagnostic marker of RA and is present in 80% of patients and indicates a more aggressive phenotype. However, RF is not specific of RA [START_REF] Nielsen | Elevated rheumatoid factor and long term risk of rheumatoid arthritis: a prospective cohort study[END_REF].

In the late 90s, antibodies directed against citrullinated proteins (ACPAs) were discovered. In its earliest stages, even before joint inflammation occurs, RA was thought to begin with a series of events in which the immune system lost its ability to distinguish between what belonged to the body (the "self") and what is foreign to the body (the "non-self"). According to a new theory, the systemic process of inflammation begins with inadequate clearance of cells as they die. As cells reach the end of their lifespan, they undergo citrullination, a process in which enzymes called peptidylarginine deaminases (PADs) convert arginine amino acid residues of proteins within the cell into citrulline ones [START_REF] Klareskog | Adaptive immunity in rheumatoid arthritis: anticitrulline and other antibodies in the pathogenesis of rheumatoid arthritis[END_REF]. This is a normal part of cell physiology. However, in most patients who develop RA, PADs leak from dying cells throughout the body and citrullinate proteins in the extracellular fluid. These citrullinated proteins are selectively recognized for their acid pH as antigens by Bcells, which produce ACPAs. High levels of ACPAs are detectable in plasma or serum of RA patients and leads to an increase of the systemic inflammatory response [START_REF] Pozsgay | In vitro eradication of citrullinated protein specific Blymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles[END_REF].

Blood levels of different inflammatory cells and pro-inflammatory cytokines were measured in RA patients and compared to healthy or osteoarthritis (OA, nonautoimmune and less inflammatory form of arthritis) controls. Higher level of Th17, but not Th1, were found in the blood of RA patients in comparison to OA patients [START_REF] Kim | Elevated levels of T helper 17 cells are associated with disease activity in patients with rheumatoid arthritis[END_REF] and healthy controls [START_REF] Zhang | Elevated circulating Th17 and follicular helper CD4(+) T cells in patients with rheumatoid arthritis[END_REF]. These results are in contrast with previous data showing no difference in Th17 in the blood of RA patients compared to controls in the early onset of arthritis [START_REF] Arroyo-Villa | Frequency of Th17 CD4+ T Cells in Early Rheumatoid Arthritis: A Marker of Anti-CCP Seropositivity[END_REF]. Moreover, the concentrations of IL-17, IL-21, and IL-22, all produced by Th17 cells, were significantly elevated in RA patients compared to healthy individuals. Furthermore IL-22 level in the serum also positively correlated with the disease activity score [START_REF] Zhang | Elevated circulating Th17 and follicular helper CD4(+) T cells in patients with rheumatoid arthritis[END_REF].

Local inflammation in RA

The reason why the systemic loss of tolerance is linked to a localized onset of inflammation in the joint is still unclear, even if the biological features of autoantigens (e.g. ACPA binding citrullinated fibrogen in joints [START_REF] Ossipova | Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint[END_REF]) or other biomechanical/microtrauma mechanisms can be involved.

The synovium, or synovial membrane, is the inner part of the joint, which function is to produce synovial fluid (SF), necessary to lubricate and to avoid cartilagecartilage contact. The synovium is composed by a two-layer structure, the subintima and the intima; the subintima is composed by connective and adipose tissues. The intima is a pauci-cellular structure composed by mesenchymal fibroblastic cells, named synoviocytes. In the normal condition synoviocytes are the main regulators of synovial fluid volume, immune response and leucocyte circulation as well as to the maintain of the membrane stability [START_REF] Mor | The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction[END_REF].

In the appropriate conditions, such as the presence of the specific HLA-DR alleles or perhaps cytokine-promoter polymorphisms, the inflammatory process modifies the structure of the synovium with a series of dynamic events: Synovial membrane hyperplasia due to the acquisition of an anti-apoptotic and aggressive phenotype by synoviocytes Synovium inflammation, with production of pro-inflammatory cytokines and chemokines to attract inflammatory cells Immune cell infiltration in the synovial cavity, principally macrophages, B lymphocytes and CD4+ T lymphocytes and further production of cytokines Neo-angiogenesis and proliferation of synovial cells responding to the produced cytokines All these changes of the synovial structure are primarily due to changes at the cellular level. It is increasingly clear that the synoviocyte, rather than being simply a passive bystander, is central to the RA process and a principal actor in RA pathogenesis. Synoviocytes hyperplasia can precede the accumulation of inflammatory cells in the synovium but whether this is primarily a cause, or a consequence of the disease, remains a matter of conjecture [START_REF] Mor | The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction[END_REF].

Mechanisms for the accumulation of synoviocytes can be different inducing decreased senescence, decreased apoptosis or hyperproliferation, but always implicating an unbalanced ratio between proliferation and apoptosis. Synoviocytes heterogeneously express a variety of pro-mitotic and anti-apoptotic mutations (as Fas or p-53 abnormalities) [START_REF] Firestein | Apoptosis in rheumatoid arthritis: p53 overexpression in rheumatoid arthritis synovium[END_REF], as well as several growth factors inducing their hyperproliferation. Moreover synoviocytes acquire an aggressive phenotype producing molecules increasing their invasive properties and contributing to the production of pro-inflammatory cytokines (as IL-6), metalloproteases (MMPs) and other enzymes responsible of cartilage destruction. The local production of cytokines induce the SDF1/CXCR4, HIF-1α pathways for cell migration and increasing the invasive phenotype of synoviocytes [START_REF] Hot | IL-17 and tumour necrosis factor α combination induces a HIF-1α-dependent invasive phenotype in synoviocytes[END_REF].

Synoviocytes produce an array of chemokines that signal cells to migrate into the joint. IL-8 produced by synoviocytes is a potent neutrophil attractant that also stimulates angiogenesis. This, along with numerous other chemokines, including RANTES, IP-10, ENA-78, and MCP-1, MIP-1α, and MIP-1β are major monocyte/macrophage recruiting proteins that are produced by synoviocytes in response to IL-1 or TNF stimulation. Release of these chemoattractants contributes to the cell mix that is subsequently recruited to the inflamed joint [START_REF] Bartok | Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis[END_REF].

Cell interactions and produced soluble factors

The interaction between synoviocytes and the immune cells stimulates the survival of the latter by maintaining survival signals and blocking apoptosis signals.

Cell interaction also induce pro-inflammatory cytokines production from immune cells (as IL-17 and TNF-α). Synoviocytes respond to the IL-17 and TNF-α synergistic stimulation by producing IL-6 [START_REF] Hot | IL-17A-versus IL-17Finduced intracellular signal transduction pathways and modulation by IL-17RA and IL-17RC RNA interference in rheumatoid synoviocytes[END_REF]. IL-6 induces chemokines (mostly monocyte attracting) as CCL2/CCL5 and further activates osteoclasts, T and B lymphocytes [START_REF] Nanki | Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis[END_REF].

Among immune cells, macrophages are major producers of TNF-α, IL-1β and IL-6. TNF-α is a primary cytokine of the inflammatory cascade, regulating the production of chemokines and IL-6 from synoviocytes. Furthermore, TNF-α contributes to the neo-angiogenesis and to the activation of the inflammatory phenotype of synoviocytes [START_REF] Maruotti | Angiogenesis in rheumatoid arthritis[END_REF] and consequently to the production of the connective tissue growth factor (CTGF). CTGF promotes aberrant activation of osteoclasts and disturbs the cartilage homeostasis, ultimately resulting in the destruction of the joint [START_REF] Nozawa | Inhibition of rheumatoid arthritis by blocking connective tissue growth factor[END_REF]. IL-1β is even more potent than TNF-α in inducing cartilage destruction [START_REF] Vervoordeldonk | Cytokines in rheumatoid arthritis[END_REF].

In the synovium, auto-reactive B cells can differentiate in plasma cells and produce autoantibodies such as RF and ACPA. B cells are also highly capable antigen-presenting cells and can contribute to auto-reactive T cell activation. B cells also produce several cytokines, such as IL-4 and IL-10, which promote leukocyte infiltration in the joint, angiogenesis and synovial hyperplasia [START_REF] Wang | The roles of B cells and their interactions with fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis[END_REF].

T-cells are one of the most abundant cells in the rheumatoid synovium and the majority of them are CD4+ Th cells. RA has traditionally been considered associated with an 'immunological shift' from Th2 to Th1, leading to a disturbance in the healthy balance between the Th derived cytokines (INF-γ from Th1 increased and IL-4 from Th2 decreased). Nowadays, data suggests the cardinal importance of Th17 cells in the pathogenesis of RA. The Th17 cells infiltrate in the synovium and their level is strongly higher in SF, in comparison to peripheral blood in RA patients [START_REF] Arroyo-Villa | Frequency of Th17 CD4+ T Cells in Early Rheumatoid Arthritis: A Marker of Anti-CCP Seropositivity[END_REF]. In the synoviumTh17 produce IL-17 and the cytokine overexpression, which is further responsible for osteoclastic bone resorption, was previously measured in RA patient SF in comparison to OA [START_REF] Kehlen | Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis[END_REF][START_REF] Kotake | IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis[END_REF].

An unbalanced ratio of Th17/Tregs cells in the SF of RA patients was also described. The unbalance was due to the IL-6 dependent suppression of the TGF-β mediated FoxP3 induction [START_REF] Noack | Th17 and regulatory T cell balance in autoimmune and inflammatory diseases[END_REF]. Recently increased levels of IL-17-producing Tregs were identified in RA patients. This Treg subset with Th17 plasticity in peripheral blood retained suppressive functions and was associated with milder inflammatory conditions, suggesting that this Treg population works as a negative regulator in RA. Contrarily, at the synovial site, they lose their suppressive activity and their expansion is induced by IL-6 [START_REF] Wang | Regulatory T cells in rheumatoid arthritis showed increased plasticity toward Th17 but retained suppressive function in peripheral blood[END_REF]. Their Th17 residual activity in the synovium may be pathogenic in RA.

In contrast to IL-17 cytokine, which is mainly produced by Th17 cells, IL-17 receptors are ubiquitously expressed. Receptor expression is increased on synoviocytes of RA patients. Moreover, CXCR4 is expressed by CD4 + memory T cells in RA synovium and its unique ligand, CXCL12 (stromal cell-derived factor-1) is expressed on synoviocytes [START_REF] Hot | Combination of IL-17 and TNFα induces a proinflammatory, pro-coagulant and pro-thrombotic phenotype in human endothelial cells[END_REF][START_REF] Nanki | Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis[END_REF].

Taken together these data sustain the description of RA as a systemic disease with a prevalent local manifestation. At this site, the interaction between synoviocytes and the cells of the immune system is pivotal in the perpetuation of inflammation, which in turn causes a transition of the synoviocytes from normal to "cancer-like" RA cells (represented in the figure 3).

Fig. 3. Inflammatory status transition from healthy to RA condition in joints. Cell composition changes between the healthy and RA conditions modify the status of joints and amplify inflammation

The role of the synoviocyte is thus central in RA pathogenesis and for RA perpetuation, even if it is still under-estimate in the choice of RA treatment.

RA Treatment

The denomination "disease modifying anti-rheumatic drugs" (DMARDs) indicates treatments able to impact the radiological progression of RA. DMARDs comprise a heterogeneous group of compounds with different target, biochemical and pharmacokinetic properties; nevertheless, they can be mainly divided in two large groups: synthetic DMARDs and biological DMARDs. The discovery of the first has traditionally been based on empirical data, in contrast with the second agents that were developed from bench to bedside.

The most commonly used synthetic DMARDs in clinical practice are methotrexate, and glucocorticoids (GC). Methotrexate is a purine synthesis antagonist and is considered to be the gold standard for treatment of RA [START_REF] Shinde | Methotrexate: a gold standard for treatment of rheumatoid arthritis[END_REF]. Since the DNA synthesis is the target of methotrexate, high rate reproducing cells, as the immune cells, are the most affected cells, even if the mechanisms of actions are still not completely clear [START_REF] Chan | Molecular action of methotrexate in inflammatory diseases[END_REF]. GCs are part of the group of corticosteroids and can be used for systemic or local therapy. They increase the production of antiinflammatory cytokines and reduce pro-inflammatory ones acting at different levels (genomic and non-genomic) [START_REF] Ayroldi | Mechanisms of the anti-inflammatory effects of glucocorticoids: genomic and nongenomic interference with MAPK signaling pathways[END_REF].

A deeper understanding of the pathophysiological and immunological mechanisms in RA has led to the development and introduction of biologic DMARDs, which gave relevant effect in patients with severe RA, difficult to control with conventional DMARDs. Currently, the available drugs in RA therapies are:

-Anti-TNF drugs (most commonly used)

-A IL-1β blocker -A B-cell depleting agent -An IL-6 blocker -A T-cell co-stimulation inhibitor

The common feature of all DMARDs, independently of their synthetic or biologic nature, is the target of immune cells, leading to reduce inflammation systemically and in the joint. Even when the treatment is used locally, the principal targets are inflammatory cells, but not mesenchymal cells.

Local treatment

Nowadays local-treatment is mainly used in mono-articular arthritis or in rheumatoid arthritis patients where only 2-3 joints are involved. Nevertheless, there is increasing evidence, from clinical trials, that more RA patients will attain the target of remission using a combination of systemic medication and local intra-articular therapy.

Synovectomy is also a form of local treatment for rheumatoid arthritis and it's the only treatment targeting synoviocytes instead of inflammatory cells. The value of synovectomy has been demonstrated by a large number of studies, for over 30 years. The procedure can be surgical (synovectomy as surgical removal of a part of synovium) or non-surgical (synoviorthesis) which involves the intra-articular administration of an agent in order to induce fibrosis in the inflamed synovium [START_REF] Rodriguez-Merchan | The technique of synoviorthesis[END_REF]; such agents may either be chemical based (osmic acid, rifampicin) or radioactive ( 90 Y isotope of yttrium or 198 Au isotope of gold). Between those two, synoviorthesis was chosen as the first choice treatment for persistent synovitis of the joints: it is a simple procedure, which eliminates the risks associated with surgery [START_REF] Rodriguez-Merchan | The technique of synoviorthesis[END_REF].

In 1973, Menkes et al. reported their experience with the use of intra-articular osmic acid (57) but their procedure never achieved such popularity and osmic acid was never approved by the French Health Authorities and registered as a drug or a medical device for use in human being (research use only, the injections were performed under responsibility of physicians) [START_REF] Ayral | Chemical synoviorthesis with osmic acid in haemophilia[END_REF]. This was due mainly to the negative benefit/risk ratio according to the European legislature about pharmaceutical quality and to the occurred complications and significant pain associated with the procedure of injection [START_REF] Swensson | Intra-articular Injections of Osmic Acid in Painful Joint Affections[END_REF]. Furthermore, osmic acid did not have a selective effect on the synovial layer and caused a brown colouration of the cartilage, which allows a certain amount of scepticism to be held in respect to the assurance that no actual damage to cartilage had been proven. Moreover, with osmic acid there was a wide discrepancy between the high success quota (90-95%)

opposed by an improvement in only 50% after one year from injection [START_REF] Menkes | Synoviorthesis with osmic acid in the hemophilic patient[END_REF][START_REF] Ayral | Chemical synoviorthesis with osmic acid in haemophilia[END_REF].

In 1980, Caruso et al. described the use of Rifampicin for the treatment of synovitis in RA but still with a high failure rate in the procedure [START_REF] Mannucci | Role of synovectomy in hemophilic arthropathy[END_REF].

Radioactive synoviorthesis was introduced as an effective, relatively simple, virtually painless and comparatively inexpensive technique for the treatment of chronic synovitis. Global results obtained with osmic acid seem to be less favourable than those with radioisotopes except for small joints where results were comparable [START_REF] Cho | Radioisotope synoviorthesis with Holmium-166-chitosan complex in haemophilic arthropathy[END_REF]. Thus, radioactive synoviorthesis was thought to be the best choice for patients with persistent synovitis but studies on the chromosomal changes that could be attributed to the radioactive material had to be performed.

After 40-years of screening, several long-term safety issues have been reported.

The central role of the synoviocyte is described in the different stages of RA pathogenesis (immune cell recruitment at the site of inflammation, inhibition of their apoptosis, perpetuation of inflammation (Figure 4A)) as well as the lack of a treatment which can specifically target it (Figure 4B). The use of non-radioactive isotopes targeting synoviocytes was evaluated in this work, with the aim of modulating hyperplasia and synovitis with an innovative local treatment.

Transition metals and group 12

The biochemical principles of life are based on both organic and inorganic chemistry. Inorganic biochemical aspects include the role of essential transition metal ions in maintaining human life and how they support growth and development. Moreover, several of these transition metal ions were shown to have functions in modulating the immune system. For that reason, the effects of several metals on the inflammatory response will be analyzed in this work with particular focus on the chronic inflammatory disease RA. The group 12 of elements, composed by zinc (Zn), cadmium (Cd) and mercury (Hg), does not strictly meet the defining properties of transition elements, but it is usually included with this group because of their similar properties. The general properties of the transition elements are:

They are usually high melting point metals.

They have several oxidation states.

They usually form colored compounds.

They are often paramagnetic.

Transition metal ions as manganese, iron, copper, and zinc are constituents of thousands of proteins and function in enzymatic catalysis and protein structure.

Transition metal ions regulate protein functions and proteins regulate their availability. For this reason the correct regulation of metal homeostasis is fundamental for cells.

Cell metal ion homeostasis requires multiple proteins for transport, sensing, chaperoning, and other functions in a network of tightly controlled interactions and with full integration into metabolism and signaling. The metal-regulatory proteins employ specific molecular mechanisms. One mechanism is the sulfur-ligand centered reactivity in zinc/thiolate coordination environments. Sulfur donors confer redox activity on the otherwise biologically redox-inert zinc ion. This coupling between zinc and redox metabolism provides a way of controlling zinc binding and protein functions [START_REF] Maret | The Function of Zinc Metallothionein: A Link between Cellular Zinc and Redox State[END_REF].

Acting on the mechanisms of homeostasis regulation of transition metals, which are integrated in cellular structure and functions, we could regulate numerous cellular activities. Altering metal ions concentration could possibly have pharmacological or toxic action, and will aid in developing strategies for preventing, diagnosing, or treating human diseases.

Metal quantification

Nowadays, several techniques are available as optical methods of chemical analysis. Many techniques perform the elemental analysis on solutions, so samples need to be at the liquid status. The liquid is then transformed or introduced in the machine as a fine aerosol.

In flame atomic absorption spectrometry (FAAS), either an air/acetylene or a nitrous oxide/acetylene flame is used to evaporate the solvent and dissociate the sample into its component atoms. When light from a hollow cathode lamp passes through the cloud of atoms, the atoms of interest absorb the light from the lamp.

This absorption is measured by a detector and used to calculate the concentration of that element in the original sample. The use of the flame limits the excitation temperature, which can be a problem for some elements and moreover the detection limit is in the parts-per-million (ppm) range and limited to 10 elements per analysis.

Inductively coupled plasma atomic emission spectrometry (ICP-AES), or simply ICP, is a multi-element analysis technique that uses an inductively coupled plasma source to dissociate the sample into its constituent atoms or ions, exciting them to a level where they emit light of a characteristic wavelength. A detector measures the intensity of the emitted light and calculates the concentration of that particular element in the sample.

Inductively coupled plasma mass spectrometry (ICP-MS) uses the same source of ICP. However, in this case, the ions themselves are detected, rather than the light that they emit. The ions are extracted from the plasma and passed into the mass spectrometer, where they are separated based on their atomic mass-tocharge ratio by a quadrupole or magnetic sector analyzer. The high number of ions produced, combined with very low backgrounds, provides the best detection limits available for most elements, normally in the parts-per trillion (ppt) range.

Quadrupole mass spectrometers in ICP-MS fulfill exacting requirements that demand the ultimate detectability and isotope ratio measurement, which were needed in our analysis. A schematic representation of the process in ICP-MS is represented in the Figure 6 below:

Use of metal isotopes

The name isotope often reminds of the use of radioactive molecules for medical treatment, as described above in the treatment dedicated chapter. More generally, the term "isotope" can be used to describe two or more variants (isotopologues) of a molecule that differ in the isotopic identity of one or more of their constituent atoms [START_REF] Eiler | Clumped-isotope " geochemistry -The study of naturallyoccurring, multiply-substituted isotopologues[END_REF]. For example, 64 Zn 66 Zn and 70 Zn are three of the naturally-occurring isotopologues of molecular Zn.

Isotopes variability between two different isotopologues of the same element is known as the "isotope effect", a term describing the mass-dependent variations of natural isotope abundances for a particular element [START_REF] Eiler | Clumped-isotope " geochemistry -The study of naturallyoccurring, multiply-substituted isotopologues[END_REF]. This phenomenon is due to an increased abundance of a specific element among coexisting molecular species, or differential partitioning through reservoirs of the human body. The distribution varies as a function of the bond strength, the ionic charge and the coordination, and it also changes with kinetics. The track of the availability of isotopes, and mainly of copper (Cu), Sulfur (S) and zinc (Zn), gave the evidence of a specific fractionation of these elements in the diseased tissues [START_REF] Balter | Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients[END_REF][START_REF] Larner | Zinc isotopic compositions of breast cancer tissue[END_REF]. The differences found between healthy and diseased subjects can be developed as a complement marker for cancer and/or inflammatory diseases. A supplementary descriptive work is needed to complement information, mainly about their transport, turnover and effect in human cells.

Preliminary investigations on the role of isotopes in healthy and diseased subjects are reviewed in the following review paper (Albarède F. et al, submitted).

Nevertheless, isotope applications are not only related to this type of analysis (fractionation normal/ill tissue) but isotopes can be used for supplementary investigations in the in vitro model, and maybe also in the in vivo model, as tracer, to avoid the application of radiotracers or fluorescent dyes.

Zinc (Zn)

Zinc is the chemical element with the atomic number 30. It is the first element of group 12 and it shares with other elements of this group a similar ion size and a common oxidation state (+2). Differently from the other elements of the group 12,

Zn is relatively stable in its divalent state and does not undergo redox changes.

Zinc is the 24th most abundant element in Earth's crust and has five stable isotopes that occur in nature. 64 Zn is the most abundant isotope (48.63% natural abundance) with a long half-life and with an ignorable radioactivity, followed by 66 Zn (28%), 68 Zn (19%), 67 Zn (4%) and at last 70 Zn (0.6%). Even if less abundant, 70 Zn conserves characteristics and half-life similar to the most abundant isotopes.

Biologic and public health importance of Zn have been highlighted in different papers and have been reviewed for this work [START_REF] Bonaventura | Zinc and its role in immunity and inflammation[END_REF]. Disturbances in Zn function and metabolism may have serious consequences for health. Actually, this element plays an important role in growth, development and functioning of all living cells. Our aim was to summarize how Zn homeostasis is regulated in the different human cells and tissues and by which mechanisms; moreover, Zn effects on the immune response were described in detail, focusing on those that are implicated in the physiopathology of rheumatoid arthritis (chapter II. Review paper: "Zinc and its role in immunity and inflammation).

Cadmium (Cd)

Another metal of the group 12, Cd, was studied in this work, with particular accent on its effect on the immune system modulation.

It is often stated that Cd is "natural" and this means that it is naturally present, in traces, everywhere in air, water, soils and food. The consumption of Cd has been increasing for years during industrialization and it cannot be degraded, causing a steady increase of its concentration in the environment [START_REF] Moulis | New perspectives in cadmium toxicity: an introduction[END_REF]. Improved technology for the production, use, and disposal of Cd and Cd-containing products is nowadays trying to reduce the emissions.

Even if contaminated water is the first source of Cd, another important source for humans is smoking, as the tobacco plant naturally accumulates relatively high concentration of Cd in its leaves. Cadmium appears as a novel risk factor, independent from smoking, contributing to cardio-vascular diseases [START_REF] Tellez-Plaza | Cadmium Exposure and Clinical Cardiovascular Disease: a Systematic Review[END_REF]. It has been recognized in the list of carcinogenic molecules and even if the exposure to high Cd-doses was previously stated as an important risk factor, there are epidemiologic evidences that even long-term exposure to low doses of Cd can have an effect. For these reasons the safe day-exposure dose to Cd according to WHO guidelines is limited to 25 μg/Kg of body weight [START_REF] Satarug | Cadmium, Environmental Exposure, and Health Outcomes[END_REF].

Liver is the first target organ following acute Cd intoxication [START_REF] Sabolić | Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs[END_REF][START_REF] Bernhoft | Cadmium Toxicity and Treatment, Cadmium Toxicity and Treatment[END_REF][START_REF] Madejczyk | Temporal changes in rat liver gene expression after acute cadmium and chromium exposure[END_REF]. High doses of Cd administered to experimental animals lead to morphological and functional changes in these organs by cellular mechanisms that will be explained in the next paragraphs (and increasing liver-damage marker as transaminases or C-reactive protein production). Moreover, Cd target kidney and bones, especially in the case of chronic exposure: kidney is targeted directly, bones can be targeted indirectly via kidney, inducing Ca 2+ excretion in urine, or directly with osteoporosis induction (73).

Transport and effects of cadmium in cells

Most chemical forms of cadmium are ionic, which prevents passive diffusion through biological membranes. A variety of trans-membrane molecules have been proposed to participate to Cd transport, but in mammalian cells, it mainly uses Zn transporters [START_REF] Guerinot | The ZIP family of metal transporters[END_REF][START_REF] Himeno | The role of zinc transporters in cadmium and manganese transport in mammalian cells[END_REF]. Cadmium, differently from other metals as Cu and Zn is not work stated that clustered Cd-MTs are more resistant than apo-MTs to proteolysis [START_REF] Pinter | Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange[END_REF].

Competitive role of Zn and Cd

Even if they share many physico-chemical properties and they use the same transporters, Cd 2+ and Zn 2+ ion competition at different levels is still not clear and depends on the model.

ZIP importers, for the uptake into cells, are not selective, except for Cu, which is not transported by ZIPs, but has other specific transporters. This means that the ratio of Zn/Cd ions transported only depends on the availability of the metal. In this way, one of the metals can influence the uptake and action of the other, depending on their levels.

For intracellular binding sites, Cd may displace Zn in a number of biological processes, due to its higher affinity to several sites. Depending on the biological systems, Cd and Zn are linked to macromolecules, primarily through sulphur (S), oxygen (O) and nitrogen (N) and interact readily with S-, O-and N-donors [START_REF] Sharma | Biomedical Implications of Heavy Metals Induced Imbalances in Redox Systems, Biomedical Implications of Heavy Metals Induced Imbalances in Redox Systems[END_REF].

They bind preferentially to the same proteins -albumin in the bloodstream and MTs and other proteins in tissues [START_REF] Plum | The Essential Toxin: Impact of Zinc on Human Health[END_REF]. Although both metals have a high affinity to biological structures (proteins, enzymes) containing -SH (sulphydryl) groups, the affinity of Cd to S-ligands as well as to N-donors is greater than the one of Zn [START_REF] Moulis | New perspectives in cadmium toxicity: an introduction[END_REF].

Thus, many toxic effects of Cd occur through a disruption of Zn-mediated or Zndependent metabolic processes, including cellular production of DNA, RNA and protein.

The binding of Zn and Cd to MTs have long been considered isomorph, but the Cd-detoxifying role of MTs occurs through the replacement of Zn-MT by Cd-MT demonstrating a competitive role of the two metals, in favor of Cd binding.

Even if the affinity of Cd for MTs is higher, there are evidences of a protection role afforded by Zn-supplementation against the deleterious effect of Cd. Numerous data, for example, indicate that some of the adverse acute and chronic effects of Cd in laboratory animals can be prevented or markedly reduced by prior, post or concomitant Zn supply [START_REF] Hammouda | Reversal of cadmium-induced thyroid dysfunction by selenium, zinc, or their combination in rat[END_REF][START_REF] Rogalska | Enhanced zinc consumption prevents cadmium-induced alterations in lipid metabolism in male rats[END_REF]. This can be due to the reduced import of Cd when a higher level of Zn is given. For the same reason, low dietary Zn is an important factor increasing Cd absorption, retention and accumulation in the organism, or The essential trace element Zn is required for human health [START_REF] Krebs | Zinc metabolism and homeostasis: the application of tracer techniques to human zinc physiology[END_REF] and it has a major role in the regulation of the immune system and inflammation as described in our previous review paper [START_REF] Bonaventura | Zinc and its role in immunity and inflammation[END_REF]. Zinc is involved in essential cell mechanisms and it is part of the structure of numerous proteins [START_REF] Dollery | Matrix metalloproteinases and cardiovascular disease[END_REF]. Although Zn importance in the pathogenesis of chronic inflammatory diseases is fully appreciated systemically [START_REF] Maruotti | Angiogenesis in rheumatoid arthritis[END_REF][START_REF] Nozawa | Inhibition of rheumatoid arthritis by blocking connective tissue growth factor[END_REF], its implication at the cellular level is not well known and needs further investigations.

As Zn cannot be imported as a divalent ion, it is imported in synoviocytes mainly

Results:

Zinc uptake is increased by the combination of IL-17 and TNF-α

The uptake of Zn was measured in the medium and in the cells (both OA and RA synoviocytes) exposed to 0.9 ppm of Zn alone or in combination with the pro inflammatory cytokines IL-17 and TNF-α.The effect of cytokine addition was not measurable in the medium, while there was evidence of a higher Zn accumulation in cells exposed to the cytokine combination, after 120 hours. The equilibrium of the 70 Zn tracer between cells and medium indicated an isotopic equilibrium between intake and efflux

TNF-α and IL-17 combination enhances zinc importer gene expression and modifies other trafficking molecule gene expression

The gene expression of the importer ZIP-8 was quantified after overnight exposure to IL-17A and TNF-α alone or in combination demonstrating that there is a synergy between those cytokines in inducing ZIP-8 expression.

The gene expression of other molecules involved in Zn trafficking were analyzed after exposure to cytokines, Zn, or both. ZIP-8 expression was enhanced by inflammation only, while the exporter ZnT1 by Zn. MT expressions were increased by Zn exposure but inflammation enhanced this effect.

Exposure to zinc and pro-inflammatory cytokines synergistically enhances IL-6 production by synoviocytes

In agreement with earlier experiments addition of the combination of IL-17 and TNF-α stimulated the production of IL-6 relative to control. The comparison of the results without IL-17 and TNF-α addition showed that Zn addition enhanced IL-6

expression. The presence of both Zn and cytokines amplified the inflammatory effect on RA synoviocytes.

A model of cytokine-induced zinc homeostasis regulation

Using the data obtained, a mathematical model of the cell was developed with the aim to extend the findings to other cell types. In simple terms, the mathematical model expresses that inflammation opens the gates for Zn uptake and prepares the cell for Zn storage, regardless of the amount of Zn actually present in the extracellular medium. On the contrary, inflammation does not prepare the cell to excrete Zn rapidly, leading to the accumulation and potential intoxication of the concerned tissue

V. INFLAMMATION-INDUCED CADMIUM UPTAKE BY SYNOVIOCYTES PROTECTS JOINTS FROM ARTHRITIS DESTRUCTION

Paola Bonaventura, Guillaume Courbon, Aline Lamboux, Giulia Benedetti, Fabien Lavocat, Hubert Marotte, Francis Albarède and Pierre Miossec.

PNAS 2016 (submitted)

Objective: The aim of this work was to study the inflammation-induced alterations of Cd balance in RA in vitro and ex vivo models and using the Cd effects as a local treatment against hyperplasia and inflammation in vivo.

Zinc (Zn) and cadmium (Cd) belong to the group 12 of divalent metals and they are closely associated for their chemical properties [START_REF] Tang | Cadmium-zinc exchange and their binary relationship in the structure of Zn-related proteins: a mini review[END_REF] as well as for their specific transport mechanisms in cells. However, while Zn is known to be necessary for the normal function of the immune response and for cell proliferation [START_REF] Bonaventura | Zinc and its role in immunity and inflammation[END_REF], the role of Cd on those pathways is still not clear regarding the induction of cell proliferation versus apoptosis [START_REF] Mohammadi-Bardbori | Arsenic, cadmium, mercury and nickel stimulate cell growth via NADPH oxidase activation[END_REF][START_REF] Aimola | Cadmium induces p53-dependent apoptosis in human prostate epithelial cells[END_REF].

In the previous chapter we showed how IL-17/TNF dependant inflammation can enhance the entry and the accumulation of Zn inside the cells [START_REF] Bonaventura | A Feedback Loop between Inflammation and Zn Uptake[END_REF]. In this paper the analysis will be enlarged to Cd, always using RA as the chronic inflammation model.

RA is characterized by hyperproliferation of synoviocytes at the site of inflammation, which are refractory to apoptosis. The inflammation in joints is sustained by a continuous production of IL-17, TNF (by the immune cells) and IL-6

(by synovioctytes). Synoviocyte selectivity for Cd will be used to reduce inflammation and hyperplasia and was tested as potential local treatment on the model of rat adjuvant-induced arthritis.

Results:

Cd is preferentially absorbed from synoviocytes in comparison to zinc and other metals

Synoviocytes were exposed to a cocktail of metals in order to measure their differential intake. Zinc was added to cultures in a 'tracerless' form using minute quantities of a stable 70 Zn spike. The affinity ratio (K D ) of metals, between cells and medium are referred to 70 The impact of Cd on bone loss was evaluated, as the reduction of bone loss and joint destruction remains the key target when applied RA patients [START_REF] Marotte | Prevention of bone mineral density loss in patients with rheumatoid arthritis treated with anti-TNFalpha therapy[END_REF]. Cd injection stopped bone loss in ankle bones and the increased density was proportional to the concentration of Cd injected

DISCUSSION

The general scope of this study was to provide new information on the mechanisms that can play a role on changes in metal homeostasis, which are altered during chronic inflammation. At the same time, after studying the differential effect of Zn and Cd on the immune system function, we could provide an innovative tool to control synoviocyte contribution to inflammation.

In the first study the positive role of IL-17/TNF inflammation on the increased uptake and accumulation of Zn was demonstrated. The changes in Zn homeostasis induced by IL-17/TNF inflammation then contribute to inflammation self-activation and may play a strong role in the transition to the chronic stage of the disease. This concept seems sufficiently general to be applicable to other cell types, for that reason the results obtained were summarized in a mathematical model.

In the second study, the effect of exposure to Cd was tested in the cellular model of the chronic inflammatory disease rheumatoid arthritis, on synovial tissue from RA patients and in the AIA rat model. On the in vitro cellular model using synoviocytes, exposure to Cd had an anti-proliferative and immune-modulatory effect, resulting in reduced cell viability and inflammation. The results were confirmed using synovial tissue, where the reduced proliferation of cells from biopsies exposed to Cd was associated with a lower IL-6 production. Finally the intra-articular injection of Cd in the arthritic rat model resulted in a less inflammatory disease and moreover in reduced bone loss and joint destruction.

Our results suggest that both the up-regulated expression of the importer ZIP-8 and of the homeostasis regulators MTs, in the presence of inflammation, contribute to Zn accumulation in cells. Our mathematical model expresses that inflammation results could imply a stronger susceptibility of subjects undergoing inflammatory events to the environmental factor, depending on its metal enrichment.

Another important point of our work consisted in analyze the effect of metal accumulation in cells on their viability and inflammation. An association was found between the accumulation of Zn in the cell and the induction of inflammation while cell viability was not modified at low doses (data not shown). We focused on the production of IL-6, the main pro-inflammatory cytokine produced by synoviocytes.

The effect of Zn on IL-6 production indicated a feedback loop, where the production of IL-17 and TNF-α upon original inflammation increases Zn uptake, leading to excess accumulation of Zn that promotes IL-6 production. Such self-sustained inflammation therefore has the potential of triggering chronicity.

On the contrary, Cd accumulation in the cell led to reduced cell viability, measured as apoptosis. This is stated as a consequent exacerbation of the toxic effect of the formation and persistence of clustered Cd-MTs, recently described in literature in substitution of the classical detoxyfing role always attributed to MTs [START_REF] Menkes | Synoviorthesis with osmic acid in the hemophilic patient[END_REF]. Regarding inflammation, Cd addition reduced IL-6 levels in supernatants of cultures and co-cultures, an effect probably linked to the reduced cell viability and confirming that cells undergo apoptosis without the release of other proinflammatory cytokines typical of other process of cell death as pyroptosis.

Interestingly, addition of Zn to the Cd-treated cultures could partially reverse the effects of Cd on synoviocytes as cell viability was increased and the production of IL-6 restored. This protective effect is a consequence of the non-selective ZIP-8 import, which, in the presence of the two metals, led to a reduction of the final concentration of Cd in the cell and a consequent reduction of the antiproliferative/anti-inflammatory effects.

The same and higher concentrations of Cd were used on synovial biopsies to establish a dose response. As the number of cell in the biopsy is very large in comparison to cultures, higher doses of Cd are needed to achieve the expected results: the reduction of cell proliferation from the biopsy to the plate layer and a strong reduction of IL-6 production could be measured with Cd used at 1 and 10 ppm.

To verify our findings about the immune-modulatory effect of low-dose Cd, an in vivo model of arthritis was used. In our model the local intra-articular injection of Cd was performed after AIA development to evaluate the therapeutic anti-proliferative and immuno-modulatory effects, as would be the case in a patient with arthritis. The results showed a clear effect on inflammation reduction with increasing Cd concentration, always keeping exposure at sub-toxic doses. The recommended tolerable weekly intake for Cd according to the WHO guidelines is fixed at 25 μg/kg/body weight/week. This concentration is 4 fold higher of the effective dose we used for the local injection. Further pharmacokinetics studies need to be performed to know the duration of action of Cd-treatment and to better evaluate the potential toxicity.

Nevertheless a previous screening of the possible toxic effect was verified on liver, one of the major sites of accumulation after acute exposure to Cd (103). Cd liver concentration was detectable only in the experimental group treated with 10 ppm (data not shown). Levels of transaminases remained unchanged suggesting that Cd therapy would remain harmless to the liver (data not shown). This represents a favorable starting point for further studies to establish the toxic dose.

Another important result is the reduction of bone loss in the Cd-treated group, as the reduction of joint destruction and bone loss remains the key target when applied to RA patients. This result may mean that there is no specific toxic effect on osteoblasts and osteocytes and that the principal target of Cd remains synoviocytes.

As the use of the intrinsic toxicity of nanomaterials for anticancer therapy is an emerging concept (104) this approach could be extended to RA and other diseases such as pigmented villo-nodular synovitis (PVNS), or mono-articular arthritis. The use of Cd-enriched quantum-dots (QDs) linked to a specific antibody could help to target specifically synoviocytes. The magnetic properties of QDs could help in the regulation and maintaining at the site of inflammation as well as the development of viscous preparations able to maintain the particles in situ, until the absorbance by synoviocytes is completed.

  Cd ont été testés dans le modèle d'arthrite chez le rat où une seule injection intra-articulaire de Cd à faible dose prévient la perte osseuse et réduit les scores cliniques d'arthrite. Cela pourrait représenter une nouvelle approche thérapeutique pour le traitement de la PR et d'autres pathologies caractérisées par une hyperplasie et une inflammation localisées.

Faculté

  Zn stimulated the inflammatory response, while addition of Cd can reduce both viability and inflammation. The anti-proliferative and anti-inflammatory properties of Cd were used in the rat model of arthritis as intra-articular treatment to reduce local inflammation and joint destruction and it may represent a new local therapeutic approach for RA treatment. UNIVERSITE CLAUDE BERNARD -LYON 1 Président de l'Université Vice-président du Conseil d'Administration Vice-président du Conseil des Etudes et de la Vie Universitaire Vice-président du Conseil Scientifique Directeur Général des Services M. François-Noël GILLY M. le Professeur Hamda BEN HADID M. le Professeur Philippe LALLE M. le Professeur Germain GILLET M. Alain HELLEU COMPOSANTES SANTE Faculté de Médecine Lyon Est -Claude Bernard Faculté de Médecine et de Maïeutique Lyon Sud -Charles Mérieux Faculté d'Odontologie Institut des Sciences Pharmaceutiques et Biologiques Institut des Sciences et Techniques de la Réadaptation Département de formation et Centre de Recherche en Biologie Humaine Directeur : M. le Professeur J. ETIENNE Directeur : Mme la Professeure C. BURILLON Directeur : M. le Professeur D. BOURGEOIS Directeur : Mme la Professeure C. VINCIGUERRA Directeur : M. le Professeur Y. MATILLON Directeur : Mme. la Professeure A-M. SCHOTT COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE Techniques des Activités Physiques et Sportives Observatoire des Sciences de l'Univers de Lyon Polytech Lyon Ecole Supérieure de Chimie Physique Electronique Institut Universitaire de Technologie de Lyon 1 Ecole Supérieure du Professorat et de l'Education Institut de Science Financière et d'Assurances Directeur : M. F. DE MARCHI Directeur : M. le Professeur F. FLEURY Directeur : Mme Caroline FELIX Directeur : M. Hassan HAMMOURI Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur Georges TOMANOV Directeur : M. le Professeur H. BEN HADID Directeur : M. Jean-Claude PLENET Directeur : M. Y.VANPOULLE Directeur : M. B. GUIDERDONI Directeur : M. le Professeur E.PERRIN Directeur : M. G. PIGNAULT Directeur : M. le Professeur C. VITON Directeur : M. le Professeur A. MOUGNIOTTE Directeur : M. N. LEBOISNE Aknowledgements Index I. INTRODUCTION ......................................................................................................12 1. Inflammation ....................................................................................................................................................... 1.1 Pro-inflammatory cytokines ........................................................................................................................................... 1.2 Chronic inflammation ........................................................................................................................................................ 1.2.1 Chronic inflammatory autoimmune diseases ........................................................................................... 1.3 Rheumatoid arthritis (RA) ............................................................................................................................................... 1.3.

Fig. 1 :

 1 Fig. 1: Feedback loop of inflammation. The production of IL-17, IL-1 and TNF-α by immune cells (1) enhances the production of IL-6, IL-8 and CCL-20 by mesenchymal cells (2) which induces a feedback loop on inflammation (3), resulting in chronicity

Fig. 2 :

 2 Fig. 2: Acute versus chronic inflammation. Shift of the cells at the site of inflammation induces the production of many inflammatory mediators and changes in cells

Fig. 4 :

 4 Fig. 4: Cell involvement and current treatment in RA. Stages of inflammation in RA. (A) On the left panel the acute (resolving) inflammation, on the right the chronic (non-resolving) inflammation. (B) Current treatment for chronic inflammation targeting immune system (cells and produced cytokines) but not synoviocytes

5 )Fig. 5 :

 55 Fig. 5: the group 12 of transition metals. Transition metals are represented in pink on the table of elements. The group 12 is part of transition metals and composed by Zinc, Cadmium and Mercury (Adapted from Bodner research web)

Fig. 6 :

 6 Fig. 6: Inductively coupled plasma mass spectrometry process. Liquid samples are evaporated using a plasma torch inductively activated using the argon flux. Compounds of the solute are dissociated in atoms, and then ions, which can be extracted into the mass spectrometer. The quadrupole mass spectrometer divides ions on the basis of their m/z ratio and a channel electron multiplier (CEM) detects ions passed by the mass filter and send the values to the system computer for analysis.

  All these reactions elicited by Cd inside the cell may lead to death or stress adaptation, survival or cancer. These processes are mediated by signaling pathways which induce up-regulation of various stress-inducing or protective molecules (e.g. glutathione). Moreover, Cd inhibit or damage DNA repair enzymes, cell cycle control proteins, pro-apoptotic and tumor-suppressing proteins (e.g., PARP-1, p53), Zn-containing transcription factors, enzymes mediating DNA methylation, and also E-cadherin[START_REF] Waisberg | Molecular and cellular mechanisms of cadmium carcinogenesis[END_REF]. Nevertheless, its role on cell proliferation and apoptosis is still not clear and depends on the cell type.Zinc transporter proteins (ZnTs) facilitate the removal of cytosolic free metal ions either by exporting them through the plasma membrane or by sequestering them in vesicles; ZnT1 is the only transporter expressed ubiquitously and the only one among ZnTs expressed on the cellular membrane and mediating metal export to the extracellular fluid. Zn is known to regulate the expression of this transporter through MTF-1 in several cell types. Nevertheless the expression of this exporter on synoviocytes and the ability of Zn and other metals in modulating its expression need further investigations.

through the importer ZIP- 8 .

 8 The zinc pool in cells is regulated by MTs and the export in the extracellular fluid is mediated by the ZnT1 exporter on the cellular membrane. The correct function of these mechanisms is necessary to maintain the correct homeostasis of Zn for the survival of the cell. Based on this knowledge this work aimed to describe how Zn is regulated by IL-17/TNF induced inflammation at the cellular level, eventually contributing to chronicity. Moreover a model of diffusion-controlled Zn transport was developed to account for the observed variations of the Zn flux in the inflammatory condition against normal conditions.

  Zn isotope as tracer. The obtained values of K D indicated that Cd is highly absorbed by synoviocytes in comparison to Zn and other metals.ZIP-8 and MT1 drive Cd accumulation in synoviocytes exposed to IL-17 and TNF-α combinationAn increased absorbance of Cd in synoviocytes exposed to IL-17 and TNF-α in comparison to Cd alone was measured. In both conditions, even after cell wash with normal medium, Cd was not excreted. The increased Cd content in cytokinestimulated cells was due to the increased expression of the ZIP-8 importer and MTs after IL-17/TNF-α exposure. Cadmium binds to MTs with a high affinity[START_REF] Pinter | Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange[END_REF]. Even if the exporter ZnT1 was overexpressed in the presence of Cd, it was not excreted but stayed linked to MTs, making ZnT1 export non-functional.Low-dose Cd exposure reduces cell viability of inflammatory synoviocytesand decreases the production of IL-6 in cultures and biopsies as local treatment. Cadmium dissolved in a physiological solution of 0.9% NaCl was injected into the rat ankles at the stage of active arthritis resulting in a reduction of clinical scores and ankle perimeters in comparison to controls.

II. AIMS OF THE WORK ..............................................................................................76 III. ZINC AND ITS ROLE IN AUTOIMMUNITY AND INFLAMMATION ..............................77 IV. A FEEDBACK LOOP BETWEEN INFLAMMATION AND ZINC UPTAKE ........................88 V. INFLAMMATION-INDUCED CADMIUM UPTAKE BY SYNOVIOCYTES PROTECTS JOINTS FROM ARTHRITIS DESTRUCTION .............................................................................. DISCUSSION ............................................................................................................ ANNEXE 1 ................................................................................................................ BIBLIOGRAPHY ........................................................................................................ List of the abbreviations used in the manuscript

  1 Systemic inflammation in RA ........................................................................................................................... 1.3.2 Local inflammation in RA ................................................................................................................................... 1.3.2.1 Cell interactions and produced soluble factors .............................................................................. 1.3.3 RA Treatment .......................................................................................................................................................... 1.3.3.1 Local treatment ............................................................................................................................................. ........... 2.4.1 Transport and effects of cadmium in cells ................................................................................................. 2.4.2 Competitive role of Zn and Cd ..........................................................................................................................

	I. INTRODUCTION 1.1 Pro-inflammatory cytokines
	Cytokines are small, nonstructural proteins with molecular weights ranging from 8 IL: interleukin 1. Inflammation TNF-α: tumour necrosis factor-alpha IFN-γ: interferon-gamma to 40,000 Da. Almost all nucleated cells are capable of synthesizing these proteins
	IL-17: interleukin-17
	IL-1β: interleukin-1-beta Th: T helper The word inflammation derives from Latin inflammo which means "I ignite" and and, in turn, of responding to them. There is no amino acid sequence motif or three-
	Th17: T helper 17
	Th1: T helper 1 IL-23: interleukin-23 describes the processes of the body tissues for self-protection to harmful stimuli, dimensional structure that links cytokines. Even if they can be divided according to
	IL-21: interleukin-21 IL-22: interkleukin-22 such as pathogens, damaged cells, or irritants. Inflammation is characterized by five their principal biological activities into pro-inflammatory and anti-inflammatory
	IL-17RA: interleukin-17 receptor A
	IL-17RC: interleukin-17 receptor C PGE-2: prostaglandins-2 cardinal signs: redness (rubor) resulting from vasodilation and the increased blood cytokines (1), their real effect is dependent on the disease context.
	NOS: nitric oxide synthase
	Pro-IL-1β: preform (precursor) IL-1β ICE: IL-1β converting enzyme flow for cell recruitment , increased heat (calor) which manifests itself as swelling Pro-inflammatory cytokines, including interleukins (IL), tumor necrosis factor α TNFR1: TNF-receptor-1 TNFR2: TNF-receptor-2 IL-8: interleukin-8 (tumor) and pain (dolor) with a possible loss of the function of the system (function (TNF-α), and interferon γ (INF-γ) are produced predominantly by macrophages and
	IL-6: interleukin-6 CCL-20 : Chemokine (C-C motif) ligand 20 CCR-6: Chemokine (C-C motif) receptor 6 laesa). Inflammation can be either acute or chronic, depending on the self-sustained lymphocytes but can be synthesized by other cell types. These polypeptides
	IL-10 : Interleukin-10 CXCR1: Chemokine (C-X-C motif) receptor 1 CXCR2: Chemokine (C-X-C motif) receptor 2 character of the disease. modulate the activity and function of other cells to coordinate and control the
	IL-1RA: Interleukin receptor 1 IL-12: Interleukin-12 Acute inflammation is the initial, beneficial, short term response of the body inflammatory response.
	Gp130: Glycoprotein 130
	IL-6Rα: Interleukin-6 receptor-alpha sIL-6Rα: soluble interlukin-6 receptor-alpha against the harmful stimulus and it is a reversible self-limited mechanism leading to
	sgp130: soluble glycoprotein 130
	ROS: Radical oxygen species RA: Rheumatoid arthritis resolution at inflammatory sites. This process can also become harmful, because
	SLE: Systemic lupus erythematosus MS: Multiple sclerosis the components of inflammation can hurt bystander tissues. At the cellular level,
	IBD: Inflammatory bowel disease
	RF: Rheumatoid factor ACPAs: Anti-citrullinated protein antibodies acute inflammation can mainly be considered a part of the innate immunity
	PADs: Peptidyl arginine deaminase
	OA: Osteoarthritis CTGF: connective tissue growth factor response and it is divided into different stages of cell recruitment, from the blood
	CXCR4: Chemokine (C-X-C motif) receptor 4 CXCL12: Chemokine (C-X-C motif) ligand 12 into the injured tissues: during the early stage there is the activation of the innate
	CCRs: Chemokine (C-C motif) receptors
	CCLs: Chemokine (C-C motif) ligands DMARDs: Disease modulating anti-rheumatic drugs immunity, where mainly neutrophils are recruited, first followed by monocytes-
	GC: glucocorticoids
	MTX: Metotrexate FAAS: In flame atomic absorption spectrometry macrophages and finally by lymphocytes for the activation of the adaptive immune
	ICP-AES: Inductively coupled plasma atomic emission spectrometry ICP-MS: Inductively coupled plasma mass spectrometry response. Those cells can interact with other cells at the site of inflammation and
	Zn: Zinc
	Cd: Cadmium MTF-1: Metallothionein transcription factor-1 undergo activation, with the release of cellular mediators of inflammation. Pro-
	MTs: Metallothioneins
	Apo-MT: Apo-metallothionein Holo-MT: Holo-metallothionein inflammatory cytokines are responsible, together with other chemical mediators
	ZIPs: Znt-Irt like proteins ZnTs: Zinc transporters (complement, leukotriene, prostaglandin, thromboxane, platelet activating factor),
	for the inflammatory clinical status.

2. Transition metals and group 12 .................................................................................................................. 2.1 Metal quantification ............................................................................................................................................................ 2.2 Use of metal isotopes ......................................................................................................................................................... 2.3 Zinc (Zn) ................................................................................................................................................................................... 2.4 Cadmium (Cd) ............................................................................................................................................................

III. ZINC AND ITS ROLE IN AUTOIMMUNITY AND INFLAMMATION IV. A FEEDBACK LOOP BETWEEN INFLAMMATION AND ZINC UPTAKE

	Bonaventura P, Lamboux A, Albarède F. and Miossec P.
	PLoS One 2016; 11(2):e0147146. doi:10.1371/journal.pone.0147146
	Objective: investigate how IL-17/TNF induced inflammation could modify Zn
	metabolism at the synoviocyte level eventually contributing to chronicity.

needed by mammalian cells for living; nevertheless, and probably through a process of ionic mimicry, it can substitute other metal ions (mainly Zn 2+ , Cu 2+ and Ca 2+ ) on the access-mechanisms into the cells and on enzymes inside the cells: this process is at the basis of Cd toxicity.

Zn transport is described in detail in the review paper in the chapter II [START_REF] Bonaventura | Zinc and its role in immunity and inflammation[END_REF] and the used transporters are the same for Cd. The expression of the Zrt/IRT-like proteins (ZIP) importers is tissue specific. For that reason, Cd absorbed through the respiratory tract, through smoking, or through the gastro-intestinal tract, through water and food, will use different and cell-specific transporters of the tissue on which it deposit. In the case of the synoviocytes the specific importer is ZIP-8 and the cell surface concentration of ZIP-8 seems to follow iron availability by a posttranscriptional mechanism [START_REF] Maret | Binding, Transport and Storage of Metal Ions in Biological Cells[END_REF].

Once it entered the cell, Cd induces the metal-regulatory transcription factor 1 (MTF-1) which triggers metallothioneins (MTs) transcription. MTs are small, cysteine rich metal binding proteins that function as the regulator of metal homeostasis in cells. The MT molecule is composed of two domains: the α-domain binds four atoms of metals, while the β-domain binds three atoms. In various organs and intracellular organelles, the different localization and physiological functions of MTs, including those of the metal-free (apo-MT) and metal-bound (holo-MT) forms, impact the redox and energy status of the cells.

In the cell, MTs act as a Zn-chaperone protein by delivering metal to metalloenzymes, which require a constant shuffling of metal ions, and control the response to the stress conditions, in particular detoxification mechanisms in response to heavy metals [START_REF] Freisinger | Cadmium in metallothioneins[END_REF], with a potential protective role for the cell. A recent vice-versa elevated level of Cd in blood are often accompanied by changes in levels of some essential elements, including Zn (84-86). The mean values of Arsenic, Lead and Cd were found higher in blood, scalp hair, and urine samples of arthritis patients as compared to those values obtained in agematched referent subjects [START_REF] Afridi | Evaluation of status of arsenic, cadmium, lead and zinc levels in biological samples of normal and arthritis patients of age groups (46 -60) and (61 -75) years[END_REF]. The concentration of Zn was lower in the biological samples of rheumatoid arthritis patients of both genders with respect to non-arthritic subjects and negatively correlated with the concentration of Cd [START_REF] Afridi | Evaluation of status of arsenic, cadmium, lead and zinc levels in biological samples of normal and arthritis patients of age groups (46 -60) and (61 -75) years[END_REF]. Moreover a case-report study described a patient affected by heavy metal intoxication, with evidence of Cd presence, who developed rheumatoid arthritis [START_REF] Bamonti | Metal chelation therapy in rheumathoid arthritis: a case report. Successful management of rheumathoid arthritis by metal chelation therapy[END_REF]. Zn supplementation potential role in mitigate the effects of Cd intoxication was already proposed [START_REF] Mccarty | Zinc and multi-mineral supplementation should mitigate the pathogenic impact of cadmium exposure[END_REF].

II. AIMS OF THE WORK

The general aim of the work was to investigate how chronic inflammation can modify metal homeostasis using the RA model. Moreover we analyzed the effect of RA synoviocytes are refractory to apoptosis and their proliferation and invasiveness is increased in the presence of pro-inflammatory cytokines [START_REF] Hot | IL-17 and tumour necrosis factor α combination induces a HIF-1α-dependent invasive phenotype in synoviocytes[END_REF], as well as their production of IL-6.

After 5 days of low-dose Cd exposure a reduction of cell viability was measured, which was further decreased in the presence of the pro-inflammatory cytokines IL-17 and TNF-α. After IL-17/TNF-α activation, Cd addition also reduced cell viability in co-cultures of synoviocytes and PBMCs and cell proliferation from ex vivo cultured biopsies of synovial tissue.

Cadmium addition was found to reduceIL-17/TNF-α induced IL-6 production of synoviocytes cultured alone or in co-culture with PBMCs and of synovial biopsies.

Zinc reduces Cd-induced effects on synoviocytes

The competition between Zn and Cd is well-known, especially the protective role of Zn on Cd toxicity [START_REF] Moulis | Cellular mechanisms of cadmium toxicity related to the homeostasis of essential metals[END_REF][START_REF] Waalkes | Relative in vitro affinity of hepatic metallothionein for metals[END_REF][START_REF] Sahmoun | Cadmium and prostate cancer: a critical epidemiologic analysis[END_REF]. The addition of Zn to Cd treated cultures partially reversed the effects of Cd on cell viability and reduced Cd-induced detachment of cells from the plate layer.

The simultaneous exposure of synoviocytes to Zn and Cd resulted in a reduced expression of MTs relative to Cd-only exposed cells, probably due to the higher resistance of the clustered Cd-MT. Moreover, the exposure to Zn in the presence of Cd reduced the effects of Cd on IL-6 production in both cultures of synoviocytes and co-cultures of synoviocytes with PBMCs.

Cadmium intra-articular injection improves adjuvant induced arthritis in the

Lewis rat model

prepares the cell for metal storage by opening the gates of metal import, regardless the amount of Zn actually present in the medium. Several previous studies demonstrated the role of inflammation in triggering importers and homeostasis regulators expression in human cells, in agreement with our results (100, 101). This may lead to new therapeutic options by targeting Zn-binding groups to control inflammation.

Moreover, ZIP-8 importer is not selective for the metal it imports. This means that the ratio of Zn ions imported only depends on Zn ions availability, in comparison to the availability of other metal ions present in the extracellular fluid. In this way, the presence of other metals can influence the uptake and action of Zn, and vice-versa depending on their levels.

In contrast inflammation seems not preparing the cell efficiently for metal excretion, but only the metal excess does. This results at early stage in the increased metal content in the inflammatory cells. The increased cytokine production may signal the potential intoxication of the inflamed tissues if metal levels become too high in the extra-cellular fluid. This result indicates that cell mechanisms for metal excretion only respond to metal excess and, importantly, that those are late-stage mechanisms, which reduce the efficacy in protecting the cell in the case of metal intoxication Nevertheless, ZnT1 dependent Zn export is effective: after inflammation-induced import, cell metal sensing can bring the status back to the equilibrium of import/export through ZnT1, without the formation of a real Zn pool in the cell.

On the basis of the results obtained in the first paper, experiments were performed exposing synoviocytes to Cd in the presence or not of inflammation. As inflammation increase the ZIP-8-dependent import, Cd accumulates inside the cell.

Moreover the expression of MT was highly enhanced by Cd exposure and further increased by inflammation. As a recent work stated, clustered Cd-MTs are more resistant than apo-MTs to proteolysis and tend to persist [START_REF] Pinter | Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange[END_REF]. This, as in our case, may exacerbate Cd persistence in mammalian cells.

The exposure to Cd, as well as Zn exposure, could slightly increase the expression of the exporter ZnT1. Nevertheless its increased expression resulted non-functional in our experiments: after 48-hour exposure to Cd and the following cell wash, no Cd was excreted in the medium but even residual traces of the metal were absorbed. This was not the same for Zn, which can be partially re-excreted in the medium after wash (data not shown). This can be due to the lower affinity of Zn for MTs in comparison to other metals, as Cd.

The reduced accumulation of Cd in cells in the presence of Zn in comparison to

Cd-alone was previously demonstrated (102). The simultaneous addition of both Cd and Zn in the extracellular medium did not change the expression of the importer and the exporter but reduced the expression of MTs compared to the expression of Cd-only exposed cells. This meant that ZIP-8 imported the two metals in a 1 to 1 ratio, but the MTs that persisted in the cell were mainly in the Cd-MTs clustered form. Quantification of MTs at earlier time points could be useful to understand better this mechanism.

In conclusion inflammation drives the accumulation of metals inside the cells in a non-selective way and helps in the regulation of MTs. Metals are the main regulator of MTs, but their persistence in the cell depends on metal-MTs affinity. Also the export depends on metal levels but it can result non-functional depending on the preceding step of metal machinery, MT binding. Moreover taken together these