Dr Estelle Louiset
email: estelle.louiset@univ-rouen.fr

Céline Duparc

Hervé Lefebvre

Role of serotonin in the paracrine control of adrenal steroidogenesis in physiological and pathophysiological conditions

Keywords: Adrenal, Steroidogenesis, Serotonin, Primary aldosteronism, Cushing's syndrome, Paracrine communication, PKA pathway

Serotonin (5-HT) and 5-HT receptors are present in adrenals from various species. In rat and human, 5-HT mainly stimulates aldosterone secretion through activation of 5-HT7 and 5-HT4 receptors, respectively. The 5-HT-ergic control of adrenal steroidogenesis is strengthened in extreme physiological conditions, like sodium depletion and chronic stress. Especially, the emergence of a 5-HT regulatory loop in glucocorticoid producing cells may be regarded as an adaptive mechanism aimed at potentiating the glucocorticoid response to stress. In human, upregulation of the 5-HT signalling pathway occurs in aldosterone-producing adenomas and cortisolproducing hyperplasias and adenomas in response to overactivation of protein kinase A. These data suggest that the intraadrenal 5-HT-ergic regulatory system represents a potential target for pharmacological treatments of primary adrenocortical diseases.

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) is synthesized from tryptophan which is first converted into 5-hydroxy-tryptophan (5-HTP) by the rate-limiting enzyme tryptophan hydroxylase (TPH). Then, 5-HTP is metabolized into 5-HT by a ubiquitous aromatic L-amino acid decarboxylase. TPH exists as two isoforms, namely TPH1 and TPH2, that differ in their distribution patterns [1,2]. TPH1 is expressed in nonneuronal tissues whereas the presence of TPH2 is restricted to neurons. 5-HT is an important neurotransmitter involved in the regulation of multiple brain functions.

However, only 5% of the endogenous 5-HT body content is found in the central nervous system [1]. In fact, the remaining 5-HT is localized in peripheral organs, especially in enterochromaffin cells of the gastrointestinal tract. In addition, intensive studies performed for the last decade, have demonstrated that 5-HT acts as a paracrine factor to regulate various physiological functions including adaptation of the pancreatic β-cell mass during pregnancy and placental development [3,4]. In both human central nervous system and peripheral organs, 5-HT actions are mediated by 13 receptor subtypes classified in 7 types (5-HT1 to 5-HT7). Six of the seven types of receptors belong to the G protein coupled receptor family [5].

In mammals, the adrenal gland is composed of two distinct structures, i.e. the central medulla formed by chromaffin cells producing catecholamines, and at the periphery, the cortex constituted by steroidogenic cells. In humans, the adult cortex is organized into three distinct layers, named glomerulosa, fasciculata and reticularis zonae. Zona glomerulosa cells synthesize aldosterone, a mineralocorticoid involved in sodium and potassium homeostasis. The zona fasciculata contains cells that produce glucocorticoids essential for glucose homeostasis, stress response and immunomodulation, while zona reticularis cells mainly secrete the androgen dehydroepiandrosterone (DHEA) and its sulfate.

It is well established that adrenal steroidogenesis is stimulated by circulating factors. In particular, production of aldosterone is activated by the renin angiotensin system (RAS), whereas secretion of cortisol is regulated by corticotropin (ACTH) released by pituitary cells in response to corticotropin-releasing hormone (CRH) produced by hypothalamic neurons. Some data have provided evidence that 5-HT also regulates adrenal steroidogenesis through both indirect and direct actions. In particular, clinical trials have revealed that administration of 5-HT precursors, 5-HT or 5-HT reuptake inhibitors to healthy volunteers and/or depressed patients results in an increase in plasma renin activity, that secondarily enhances aldosterone production [6][7][8]. 5-HT also exerts an indirect effect on glucocorticoid secretion by stimulating CRH release from hypothalamic neurons and ACTH secretion by pituitary corticotrophs [9]. In addition, numerous data indicate that 5-HT produced in the adrenal gland directly stimulates steroidogenesis by adrenocortical cells through a paracrine mechanism. Interestingly, reinforcement of the 5-HT signalling pathway has been reported in adrenal lesions responsible for steroid hypersecretion, suggesting that the 5-HT-ergic control of corticosteroidogenesis may play a role in the pathogenesis of primary adrenal diseases [10][11][12][13][14].

The present review summarizes our current knowledge on the implication of 5-HT in the regulation of adrenal steroid hormone production in physiological and pathological conditions. It also discusses the potential interest of targeting the intraadrenal 5-HT-ergic regulatory system for clinical management of patients with adrenocortical disorders.

Physiological role of 5-HT in adrenal steroidogenesis

Occurrence of 5-HT in normal adrenal

The presence of 5-HT in adrenal glands has been demonstrated in various species including fish, frog, mouse, rat, pig, cattle and human [START_REF] Delarue | Immunohistochemical and biochemical evidence for the presence of serotonin in amphibian adrenal chromaffin cells[END_REF][START_REF] Fernández-Vivero | Immunocytochemical distribution of serotonin and neuropeptide Y (NPY) in mouse adrenal gland[END_REF][START_REF] Kong | Differential distribution of neuropeptides and serotonin in pig adrenal glands[END_REF][START_REF] Lefebvre | Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT[END_REF][START_REF] Lim | Serotonin directly stimulates cortisol secretion from the interrenals in goldfish[END_REF][START_REF] Verhofstad | Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat[END_REF][START_REF] Wojnicz | Simultaneous monitoring of monoamines, amino acids, nucleotides and neuropeptides by liquid chromatography-tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells[END_REF]. The cell types that contain 5-HT differ among species. For instance, 5-HT is exclusively detected in adrenal subcapsular mast cells in human, whereas the monoamine is localized in chromaffin cells in fish, frog, cattle and pig, in both mast cells and chromaffin cells in rats, as well as nerve fibres in mice [14- [START_REF] Fernández-Vivero | Immunocytochemical distribution of serotonin and neuropeptide Y (NPY) in mouse adrenal gland[END_REF][START_REF] Lim | Serotonin directly stimulates cortisol secretion from the interrenals in goldfish[END_REF][START_REF] Verhofstad | Immunohistochemical and neurochemical evidence for the presence of serotonin in the adrenal medulla of the rat[END_REF][START_REF] Hinson | Adrenal mast cells modulate vascular and secretory responses in the intact adrenal gland of the rat[END_REF] (Figure 1). 5-HT can be taken up from the plasma through the serotonin transporter (SERT) which is expressed in mouse, rat and human adrenals, and/or de novo synthesized by the adrenal tissue from tryptophan under the influence of TPH [14, [START_REF] Linder | Body distribution of infused serotonin in rats[END_REF][START_REF] Brindley | An interplay between the serotonin transporter (SERT) and 5-HT receptors controls stimulus-secretion coupling in sympathoadrenal chromaffin cells[END_REF]. In fact, in vivo experiments have demonstrated that the adrenal 5-HT content is dramatically reduced in SERT-deficient mice or rats [START_REF] Linder | Body distribution of infused serotonin in rats[END_REF][START_REF] Brindley | An interplay between the serotonin transporter (SERT) and 5-HT receptors controls stimulus-secretion coupling in sympathoadrenal chromaffin cells[END_REF]. In addition, internalization of 5-HT in frog adrenal cells has been confirmed in vitro by using fluoxetine, an inhibitor of 5-HT uptake [START_REF] Delarue | Serotonin synthesis in adrenochromaffin cells[END_REF]. Because SERT is predominantly expressed in chromaffin cells, 5-HT uptake mainly occurs in medulla. However, frog chromaffin cells are also able to synthesize 5-HT from 3 H-labelled tryptophan [START_REF] Delarue | Serotonin synthesis in adrenochromaffin cells[END_REF]. Finally, the occurrence of TPH1 and 5-HT has been reported in adrenocortical cells from rats exposed to chronic stress [START_REF] García-Iglesias | Sensitization of restraint-induced corticosterone secretion after chronic restraint in rats: involvement of 5-HT₇ receptors[END_REF].

Role of 5-HT in normal adrenal

5-HT secreted by the different cell types, controls adrenal steroidogenesis through diverse mechanisms. For instance, the monoamine released by nerve fibres or mast cells in the vicinity of blood vessels [START_REF] Fernández-Vivero | Immunocytochemical distribution of serotonin and neuropeptide Y (NPY) in mouse adrenal gland[END_REF][START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF], modulates blood flow, which is known to influence steroid synthesis [START_REF] Vinson | The mammalian adrenal circulation and the relationship between adrenal blood flow and steroidogenesis[END_REF]. Due to the intermingling of chromaffin and steroidogenic cells in fish and amphibian adrenals, 5-HT secreted by chromaffin cells exerts a paracrine control on steroidogenesis [START_REF] Lim | Serotonin directly stimulates cortisol secretion from the interrenals in goldfish[END_REF] (Figure 1). In this regard, it has been demonstrated that 5-HT stimulates cortisol production in fish, and increases both aldosterone and corticosterone secretion in frog [START_REF] Lim | Serotonin directly stimulates cortisol secretion from the interrenals in goldfish[END_REF][START_REF] Delarue | Serotonin stimulates corticosteroid secretion by frog adrenocortical tissue in vitro[END_REF]. It must be noticed that such intercellular communication system may not occur in higher vertebrates because of the zonation of the adrenal gland and the centripetal direction of the blood flow. Conversely, cell-to-cell interactions exist between mast cells immunoreactive to TPH1 and 5-HT, and aldosterone-producing cells in the human zona glomerulosa [START_REF] Lefebvre | Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT[END_REF][START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF]. Indeed, we have observed that mast cell secretory products upregulate expression of CYP11B2, encoding aldosterone synthase, and activate aldosterone secretion by the adrenocortical cell line H295R [START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF]. The decrease in the aldosterone response to mast cell-conditioned medium induced by concomitant application of 5-HT receptor antagonists has revealed the important contribution of 5-HT to this intercellular communication process [START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF]. Similarly, experiments conducted ex vivo on explants of human adrenals have demonstrated that degranulation of intraadrenal mast cells stimulates aldosterone production through release of 5-HT [START_REF] Lefebvre | Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT[END_REF] (Figure 1). Consistently, 5-HT turns out to be more efficient on aldosterone than cortisol or DHEA secretion from cultured adrenocortical cells [START_REF] Duparc | l-Lysine Acts as a Serotonin Type 4 Receptor Antagonist to Counteract In Vitro and In Vivo the Stimulatory Effect of Serotonergic Agents on Aldosterone Secretion in Man[END_REF][START_REF] Louiset | Role of cAMP/PKA pathway and T-type calcium channels in the mechanism of action of serotonin in human adrenocortical cells[END_REF][START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF]. In addition, an increase in adrenal mast cell density precedes aldosterone synthase expression in the human fetal adrenal gland, suggesting that mast cells favour differentiation of aldosterone-producing cells during adrenal development [START_REF] Naccache | Temporal and spatial distribution of mast cells and steroidogenic enzymes in the human fetal adrenal[END_REF]. Finally, it has been reported that the steroidogenic effects of 5-HT on the human adult adrenal is counteracted by monoamine oxidase type A which metabolizes 5-HT into inactive compounds as observed in serotonergic synapses [START_REF] Lefebvre | Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT[END_REF].

Up to now, the physiological stimulus eliciting 5-HT release by intraadrenal mast cells remains unknown. The observation that nerve fibres establish connections with adrenal mast cells suggests that 5-HT may be produced in response to activation of the sympathetic system [START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF]. On the other hand, expression of angiotensin 2 receptors (AT2R) by rat mast cells [START_REF] Carvalho | Angiotensin II potentiates inflammatory edema in rats: Role of mast cell degranulation[END_REF] leads us to speculate on a potential role of RAS on 5-HT secretion by adrenal mast cells. In support of this hypothesis, our recent in vivo experiments conducted in mice have revealed that activation of circulating RAS by low sodium diet is associated with adrenal upregulation of Tpsb2 gene encoding tryptase, a mast-cell specific protease which reflects mast cell activation [START_REF] Boyer | Dysregulation of Aldosterone Secretion in Mast Cell-Deficient Mice[END_REF]. Moreover, Tpsb2 transcript level is positively correlated with Cyp11b2 gene expression, suggesting a common mechanism controlling these genes or an interdependent relationship between the two.

Interestingly, the mast cell-deficient Kit W-sh/W-sh mouse strain develops an exaggerated aldosterone response to sodium restriction, consecutive to adrenal renin and angiotensin type 1 receptor overexpression [START_REF] Boyer | Dysregulation of Aldosterone Secretion in Mast Cell-Deficient Mice[END_REF]. Activation of adrenal RAS may be regarded as a compensatory mechanism enhancing aldosterone production in the absence of mast cells. It is not known whether similar regulatory system also occurs in human adrenal. Thus, it would be relevant to evaluate the mineralocorticoid function in response to sodium restriction in patients treated with mast cell stabilizers to prevent allergic disorders, or in subjects with mast cell deficiency due to inactivating KIT mutation (i.e. patients with piedbaldism).

Altogether, the present data indicate that adrenal mast cells contribute to the control of mineralocorticoid synthesis in concert with circulating RAS. Nevertheless, the implication of 5-HT in this regulatory system remains to be investigated. Thus, it would be interesting to examine the aldosterone response to sodium restriction in 5-HT-deficient animals, like TPH inhibitor-treated or TPH1-knockout mice [3]. Similarly, it would be worthy of note to test the effect of peripheral TPH inhibitors, currently used in the treatment of carcinoid syndrome, on plasma aldosterone concentration in healthy volunteers under normal and low sodium diets.

5-HT receptors in normal adrenal

Combinations of RT-PCR, immunohistochemical, and pharmacological experiments have revealed species specificities in the adrenal expression of 5-HT receptor subtypes. In particular, the steroidogenic effect of 5-HT is mediated by 5-HT4 receptors in fish, frog and human, whereas 5-HT7 receptors are involved in the aldosterone response to 5-HT in rat [START_REF] Lim | Serotonin directly stimulates cortisol secretion from the interrenals in goldfish[END_REF][START_REF] Lefebvre | Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype[END_REF][START_REF] Contesse | Activation of 5hydroxytryptamine4 receptors causes calcium influx in adrenocortical cells: involvement of calcium in 5-hydroxytryptamine-induced steroid secretion[END_REF][START_REF] Lenglet | Activation of 5-HT(7) receptor in rat glomerulosa cells is associated with an increase in adenylyl cyclase activity and calcium influx through T-type calcium channels[END_REF] (Figure 1). In contrast, the 5-HT receptors expressed in mouse adrenal have not yet been identified. In all species studied, both adrenal 5-HT4 and 5-HT7 receptors are positively coupled to AMPc/protein kinase A and membrane calcium channels, two transduction pathways that are known to activate steroidogenesis [START_REF] Louiset | Role of cAMP/PKA pathway and T-type calcium channels in the mechanism of action of serotonin in human adrenocortical cells[END_REF][START_REF] Contesse | Activation of 5hydroxytryptamine4 receptors causes calcium influx in adrenocortical cells: involvement of calcium in 5-hydroxytryptamine-induced steroid secretion[END_REF][START_REF] Lenglet | Activation of 5-HT(7) receptor in rat glomerulosa cells is associated with an increase in adenylyl cyclase activity and calcium influx through T-type calcium channels[END_REF].

In human, the distribution of the 5-HT4 receptor, which is abundant in the zona glomerulosa and weakly expressed in zonae fasciculata and reticularis, explains the higher efficacy of 5-HT to stimulate mineralocorticoid than glucocorticoid and androgen production [11,14,[START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF]. The predominant effect of 5-HT on mineralocorticoid synthesis has been confirmed by clinical trials showing that 5-HT4 receptors agonists, such as metoclopramide, cisapride, zacopride, tegaserod and mosapride, increase plasma aldosterone levels without modifying circulating cortisol concentration in healthy volunteers [START_REF] Edwards | In vivo and in vitro studies on the effect of metoclopramide on aldosterone secretion[END_REF][START_REF] Gale | The effect of repeat dosing of mosapride on plasma aldosterone in healthy Japanese volunteers[END_REF][START_REF] Lefebvre | The serotonin-4 receptor agonist cisapride and angiotensin-II exert additive effects on aldosterone secretion in normal man[END_REF][START_REF] Lefebvre | Effect of the serotonin-4 receptor agonist zacopride on aldosterone secretion from the human adrenal cortex: in vivo and in vitro studies[END_REF]. Inhibition of the plasma aldosterone response to cisapride by piboserod, a 5-HT4 receptor antagonist, gave evidence for expression of functional 5-HT4 receptors in aldosterone-producing cells [START_REF] Bharucha | Effects of a serotonin 5-HT(4) receptor antagonist SB-207266 on gastrointestinal motor and sensory function in humans[END_REF]. Unfortunately, the current lack of specific 5-HT4 receptor antagonists available for clinical use hampers in vivo investigation aimed at understanding the physiological role of adrenal 5-HT4 receptors. This problem could be circumvented by submitting healthy volunteers to a diet enriched with L-lysine which has been shown to act as a weak 5-HT4 receptor antagonist [START_REF] Smriga | L-Lysine acts like a partial serotonin receptor 4 antagonist and inhibits serotonin-mediated intestinal pathologies and anxiety in rats[END_REF]. We observed that L-lysine supplementation expectedly decreased the plasma aldosterone response to the 5-HT4 receptor agonist metoclopramide but had no impact on plasma aldosterone adaptation to low sodium diet and upright position [START_REF] Duparc | l-Lysine Acts as a Serotonin Type 4 Receptor Antagonist to Counteract In Vitro and In Vivo the Stimulatory Effect of Serotonergic Agents on Aldosterone Secretion in Man[END_REF]. Thus, the physiological role of adrenal 5-HT released by mast cell and 5-HT4 receptors remains to be established by conducting clinical studies with potent and selective 5-HT receptor antagonists.

It is possible that a serotonergic control of glucocorticoid synthesis may be activated during specific physiological stimuli. In fact, ectopic synthesis of 5-HT and 5-HT7 receptor overexpression in zona fasciculata has been described in rats submitted to chronic immobilization stress [START_REF] García-Iglesias | Sensitization of restraint-induced corticosterone secretion after chronic restraint in rats: involvement of 5-HT₇ receptors[END_REF] (Figure 1). This process might result from activation of the adrenal cortex by the high plasma ACTH levels related to stress. In agreement with this interpretation, adrenals removed from patients with increased plasma ACTH concentrations, i.e. patients with 21-hydroxylase deficiency, Cushing's disease or paraneoplastic Cushing's syndrome, harbour overexpression of TPH and 5-HT4/6/7 receptors [START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF]. Recent data obtained in the rat adrenal suggest that 5-HT synthesis and 5-HT7 receptor expression in the zona fasciculata may also result from the increase in glucocorticoid production induced by stress [START_REF] Saroj | Effect of chronic corticosterone treatment on expression and distribution of serotonin 5-HT7 receptors in rat adrenal glands[END_REF].

Upregulation of the 5-HT signalling pathway in the adrenal may thus be regarded as an adaptive mechanism aimed at potentiating the glucocorticoid response to stress through recruitment of intraadrenal stimulatory signals whose action adds to that of circulating ACTH (Figure 2). Similar serotonergic paracrine inducible systems have been previously reported in other organs. For instance, prolactin and placental lactogen produced during pregnancy, stimulate production of TPH and 5-HT in βpancreatic cells, a process which results in an increase in β-pancreatic cell mass and activation of insulin secretion [3]. In addition, estrogens upregulate the serotonergic signalling pathway in placenta, as glucocorticoids in adrenals [START_REF] Thibeault | Serotonin-estrogen interactions: What can we learn from pregnancy?[END_REF].

Globally, the observation that the adrenocortical function is controlled by 5-HT in adrenals of all studied species, emphasizes its physiological importance. The action of 5-HT seems to be crucial for mineral and/or metabolic homeostasis in particular conditions linked to extreme events, like sodium depletion or chronic stress.

Pathophysiological role of 5-HT in adrenal tumors

Role of 5-HT in aldosterone-producing adenoma

There is increasing evidence that 5-HT plays a significant role in the aldosterone excess in patients with primary aldosteronism, in whom mineralocorticoid secretion is independent of the RAS. In particular, an increase in mast cell density has been reported in aldosterone-producing adenomas (APA), that likely leads to an enhancement of intraadrenal production of 5-HT [11,[START_REF] Duparc | Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas[END_REF] (Figure 3). The strengthening of the 5-HT-ergic control of the mineralocorticoid function in APAs is also due to abnormal synthesis of 5-HT by a subpopulation of steroidogenic cells [11]. Furthermore, in vitro and in vivo studies revealed that adenoma cells are overresponsive to 5-HT. Indeed, patients with APA show exaggerated plasma aldosterone responses to the 5-HT4 receptor agonists cisapride, metoclopramide and tegaserod in comparison with healthy volunteers [START_REF] Lefebvre | Characterization of serotonin(4) receptors in adrenocortical aldosterone-producing adenomas: in vivo and in vitro studies[END_REF][START_REF] Zwermann | Screening for membrane hormone receptor expression in primary aldosteronism[END_REF][START_REF] Lampron | Regulation of aldosterone secretion by several aberrant receptors including for glucosedependent insulinotropic peptide in a patient with an aldosteronoma[END_REF][START_REF] Grunenwald | In vivo systematic screening for hormone receptors in primary aldosteronism reveals frequent aberrant hormone responsiveness[END_REF]. Hypersensitivity of APAs to 5-HT4 receptor ligands results from overexpression of 5-HT4 receptors. For instance, many reports have described upregulation of 5-HT4 receptors in APAs [11,[START_REF] Lampron | Regulation of aldosterone secretion by several aberrant receptors including for glucosedependent insulinotropic peptide in a patient with an aldosteronoma[END_REF][START_REF] Ye | G-protein-coupled receptors in aldosterone-producing adenomas: a potential cause of hyperaldosteronism[END_REF][START_REF] Williams | Teratocarcinoma-derived growth factor-1 is upregulated in aldosterone-producing adenomas and increases aldosterone secretion and inhibits apoptosis in vitro[END_REF][START_REF] Azizan | Microarray, qPCR, and KCNJ5 sequencing of aldosterone-producing adenomas reveal differences in genotype and phenotype between zona glomerulosa-and zona fasciculata-like tumors[END_REF][START_REF] Itcho | Aberrant G protein-receptor expression is associated with DNA methylation in aldosteroneproducing adenoma[END_REF], that may be explained by hypomethylation of the promoter region of the HTR4 gene, encoding the 5-HT4 receptor [START_REF] Itcho | Aberrant G protein-receptor expression is associated with DNA methylation in aldosteroneproducing adenoma[END_REF]. Hypersensitivity of APAs tissues to 5-HT4 receptor ligands may also be consecutive to abnormal splicing of HT4R transcripts giving rise to an expression pattern of 5-HT4 receptor isoforms different to that observed in normal adrenals [11]. Collectively, these data suggest that targeting the 5-HT pathway may represent an efficient therapeutic approach to decrease aldosterone production in primary aldosteronism. Especially, clinical trials should be conducted to evaluate the effect of peripheral TPH inhibitors and/or 5-HT4 receptor antagonists (when available for human clinical studies) on plasma aldosterone concentrations in patients with APA. Finally, clinical scientists may also take advantage of overexpression of 5-HT4 receptors in APAs to develop new adrenal imaging methods in vivo aimed at determining the lateralization of aldosterone secretion in primary aldosteronism. In fact, positron emission tomography (PET)-scan imaging with radiolabeled 5-HT4R specific ligands has already been used for functional brain imaging in man and adrenals have been successfully visualized in monkeys through this approach [START_REF] Tavares | In vivo evaluation of 18F-MNI698: an 18F-labeled radiotracer for imaging of serotonin 4 receptors in brain[END_REF].

Role of 5-HT in primary Cushing's syndrome

Due to the weak effect of 5-HT on cortisol synthesis in physiological conditions, it was not expected that a 5-HT-ergic regulatory system could significantly influence glucocorticoid secretion by benign adrenal lesions in patients with primary Cushing's syndrome. However, ectopic 5-HT production has been reported in steroidogenic cells in cortisol-producing adenomas (CPA), primary bilateral macronodular adrenal hyperplasia (PBMAH) and primary pigmented nodular adrenocortical disease (PPNAD), responsible for cortisol overproduction [12,14,[START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF][START_REF] Contesse | Abnormal sensitivity of cortisol-producing adrenocortical adenomas to serotonin: in vivo and in vitro studies[END_REF]. This observation seems to result from local synthesis of the monoamine, as shown by abnormal expression of TPH in the adrenal tissues [14,[START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF].

There is now a growing body of evidence showing that locally-produced 5-HT exerts a paracrine stimulatory tone on glucocorticoid secretion: 1) 5-HT is detectable in the incubation medium of PPNAD tissue explants demonstrating the ability of steroidogenic cells to release the amine [14]; 2) 5-HT dose-dependently increases cortisol synthesis in cultured cells derived from patients with CPA, PBMAH and PPNAD, with higher potency and/or efficacy in comparison with normal adrenal [12,14,[START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF][START_REF] Contesse | Abnormal sensitivity of cortisol-producing adrenocortical adenomas to serotonin: in vivo and in vitro studies[END_REF]; 3) the TPH inhibitor p-chlorophenylalanine reduces cortisol production from CPA and PPNAD tissues in vitro [14,[START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF]; 4) the action of 5-HT can be ascribed to abnormal expression of 5-HT4, 5-HT6 and/or 5-HT7 receptors evidenced by RT-Q-PCR and immunohistochemical studies [14, [START_REF] Mestre | Illicit upregulation of serotonin signaling pathway in adrenals of patients with high plasma or intraadrenal ACTH levels[END_REF][START_REF] Louiset | Expression of serotonin7 receptor and coupling of ectopic receptors to protein kinase A and ionic currents in adrenocorticotropin-independent macronodular adrenal hyperplasia causing Cushing's syndrome[END_REF] In vivo, the role of the 5-HT4 receptor in the control of glucocorticoid production was first evidenced by André Lacroix and coworkers in a patient with PBMAH who showed an abnormal plasma cortisol response to the 5-HT4 receptor agonists cisapride and metoclopramide [10]. Similar observations were subsequently reported in series of patients with CPA and PBMAH associated with subclinical or overt hypercortisolism [12,[START_REF] Bourdeau | Aberrant membrane hormone receptors in incidentally discovered bilateral macronodular adrenal hyperplasia with subclinical Cushing's syndrome[END_REF][START_REF] Dall'asta | Assessing the presence of abnormal regulation of cortisol secretion by membrane hormone receptors: in vivo and in vitro studies in patients with functioning and non-functioning adrenal adenoma[END_REF][START_REF] Feelders | Luteinizing hormone (LH)-responsive Cushing's syndrome: the demonstration of LH receptor messenger ribonucleic acid in hyperplastic adrenal cells, which respond to chorionic gonadotropin and serotonin agonists in vitro[END_REF][START_REF] Mannelli | Cushing's syndrome in a patient with bilateral macronodular adrenal hyperplasia responding to cisapride: an in vivo and in vitro study[END_REF][START_REF] Reznik | Aberrant adrenal sensitivity to multiple ligands in unilateral incidentaloma with subclinical autonomous cortisol hypersecretion: a prospective clinical study[END_REF]. Due to the lack of specific ligands for 5-HT6 and 5-HT7 receptors available for clinical studies, the contribution of these receptor subtypes to cortisol overproduction remains unknown.

Recent studies have focused on the mechanism underlying the formation of illicit 5-HT regulatory loop in adrenal lesions. We have previously mentioned that the 5-HT signalling pathway in zona fasciculata represents an inducible system induced by high plasma ACTH levels. Since ACTH acts through melanocortin 2 receptors (MC2R) positively coupled to the cAMP/PKA pathway, we have hypothesized that chronic stimulation of the cAMP/PKA pathway may also trigger this adaptive process (Figure 4). In this respect, it is well established that CPA, PBMAH or PPNAD are frequently associated with constitutive activation of PKA resulting from germline or somatic mutations affecting proteins of the pathway. These mutations include gain-offunction mutations of MC2R, GNAS, PRKACA, PRKACB genes, and inactivation mutations of the PRKAR1A, PDE11A and PDE8B genes [START_REF] Beuschlein | Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome[END_REF][64][START_REF] Stergiopoulos | Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease![END_REF]. Stimulation of the cAMP/PKA pathway in PBMAH can also result from activation of both MC2R by locally produced ACTH [START_REF] Louiset | Intraadrenal corticotropin in bilateral macronodular adrenal hyperplasia[END_REF] and illicit receptors by their natural ligands [13]. It is also possible that, as in rat under chronic stress, glucocorticoid overproduction in adrenal lesions may favour the emergence of the local 5-HT regulatory system. This process seems to be particularly probable in PPNAD tissues, in which glucocorticoid receptors are overexpressed [START_REF] Bourdeau | Primary pigmented nodular adrenocortical disease: paradoxical responses of cortisol secretion to dexamethasone occur in vitro and are associated with increased expression of the glucocorticoid receptor[END_REF]. In addition, the presence of glucocorticoid responsive elements in the promoter of the HTR6 gene might lead to its upregulation in cortisol-producing adrenal neoplasia [START_REF] Falkenberg | Identification of a potential molecular link between the glucocorticoid and serotonergic signaling systems[END_REF].

In conclusion, all these data indicate that the illicit paracrine serotonergic mechanism occurring in benign adrenal neoplasias, may play a role in the pathogenesis of corticosteroid excess and consequently represent a potential target for pharmacological treatments of hypercorticisms. In this respect, since different 5-HT receptor types are expressed in cortisol-producing adenomas and hyperplasias, the most efficient therapeutic strategy aimed at counteracting in vivo the 5-HTergic tone on cortisol production would be to reduce 5-HT synthesis by using TPH inhibitors rather than blocking 5-HT receptors by specific antagonists. In contrast, 5-HT4 receptor antagonists may be regarded as new potential antialdosterone drugs for the clinical management of primary aldosteronism. This study was the first to identify 5-HT as a paracrine factor stimulating pancreatic beta cell mass during pregnancy. The pancreatic 5-HT regulatory loop represents an adaptive mechanism linking beta cell mass to insulin need during pregnancy. This study describes a causative link between germline PRKAR1A mutations stimulating protein kinase activity, and occurrence of a 5-HT regulatory loop in adrenals.

Legends to figures

  P, Sibon D, Côté F: Life without peripheral serotonin: insights from tryptophan hydroxylase 1 knockout mice reveal the existence of paracrine/autocrine serotonergic networks. ACS Chem Neurosci 2013, 4:64-71. 2. Swami T, Weber HC: Updates on the biology of serotonin and tryptophan hydroxylase. Curr Opin Endocrinol Diabetes Obes 2018, 25:12-21. 3. ** Kim H, Toyofuku Y, Lynn FC, Chak E, Uchida T, Mizukami H, Fujitani Y, Kawamori R, Miyatsuka T, Kosaka Y, et al.: Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med 2010, 16:804-808.

Figure 1 .

 1 Serotonergic control of adrenal steroidogenesis in different species.A, In fish and amphibian adrenals, serotonin (5-HT) produced by chromaffin cells stimulates steroid secretion through a paracrine mechanism involving the 5-HT4 receptor. B-C, In rodent and human adrenals, 5-HT released by mast cell located in zona glomerulosa (ZG) increases aldosterone synthesis via activation of 5-HT7 and 5-HT4 receptors, respectively. In mice, sodium depletion provokes activation of mast cells which might reinforce 5-HT secretion, and subsequently aldosterone production (B). In mammal adrenals, 5-HT influences little or not glucocorticoid secretion in basal conditions. By contrast, huge and prolonged increase in plasma ACTH concentration, due to chronic stress or Cushing's disease, favours expression of genes encoding the rate-limiting enzyme tryptophan hydroxylase (TPH) and 5-HT4, 5-HT6 and/or 5-HT7 receptors in the zona fasciculata (ZF). Upregulation of the 5-HT signalling pathway may thus represent an adaptive mechanism potentiating the glucocorticoid response to ACTH. ZR, zona reticularis; MC2R, melanocortin type 2 receptor, ACTH receptor; DHEA, dehydroepiandrosterone.

Figure 2 .

 2 Figure 2. The serotonergic signalling pathway as an inducible paracrine process in endocrine tissues. A, In response to chronic stress, the increase in plasma ACTH concentration upregulates the 5-HT signalling pathway in mammalian adrenals. Activation of the 5-HT regulatory loop enhances glucocorticoid synthesis. B, During pregnancy, prolactin and placental lactogen stimulate the expression of TPH and 5-HT receptors in β-cells, a phenomenon which results in increased cell proliferation and insulin secretion. Upregulation of 5-HT synthesis and serotonergic receptors represents an adaptive mechanism aimed to potentiate the glucocorticoid response to stress through recruitment of intraadrenal stimulatory signals, and to enhance insulin production in order to counteract insulin resistance related to pregnancy, respectively.

Figure 3 .

 3 Figure 3. Serotonergic control of aldosterone secretion in primary aldosteronism. In aldosterone-producting adenoma (APA), the intraadrenal 5-HT signalling pathway is enhanced as a result of mast cells proliferation, ectopic synthesis of 5-HT in steroidogenic cells, and overexpression of the 5-HT4 receptor.

Figure 4 .

 4 Figure 4. Serotonergic control of cortisol secretion in primary adrenal Cushing's syndrome.A, In physiological conditions, ACTH produced by pituitary cells stimulates cortisol synthesis through activation of MC2-R positively coupled to the adenylyl cyclase (AC)/protein kinase A (PKA) pathway. PKA is an inactive tetramer composed by regulatory (R1α) and catalytic (Cα or Cβ) subunits. In response to ACTH, cAMP produced by AC, binds to regulatory subunits resulting in the release of catalytic subunits. Catalytic subunits then phosphorylate proteins leading to an increase in cortisol synthesis and transcription of target genes. Phosphodiesterases (PDE) degrade cAMP in order to stop signal transduction. B, In patients with primary adrenal Cushing's syndrome, the adrenal cAMP/PKA pathway is reinforced due to i) gain-of-function mutations of PRKACA, PRKACB genes (encoding Cα and Cβ), ii) inactivating mutations of PRKAR1A, PDE11A, PDE8B genes (encoding R1α and PDEs), iii) intraadrenal ACTH production in PBMAH, iv) activation of illicit receptors (LH or GIP receptors). Reinforcement of the cAMP/PKA pathway stimulates expression of genes encoding TPH, 5-HT4, 5-HT6 and/or 5-HT7 receptors giving rise to an illicit 5-HT stimulatory loop that participates in activation of cortisol synthesis.

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Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Conseil Régional de Normandie, the European Regional Development Fund and the Société Française d'Endocrinologie.


Pierre-Jean Bertrand

Yvan Jamilloux

René Ecochard

Gaelle Richard-Colmant

Mathieu Gerfaud-Valentin

Martin Guillaud

Philippe Denis

Laurent Kodjikian

Pascal Sève
email: pascal.seve@chu-lyon.fr

Prof Pascal Sève

Uveitis: Autoimmunity... and beyond

Keywords: epidemiology, uveitis, diagnosis

Highlights

1. Uveitis referred to internal medicine for the diagnostic work-up remains idiopathic in nearly half of the cases.

2. Systemic diseases are responsible for more than one-third of uveitis 3. A multidisciplinary approach is required for the diagnostic work-up.

4. Etiologic diagnosis of uveitis should be guided by anatomic localization.

INTRODUCTION

Uveitis is defined as an inflammation of the iris, ciliary body, vitreous, retina and/or choroid.

Its incidence is 17-52/100,000 person-years while its prevalence is 38-284/100,000 individuals [START_REF] Prete | Autoimmune uveitis: clinical, pathogenetic, and therapeutic features[END_REF][START_REF] Gritz | Incidence and prevalence of uveitis in Northern California; the Northern California Epidemiology of Uveitis Study[END_REF][START_REF] Rim | Incidence and prevalence of uveitis in South Korea: a nationwide cohort study[END_REF][START_REF] Selmi | Diagnosis and classification of autoimmune uveitis[END_REF]. In the USA, a medical insurance claims-based analysis of 4 million individuals reported a prevalence of 133/100,000 persons, with predominantly non-infectious uveitis (90.7%) and anterior uveitis (80%) [START_REF] Thorne | Prevalence of Noninfectious Uveitis in the United States: A Claims-Based Analysis[END_REF]. About 10-15% of preventable blindness in Western countries is caused by uveitis or its complications. A major vision loss (i.e. bestcorrected visual acuity ≤20/50) was reported in 20-70% of the patients in uveitis referral centers [START_REF] Dick | Risk of Ocular Complications in Patients with Noninfectious Intermediate Uveitis, Posterior Uveitis, or Panuveitis[END_REF]. About 60 causes of uveitis have been described and can be classified into five groups including pure ophthalmological entities, infectious and inflammatory diseases, masquerade syndromes, and drug-related uveitis. The epidemiology varies depending on genetic and ethnic factors (e.g. HLA-B27, Behcet's disease and sarcoidosis), environmental factors (e.g. tuberculosis), the disease definition (e.g. requirement of a histological proof for sarcoid uveitis), the inclusion of some ophthalmological entities in the idiopathic uveitis group (e.g. pars planitis), the paraclinical investigations that were performed (e.g. nuclear imaging), and the patients' referral (e.g. tertiary centers). Such variability explains the great heterogeneity in epidemiological studies.

The epidemiology also varies according to geographical areas; such as Vogt-Koyanagi-Harada disease (VKH) and sarcoidosis in Japan [START_REF] Kitamei | Clinical features of intraocular inflammation in Hokkaido, Japan[END_REF][START_REF] Keino | Frequency and clinical features of intraocular inflammation in Tokyo[END_REF], Behcet's disease in the Mediterranean Rim and Asia [START_REF] Mahr | Population-based prevalence study of Behçet's disease: differences by ethnic origin and low variation by age at immigration[END_REF], tuberculosis in India [START_REF] Dogra | Epidemiology of Uveitis in a Tertiary-care Referral Institute in North India[END_REF], and toxoplasmosis in South America [START_REF] Fernandez | Uveitis in São Paulo, Brazil: 1053 New Patients in 15 Months[END_REF][START_REF] De La Torre | Clinical patterns of uveitis in two ophthalmology centres in Bogota, Colombia[END_REF]. In Western countries, approximately one-quarter of the cases are related to ophthalmological diseases, one-quarter to systemic diseases fulfilling consensual diagnostic criteria, onequarter to presumed systemic diseases, and one-quarter remain unexplained [START_REF] Abad | Guidelines for the management of uveitis in internal medicine[END_REF]. According to recent studies from western countries and Japan, 23-44% of uveitis are idiopathic [START_REF] Keino | Frequency and clinical features of intraocular inflammation in Tokyo[END_REF][START_REF] Luca | Changes in patterns of uveitis at a tertiary referral center in Northern Italy: analysis of 990 consecutive cases[END_REF][START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF][START_REF] Llorenç | Epidemiology of uveitis in a Western urban multiethnic population. The challenge of globalization[END_REF][START_REF] Jakob | Uveitis subtypes in a german interdisciplinary uveitis center-analysis of 1916 patients[END_REF][START_REF] Lopalco | Cantarini L Epidemiological profile of non-infectious uveitis from the rheumatologist's perspective: a survey from two tertiary referral centres in Italy[END_REF][START_REF] Jones | The Manchester Uveitis Clinic: the first 3000 patients--epidemiology and casemix[END_REF][START_REF] Grajewski | Spectrum of Uveitis in A German Tertiary Center: Review of 474 Consecutive Patients[END_REF][START_REF] Zagora | Etiology and Clinical Features of Ocular Inflammatory Diseases in a Tertiary Referral Centre in Sydney, Australia[END_REF][START_REF] Oruc | Uveitis referral pattern in a Midwest University Eye Center[END_REF][START_REF] Bajwa | Epidemiology of uveitis in the mid-Atlantic United States[END_REF][START_REF] Barisani-Asenbauer | Uveitis-a rare disease often associated with systemic diseases and infections-a systematic review of 2619 patients[END_REF][START_REF] Cimino | The causes of uveitis in a referral centre of Northern Italy[END_REF].

Since the beginning of the twenty-first century, there were seven studies on the distribution, clinical patterns, and etiologies of uveitis [START_REF] Luca | Changes in patterns of uveitis at a tertiary referral center in Northern Italy: analysis of 990 consecutive cases[END_REF][START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF][START_REF] Llorenç | Epidemiology of uveitis in a Western urban multiethnic population. The challenge of globalization[END_REF][START_REF] Jakob | Uveitis subtypes in a german interdisciplinary uveitis center-analysis of 1916 patients[END_REF][START_REF] Cimino | The causes of uveitis in a referral centre of Northern Italy[END_REF], mostly in ophthalmological centers, in Western Europe. There were few studies in France, with only a large study involving 927 patients with chronic severe uveitis who were referred to a tertiary ophthalmological center, between 1991 and 1996 [START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF].

The aim of this work was to describe the etiological spectrum, and anatomical patterns of uveitis in a large cohort of consecutive patients referred to our department of internal medicine for the etiological diagnosis of uveitis. Moreover, we analyzed the diagnostic classification according to demographic data and anatomical localization.

PATIENTS AND METHODS

Patients

This study was a retrospective analysis of the records of 1341 patients with "uveitis" referred by the department of ophthalmology (Lyon University Hospital, Lyon, France) or out hospital ophthalmologists to the department of internal medicine (Lyon University Hospital, Lyon, France) between July, 2002 and December, 2018. The diagnosis of uveitis was always confirmed by an ophthalmological examination. Uveitis related to pure ophthalmological entities, diagnosed by an ophthalmological examination only and referred for the treatment or to rule out differential diagnoses, were excluded (n=205), as well as uveitis occurring in the course of previously diagnosed diseases (n=122). According to French law (no. 2004-806, August 9, 2004), this study did not require the research ethics committee approval because data were collected retrospectively and the patients' management was not modified.

Diagnostic work-up and definitions

All the patients underwent a standard screening protocol for uveitis, which included a tuberculosis skin test, a C-reactive protein and erythrocyte sedimentation rate (ESR), a complete blood cell count (CBC), a serological tests for syphilis, and a chest X-ray [START_REF] De Parisot | Randomized Controlled Trial Evaluating a Standardized Strategy for Uveitis Etiologic Diagnosis (ULISSE)[END_REF].

Human leucocyte antigen (HLA)-B27 typing was performed in patients with acute anterior uveitis. In chronic anterior uveitis or granulomatous uveitis, an angiotensin-converting enzyme (ACE) and a chest computerized tomography (CT) scan were performed.

The diagnostic screening for sarcoidosis included conjunctival or skin biopsies if there were clinically suggestive features. Some patients underwent a minor salivary gland biopsy (MSGB), a transbronchial lung biopsy, a bronchoalveolar lavage (BAL), a cerebral magnetic resonance imaging (MRI) or a nuclear imaging. This work-up was completed in some patients by an anterior chamber paracentesis (with a polymerase chain reaction for Herpesvirus, Toxoplasma or RNA16S, and sometimes an interleukin-10 measurement), a vitreous biopsy, and/or a lumbar puncture, when appropriate.

The Standardization of Uveitis Nomenclature was used throughout this study for the anatomical classification of uveitis [START_REF] Selmi | Diagnosis and classification of autoimmune uveitis[END_REF][START_REF] Jabs | Standardization of Uveitis Nomenclature (SUN) Working Group. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop[END_REF].

Briefly, the diagnostic criteria used were:

• the international study group for Behcet's disease criteria [START_REF]Criteria for diagnosis of Behçet's disease. International Study Group for Behçet's Disease[END_REF];

• the Levinson's criteria [START_REF] Levinson | Research criteria for the diagnosis of birdshot chorioretinopathy: results of an international consensus conference[END_REF] or the global diagnostic criteria for birdshot retinochoroidopathy [START_REF] Bonfioli | Fuchs' heterochromic cyclitis[END_REF]. All birdshot retinochoroidopathy were HLA-A29 positive;

• the 2010 revised McDonald's criteria for multiple sclerosis (MS) [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria[END_REF] and Okuda's criteria for radiologically isolated syndromes (RIS) [START_REF] Okuda | Incidental MRI anomalies suggestive of multiple sclerosis: the radiologically isolated syndrome[END_REF]. All MS diagnoses or RIS were made in partnership with a neurologist.

• The international criteria for the diagnosis of sarcoidosis [START_REF] Costabel | European Respiratory Society. World Association for Sarcoidosis and Other Granulomatous Disorders[END_REF] (with Zajicek's classification for neurosarcoidosis) [START_REF] Zajicek | Central nervous system sarcoidosis--diagnosis and management[END_REF]. In the absence of histological proof, we used Abad's modified criteria [START_REF] Abad | Association of peripheral multifocal choroiditis with sarcoidosis: a study of thirty-seven patients[END_REF]. Patients had presumed sarcoid uveitis if they had at least 2 of the following 4 criteria: typical changes on chest X-ray or CT scan, a predominantly CD4 lymphocytosis on BAL fluid analysis, an elevated ACE or an 18-fluorodeoxyglucose (18-FDG) uptake on scintigraphy.

They had indeterminate sarcoid uveitis when only one criterion was met.

• the Assessment of SpondyloArthritis international Society (ASAS) criteria for spondyloarthritis [START_REF] Rudwaleit | The Assessment of SpondyloArthritis International Society classification criteria for peripheral spondyloarthritis and for spondyloarthritis in general[END_REF]. HLA-B27 positive patients with acute non-granulomatous anterior uveitis without clinical and/or radiological features of spondyloarthritis were diagnosed as HLA-B27-related anterior uveitis;

• the revised diagnostic criteria for VKH disease [START_REF] Read | Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: report of an international committee on nomenclature[END_REF];

• Gupta's criteria for the diagnosis of intraocular tuberculosis [START_REF] Gupta | Classification of intraocular tuberculosis[END_REF].

Whenever the intraocular inflammation could not be assigned to a specific diagnosis, the uveitis was considered as idiopathic.

Data collection

We collected patients' demographic data, follow-up times, and the following ophthalmological characteristics at diagnosis: anatomical type of uveitis (anterior, intermediate, posterior, panuveitis) [START_REF] De Parisot | Randomized Controlled Trial Evaluating a Standardized Strategy for Uveitis Etiologic Diagnosis (ULISSE)[END_REF] and anterior chamber examination (tonometry, slit lamp, biomicroscopy to assess whether the uveitis was granulomatous or not). We also reported whether the uveitis was acute or chronic, and uni-or bilateral.

Statistical analysis

Data are described as frequencies and/or percentages for categorical variables and as medians and minimum-maximum ranges for quantitative variables. Most of the time, data were not compared between subgroups because of the great heterogeneity of sample sizes.

All analyses were performed using an R-software, version 3.1.1 (R Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Demographic data

The department of ophthalmology is a referral center for uveitis in Rhône-Alpes (8.5 million inhabitants). After excluding 102 patients because of insufficient data, 912 patients were included, of which 545 (59.7%) women. 832 patients were actually referred by the department of ophthalmology. Patients' characteristics are shown in Table 1. Overall, the mean age at diagnosis was 48.8 (4-92) years. 199 (21.8%) patients were non-Caucasian, including patients from North Africa (15.7%), Sub-Saharan Africa (2.5%), Asia (1.6%) and the West Indies (0.8%).

Anatomical diagnosis

The anatomical distribution of uveitis was: anterior uveitis (AU, 40.6%), intermediate uveitis (IU, 9%), posterior uveitis (UP, 18.8%), and panuveitis (PU, 31.7%).

Among the 912 patients, 58.9% had a bilateral disease and 66.7% had a chronic course.

Most were non-hypertensive (93.5%) or non-granulomatous (60.3%) (Table 1).

† † Medications: BCG-therapy (n=2), immune check-point inhibitors (n=2), etanercept (n=2), rifabutine (n=1), interferon (n=1).

Classification by anatomical type

Uveitis by age, sex, and ethnicity

Within patients with a definite etiology, young patients (aged <29 years at uveitis onset) had more frequently Behcet's disease, HLA-B27-related anterior uveitis (10.3% each) and spondyloarthritis (8.6%) (Table 4). Between 30 and 59 years old, sarcoidosis was the most frequent etiology (14%), followed by spondyloarthritis (8.6%), tuberculosis (7.5%) and HLA-B27-related anterior uveitis (7%). In patients >60 years old, sarcoidosis was the most frequent etiology (30.3%) followed by tuberculosis (11%). Sarcoidosis was more frequently histologically proven in patients aged <60 years (70.1% vs 42%). The male/female ratio was 0.67 in the whole cohort. Behcet's disease, tuberculosis, and syphilis were more frequent in men, while sarcoidosis tended to be more frequent in women (Table 5). patients with non-infectious uveitis were evaluated in two tertiary referral rheumatology departments in Italy [START_REF] Lopalco | Cantarini L Epidemiological profile of non-infectious uveitis from the rheumatologist's perspective: a survey from two tertiary referral centres in Italy[END_REF]. An associated disease was identified in 41.7% of the patients, mostly Behçet's disease, ankylosing spondylitis and juvenile idiopathic arthritis. In our practice, most patients are referred to an internist for the diagnostic work-up of uveitis if there is no obvious diagnosis, such as post-surgical or traumatic uveitis, a previously known disease likely to be the cause of the uveitis and/or a specific ocular diagnosis (diagnosed by ophthalmological examination, such as toxoplasmosis infection, Herpesvirus infection…) [START_REF] De Parisot | Randomized Controlled Trial Evaluating a Standardized Strategy for Uveitis Etiologic Diagnosis (ULISSE)[END_REF].

In two previous French studies, 34% of uveitis were unclassified [START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF][START_REF] Hadjadj | Relevance of diagnostic investigations in patients with uveitis: Retrospective cohort study on 300 patients[END_REF]. In the first study, "open strategy" group [START_REF] De Parisot | Randomized Controlled Trial Evaluating a Standardized Strategy for Uveitis Etiologic Diagnosis (ULISSE)[END_REF]. As in our work, patients who had a surgery or a trauma, a known pathology likely to be the cause of the uveitis, a specific ocular disease diagnosed by ophthalmic examination only were excluded. Despite progress in diagnostic tests and clinical research, and the development of multidisciplinary specialized departments, the rate of unclassified uveitis remains high (23 to 40% in Western Europe) [14-19, 24, 27] (Table 7). In this setting, immunotherapeutic interventions have led to significant improvements and studies to evaluate new options are ongoing [START_REF] Papotto | Immunotherapeutic strategies in autoimmune uveitis[END_REF][START_REF] Mesquida | Targeting interleukin-6 in autoimmune uveitis[END_REF]. Basic research should also help understanding the immune mechanisms involved in autoimmune uveitis and lead to the preclinical testing of potential new therapies [START_REF] Bose | Dry eye disease and uveitis: A closer look at immune mechanisms in animal models of two ocular autoimmune diseases[END_REF].

Compared to recent series in Western Europe, there were less anterior uveitis but more idiopathic uveitis as well as posterior uveitis and panuveitis in our study. The low proportion of uveitis patients with an etiological diagnosis may be explained by the exclusion of specific ocular entities diagnosed by ophthalmological examination only, which were included in other studies. With regards to anatomical distribution, the difference is certainly explained by the referral of patients that selects more severe uveitis. Thus, the anatomical distribution in our population is close to that of other published series in tertiary referral centers where anterior uveitis accounted for 29-60% of the cases [START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF][START_REF] Hadjadj | Relevance of diagnostic investigations in patients with uveitis: Retrospective cohort study on 300 patients[END_REF]. Intermediate uveitis was the less frequent anatomical type (9%), as in previous studies, except for Jakob's (23%) [START_REF] Jakob | Uveitis subtypes in a german interdisciplinary uveitis center-analysis of 1916 patients[END_REF]. In our study, intermediate uveitis remained unclassified in 62.1% of the cases, but we excluded pars planitis. Sarcoidosis, primary intraocular lymphoma, multiple sclerosis and tuberculosis were the main etiologies among intermediate uveitis. This is consistent with previous studies [START_REF] Luca | Changes in patterns of uveitis at a tertiary referral center in Northern Italy: analysis of 990 consecutive cases[END_REF][START_REF] Llorenç | Epidemiology of uveitis in a Western urban multiethnic population. The challenge of globalization[END_REF][START_REF] Jakob | Uveitis subtypes in a german interdisciplinary uveitis center-analysis of 1916 patients[END_REF][START_REF] Lopalco | Cantarini L Epidemiological profile of non-infectious uveitis from the rheumatologist's perspective: a survey from two tertiary referral centres in Italy[END_REF][START_REF] Hadjadj | Relevance of diagnostic investigations in patients with uveitis: Retrospective cohort study on 300 patients[END_REF], except for ocular lymphoma, which was often excluded or considered as a masquerade syndrome in other studies, although in our experience the diagnostic work-up is initially similar to conventional uveitis. There was no Behcet's disease in this subgroup unlike in other larger cohorts in different countries [START_REF] Luca | Changes in patterns of uveitis at a tertiary referral center in Northern Italy: analysis of 990 consecutive cases[END_REF][START_REF] Jakob | Uveitis subtypes in a german interdisciplinary uveitis center-analysis of 1916 patients[END_REF].

Overall, the most frequent diagnosis in our study was sarcoidosis (45.8% of systemic diseases). The prevalence of sarcoidosis was higher than previously reported in Western

European series where it ranged from 2.4-9.7% [START_REF] Keino | Frequency and clinical features of intraocular inflammation in Tokyo[END_REF]. Uveitis occurring in the course of previously diagnosed diseases were excluded to avoid a referral center bias. Other factors may have contributed to this difference, such as the diagnostic criteria for sarcoidosis. These criteria vary among studies and a histological proof may be required or not to establish the diagnosis [START_REF] Luca | Changes in patterns of uveitis at a tertiary referral center in Northern Italy: analysis of 990 consecutive cases[END_REF][START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF]. In our series, we used Abad's modified criteria with 18-FDG PET/CT instead of gallium scintigraphy, since it is nowadays considered as superior for the diagnosis of sarcoidosis [START_REF] Morgenthau | Recent advances in sarcoidosis[END_REF]. The actual IWOS (International Workshop on Ocular Sarcoidosis) criteria were not used, as their relevance was recently questioned. In a recent study, Nisha et al.

showed that the IWOS clinical findings and investigational tests had low sensitivities, except for bilateral hilar lymphadenopathy [START_REF] Abad | Guidelines for the management of uveitis in internal medicine[END_REF]. The high proportion of sarcoidosis in our population may also be explained by the exhaustiveness of investigations, as 20 patients were considered as having sarcoid uveitis on the basis of an positive 18-FDG PET/CT only while 4

patients had an isolated lymphocytic alveolitis [START_REF] Jamilloux | Sarcoidosis and uveitis[END_REF]. We previously showed that increasing age at diagnosis of uveitis, the presence of posterior synechiae and the positivity of a highresolution chest CT are associated with 18-FDG PET/CT signs consistent with sarcoidosis [START_REF] Jamilloux | Sarcoidosis and uveitis[END_REF]. We suppose that there might be a less important hilar or mediastinal involvement in the elderly, that is identified by a chest CT and/or an 18-FDG PET/CT only [START_REF] Chauvelot | 18 F-fluorodeoxyglucose positron emission tomography is useful for the diagnosis of intraocular sarcoidosis in patients with a normal CT scan[END_REF].

HLA-B27-related uveitis and spondyloarthritis were the second most common causes of uveitis (11.4%), as in other studies. This relatively high proportion, despite a low rate of anterior uveitis, may be explained by the systematic HLA typing of patients with anterior uveitis [START_REF] Wach | Acute anterior uveitis and undiagnosed spondyloarthritis: usefulness of Berlin criteria[END_REF]. Moreover, patients with low back pain were systemically investigated for spondyloarthritis. Haroon et al. recently developed a diagnostic algorithm for the early diagnosis of spondyloarthritis in patients presenting with acute anterior uveitis [START_REF] Haroon | A novel evidence-based detection of undiagnosed spondyloarthritis in patients presenting with acute anterior uveitis: the DUET (Dublin Uveitis Evaluation Tool)[END_REF]. These criteria were: back pain for >3 months or arthralgia before 45 years old and (ii) HLA-B27 positivity or psoriasis. This tool (DUET, Dublin Uveitis Evaluation Tool) was 96%-sensitive and 87%-specific for the diagnosis of spondyloarthritis.

The most frequent infection in our patients was tuberculosis, with a higher prevalence than reported in recent Western European series [START_REF] Jones | The Manchester Uveitis Clinic: the first 3000 patients--epidemiology and casemix[END_REF], due to interferon-γ releasing assays (IGRA) and a better awareness of 'mimicking' forms of uveitis (including serpiginous-like uveitis) [START_REF] Ang | Cost-effectiveness of alternative strategies for interferon-γ release assays and tuberculin skin test in tuberculous uveitis[END_REF].

The frequency of toxoplasmosis and herpesvirus infections was lower in our study than previously reported in Western Europe (Table 7), as these diseases may have been quite easily diagnosed by ophthalmologists, and therefore not included. The diagnosis was established later on in six patients on the basis of a successful valaciclovir treatment or after a new ophthalmological examination (toxoplasmosis). We recently reported that repeating ophthalmological examinations may be useful in patients with idiopathic uveitis [START_REF] Colmant | Uveitis of Unknown Etiology: Clinical and Outcome features. A Retrospective Analysis of 355 Patients[END_REF].

Birdshot retinochoroidopathy was the most frequent ocular syndrome, with a lower prevalence than in Bodaghi's study [START_REF] Bodaghi | Chronic severe uveitis: etiology and visual outcome in 927 patients from a single center[END_REF]. Most of these patients were referred to the department of internal medicine with a suspected sarcoidosis or retinal vasculitis. Several authors consider that HLA-A29 typing may help diagnose atypical cases of birdshot retinochoroidopathy. For Herbort et al, HLA-A29 positivity should be an essential diagnostic criterion, as the association with birdshot retinochoroidopathy might be close to 100% when using PCR testing coupled with indocyanine green angiography. This approach could lead to an earlier diagnosis and treatment, as the pathognomonic birdshot fundus lesions are probably scar tissue, likely to be missing at disease onset [START_REF] Herbort | Why birdshot retinochoroiditis should rather be called 'HLA-A29 uveitis[END_REF]. On the opposite, the absence of HLA-A29, given its strong negative predictive value, should encourage to search for other diagnoses, especially sarcoidosis [START_REF] Sève | Uveitis: Diagnostic work-up. A literature review and recommendations from an expert committee[END_REF].

Finally, our study confirmed a strong demographical and ethnical burden. The most frequent diagnoses among young and middle-aged adults were Behcet's disease and HLA-B27related anterior uveitis (10% each), followed by spondyloarthritis (8.6%) and sarcoidosis (7.5%). Our study also confirmed the high prevalence of sarcoid uveitis in patients aged over 60 years presenting with uveitis, especially women.

Tuberculosis and sarcoidosis were frequent in patients from North Africa or from Sub-Saharan Africa. On the other hand, birdshot retinochoroidopathy was only seen in Caucasian patients. In Llorenç et al. series of 1022 patients in Spain, there were similar results with a large variety of etiologies and a strong geographical burden in a multi-ethnic population [START_REF] Llorenç | Epidemiology of uveitis in a Western urban multiethnic population. The challenge of globalization[END_REF].

The main limit of our study is a selection bias due to the referral of patients who were certainly not completely representative of the general population. Most patients with obvious diagnoses or acute (anterior) uveitis did not require a multidisciplinary approach and were not referred to our department. Thus, our cohort represents a subset of patients with more severe, chronic and complex uveitis. Nevertheless, the large number of cases (912 patients)

can counteract, at least in part, this selection bias. Another limit is that >80% of the patients were Caucasians and our conclusions may not apply outside Western Europe. Finally, the study design was retrospective and some data were missing.

  Bodaghi et al. analyzed a series of 927 consecutive patients referred to their department of ophthalmology during 5 years, for the diagnosis and/or therapeutic management of severe uveitis [15]. Hadjadj et al. recently conducted a retrospective study of adult patients referred by the department of Ophthalmology during 6 years (tertiary referral center) to the department of Internal Medicine for the diagnostic work-up of uveitis [40, 41]. The ULISSE study (Uveitis cLInical and medicoeconomic evaluation of a Standardized Strategy of the Etiological diagnosis), was performed in 21 French centers and the aim was to assess the efficiency of a standardized diagnostic approach compared to an open strategy. There was an etiological diagnosis in 50.4% of the cases in the standardized group versus 54.4% in the

Table 1 . Characteristics of the 912 patients with uveitis

 1

	Characteristics	n=912 (%)
	Sex, n
	Men	367 (40.3)
	Women	545 (59.7)
	Mean age at diagnosis (years)	48.8
	Ethnicity, n
	Caucasian	713 (78.1)
	North-African	144 (15.7)
	Sub-Saharan	23 (2.5)
	Asian	15 (1.6)
	West Indies	7 (0.7)
	Others	10 (1.0)
	Characteristics of uveitis
	Bilateral, n	537 (58.8)
	Chronic, n	608 (64.5)
	Non-granulomatous, n	649 (71.1)
	Non-hypertensive, n	853 (93.5)
	Anatomical type, n
	Anterior	370 (40.6)
	Intermediate	82 (9.0)
	Posterior	171 (18.7)
	Panuveitis	289 (31.7)
	2.3 Etiological diagnosis
	B27-related anterior uveitis (6.3%), spondyloarthritis (6.3%), Behcet's disease (3.1%),
	multiple sclerosis or radiologically isolated syndromes (2%), and VKH disease (1%). Twenty
	patients were considered as having presumed sarcoid uveitis on the basis of a positive 18-F-
	FDG PET/CT suggestive of sarcoidosis, 4 had an isolated alveolar lymphocytosis, while 3
	patients had a possible neurosarcoidosis. The most common purely ophthalmological entity

A definite etiological diagnosis was made in 484 (53.1%) patients. 37% of uveitis were associated with a definite or presumed systemic diseases, 2.3% were purely ophthalmological entities, 10.8% were of infectious origin, 1.8% were related to a lymphoma and 0.8% were related to drug side effects (Table

2

). The most commonly identified systemic diseases were: sarcoidosis (17.1%, n=156), of which 85 were histologically proven, HLA-

Table 2 . Etiological distribution of the 912 uveitis

 2

	Etiology

Table 3

 3 shows the etiological diagnoses according to anatomical localization. The most common diagnoses for anterior uveitis were idiopathic (45.9%), spondyloarthritis (14.8%),

	HLA-B27-related uveitis (14.6%), and sarcoidosis (10.8%). Intermediate uveitis was most
	commonly idiopathic (62.1%). Sarcoidosis (18.3%), oculocerebral lymphoma (11%) and
	multiple sclerosis (6.1%) were the most common diseases associated with intermediate
	uveitis. For posterior uveitis, the most frequent diagnoses were idiopathic (50.3%),
	tuberculosis (12.4%), sarcoidosis (11.7%) and birdshot retinochoroidopathy (5.8%). For
	patients with panuveitis, a specific diagnosis could be established in 55.1% of the cases;
	including sarcoidosis (22.9%), tuberculosis (7.3%), and Behcet's disease (5.5%).

Table 3 . Etiological distribution according to the anatomical type Etiology Anatomical type Anterior Intermediate Posterior Panuveitis

 3

	Idiopathic, %	45.9	62.1	50.3	44.9
	Systemic diseases, %
	Sarcoidosis	10.8	18.3	11.7	26.9
	Proven sarcoidosis	5.6	11	5.3	14.2
	HLA-B27-related uveitis	14.6	0	0	1
	Spondyloarthritis	14.8	0	0	1
	Behcet's disease	0.8	0	5.3	5.5
	Vogt-Koyanagi-Harada	0	0	0.6	2.4
	Multiple sclerosis	1.1	6.1	1.2	2.4
	Others	2.1	0	0.6	2.4
	Ophthalmological entities, %
	Birdshot retinochoroidopathy	0	2.4	5.8	0.7
	Fuchs uveitis	0. 3	0	0	0
	Others	0	0	2.3	.0
	Infections, %
	Tuberculosis	5.7	4.8	12.9	7.3
	Herpesvirus	1.3	0	0	0
	Toxoplasmosis	0	0	0	0.7
	Lyme disease	0.3	2.4	2.9	0.6
	Syphilis	0	0	2.3	2.1
	Other	0	0	0	1.4
	Lymphoma, %	0.5	11	1.7	0.6

Table 4 . Etiological distribution by age Etiology <29

 4

	Age
	30-59	>60

Table 5 . Etiological distribution by gender

 5

	Et Etiology

Table 6 . Etiological distribution by ethnicity

 6

	Etiology

radiographs were performed. In this study, sarcoidosis (4.9%) and ankylosing spondylitis (4.9%) were the most frequent associated systemic diseases. In

Lopalco et al. study, 278

Table 7 . Main Previous epidemiological studies in Western Europe

 7

	First author	Bodaghi Cimino	Jakob	Barisani-Asenbauer	Jones Llorenç Luca	Our study
	Country	France	Italy Germany	Austria	UK	Spain	Italy	France
	Population, n	927	1064	1686	2619	3000	1022	990	912
	Uveitis, %
	Anterior	28.5	51.2	45.4	59.9	46.0	52.0	53.5	40.6
	Intermediate	15	5.8	22.9	14.8	11.1	9.0	7.5	9
	Posterior	21.6	23.4	13.5	18.3	21.8	23.0	16.2	18.75
	Panuveitis	35	19.6	6.2	7.0	21.1	15.0	22.8	31.7
	Etiology, %
	Idiopathic	34	26	35.3	39.3	31.2	26	23	46.9
	Sarcoidosis	6.4	3	4.9	2.4	9.7	3	4.3	17.1
	B27+ or SPA	5	7	14	18.3	4.5	10	7.7	12.5
	Behcet's disease	6.1	5.3	2.2	1.8	2.7	5	4.8	3
	VKH	2	2.5	NA	0.4	0.8	1	4.1	1
	Multiple sclerosis	1.7	NA	3.4	1	0.6	0.8	NA	2
	Birdshot	4.4	NA	NA	0.4	1.2	3	0.8	1.6
	Pars planitis	11.3	NA	NA	NA	NA	1	3.3	NA
	Fuchs uveitis	2.7	22.7	8.5	3.4	11.5	1	9.7	0.1
	Toxoplasmosis	11.9	6.9	5.1	7.5	6.9	7	4.7	0.2
	Tuberculosis	4.1	4.5	NA	NA	3.3	5	5.7	7.5
	Herpesvirus	8.2	9.9	6.3	8.6	3.5	12	15.6	0.5
	Lyme disease	1.1	NA	1.4	NA	NA	NA	NA	1.1
	Syphilis	0.6	NA	NA	NA	0.3	NA	0.8	1.1
	B27+: HLA-B27 positive
	SPA: ankylosing spondylitis
	VKH: Vogt-Koyanagi-Harada disease
	NA: Not Available

CONCLUSION

This is the largest cohort study of patients with uveitis referred to a department of internal medicine for the diagnostic work-up of uveitis. Despite a huge improvement in the diagnostic strategy, a large number of uveitis remains idiopathic. A multidisciplinary approach, involving the ophthalmologist, the internist and/or the rheumatologist, may increase the diagnostic rate, especially for complex, chronic or severe uveitis.