J Buffault

A Labbé

P Hamard

F Brignole-Baudouin D E

C Baudouin

The trabecular meshwork: structure, function and clinical implications. A review of the literature

Keywords: trabecular meshwork, glaucoma, intraocular pressure, ocular hypertension, iridocorneal angle, trabecular meshwork cells, aqueous humor, primary open angle glaucoma, secondary glaucoma, steroid-induced glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, glaucoma medications, trabeculoplasty, glaucoma surgery Outline Introduction Structure and function Anatomy Trabecular meshwork cells Extracellular matrix Aqueous outflow and regulation of intraocular pressure Trabecular degeneration in glaucoma Primary open angle glaucoma Angle closure glaucoma Steroid-induced glaucoma Pigmentary glaucoma Pseudoexfoliative glaucoma Uveitic glaucoma Trabecular toxicity of glaucoma medications Therapeutic approaches Medical Laser Surgical Filtering surgery

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, threedimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

Introduction

Glaucoma is a blinding optic neuropathy affecting approximately 70 million individuals world-wide [START_REF] Tham | Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and metaanalysis[END_REF]. Its main risk factor is elevated intraocular pressure (IOP) [START_REF] Gordon | The Ocular Hypertension Treatment Study: design and baseline description of the participants[END_REF]. The trabecular meshwork, located within the iridocorneal angle, constitutes the main pathway for drainage of aqueous humor out of the eye. It is a fenestrated three-dimensional structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM) [START_REF] Stamer | The many faces of the trabecular meshwork cell[END_REF]. The trabecular meshwork controls the IOP by regulating outflow of aqueous humor from the anterior chamber of the eye into the adjacent Schlemm's canal (SC) and then via aqueous vein collector channels into the venous system. Dysfunction of the trabecular meshwork is the cause of IOP elevation. The purpose of this literature review is to provide an overview of current understanding of this complex structure, which plays a key role in the pathophysiology of glaucoma.

Structure and function Anatomy

Embryologically, the trabecular meshwork is derived from a mixed mesodermal origin and a wave of mesenchymal cells coming from the neural crest between the 15 th and 20 th week of development [START_REF] Vranka | Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma[END_REF][START_REF] Cvekl | Anterior eye development and ocular mesenchyme[END_REF].

It is a sieve-like structure which acts as a filter between the anterior chamber and Schlemm's canal (SC), a circular canal which collects the aqueous and evacuates it into the extraocular circulation (figure 1a). The trabecular meshwork, which bridges the scleral sulcus, thus isolating SC within the scleral sulcus, inserts anteriorly into the peripheral cornea at the level of Schwalbe's line, while posteriorly, the trabecular lamellae are connected to the junction between the ciliary body, iris and scleral spur. We make a distinction between two different functional portions: the anterior, or nonfiltering, trabecular meshwork, which is not in communication with SC, and the posterior, or filtering, trabecular meshwork, which is in communication with SC [START_REF] Lütjen-Drecoll | Functional morphology of the trabecular meshwork in primate eyes[END_REF].

The anterior trabecular meshwork is a transition zone between Schwalbe's line and the posterior trabecular meshwork. It is formed of 4 to 5 trabecular lamellae covered with TMC. Within the transition zone between the non-filtering and filtering trabecular meshwork, electron microscopy reveals spaces organized into microscopic canals whose significance is poorly understood but which may serve in fluid transport or access to accessory cells necessary for cellular regeneration.

The posterior trabecular meshwork is known as filtering, since it is in communication with SC. It represents the trabecular filter proper. It consists of three anatomically distinct regions, from inner to outer: the uveal meshwork, the corneoscleral meshwork, and the juxtacanalicular or cribriform meshwork, whose outermost aspect is in communication with SC [START_REF] Gong | Morphology of the aqueous outflow pathway[END_REF][START_REF] Buller | Segmental variability of the trabecular meshwork in normal and glaucomatous eyes[END_REF] (figure 1b).

-The uveal meshwork is in direct communication with the aqueous humor. It is organized into fine, interwoven cords or pillars extending from the iris root and ciliary body to Schwalbe's line. These pillars consist of collagen and elastin fibers and are covered with TMC resting on a basement membrane. The meshwork of these pillars creates orifices allowing for outflow of aqueous. -The corneoscleral meshwork, which represents the majority of the trabecular meshwork, is formed by a superimposition of joined lamellae traversed by orifices which extend and grow in number from the anterior wall of the scleral sulcus to the scleral spur. These lamellae consist of collagen and elastin fibers covered with a single layer of TMC resting on a basement membrane. The interlamellar spaces become more narrow near the juxtacanalicular meshwork but do not constitute an obstacle to aqueous outflow. -The juxtacanalicular meshwork, the outermost portion of the posterior filtering trabecular meshwork, is histologically different from the other two portions of the trabecular meshwork. It is formed of loose, unstratified connective tissue and 2 to 5 layers of TMC dispersed within the extracellular matrix and arranged in a network with the help of their cytoplasmic extensions [START_REF] Stamer | The many faces of the trabecular meshwork cell[END_REF]. Its outermost portion corresponds to the endothelium of the inner wall of SC [START_REF] Johnson | How does nonpenetrating glaucoma surgery work? Aqueous outflow resistance and glaucoma surgery[END_REF]. This is made up of a continuous layer of endothelial cells spaced at 15 to 20 nm intervals and connected by junctional complexes. Inside these cells, giant cytoplasmic vacuoles are described, as well as pores 0.5 to 2 µm in diameter, allowing flow through the inner wall of Schlemm's canal [START_REF] Renard | Mécanismes de l'écoulement de l'humeur aqueuse[END_REF].

The porosity of the posterior filtering trabecular meshwork decreases from inside to outside [START_REF] Tamm | The trabecular meshwork outflow pathways: structural and functional aspects[END_REF], but under physiologic conditions, the uveal and corneoscleral meshwork do not offer resistance to aqueous outflow.

a.

b. Figure 1

Trabecular meshwork cells

Trabecular meshwork cells (TMC) are special cells combining properties of endothelial cells, myofibroblasts and macrophages (figure 2). They are responsible for regulation of aqueous outflow resistance. The properties of TMC differ depending on their location within the trabecular meshwork and are summarized in table 1. Functioning as endothelial cells in the corneoscleral and uveal portions, TMC produce large quantities of antithrombotic substances, such as heparin sulfate and tissue plasminogen activator (tPA) [START_REF] Shuman | Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor[END_REF]. Similarly to endothelial cells, TMC in the inner portion appear to participate in mediation of inflammation. The hypothesis that these cells might play a role in antigen presentation was proposed in the 1990's when class I and II major histocompatibility complexes (MHC) were detected on frozen sections of trabecular meshwork and cultured TMC [START_REF] Lynch | Expression of HLA class I and II antigens on cells of the human trabecular meshwork[END_REF][START_REF] Latina | Immunohistochemical staining of the human anterior segment. Evidence that resident cells play a role in immunologic responses[END_REF][START_REF] Coupland | Immunohistochemistry study of the glaucomatous and normal human trabecular meshwork[END_REF][START_REF] Tripathi | Expression of HLA by the human trabecular meshwork and corneal endothelium[END_REF]. More recently, studies have shown that these cells may secrete a certain number of factors such as enzymes and cytokines which modulate trabecular cell and ECM functions. Shifera et al. reported that cultured human TMC secrete significant quantities of chemotactic cytokines IL-8, CXCL6 and MCP1 in the absence of any stimulation [START_REF] Shifera | Constitutive secretion of chemokines by cultured human trabecular meshwork cells[END_REF]. Secretion of these cytokines increased under the influence of the pro-inflammatory cytokines TNFα and IL1β. They were also able to demonstrate the monocytes, apparently under the influence of these chemotactic signals, circulated through the trabecular meshwork [START_REF] Alvarado | Monocyte modulation of aqueous outflow and recruitment to the trabecular meshwork following selective laser trabeculoplasty[END_REF]. TMC also have activity similar to that of macrophages for elimination of cellular debris present within the aqueous [START_REF] Li | Gap junction connexin43 is a key element in mediating phagocytosis activity in human trabecular meshwork cells[END_REF][START_REF] Peotter | Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells[END_REF]. Consistent with their role in regulating aqueous outflow resistance, TMC in the juxtacanalicular region have both fibroblastic and contractile properties. Their phenotype is quite different, with a spindle-shaped morphology. They secrete a certain number of ECM proteins (collagens, fibronectin, elastin, fibrillins and proteoglycans) and their degradative enzymes (matrix metalloproteinases (MMP-1, -2, -3, -9, -12, et -14)), so as to support continuous remodeling of the ECM [START_REF] Weinreb | Matrix Metalloproteinases and Glaucoma Treatment[END_REF]. They are contractile, expressing smooth muscle actin and myosin, which are important in mechanotransduction [START_REF] Stamer | The many faces of the trabecular meshwork cell[END_REF]. A number of authors have suggested the hypothesis that the non-filtering region of the trabecular meshwork, near Schwalbe's line, might serve as a niche for stem cells capable of dividing and recolonizing the trabecular meshwork after an injury [START_REF] Yun | Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure[END_REF][START_REF] Du | Multipotent stem cells from trabecular meshwork become phagocytic TM cells[END_REF].

Phenotype

Extracellular matrix (ECM)

The ECM is made up of fibrillar components and an amorphous ground substance composed of collagens, hyaluronate and proteoglycans [START_REF] Tamm | Chapter Eighteen -Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways[END_REF]. In the uveal and corneoscleral meshwork, the connective tissue and/or ECM lamellae are covered with TMC. In the juxtacanalicular region, the cells live relatively freely and are integrated within the ECM (figure 3) [START_REF] Knepper | Chapter 22 -Abnormal trabecular meshwork outflow[END_REF]. The juxtacanalicular tissue is composed of a network of elastic fibers, the cribriform plexus, which extends between the corneoscleral meshwork and the inner wall of SC. The elastic fibers of the cribriform plexus are composed of elastin fibers sheathed with a material containing type VI collagen, laminin and fibronectin, responsible for adherence of cells to their substrates [START_REF] Ueda | Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes[END_REF]. The cribriform plexus fibers are connected to the SC endothelial cells by fine elastic fibrils emerging from the sheath material [START_REF] Keller | Extracellular matrix turnover and outflow resistance[END_REF].

Aqueous outflow and IOP regulation

Aqueous exits the eye via two pathways: the primary pathway, trabecular, also known as conventional, (40-96% of the outflow [START_REF] Fautsch | Aqueous humor outflow: what do we know? Where will it lead us?[END_REF][START_REF] Winkler | Effects of prostaglandin analogues on aqueous humor outflow pathways[END_REF]), and the secondary pathway, known as uveoscleral. In trabecular outflow, the trabecular meshwork controls the IOP by regulating outflow of aqueous from the anterior chamber into the adjacent SC, then into the aqueous vein collector channels and into the venous system (Figure 1). One may think of it as a selfcleaning biologic filter whose caliber can be more or less fine depending on various regulatory factors. In man, 75% of aqueous outflow resistance occurs within the trabecular meshwork, in particular the juxtacanalicular portion, and 25% within SC [START_REF] Grant | Further studies on facility of flow through the trabecular meshwork[END_REF].

Resistance is influenced by two contractile systems, that of the anterior portion of the ciliary muscle via tendinous extensions from the ciliary muscle traversing the cribriform trabecular meshwork and inserting into the wall of SC [START_REF] Rohen | The fine structure of the cribriform meshwork in normal and glaucomatous eyes as seen in tangential sections[END_REF], and that due to myofibroblastic type contractile TMC. Thus, resistance is reduced by ciliary muscle contraction or by relaxation of the contractile cells within the trabecular outflow pathways.

Regulation of resistance occurs by a mechanism of cellular detection of stretching or deformation in the juxtacanalicular region of the trabecular meshwork [START_REF] Wang | Trabecular meshwork stiffness in glaucoma[END_REF]. The TMC in this region also secrete ECM proteins and degradative enzymes to support ECM remodeling. With input from cellular stretch receptors, adjustments in resistance thus occur through changes in ECM turnover: secretion and/or activation of proteinases, matrix cleavage, digestion of fragments and biosynthesis of its components [START_REF] Keller | Extracellular matrix turnover and outflow resistance[END_REF].

TMC also possess adaptive properties such as mechanisms of intercellular adhesion, cellmatrix interactions, cellular contractility related to their actin cytoskeleton, and expression of water channels to facilitate rapid changes in cell volume [START_REF] Wang | Trabecular meshwork stiffness in glaucoma[END_REF].

Trabecular degeneration in glaucoma

Primary open angle glaucoma

In primary open angle glaucoma (POAG), trabecular aqueous outflow resistance is abnormally elevated [START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF]. Studies have shown that this increased resistance is related to a mechanism of trabecular stiffening involving senescence and apoptosis of TMC and remodeling of the ECM [START_REF] Stamer | The many faces of the trabecular meshwork cell[END_REF][START_REF] Vranka | Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma[END_REF][START_REF] Tamm | The trabecular meshwork outflow pathways: structural and functional aspects[END_REF][START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF][START_REF] Liton | Cellular senescence in the glaucomatous outflow pathway[END_REF]. These changes are similar to age-related trabecular changes but seem to accelerate in glaucoma [START_REF] Liu | Aging and ocular tissue stiffness in glaucoma[END_REF]. Thus, Tektas et al. have described a significant increase in thickness of the sheaths of the elastic fibers compared to the trabecular meshwork of normal subjects of the same age. On transverse sections, these appear as extracellular "plaques" and are known as "sheath-derived plaques" [START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF]. This increase is due to the fibrils and other ECM components adhering to the sheaths of the elastic fibers and their connections with the endothelium of the inner wall. In eyes with POAG, there is also a marked loss of TMC, which in spots leads to fusion and thickening of the trabecular lamellae [START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF].

A confocal microscopic study by Hamard et al. has shown a very significant decrease in cell density within the outer trabecular membrane (OTM) removed during non-penetrating deep sclerectomy (NPDS) in glaucomatous subjects compared to controls (figure 4). This cell loss involves the juxtacanalicular meshwork as well as a portion of the adjacent corneoscleral meshwork [START_REF] Hamard | Confocal microscopic examination of trabecular meshwork removed during ab externo trabeculectomy[END_REF]. Tamm et al. have also described changes in juxtacanalicular connective tissue cells, which acquire contractile properties [START_REF] Cvekl | Anterior eye development and ocular mesenchyme[END_REF][START_REF] Lütjen-Drecoll | Functional morphology of the trabecular meshwork in primate eyes[END_REF]. An augmentation and reorganization of the actin cytoskeleton is seen, as well as the ECM, leading to stiffening of the inner wall of SC [START_REF] Tian | The role of the actomyosin system in regulating trabecular fluid outflow[END_REF].

Under the influence of transforming growth factor β2 (TGF-β2), changes are also seen in the trabecular meshwork itself. TGF-β2 thus seems to play a significant role in the pathogenesis of POAG. It is a profibrotic cytokine known to be present in higher concentration in the aqueous of glaucoma patients [START_REF] Prendes | The role of transforming growth factor β in glaucoma and the therapeutic implications[END_REF]. The trabecular meshwork is an endogenous source of TGF-β2 [START_REF] Webber | Crosstalk between TGFβ and Wnt signaling pathways in the human trabecular meshwork[END_REF][START_REF] Pervan | Smad-independent TGF-β2 signaling pathways in human trabecular meshwork cells[END_REF]. Tripathi et al. were able to show that TGF-β2 increased IOP by inducing synthesis by the TMC of certain matrix components not degradable by metalloproteinases [START_REF] Tripathi | Pathologic anatomy in the outflow pathway of aqueous humour in chronic simple glaucoma[END_REF][START_REF] Picht | Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development[END_REF]. These effects are mediated by induction of conventional TGF-β2 signal cascades (SMAD pathways) as well as unconventional pathways (MAPK and Rho GTPase pathways) [START_REF] Pervan | Smad-independent TGF-β2 signaling pathways in human trabecular meshwork cells[END_REF]. The conventional SMAD signaling pathways involve omnipresent signaling proteins with a number of downstream effectors implicated in, among others, healing of corneal scars and maintenance of the ocular immune privilege. The SMAD3 isoform is more systematically related to expression of mediators implicated in ocular hypertension. This implies that targeted inhibition of SMAD3 might be an interesting approach for glaucoma treatment [START_REF] Pervan | Smad-independent TGF-β2 signaling pathways in human trabecular meshwork cells[END_REF]. Another approach might be that of SMAD7 modulation, a SMAD inhibitor with functions as an intracellular antagonist to TGF-β signaling [START_REF] Fuchshofer | Gene expression profiling of TGFbeta2-and/or BMP7-treated trabecular meshwork cells: Identification of Smad7 as a critical inhibitor of TGF-beta2 signaling[END_REF].

TGF-β2 also appears to increase cellular rigidity by formation of Cross-linked Actin Networks (CLANs) via the Rho-GTPase pathway (figure 5) [START_REF] Webber | Crosstalk between TGFβ and Wnt signaling pathways in the human trabecular meshwork[END_REF]. CLANs are cytoskeletal rearrangements changing cell shape and rigidity and thus their ability to respond to external signals. The cells of the trabecular outflow pathways would thus be stimulated by TGF-β2 to take on an increasingly contractile phenotype, involving an increase in both the actin cytoskeleton and the surrounding fibrillar ECM.

Bone morphogenetic proteins (BMP) are a family of growth factors involved in regulation of the ECM and thus in regulation of aqueous outflow resistance. The BMP and TGF-β signaling pathways have opposing antifibrotic and profibrotic roles. BMPs may thus degrade matrix deposits induced by TGF-β2 [START_REF] Tovar-Vidales | Human trabecular meshwork cells express BMP antagonist mRNAs and proteins[END_REF]. It has been shown that the BMP antagonists, gremlin and noggin are more strongly expressed in glaucomatous TMC and provoke an increase in IOP in perfused anterior segments [START_REF] Hernandez | BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork[END_REF][START_REF] Wordinger | Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma[END_REF][START_REF] Mcdowell | Gremlin Induces Ocular Hypertension in Mice Through Smad3-Dependent Signaling[END_REF]. Gremlin also apparently blocks BMP4 repression of the fibronectin synthesis induced by TGF-β [START_REF] Wordinger | Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma[END_REF][START_REF] Mody | Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells[END_REF]. Deregulation of this factor, probably under the influence of mutations or induced genetic polymorphisms, may thus explain the overexpression of TGF in primary glaucoma.

Oxidative stress may also play a significant role in the pathophysiology of glaucoma. A meta-analysis published in 2016 reported that markers of oxidative stress were overexpressed in the serum and aqueous humor of glaucoma patients [START_REF] Benoist D'azy | Oxidative and Anti-Oxidative Stress Markers in Chronic Glaucoma: A Systematic Review and Meta-Analysis[END_REF]. The production of reactive oxygen species (ROS) in the trabecular meshwork is both endogenous (generated by mitochondria) and exogenous. It may increase due to age, inflammation, light, infrared or ultraviolet exposure, or certain toxins. A disturbance in the pro-oxidant/antioxidant equilibrium in the aqueous humor results in an increase in the production of ROSs, thus altering the trabecular meshwork [START_REF] Zhao | Oxidative stress in the trabecular meshwork (Review)[END_REF]. The ROSs damage proteins, lipids and DNA molecules; these processes are associated with cellular aging, chronic inflammation, apoptosis and cell death, releasing free radicals and resulting in a vicious circle. Within the trabecular meshwork, one sees a cessation of cell growth, a change in cellular permeability, rearrangements of the cytoskeleton of TMC, affecting their function and interactions with the ECM, and an accumulation of ECM [START_REF] Zhao | Oxidative stress in the trabecular meshwork (Review)[END_REF][START_REF] Nita | The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults[END_REF]. Interestingly, it has been shown that TGF-β2 provokes an increase in oxidative stress in human TMC. Pretreatment of TMC with antioxidants targeted to mitochondria (XJB-5-131 (10 μM) or MitoQ (10 nM)) decrease the TGF-β2-mediated effects on SMAD-dependent transcriptional activity, including marked reductions in stress fibers and collagen expression [START_REF] Rao | Mitochondrial-Targeted Antioxidants Attenuate TGF-β2 Signaling in Human Trabecular Meshwork Cells[END_REF].

Angle closure glaucoma

In angle closure glaucoma, the mechanism of IOP elevation is explained by apposition of the iris to the trabecular meshwork, blocking aqueous outflow. However, in certain cases, even after removal of the trabecular block, the IOP remains elevated. On gonioscopy, peripheral anterior synechiae or pigment deposits may be seen on the trabecular meshwork.

A histologic study by Hamanaka et al. on trabecular specimens from patients undergoing trabeculectomy for angle closure has allowed observation of the damage to SC and the posterior trabecular meshwork [START_REF] Hamanaka | Histopathology of the trabecular meshwork and Schlemm's canal in primary angle-closure glaucoma[END_REF]. Persistent contact between the trabecular meshwork and the iris or peripheral anterior synechiae blocking aqueous outflow result in SC involvement and occlusion. In the posterior portion of the trabecular meshwork, a loss of TMC is seen, along with a change in their mitochondrial function, with enlargement and fusion of the trabecular beams [START_REF] Hamanaka | Histopathology of the trabecular meshwork and Schlemm's canal in primary angle-closure glaucoma[END_REF][START_REF] Sihota | Scanning electron microscopy of the trabecular meshwork: understanding the pathogenesis of primary angle closure glaucoma[END_REF]. These mechanisms can explain persistent IOP elevation even once the angle is reopened in a patient with chronic angle closure glaucoma.

Steroid-induced glaucoma

The primary finding in steroid-induced glaucoma is an accumulation of type IV collagen and fibronectin in the outer portion of the trabecular meshwork [START_REF] Tawara | Immunohistochemical evaluation of the extracellular matrix in trabecular meshwork in steroid-induced glaucoma[END_REF]. Also seen are an increase in mixed material with redundant basement membrane in a fingerprint pattern and unidentified fine fibrillar deposits arranged in bands in the subendothelial region of SC [START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF]. Steroids such as dexamethasone (DEX) are known to change the architecture of the trabecular meshwork by increasing trabecular cell rigidity. The matrix deposited by TMC under the influence of DEX is approximately 4 times more rigid, more organized, and shows a higher expression of ECM proteins generally implicated in glaucoma (decorin, myocilin and fibrillin) [START_REF] Clark | Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue[END_REF][START_REF] Raghunathan | Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix[END_REF]. Biochemical and genetic studies have suggested that changes in trabecular myocilin expression, still known as the trabecular meshwork induced glucocorticoid response (TIGR), might play a role in the development of steroid-induced glaucoma [START_REF] Polansky | Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells[END_REF][START_REF] Filla | In Vitro Localization of TIGR/MYOC in Trabecular Meshwork Extracellular Matrix and Binding to Fibronectin[END_REF]. It is interesting to note that the expression of this gene induced by exposure to steroids has a pro-apoptotic effect [START_REF] Sakai | Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork cells[END_REF][START_REF] Yam | Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma[END_REF]. Consequently, the phagocytotic capacity of the cell population remaining in the trabecular meshwork is insufficient for effective filtration of the aqueous humor, thus increasing aqueous outflow resistance. Also in this case exists a mixed mechanism, both mechanical and degenerative, which may explain why the IOP does not always return to normal upon discontinuation of the steroid in steroid-induced glaucoma.

Pigmentary glaucoma

Pigment dispersion syndrome (PDS) is due to release of pigment from the posterior aspect of the iris into the anterior segment of the eye [START_REF] Campbell | Pigmentary dispersion and glaucoma. A new theory[END_REF]. Blockage of the trabecular meshwork by this iris pigment may lead to its dysfunction and result in an IOP elevation. It is estimated that 25 to 50% of patients with PDS are at risk of developing ocular hypertension [START_REF] Scott | YAG laser peripheral iridotomy for the prevention of pigment dispersion glaucoma a prospective, randomized, controlled trial[END_REF].

In this disease, a loss of TMC is seen, more significant than in POAG, probably related to toxicity of the pigment granule overload [START_REF] Tektas | Structural changes of the trabecular meshwork in different kinds of glaucoma[END_REF]. Exposure of the TMC to the pigment also apparently leads to a reduction in phagocytic ability and cellular migration, as well as an increase in the formation of stress fibers and cellular contraction [START_REF] Wang | Impact of pigment dispersion on trabecular meshwork cells[END_REF][START_REF] Dang | A porcine ex vivo model of pigmentary glaucoma[END_REF]. Due to its role in the regulation of cell movement and shape, the Rho-ROCK signal pathway plays a central role in the formation and contraction of stress fibers, cellular adhesion, migration, phagocytosis and apoptosis. In a porcine pigmentary glaucoma model, the use of a ROCK inhibitor thus decreased IOP and increased phagocytosis [START_REF] Wang | Rho/Rho-associated kinase pathway in glaucoma (Review)[END_REF][START_REF] Dang | RKI-1447, a Rho kinase inhibitor, causes ocular hypotension, actin stress fiber disruption, and increased phagocytosis[END_REF].

Pseudoexfoliative glaucoma

Pseudoexfoliation syndrome corresponds to deposits of exfoliative material on certain organs such as the heart, vessels, lungs or meninges, but also in the anterior segment of the eye. This systemic degenerative fibrillopathy is multifactorial. Its risk factors are both genetic and environmental (age, exposure to ultraviolet) [START_REF] Schweitzer | Pseudoexfoliation syndrome and pseudoexfoliation glaucoma][END_REF]. Pseudoexfoliative glaucoma results from the accumulation of extracellular fibrillar material and pigment in the trabecular meshwork and SC, leading to an increase in IOP. Immunohistochemical and mass spectrometry analyses have revealed that this pseudoexfoliative material is a highly glycosylated protein complex extremely resistant to degradation. This protein complex is a combination of basement membrane proteins, elastic fibers, TGF-β, metalloproteinases, chaperone proteins, complement proteins, type 1 lysyl-oxidase (LOXL1) and apolipoprotein E (ApoE) [START_REF] Challa | Composition of Exfoliation Material[END_REF].

Uveitic glaucoma

In patients with uveitis, the pressure elevation seen may be due to the uveitis itself or may be a side effect of local steroid treatment. In fact, according to studies, 45 % to 62 % of uveitis patients are steroid responders [START_REF] Sharon | Uveitic Glaucoma: Long-term Clinical Outcome and Risk Factors for Progression[END_REF]. It has been shown that elevated levels of trabecular proteins reduce trabecular outflow. Inflammatory cells, free radicals, and enzymes are also prone to raising IOP [START_REF] Baneke | The Pathogenesis of Raised Intraocular Pressure in Uveitis[END_REF]. The main etiologies of hypertensive uveitides are Fuchs heterochromic iridocyclitis, herpes, Posner-Schlossman syndrome and juvenile idiopathic arthritis. Tektas et al. studied the trabecular meshwork of uveitic glaucoma patients removed during trabeculectomy [START_REF] Tektas | Morphological changes of trabeculectomy specimens in different kinds of uveitic glaucoma[END_REF]. They observed that there was an increase in ECM within the cribriform trabecular meshwork; the majority of the trabecular beams were thickened with a fibrillar material deposited between the TMC and basement membranes. Under the endothelium of the inner wall, there was an increase in "plaques" morphologically comparable to those seen in POAG patients. The trabecular meshwork of patients with juvenile idiopathic arthritis also showed accumulations of "plaques" on the inner wall and thickening of the trabecular beams, but this was due to a thickening of the basement membrane itself with collagen inclusions. The trabecular meshwork in herpes showed folds of basement membrane between the trabecular lamellae. The subendothelial region also showed an increase in plaques and fibrillar material. In addition, secondary iridocorneal angle closure may occur in uveitis by various mechanisms: posterior synechiae with pupillary block, peripheral anterior synechiae, forward rotation of the ciliary body [START_REF] Souissi | Étiopathogénie des modifications de la pression intraoculaire au cours des uvéites[END_REF]. Nearly 11% of patients develop peripheral anterior synechiae (PAS) secondary to inflammation [START_REF] Sharon | Uveitic Glaucoma: Long-term Clinical Outcome and Risk Factors for Progression[END_REF]. The risk factors for development of PAS are: a narrow iridocorneal angle, use of mydriatics, and presence of posterior synechiae. The underlying mechanism is apparently precipitation of inflammatory cells and debris in the iridocorneal angle, leading to the formation of bridges between the peripheral iris and the sclera, resulting in the formation of synechiae.

Trabecular toxicity of glaucoma medications

Since glaucoma is a chronic pathology, it is important to consider the consequences of exposure to drops and their preservatives which are administered for the long term in these patients, especially benzalkonium chloride (BAK), which is the most widely utilized preservative in multidose glaucoma drops and which has shown toxic, pro-oxidant and proinflammatory effects on the ocular surface as well as the interior of the eye, particularly in the trabecular meshwork [START_REF] Baudouin | Ocular surface inflammatory changes induced by topical antiglaucoma drugs: human and animal studies[END_REF]. In fact, Brignole-Baudouin et al. have demonstrated in rabbits, with mass spectrometry imaging, that BAK may penetrate into the trabecular meshwork after repeated ocular instillation [START_REF] Brignole-Baudouin | A new safety concern for glaucoma treatment demonstrated by mass spectrometry imaging of benzalkonium chloride distribution in the eye, an experimental study in rabbits[END_REF]. Baudouin et al. have also shown that BAK induces apoptosis, a stress oxidant, and expression of the cytokine, fractalkine. In addition, BAK participates in the cleavage of SDF1 (CXCL12) into a truncated form, SDF-1(5-67); SDF1 has protective effects via its classic GPCR receptor, CXCR4 and its truncated form are apparently responsible for cell death through involvement of another GPCR, CXCR3.

The same team also demonstrated a pro-apoptotic effect of BAK, with inhibition of Bc12, on cultured human TMC [START_REF] Hamard | In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells[END_REF][START_REF] Baudouin | In vitro and in vivo experimental studies on trabecular meshwork degeneration induced by benzalkonium chloride (an American Ophthalmological Society thesis)[END_REF][START_REF] Denoyer | CXCR3 antagonism of SDF-1(5-67) restores trabecular function and prevents retinal neurodegeneration in a rat model of ocular hypertension[END_REF].

BAK also induces a pro-inflammatory effect, with increased expression of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 (CXCL8) in a three-dimensional trabecular meshwork model [START_REF] Bouchemi | Effect of benzalkonium chloride on trabecular meshwork cells in a new in vitro 3D trabecular meshwork model for glaucoma[END_REF]. This work thus suggests that BAK aggravates all the characteristics of trabecular degeneration described in glaucoma: trabecular cell apoptosis, stress oxidants and induction of inflammatory chemokines.

The trabecular meshwork as therapeutic target

The only currently proven treatment strategy aimed at slowing the progression of glaucomatous optic neuropathy consists of lowering IOP [START_REF] Kass | The Ocular Hypertension Treatment Study: a randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma[END_REF][START_REF] Heijl | Reduction of intraocular pressure and glaucoma progression: results from the Early Manifest Glaucoma Trial[END_REF][START_REF]The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration.The AGIS Investigators[END_REF]. This relies on medical, physical (such as lasers) or surgical approaches. The trabecular meshwork, as the main site of aqueous outflow resistance, is therefore widely targeted in glaucoma treatment.

Medical approach

While trabecular degeneration appears to be at the origin of glaucoma, medications acting directly on the trabecular meshwork are rare. Among medical glaucoma treatments, prostaglandin analogs are the most utilized, most effective, and best tolerated medications. The pressure lowering mechanism of action of the prostaglandins is still not perfectly understood. It appears to be mainly due to an increase in aqueous outflow through the uveoscleral pathway. However, more recent results might suggest a direct action of prostaglandins on the trabecular meshwork, with a remodeling of the trabecular ECM [START_REF] Weinreb | Matrix Metalloproteinases and Glaucoma Treatment[END_REF][START_REF] Winkler | Effects of prostaglandin analogues on aqueous humor outflow pathways[END_REF][START_REF] Bahler | Prostaglandins Increase Trabecular Meshwork Outflow Facility in Cultured Human Anterior Segments[END_REF]. Kalouche et al. have shown that latanoprost, an analog of prostaglandin F2α, decreased trabecular ECM collagen accumulation but favored a contractile cell phenotype. However, butaprost, a prostanoid EP2 receptor antagonist, decreased both contraction of TMC and collagen deposition, thus inhibiting myofibroblastic cell transformation [START_REF] Kalouche | Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells[END_REF]. The concept of medications acting directly on the trabecular cytoskeleton has thus been proposed.

Rho-kinase (ROCK) inhibitors constitute a particularly interesting example, since they accurately target the profibrotic pathway which results in the trabecular ECM changes. The Rho/ROCK signal pathway plays an important role indeed in the modulation of the cytoskeleton and synthesis of the ECM [START_REF] Amano | Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity[END_REF]. The Rho family includes small proteins that bind to guanosine triphosphate (GTP), which regulate the shape, motility, proliferation and apoptosis of cells throughout the body. As is shown in the schematic in figure 6, after binding of TGF-β2 to its receptor, Rho-GTP activates its effector molecules (Rho-kinase ROCK 1 and 2). The ROCKs thus inhibit myosin light chain phosphatase by phosphorylating the myosinbinding subunit, thus inducing changes in the actin cytoskeleton. The ROCKs activate LIM kinases via phosphorylation, which stabilizes filamentous actin and reduces cellular migration. Activation of this pathway leads to increased aqueous outflow resistance, while inhibition of it decreases the IOP [START_REF] Wang | Rho/Rho-associated kinase pathway in glaucoma (Review)[END_REF][START_REF] Pattabiraman | Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells[END_REF]. The Rho-kinase inhibitors represent a new treatment strategy in glaucoma, targeting the TGF-β pathway that causes ECM changes [START_REF] Torrejon | TGFβ2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor[END_REF]. A ROCK inhibitor, Ripasudil, has been approved by the Japanese health authorities for the treatment of glaucoma or ocular hypertension, as second-line therapy after standard treatment. The first study published on the efficacy of Ripasudil shows moderate efficacy on IOP, going from 15.1 ± 4.6 mmHg prior to treatment to 13.3 ± 3.0 mmHg at 24 months (p<0.05) [START_REF] Maruyama | Safety and Efficacy of Long-Term Ripasudil 0.4% Instillation for the Reduction of Intraocular Pressure in Japanese Open-Angle Glaucoma Patients[END_REF]. Another molecule in the same family, Netarsudil 0.02% (Rhopressa®, Aerie Pharmaceuticals, Inc., USA), received FDA approval in December 2017 for lowering IOP in patients with POAG [89] and European market approval in late 2019. In three phase III trials in patients with elevated IOP, the ocular hypotensive efficacy of Netarsudil 0.02% instilled once a day met criteria of non-inferiority compared to timolol 0.5% instilled twice a day [START_REF] Kopczynski | Netarsudil ophthalmic solution 0.02% for the treatment of patients with open-angle glaucoma or ocular hypertension[END_REF]. One may nonetheless be bothered by frequent adverse effects: blepharitis, conjunctival hyperemia, subconjunctival hemorrhages, cornea verticillata type corneal opacities [START_REF] Maruyama | Safety and Efficacy of Long-Term Ripasudil 0.4% Instillation for the Reduction of Intraocular Pressure in Japanese Open-Angle Glaucoma Patients[END_REF][START_REF] Serle | Two Phase 3 Clinical Trials Comparing the Safety and Efficacy of Netarsudil to Timolol in Patients With Elevated Intraocular Pressure: Rho Kinase Elevated IOP Treatment Trial 1 and 2 (ROCKET-1 and ROCKET-2)[END_REF][START_REF] Sakamoto | Evaluation of offset of conjunctival hyperemia induced by a Rho-kinase inhibitor; 0.4% Ripasudil ophthalmic solution clinical trial[END_REF]. The combination of a ROCK inhibitor with a prostaglandin has been proposed and appears to obtain synergistic results on IOP lowering, with IOP reductions of 30% or more seen in 59 to 65% of subjects treated with the combination of netarsudil and latanoprost, vs. 29 to 37% of subjects treated with latanoprost alone and 21 to 29% of subjects treated with netarsudil alone (p<0.0001) [START_REF] Asrani | Fixed-Dose Combination of Netarsudil and Latanoprost in Ocular Hypertension and Open-Angle Glaucoma: Pooled Efficacy/Safety Analysis of Phase 3 MERCURY-1 and -2[END_REF][START_REF] Radell | Netarsudil/latanoprost fixed-dose combination for the treatment of open-angle glaucoma or ocular hypertension[END_REF].

Laser treatment

Selective laser trabeculoplasty (SLT), described by Latina and Park in 1995 [START_REF] Latina | Selective targeting of trabecular meshwork cells: in vitro studies of pulsed and CW laser interactions[END_REF], is performed with a pulsed, frequency-doubled (Q-switched) Nd:YAG laser. It has been called "selective" due to its targeting of pigmented TMC while leaving the trabecular meshwork intact. The increase in aqueous outflow from the eye after an SLT may be explained by several mechanisms, notably mechanical traction on the uveoscleral trabecular meshwork and SC, cellular mechanisms which stimulate cell division, and biochemical mechanisms which alter cytokines and stimulate the macrophage properties of the TMC [START_REF] Alvarado | A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells[END_REF]. A histologic study of eyes which had undergone SLT showed only minimal mechanical damage [START_REF] Kramer | Comparison of the morphologic changes after selective laser trabeculoplasty and argon laser trabeculoplasty in human eye bank eyes[END_REF]. The intraocular pressure (IOP) lowering effect of SLT may thus be explained above all by biochemical and cellular changes more so than mechanical effects [START_REF] Latina | Selective laser trabeculoplasty[END_REF].

Surgical treatment

Since elevated IOP stems from a change in aqueous outflow, surgical glaucoma treatments aim to improve outflow through existing physiologic pathways or to divert the aqueous through new non-physiologic pathways (such as subconjunctival outflow).

Filtering surgeries

For IOP lowering, the surgeries most often performed in France are trabeculectomy [START_REF] Landers | A twenty-year follow-up study of trabeculectomy: risk factors and outcomes[END_REF] and non-penetrating deep sclerectomy (NPDS) [START_REF] Hamard | Non penetrating filtering surgery, evolution and results[END_REF]. These are both filtering surgeries, the principle of which is to create a pathway for aqueous outflow from the anterior chamber of the eye to a subconjunctival space (the filtering bleb) by bypassing all or part of the trabecular meshwork. Trabeculectomy consists of the en bloc removal of full thickness trabecular meshwork. It offers IOP lowering of approximately 46 to 51% at 2 years according to studies [START_REF] Kirwan | Trabeculectomy in the 21st Century: A Multicenter Analysis[END_REF][START_REF] Gedde | Treatment Outcomes in the Primary Tube Versus Trabeculectomy Study after 1 Year of Follow-up[END_REF][START_REF] Cillino | Deep sclerectomy versus trabeculectomy with low-dosage mitomycin C: four-year follow-up[END_REF]. The NPDS technique was developed to minimize the complications of trabeculectomy. It consists of the selective removal of the outer trabecular membrane (juxtacanalicular trabecular meshwork and inner wall of SC), where the main component of increased aqueous outflow resistance is located. Cillino et al. [START_REF] Cillino | Deep sclerectomy versus trabeculectomy with low-dosage mitomycin C: four-year follow-up[END_REF] compared IOP lowering at one year between trabeculectomy and NPDS and found a decrease of 51% for trabeculectomy and 42.5% for NPDS. The rates of complications such as hypotony (38.1% vs. 0%) and hypothalamia (33.3% vs. 5.2%) were significantly lower in the NPDS group.

Trabecular MIGS In the last several years, the options for surgical management of glaucoma have multiplied, in particular with the appearance of MIGS, or minimally invasive glaucoma surgeries. With these techniques, the goal of IOP lowering is more modest than with classic filtering surgery, but with lower risk of complications and a more rapid visual recovery. They are most often performed in association with cataract surgery. The MIGS utilize various pathways to encourage aqueous outflow: trabecular, suprachoroidal or subconjunctival. The trabecular devices aim to re-establish the natural pathway for aqueous outflow from the anterior chamber to SC. These procedures are based on the fact that the juxtacanalicular portion is the site of greatest resistance to aqueous outflow in the majority of open angle glaucoma patients. The trabecular MIGS increase trabecular outflow by one or more of the four following mechanisms: 1. Trabecular bypass and direct communication between the anterior chamber and SC by way of a stent; 2. Maintenance of the lumen of SC; 3. Dilation of the collector channels; 4. Surgical opening of the inner wall of the canal [START_REF] Andrew | A review of aqueous outflow resistance and its relevance to microinvasive glaucoma surgery[END_REF]. The iStent and iStent Inject (Glaukos Corp., San Clemente, CA) are trabecular stents which act by locally bypassing the trabecular meshwork to empty into SC [START_REF] Lavia | Minimally-invasive glaucoma surgeries (MIGS) for open angle glaucoma: A systematic review and meta-analysis[END_REF][START_REF] Wellik | A review of the iStent(®) trabecular micro-bypass stent: safety and efficacy[END_REF]. A limitation of this approach is that the stent may not necessarily be positioned near one of the 25 to 30 collector channels of SC. In addition, certain collector channels may be more active than others. The Hydrus Microstent (Ivantis Inc., Irvine, CA) is an 8 mm long stent which is implanted in the nasal portion of SC. It acts by both opening and stretching the trabecular meshwork. It thus holds the SC lumen open [START_REF] Pfeiffer | A Randomized Trial of a Schlemm's Canal Microstent with Phacoemulsification for Reducing Intraocular Pressure in Open-Angle Glaucoma[END_REF]. Excimer Laser Trabeculotomy (ELT) allow the creation of full thickness openings in the trabecular meshwork and inner wall of SC so as to decrease trabecular resistance [START_REF] Andrew | A review of aqueous outflow resistance and its relevance to microinvasive glaucoma surgery[END_REF]. The Kahook Dual Blade (New World Medical Inc, Rancho Cucamonga, CA) works by incising the trabecular meshwork and inner wall of SC to open 90° of the trabecular meshwork [START_REF] Dorairaj | A multicenter retrospective comparison of goniotomy versus trabecular bypass device implantation in glaucoma patients undergoing cataract extraction[END_REF]. The Trabectome (NeoMedix Corp., Tustin, CA) is a handpiece which makes it possible to perform an electrosurgical circumferential trabeculotomy for 60 to 120° of the trabecular meshwork and inner wall of SC [START_REF] Andrew | A review of aqueous outflow resistance and its relevance to microinvasive glaucoma surgery[END_REF]. Ab interno canaloplasty aims to dilate SC by injecting viscoelastic device through a microcatheter introduced ab interno through a goniotomy. It thus creates a microperforation in the trabecular meshwork which may increase its permeability [START_REF] Körber | Canaloplasty ab interno -a Minimally Invasive Alternative[END_REF]. Gonioscopy-assisted Transluminal Trabeculotomy (GATT) aims to insert the same illuminated catheter used for ab interno canaloplasty into SC, but, once the catheter has been fed through the entire 360°, the two ends of the catheter are externalized to create a complete ab interno trabeculotomy [START_REF] Grover | Gonioscopy-assisted transluminal trabeculotomy, ab interno trabeculotomy: technique report and preliminary results[END_REF]. The success of these trabecular MIGS may also be limited by postoperative scarring at the SC site. Finally, these procedures reduce juxtacanalicular resistance but do not reduce the distal sites more resistant to flow, such as elevated episcleral venous pressure, which may be more significant in certain patients. The postoperative IOP result after these procedures does not decrease below the episcleral venous pressure. This is why the efficacy of trabecular techniques is generally less than that of subconjunctival filtration techniques, which create a non-physiologic pathway for aqueous outflow [START_REF] Richter | Minimally invasive glaucoma surgery: current status and future prospects[END_REF].

Perspectives New techniques under investigation

In clinical practice, examination of the trabecular meshwork relies on gonioscopy. The trabecular meshwork has a translucent appearance and is often dull gray or brown (figure 7a). The anterior portion of the trabecular meshwork is generally less pigmented and is considered the non-filtering portion of the meshwork. The posterior portion is larger in size and covers SC. The trabecular meshwork is pigmented and can accumulate pigment with age and specific ocular diseases such as pigment dispersion or pseudoexfoliation. Trauma, uveitis and surgery, including laser (iridotomy in particular) are also causes of pigment deposition in the angle. Clinical gonioscopy permits an appreciation of the openness of the angle, the amount of pigmentation, and anatomic features such as iris processes. New techniques of clinical investigation of the trabecular meshwork are being developed to more precisely image this key structure. Indeed, a better resolution, on the order of microns, would aid in understanding the changes brought on by aging of the trabecular meshwork and would allow evaluation of glaucoma medications targeting the trabecular meshwork so as to improve evaluation of the results and causes of failure of classic or minimally invasive glaucoma surgeries (MIGS). Optical coherence tomography (OCT) of the anterior segment provides certain details, but represents only a crude approach to the trabecular meshwork and SC (figure 7b). OCT coupled with gonioscopy offers resolution superior to goniophotography and also allows a more in depth investigation [START_REF] Perinchery | High resolution iridocorneal angle imaging system by axicon lens assisted gonioscopy[END_REF][START_REF] Yasuno | Visibility of trabecular meshwork by standard and polarization-sensitive optical coherence tomography[END_REF]. Thus, King et al. have developed a high-resolution imaging system based on an adaptive optics scanning laser ophthalmoscopic (AOSLO) system, originally conceived for retinal imaging, then coupled to a gonioscopic lens. This provides imaging of the trabecular meshwork on a micron scale in vivo in humans. The images obtained show the trabecular beams and endothelial cells [START_REF] King | High-Resolution, Adaptive Optics Imaging of the Human Trabecular Meshwork In Vivo[END_REF]. The development of this technique could allow direct in vivo measurements, on a micron scale, of the changes occurring in the human trabecular meshwork in glaucoma as well as after a therapeutic procedure.

Trabecular protection strategy

Oxidative stress induces damage to the trabecular meshwork and is one of the pathophysiologic mechanisms at the origin of glaucoma [START_REF] Zhao | Oxidative stress in the trabecular meshwork (Review)[END_REF].

Regulation of the oxidation/reduction balance thus constitutes a therapeutic approach, in the form of trabecular protection to prevent glaucoma [START_REF] Rao | Mitochondrial-Targeted Antioxidants Attenuate TGF-β2 Signaling in Human Trabecular Meshwork Cells[END_REF][START_REF] Lin | Curcumin Protects Trabecular Meshwork Cells From Oxidative Stress[END_REF]. Kalouche et al. have shown that activation of the E prostanoid sub-type 2 (EP2) receptors protects the endoplasmic reticulum from apoptosis induced by stress through a down-regulation of p53 [START_REF] Kalouche | Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53[END_REF]. Another therapeutic approach has been suggested by Denoyer et al. [START_REF] Denoyer | CXCR3 antagonism of SDF-1(5-67) restores trabecular function and prevents retinal neurodegeneration in a rat model of ocular hypertension[END_REF]: blockage of the interaction between the truncated form of CXCL12 (SDF1(5-67)) and CXCR3, which causes an induction of apoptosis in TMC. Treatment with a CXCR3 antagonist in a rat ocular hypertension model reduced IOP. Inhibition of this pathway might constitute an innovative therapeutic approach to restore trabecular function in patients with POAG.

Cell therapy

The decrease in number of TMC due to age and disease, accelerated apoptosis and senescence are associated with an increased resistance to aqueous outflow and thus an increase in IOP [START_REF] Liton | Cellular senescence in the glaucomatous outflow pathway[END_REF]. Due to their phagocytotic properties, TMC have the ability to eliminate debris potentially obstructing the trabecular filter, while they have a role in the synthesis and degradation of the components of the ECM [START_REF] Stamer | The many faces of the trabecular meshwork cell[END_REF]. The reduction in trabecular cell density thus affects renewal of the ECM and provokes the accumulation of debris, leading to increased outflow resistance in glaucomatous eyes. Theoretically, recolonization of the trabecular meshwork from stem cells could compensate for the cell loss in glaucomatous eyes and allow the trabecular meshwork to regain its function, thus reducing IOP [START_REF] Yun | Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure[END_REF][START_REF] Wang | Stem cells from trabecular meshwork cells can secrete extracellular matrix[END_REF].

Cell therapies based on stem cells for regeneration of the trabecular meshwork provide promising treatment perspectives. The trabecular stem cells are located in the region of the insertion of the trabecular meshwork into Schwalbe's line [START_REF] Kelley | Stem cells in the trabecular meshwork: present and future promises[END_REF]. They can be isolated by cell sorting, monoclonal or sphere culture. Trabecular stem cells are multipotent, with the ability to colonize the trabecular region and differentiate into TMC in vivo. Other types of stem cells, such as adipocyte-derived stem cells, mesenchymal stem cells and induced pluripotent stem cells have been discovered for differentiation and regeneration of TMC [START_REF] Du | Multipotent stem cells from trabecular meshwork become phagocytic TM cells[END_REF]. Roubeix et al. have shown that, in the rat, injection of mesenchymal stem cells isolated from femoral bone marrow into the anterior chamber decreased IOP in an ocular hypertension model created by cauterization of the episcleral veins, and demonstrated a protective effect on the trabecular meshwork. They essentially observed an increase in trabecular cell survival with activation of the anti-apoptotic Akt pathway, relaxation of the TMC, and inhibition of the profibrotic phenotype induced by TGF-β2 [START_REF] Roubeix | Intraocular pressure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma[END_REF].

Another therapeutic approach is that of orientation of induced pluripotent stem cells (iPSC). Ding et al. have succeeded in obtaining iPSC cells with a phenotype close to that of human TMC after placing them in co-culture with human TMC [START_REF] Ding | Induction of Trabecular Meshwork Cells From Induced Pluripotent Stem Cells[END_REF]. Transplantation of these cells into perfused human anterior segments or into animal models stimulated proliferation of the endogenous TMC [START_REF] Zhu | Transplantation of iPSC-TM stimulates division of trabecular meshwork cells in human eyes[END_REF] and restored aqueous outflow, thus lowering IOP [START_REF] Zhu | Restoration of Aqueous Humor Outflow Following Transplantation of iPSC-Derived Trabecular Meshwork Cells in a Transgenic Mouse Model of Glaucoma[END_REF]. This type of iPSC based therapy is a promising strategy for IOP regulation in glaucoma patients [START_REF] Castro | Trabecular Meshwork Regeneration -A Potential Treatment for Glaucoma[END_REF].

Conclusion

The trabecular meshwork is a complex structure central to the pathophysiology of glaucoma. A deeper knowledge of the mechanisms at the origin of its degeneration and a better visualization of its architectural changes may change both the medical and surgical management of glaucoma.

  : a. Schematic representation of aqueous flow within the anterior segment of the eye. (Rapport SFO 2014 : Glaucome primitif à angle ouvert J-P Renard [10]) b. Schematic of trabecular meshwork organization.

Figure 2 :

 2 Human trabecular meshwork cells (TMC). The actin cytoskeleton is stained with phalloidin (red) and the nucleus with DAPI (blue). a. stellate morphology b. spindle morphology (200X).

Figure 3 :

 3 Figure 3: Human trabecular meshwork cells (TMC) within their extracellular matrix (ECM). The ECM is stained with an anti-fibronectin antibody (green) and the nuclei are stained with DAPI (blue) (100X magnification).

Figure 4 :

 4 Figure 4: a. Confocal microscopic image of normal trabecular meshwork: the nuclei are stained red, elastin shows a mild green autofluorescence allowing visualization of the ECM, and the cells of the cribriform trabecular meshwork have a dendritic appearance and are strongly fluorescent after staining with an anti-vimentin antibody. b. Glaucomatous trabecular meshwork on confocal microscopy: significant rarefaction of the trabecular meshwork cells (red) and major increase in ECM (autofluorescent green fibers) in the corneoscleral meshwork.

Figure 5 :

 5 Figure 5: Cross-linked Actin Networks (CLANs) within human trabecular meshwork cells. Cytoskeletal rearrangement with interconnection of actin fibers leading to a change in cellular shape and rigidity. The actin cytoskeleton is stained with an anti-alpha-smooth muscle actin antibody (green) and the nucleus with DAPI (blue) (20X).

Figure 6 :

 6 Figure 6: Rho-kinase signal pathway. The activated TGF β receptor (TRF-β RI-II) binds guanosine triphosphate (GTP) to the protein Rho. Rho-GTP activates its effector molecules, the ROCKs (Rho-kinases ROCK 1 and 2). The ROCKs inhibit myosin light chain phosphatase (MLCP). Phosphorylation of the binding subunit to myosin induces changes in the actin cytoskeleton. The ROCKs also activate LIM kinases, which phosphorylate cofilin, which stabilizes actin.

Figure 7 :

 7 Figure 7: a. Goniophotograph of the iridocorneal angle. The anterior portion of the trabecular meshwork (ant. TM) is generally less pigmented; the posterior portion (post. TM) is larger in size and covers Schlemm's canal. The structures are only visible as bands

Table 1 : Phenotypes and properties of trabecular meshwork cells (TMC).

 1

		Location	Cellular behavior	Role
	Endothelial	Corneoscleral and	Endothelium	Maintenance of
		uveal portions		permeability
	Round to oval,			Neutralization of reactive
	large cell body			oxygen species
			Macrophage	Biologic filter/
				phagocytosis
				Immune mediation
	Fibroblastic	Juxtacanalicular	Fibroblast	ECM renewal/
				tissue repair
	Elongated shape		Smooth muscle cell	Contractility
				Mechanotransduction


Fatma Kort

Laure Fournier

Le Ray

Aurélie Chauffour

Vincent Jarlier

Nacer Lounis

Koen Andries

Alexandra Aubry

Lorenzo Guglielmetti

Nicolas Veziris
email: nicolas.veziris@sorbonne-universite.fr

Laure Fournier Le Ray

Fully weekly antituberculosis regimen: a proof-of-concept study

Keywords: Tuberculosis, Bedaquiline, Rifapentine, Directly Observed Therapy, Compliance

come

INTRODUCTION

The treatment of drug-susceptible tuberculosis (TB) is based on a four-drug combination given for 6 months that has not changed for the last 40 years despite evidence that it was not optimally designed [START_REF] Van Ingen | Why Do We Use 600 mg of Rifampicin in Tuberculosis Treatment?[END_REF]. The complexity and length of this regimen limits treatment compliance and this can be the cause of treatment failure and emergence of drug resistance. Indeed, a study has shown that, in a daily regimen, ≤90% adherence significantly increases the rate of unfavorable outcome [START_REF] Imperial | A patient-level pooled analysis of treatment-shortening regimens for drug-susceptible pulmonary tuberculosis[END_REF][START_REF] Srivastava | Pharmacokinetic Mismatch Does Not Lead to Emergence of Isoniazid-or Rifampin-Resistant Mycobacterium tuberculosis but to Better Antimicrobial Effect: a New Paradigm for Antituberculosis Drug Scheduling ▿[END_REF].

In recent years, multiple clinical trials have been conducted in order to reduce the duration of TB treatment. These efforts have not been successful with fluoroquinolones [START_REF] Gillespie | Four-month moxifloxacin-based regimens for drug-sensitive tuberculosis[END_REF][START_REF] Merle | OFLOTUB/Gatifloxacin for Tuberculosis Project. A four-month gatifloxacincontaining regimen for treating tuberculosis[END_REF][START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF] and are currently ongoing with daily rifapentine and high-dose rifampin [START_REF] Boeree | High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis: a multi-arm, multi-stage randomised controlled trial[END_REF][START_REF] Dorman | Daily rifapentine for treatment of pulmonary tuberculosis. A randomized, dose-ranging trial[END_REF][START_REF] Mourik | Improving treatment outcome assessment in a mouse tuberculosis model[END_REF], as well as with new drugs such as bedaquiline and pretomanid [START_REF] Dawson | Efficiency and safety of the combination of moxifloxacin, pretomanid (PA-824), and pyrazinamide during the first 8 weeks of antituberculosis treatment: a phase 2b, open-label, partly randomised trial in patients with drug-susceptible or drug-resistant pulmonary tuberculosis[END_REF][START_REF] Tweed | Bedaquiline, moxifloxacin, pretomanid, and pyrazinamide during the first 8 weeks of treatment of patients with drug-susceptible or drug-resistant pulmonary tuberculosis: a multicentre, open-label, partially randomised, phase 2b trial[END_REF]. However, reducing the duration of treatment is not the only way to simplify the treatment and its supervision. Another approach is to space out drug administration.

Current treatment regimens are given either daily or twice-or thrice-weekly. These intermittent regimens are based on old studies and may not be as active as the daily one. Efforts have been conducted in order to develop once-weekly regimens based on drugs with a long half-life [START_REF] Grosset | Onceweekly rifapentine-containing regimens for treatment of tuberculosis in mice[END_REF][START_REF] Lenaerts | Evaluation of rifapentine in long-term treatment regimens for tuberculosis in mice[END_REF][START_REF] Dhillon | Activity of two longacting rifamycins, rifapentine and FCE 22807, in experimental murine tuberculosis[END_REF]. Some of these studies have been designed and conducted based on the use of a long half-life rifamycin, rifapentine. The first studies have not been successful because rifapentine was combined with isoniazid, a drug with short half-life. Thus, the once-weekly combination of isoniazid and rifapentine was indeed a rifapentine monotherapy for 6 days out of 7 and has led to treatment failure with selection of rifamycin-resistant mutants [START_REF] Vernon | Acquired rifamycin monoresistance in patients with HIV-related tuberculosis treated with onceweekly rifapentine and isoniazid. Tuberculosis Trials Consortium[END_REF]. In order to avoid this risk, rifapentine should be combined with at least one companion drug with a long half-life, or be used only in cases with low bacillary burden to reduce the risk of treatment failure and selection of drug resistant mutants. Two different studies have demonstrated the efficacy of such strategies. In the first one, a randomized controlled clinical trial, an experimental arm containing rifapentine combined with moxifloxacin once-weekly during the 4 months of treatment continuation achieved non-inferiority compared to the daily control regimen [START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF]. In a second clinical trial, a rifapentineisoniazid weekly combination was successfully used for the treatment of latent TB infection [START_REF] Sterling | Three months of rifapentine and isoniazid for latent tuberculosis infection[END_REF][START_REF] Migliori | ERS/ECDC Statement: European Union standards for tuberculosis care, 2017 update[END_REF][START_REF] Rosales-Klintz | Guidance for programmatic management of latent tuberculosis infection in the European Union/European Economic Area[END_REF]. Bedaquiline is a new anti-TB drug which has been mainly evaluated for the treatment of drug-resistant TB [START_REF] Vasilyeva | on of the TMC207TBC3001 study group. Early access to bedaquiline for extensively drug-resistant (XDR) and pre-XDR tuberculosis[END_REF][START_REF] Ndjeka | High treatment success rate for multidrug-resistant and extensively drug-resistant tuberculosis using a bedaquiline-containing treatment regimen[END_REF][START_REF] Migliori | European Union standard for tuberculosis care on treatment of multidrug-resistant tuberculosis following new World Health Organization recommendations[END_REF][START_REF] Borisov | Effectiveness and safety of bedaquiline-containing regimens in the treatment of MDR-and XDR-TB: a multicentre study[END_REF]. However, it has a very long half-life and could thus be a good candidate for a once-weekly regimen in combination with rifapentine, despite the known drug-drug interactions between bedaquiline and rifamycins. Since for both bedaquiline and rifapentine the main parameter predicting efficacy is the area under curve (AUC), a weekly regimen should be as active as daily one given that the weekly AUC is the same [START_REF] Rouan | Pharmacokinetics and Pharmacodynamics of TMC207 and Its N-Desmethyl Metabolite in a Murine Model of Tuberculosis[END_REF][START_REF] Sirgel | The early bactericidal activities of rifampin and rifapentine in pulmonary tuberculosis[END_REF][START_REF] Jayaram | Pharmacokineticspharmacodynamics of rifampin in an aerosol infection model of tuberculosis[END_REF].

We previously demonstrated in a murine model of TB that once-weekly regimens based on the rifapentine-bedaquiline combination have higher bactericidal activity than the standard daily regimen after 1 and 2 months [START_REF] Veziris | A Once-Weekly R207910-containing Regimen Exceeds Activity of the Standard Daily Regimen in Murine Tuberculosis[END_REF]. The objective of the present study was to evaluate the sterilizing activity of such regimens over the whole duration of treatment.

METHODS

Antimicrobial Agents

Bedaquiline was provided by Janssen. The other compounds were purchased from the following manufacturers: rifampin from Sandoz, isoniazid from Galien, pyrazinamide from Sanofi, moxifloxacin from Eurogenerics, clofazimine from Sigma and rifapentine from Carbosynth.

Drugs were diluted in water containing 0.05% agar, except for bedaquiline which was diluted in aqueous 20% 2-hydroxypropyl-β-cyclodextrin.

Drugs suspensions were prepared weekly except for rifampin (once every two weeks) and bedaquiline (once for the whole experiment), and were stored at 4°C.

Ethics

The study was reviewed by ethical committee Charles Darwin and conducted under approval of Ministère de l'Enseignement Supérieur de la Recherche et de l'Innovation N° 123802017112809414820v3.

Mycobacterium tuberculosis strain

The H37Rv strain of M. tuberculosis was grown on Lowenstein-Jensen medium. This strain was obtained from a lung of an untreated mouse of a previous experiment stored at -80°C [START_REF] Poissy | Should moxifloxacin be used for the treatment of extensively drugresistant tuberculosis? An answer from a murine model[END_REF]. Minimal inhibitory concentrations of drugs used in this experiment are presented in table S1.

Colonies were subcultured in Dubos broth plus 10% oleic acid-albumin-dextrosecatalase (OADC) for 7 days at 37°C. The turbidity of the resulting suspension was adjusted with normal saline to match that of standard 1 mg/ml suspension of Mycobacterium bovis BCG and was further diluted with normal saline to obtain a suspension for mouse inoculation containing 2.06x10 6 Colony Forming Unit (CFU)/ml.

Intravenous infection

Three hundred six-week-old Swiss female mice were purchased from the Janvier Breeding Center. They were intravenously infected in the tail vein with 0.5 ml of bacterial suspension containing 1.03x10 6 CFU of Mycobacterium tuberculosis H37Rv. Intravenous infection has been historically used in the laboratory and has shown a better correlation with relapses in clinical trials than aerosol infection ( [START_REF] Ibrahim | Sterilizing activity of R207910 (TMC207)-containing regimens in the murine model of tuberculosis[END_REF][START_REF] Nuermberger | Moxifloxacin-containing regimens of reduced duration produce a stable cure in murine tuberculosis[END_REF]).

Chemotherapy

After infection, mice were randomized into eight groups (Table 1). The negative control group contained 40 untreated mice, of which 10 were sacrificed the day after infection (D-27), 20 were sacrificed at the beginning of the treatment (D0) and 10 were sacrificed at 2 months of treatment. In the positive control group, mice received the daily standard regimen of drug-susceptible TB: 2 months of rifampin (R), isoniazid (H) and pyrazinamide (Z) followed by 2 or 4 months of R and H. In the experimental groups mice were treated once-weekly with: a) bedaquiline (B), rifapentine (P) and pyrazinamide (Z) (BPZ); b) BPZ plus moxifloxacin (BPZM); c) BPZM plus clofazimine (BPZMC). Each experimental regimen was tested for two treatment durations: 4 and 6 months.

Treatment began 4 weeks after infection (D0). Drugs were administrated by gavage, either 5 days per week or once-weekly, at the following dosing: R 10mg/kg, H 25mg/kg, Z 150mg/kg once-a-day (daily control) or 300mg/kg once-a-week (experimental regimens); B 62.5mg/kg; P 20mg/kg; M 200mg/kg; C 20mg/kg. R was administered one hour before the other drugs to avoid drug-drug interactions. Drug dosing were selected to provide areas under the curve (AUC) values in mice comparable with those achievable in human (table S2). For bedaquiline, since the AUC is the main parameter predictive of efficacy, a weekly dosing generates an efficacy equivalent to that of daily dosing given that the total weekly amount of drug is the same. As a consequence, and as demonstrated previously a 125 mg/kg dosing once-weekly generates the same bactericidal activity as a 25 mg/kg dosing 5 times a week [START_REF] Rouan | Pharmacokinetics and Pharmacodynamics of TMC207 and Its N-Desmethyl Metabolite in a Murine Model of Tuberculosis[END_REF][START_REF] Veziris | A Once-Weekly R207910-containing Regimen Exceeds Activity of the Standard Daily Regimen in Murine Tuberculosis[END_REF]. The 25 mg/kg dose in the mouse generates an AUC of 19 mcg • h/ml, thus a 125 mg/kg dosing should generate an AUC around 100 mcg • h/ml. Since rifapentine reduces by 50% the AUC of bedaquiline in human and since this interaction does not exist in mouse, we used a half weekly dosing of bedaquiline in order to mimic the interaction seen in human [START_REF] Rosenthal | Dose-Ranging Comparison of Rifampin and Rifapentine in Two Pathologically Distinct Murine Models of Tuberculosis[END_REF][START_REF] Healan | Impact of Rifabutin or Rifampin on Bedaquiline Safety, Tolerability, and Pharmacokinetics Assessed in a Randomized Clinical Trial with Healthy Adult Volunteers[END_REF]. Such dosing should generate and AUC close to 50 mcg • h/ml after a single dose of bedaquiline which corresponds to 50% of the AUC of bedaquiline after a single dose of 700 mg of bedaquiline in human [START_REF] Van Heeswijk | Bedaquiline: a review of human pharmacokinetics and drug-drug interactions[END_REF]. Bedaquiline undergoes accumulation both in murine model and in human leading to a doubling of the AUC after multiple doses (tables S3 and S4).

Assessment of treatment efficacy

Treated mice were sacrificed through cervical elongation at 2 months and at the end of the treatment (4 or 6 months) to assess bactericidal activity. Survival rate, mean spleen weights, gross lung lesions and mean lung CFU were analyzed. Lung CFU were determined by plating three serial 10-fold dilutions of homogenized suspensions onto triplicate Lowenstein-Jensen slants that were incubated at 37°C for 6 weeks. Treated mice were sacrificed 3 months after treatment completion to assess sterilizing activity. Mean spleen weights, gross lung lesions and proportion of relapsing mice with positive lung culture (relapsing mice) were analyzed.

Statistical analysis

Mean spleen weights and lung CFU were compared using the Mann-Whitney's test.

Proportions of mice with a positive M. tuberculosis culture from lung tissue 3 months after the completion of treatment were compared using the Fisher's test. Mice with contaminated lung cultures were excluded from analysis.

Statistical tests have been performed with BioStaTGV (https://biostatgv.sentiweb.fr/).

Gene sequencing

Among mice relapsing 3 months after treatment completion in the experimental groups, gene sequencing was performed on colonies grown on Lowenstein-Jensen slants for Rv0678, atpE, rpoB, pncA, gyrA and gyrB genes as previously described (table S5).

RESULTS

Survival rates

Among untreated mice, 1/10 died on day 56, the others were sacrificed at 2 months. Among treated mice, some died of gavage accidents: 7/70 in BPZ group, 5/70 in BPZM group and 2/70 in BPZMC group. The proportion of gavage accidents was not different between daily and weekly groups, (p>0.05 Fischer's exact test).

Mean spleen weights

The day after infection (D-27), the mean spleen weight was 152 ± 20 mg and increased to 413 ± 250 mg four weeks later (D0). After 2 months of treatment, the mean spleen weight decreased, although not significantly so, across all treated groups.

The mean spleen weight did not differ significantly between mice treated 2 months and mice at the end of treatment (4 or 6 months), except between mice treated 2 months and mice treated 4 months in the BPZMC group (259 ± 76 v 164 ± 37, p= 0.006). The mean spleen weight was similar between mice sacrificed at 7 months (3 months after the 4-month treatment completion) and at 4 months. The mean spleen weight of mice sacrificed at 9 months (3 months after the 6-months treatment completion) was significantly lower than at 6 months in once-weekly regimen groups, but not in daily standard regimen group.

Bactericidal activity

The mean CFU count in the lungs was 2.30 ± 0.66 log10 the day after infection (D-27) and increased significantly to 4.60 ± 1.26 log10 at the beginning of the treatment (D0) (p=0.007) (Table 2). After 2 months of treatment, all infected and untreated mice were sacrificed and their mean lung CFU count was 6.03 ± 1.70 log10.

The bactericidal activity was significantly greater in the once-weekly regimen groups than in the daily standard regimen group after 2 months of treatment: the mean lung CFU count was 0.76 ± 0.60 log10 in the daily standard regimen group (with 6/9 positive mice), and negative in all mice in the BPZ, BPZM and BPZMC groups (p=0.008, p=0.005 and p=0.003 respectively as compared to daily control regimen).

On completion of the 4-month and 6-month treatments, all treated mice, either with the daily standard regimen or with the once-weekly regimens, had a negative lung culture.

Sterilizing activity

After 4 months of treatment and 3 months of follow-up, 9 out of 14 mice (64%) relapsed in the daily standard regimen group versus only 1 out of 19 mice (5%) in the BPZ group and none out of 20 mice in the BPZM and BPZMC groups, respectively (Figure). The mean lung CFU counts in the relapsing mice were 2.19 ± 0.75 log10 in the standard regimen group and 1.52 log10 in the positive mouse of the BPZ group.

The relapse rate 3 months after 4 months of once-weekly treatment with BPZ, BPZM and BPZMC was significantly lower than after 4 months of daily standard treatment (p<0.001) (table 2). After 6 months of treatment and 3 months of follow-up, 2 out of 15 mice (13%) relapsed in the daily standard regimen group versus 1 out of 19 (5%) in the BPZMC group, with 0.93 ± 1.31 log10 and 2.44 log10 lung CFU counts, respectively, and no relapse in the BPZ and BPZM groups. The difference in the relapse rate between 6 months of daily standard regimen and 6 months of weekly regimen with BPZ, BPZM or BPZMC (p= 0.32, p=0.32 and p=0.93, respectively), or between 6 months of daily standard regimen and 4 months of weekly regimen with BPZ, or BPZM or BPZMC (p= 0.57, p=0.18 and p=0.18, respectively), did not achieve statistical significance.

In the mouse that relapsed at 6 months of treatment and 3 months of follow-up in the BPZMC group, Rv0678, atpE, rpoB, pncA, gyrA and gyrB genes sequencing did not show any mutation.

Discussion

This study demonstrates, to our knowledge for the first time, that once-weekly 4-or 6-month regimens based on the bedaquiline-pyrazinamide-rifapentine combination are at least as active as the 6-month daily standard regimen of tuberculosis in a murine model.

In a previous experiment in this model, we showed that the bactericidal activity of bedaquiline is the same whatever the frequency of administration (once-weekly, twice-weekly or 5/week), provided that the total weekly dose is the same [START_REF] Veziris | A Once-Weekly R207910-containing Regimen Exceeds Activity of the Standard Daily Regimen in Murine Tuberculosis[END_REF].

However, in this previous study, we did not take into account the pharmacokinetic interaction between rifapentine and bedaquiline. Indeed, it has been shown later that rifapentine reduces bedaquiline AUC by 50% [START_REF] Winter | Evaluation of the Pharmacokinetic Interaction between Repeated Doses of Rifapentine or Rifampin and a Single Dose of Bedaquiline in Healthy Adult Subjects[END_REF]. As a consequence, in the current study we reduced the bedaquiline weekly dose by half, from 125 to 62.5 mg/kg, in order to mimic the AUC reduction seen in humans in presence of rifapentine and that does not exist in mouse [START_REF] Tasneen | Sterilizing Activity of Novel TMC207-and PA-824-Containing Regimens in a Murine Model of Tuberculosis▿[END_REF].

Despite this drug-drug interaction, we believe that there is a clear benefit in combining these two drugs. We showed previously that in a daily regimen, the most active combinations of bedaquiline included rifampin and pyrazinamide [START_REF] Ibrahim | Sterilizing activity of R207910 (TMC207)-containing regimens in the murine model of tuberculosis[END_REF]. A daily regimen of bedaquiline, pyrazinamide and rifapentine given for just 3 months resulted in a similar proportion of relapses as the standard regimen after 6 months [START_REF] Andries | Bactericidal potencies of new regimens are not predictive of their sterilizing potencies in a murine model of tuberculosis[END_REF]. As these drugs all have potent sterilizing activities, it is not surprising that their combination is very active.

In the current study, all three bedaquiline-rifapentine-pyrazinamide once-weekly groups showed superiority compared to standard daily regimen in terms of bactericidal activity at 2 months and of sterilizing activity at 4 months of treatment and 3 months of follow-up. At 6 months of treatment and 3 months of follow-up, the difference in bactericidal and sterilizing activity was not significant anymore, but at this point the relapse rate of the control regimen was low (13%). Showing a statistically significant difference might require a higher number of individuals per arm. In addition, it must be underlined that the relapse rate of the once-weekly test regimens after 4 months was not different from that of the control regimen after 6 months. It is therefore possible that a bedaquiline-rifapentine-pyrazinamide regimen may reduce both the frequency of drug administration and the total duration of treatment.

Based on previous studies, we hypothesized that the addition of clofazimine and moxifloxacin could increase the sterilizing activity of the bedaquiline-rifapentinepyrazinamide combination [START_REF] Tasneen | Sterilizing Activity of Novel TMC207-and PA-824-Containing Regimens in a Murine Model of Tuberculosis▿[END_REF][START_REF] Williams | Sterilizing Activities of Novel Combinations Lacking First-and Second-Line Drugs in a Murine Model of Tuberculosis[END_REF][START_REF] Veziris | Efficient Intermittent Rifapentine-Moxifloxacin-Containing Short-Course Regimen for Treatment of Tuberculosis in Mice[END_REF]. In our study, no difference was shown in the groups treated with BPZ plus M and MC. This might be partially explained by the high sterilizing activity of the BPZ regimen. At least 80 mice per group and per time-point sacrifice would be required in order to show a 20% statistical difference. Similarly, in another study testing daily treatment with BPZ, the addition of M did not increase the sterilizing activity of the regimen [START_REF] Andries | Bactericidal potencies of new regimens are not predictive of their sterilizing potencies in a murine model of tuberculosis[END_REF].

Based on our knowledge of the anti-TB chemotherapy, an initial "intensive phase" with daily treatment could be warranted before switching to once-weekly dosing.

Such daily intensive phase could rapidly reduce the bacillary load. This is unlikely to occur with a once-weekly bedaquiline-based regimen, given the delayed early bactericidal activity exerted by bedaquiline [START_REF] Rustomjee | Early Bactericidal Activity and Pharmacokinetics of the Diarylquinoline TMC207 in Treatment of Pulmonary Tuberculosis[END_REF][START_REF] Diacon | 14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial[END_REF]. In our opinion, moxifloxacin could be included in such a clinical trial regimen, despite the lack of additive effect shown in our study. Indeed, moxifloxacin has demonstrated early bactericidal activity [START_REF] Gillespie | Early bactericidal activity of a moxifloxacin and isoniazid combination in smearpositive pulmonary tuberculosis[END_REF]; in addition, the long half-life of this drug makes it a good companion drug for rifapentine [START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF]. Regarding clofazimine, at present, there has been no clinical proof of its activity in the context of drug-susceptible tuberculosis and the skin pigmentation can be a limit to its use even if it may be less pronounced if given weekly. On the other hand, data from murine studies support the use of clofazimine against drug-susceptible tuberculosis [START_REF] Williams | Sterilizing Activities of Novel Combinations Lacking First-and Second-Line Drugs in a Murine Model of Tuberculosis[END_REF][START_REF] Saini | Treatment-Shortening Effect of a Novel Regimen Combining Clofazimine and High-Dose Rifapentine in Pathologically Distinct Mouse Models of Tuberculosis[END_REF]; in addition, clofazimine has also been shown to act synergistically with bedaquiline in a murine model [START_REF] Williams | Sterilizing Activities of Novel Combinations Lacking First-and Second-Line Drugs in a Murine Model of Tuberculosis[END_REF] and to have a long half-life [START_REF] Holdiness | Clinical pharmacokinetics of clofazimine. A review[END_REF][START_REF] Swanson | Pharmacokinetics and pharmacodynamics of clofazimine in a mouse model of tuberculosis[END_REF], making it an interesting choice for weekly regimens. However, bedaquiline, clofazimine and moxifloxacin are all known to prolong the QT interval and should be used carefully in patients either in clinical trials or in the programmatic use.

When discussing findings from preclinical models, a crucial question is to what extent they are predictive for treatment outcomes in humans. The model used here (IV infected swiss mice) can be considered as a very conservative one and is therefore not expected to generate results which are too "optimistic", compared to what eventually is seen in humans [START_REF] De Groote | Comparative studies evaluating mouse models used for efficacy testing of experimental drugs against Mycobacterium tuberculosis[END_REF]. This was illustrated in the evaluation of the ability of fluoroquinolones to shorten the treatment. While in a model of aerosol-infected BALB/c mice the 4-month moxifloxacin-based regimen was as active as the standard 6-month daily regimen, this was not the case in our model [START_REF] Ibrahim | Sterilizing activity of R207910 (TMC207)-containing regimens in the murine model of tuberculosis[END_REF][START_REF] Nuermberger | Moxifloxacin-containing regimens of reduced duration produce a stable cure in murine tuberculosis[END_REF]. Ultimately, the clinical studies confirmed the inferiority of the 4-month moxifloxacin (REMOX study) and Gatifloxacin (OFLOTUB study) regimen [START_REF] Gillespie | Four-month moxifloxacin-based regimens for drug-sensitive tuberculosis[END_REF][START_REF] Merle | OFLOTUB/Gatifloxacin for Tuberculosis Project. A four-month gatifloxacincontaining regimen for treating tuberculosis[END_REF][START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF].

Another limit is the number of animals used in the study, which, although high for a murine study (300 mice), remains low compared to the expected sample size of a clinical trial (approximately 500 patients/arm). Thus this study as others conducted in murine models, must be seen as a proof-of-concept that needs to be confirmed in a clinical trial. Also toxicity in relation with high peak serum level the day of drug intake may be a limit. Finally, the bedaquiline AUC reduction in presence of rifapentine may be seen as a limitation since this reduction may vary from patient to patient and generate heterogeneous exposures to bedaquiline, requiring therapeutic drug monitoring [START_REF] Alffenaar | Should we worry about bedaquiline exposure in the treatment of multidrug-resistant and extensively drug-resistant tuberculosis?[END_REF].

A possible solution would be to replace rifapentine with another drug with a long halflife: moxifloxacin which has shown interesting activity combined with bedaquiline without rifapentine or also other newly approved drugs or under development [START_REF] Tasneen | Sterilizing Activity of Novel TMC207-and PA-824-Containing Regimens in a Murine Model of Tuberculosis▿[END_REF][START_REF] Pethe | Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis[END_REF][START_REF] Lamprecht | Turning the respiratory flexibility of Mycobacterium tuberculosis against itself[END_REF][START_REF] Salinger | Population Pharmacokinetics of the Antituberculosis Agent Pretomanid[END_REF]. Whatever the choice, the potential candidate should have at least as strong sterilizing properties as rifamycins and a preliminary pharmacokinetic study assessing the different drug interactions in human would be required before starting a therapeutic trial.

Up to now, bedaquiline development has focused on drug-resistant tuberculosis, similarly to two other recently-approved drugs, delamanid and pretomanid. We believe that these new drugs could also play a role in improving treatment of drugsusceptible TB. Overall, the development of intermittent regimens has been somewhat neglected in favor of shortened regimens. It should be recalled that to date, whereas all shorter fluoroquinolone-based regimens failed to show noninferiority to the standard daily 6-month regimen, a regimen including once-weekly rifapentine-moxifloxacin during the continuation phase managed to show noninferiority [START_REF] Jindani | High-dose rifapentine with moxifloxacin for pulmonary tuberculosis[END_REF]. If confirmed in a clinical trial, such a weekly regimen would reduce the number of days with drug intakes from 182 to 26. A daily regimen would have to be reduced to less than one month in order to reach a similar low number of drug intakes. Once weekly regimens should be explored to increase compliance.

Tables and Figures

Table 1.

  Figure: proportion (%) of mice with positive lung culture after 2, 4 and 6 months of treatment in murine tuberculosis

This work was supported by a grant from 'Fonds de dotation Recherche en Santé Respiratoire' and by a grant from Janssen.