Fighting inflammation to save cones: anti-inflammatory approaches to slow down cone degeneration in a mouse model of retinitis pigmentosa

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A te, che mi hai insegnato a guardare alle stelle

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i

I. SUMMARY

The term Retinitis Pigmentosa (RP) defines a group of inherited dystrophies characterized by progressive degeneration of photoreceptors (PRs) and abnormalities in retinal pigment epithelium (RPE). In typical RP, primary degeneration of rods is followed by secondary death of cones. Affected individuals exhibit initial night blindness and constricted visual field, while central vision is eventually lost later, as cone cells degenerate. Unfortunately, there is no cure for RP.

An important concept of RP pathophysiology is the biological link(s) between rod and cone death in this disease, where mutations are usually rod-specific and cones, genetically intact, degenerate as a consequence of a bystander effect. To note, the main cause of clinically significant vision loss is associated with cone, rather than with rod death. Although cones represent less than 5% of all PRs in the retina of most mammals, their role on human vision is crucial and their degeneration leads to a condition of irreversible vision loss. Survival of still-functioning cones following rod death enables patients with a night-blindness disease to lead normal lives for some time (Shelley et al., 2009).

Previous studies of our laboratory and based on the rd10 mouse model of human RP demonstrated with molecular tools that inflammation emerges as a relevant component of RP, overcoming any other biological process expected to occur in this pathology. Here, we hypothesized that cones, non-primarily affected by the disease-causing mutation and long lasting with respect to rods, may suffer from side effects of such inflammatory process and finally die out. In this study, we employed a protocol of synthetic steroid administration to test the hypothesis that counteracting retinal inflammation concomitantly to the acute phase of PR degeneration may improve cone fate. Indeed, we demonstrated that systemic Dexamethasone treatment resulted in decreased inflammatory response at retinal level and this event was associated to improved cone survival and preservation of visual acuity in rd10 mice. Subsequently, we postulated that different classes of Mononuclear Phagocytes (MPs, such as microglia and monocytes-derived macrophages, primarily

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v involved in the inflammatory response) played different roles in the chronic, noxious inflammatory response found at retinal level. The particular role of the CCL2 chemokine was assessed.

Altogether, our findings suggest a link between local retinal inflammation and worsening of cone fate, opening the perspective of slowing down retinal decay and vision loss in RP by using anti-inflammatory strategies.

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II. RIASSUNTO

Il termine Retinite Pigmentosa (RP) definisce un gruppo di patologie ereditarie caratterizzate dalla progressive degenerazione dei fotorecettori e da anomalie nell’epitelio pigmentato della retina (RPE). Nella forma tipica di RP, la degenerazione primaria dei bastoncelli è seguita dalla morte secondaria dei coni. Gli individui affetti inizialmente manifestano cecità notturna ed un restringimento del campo visivo mentre la visione centrale viene eventualmente persa in un secondo momento, in seguito alla degenerazione dei coni. Purtroppo non esiste cura per la RP.

Un concetto importante nella patofisiologia della RP riguarda il collegamento biologico tra la morte dei bastoncelli e quella dei coni in questa malattia dove le mutazioni sono generalmente bastoncello-specifiche e i coni, geneticamente sani, degenerano come conseguenza di un “effetto spettatore” dovuto alla prossimità con i bastoncelli durante il processo che conduce alla morte di questi ultimi.

È degno di nota il fatto che la principale causa di riduzione clinicamente significativa della vista è associata alla morte dei coni, piuttosto che a quella dei bastoncelli.

Sebbene i coni rappresentino meno del 5% di tutti i fotorecettori nella retina della maggior parte dei mammiferi, il ruolo di queste cellule nella visione dell’uomo è cruciale e la loro degenerazione conduce ad una condizione di perdita irreversibile della vista. La sopravvivenza di coni ancora funzionanti, in seguito alla morte dei bastoncelli, permette a pazienti con cecità notturna di condurre una vita relativamente normale almeno per un certo periodo.

Precedenti studi nel nostro laboratorio, condotti su topi rd10, un modello murino di RP umana, hanno dimostrato con metodi molecolari che l’infiammazione emerge come una componente rilevante nella RP, prevalendo su altri processi biologici attesi in questa patologia. Qui noi ipotizziamo che i coni, non affetti dalla mutazione causante la malattia e sopravviventi più a lungo dei bastoncelli, possano risentire delle conseguenze di questo processo infiammatorio fino a condurli alla morte.

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vii In questo studio abbiamo impiegato un protocollo di somministrazione di steroidi sintetici con l’obbiettivo di testare l’ipotesi che, contrastando l’infiammazione retinica concomitante alla fase acuta della degenerazione dei fotorecettori, sia possibile migliorare il destino dei coni.

Infatti, abbiamo dimostrato che il trattamento sistemico con Desametasone conduce ad una diminuzione della risposta infiammatoria a livello retinico e che questo evento è associato ad un incremento della sopravvivenza dei coni e della preservazione dell’acuità visiva nei topi rd10. Successivamente abbiamo ipotizzato che differenti classi di fagociti mononucleati (nello specifico cellule microgliali e macrofagi derivati da monociti circolanti, primariamente coinvolti nella risposta immunitaria) giocassero ruoli diversi nell’infiammazione, cronica e quindi particolarmente nociva per i coni, evidenziata a livello retinico. Abbiamo indagato il ruolo particolare assolto dalla chemochina CCL2.

Nell’insieme, i risultati da noi ottenuti suggeriscono un collegamento tra l’infiammazione in atto a livello locale nella retina ed il peggioramento del destino dei coni, aprendo così la prospettiva di poter rallentare il decadimento della retina e la perdita della visione mediata dai coni nella RP tramite l’utilizzo di strategie anti-infiammatorie.

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III. AIMS OF THE THESIS

The aims of this thesis are essentially two:

1. In the first part, we address the occurrence of an inflammatory response in the retina at the time cones die out in RP and we hypothesize that this response can affect cone survival. To test this hypothesis we implemented a protocol of anti-inflammatory drug administration in rd10 mice. Pharmacological treatment consisted of daily subcutaneous injection of Dexamethasone during the time window encompassing the peaks of rod and cone death in the mutant under study, starting at P23 until P45, or longer until P60. All control animals received identical injections of vehicle only.

Treatment efficacy and possible association between retinal inflammation and cone fate were tested on all treated and control mice using 3 measurable output parameters:

a) Inflammation levels, both at P45 and at P60, assessed by counts of microglia/macrophages in retinal whole mounts after antibody specific staining. Moreover, the degree of activation of the inflammatory cells was measured through the analysis of their morphology (amoeboid in activated cells or ramified in resting cells) and of the expression of CD68, a known marker of phagocytes activation. b) Survival of cones, assessed with histological analysis of retinal samples and cone cell

counts, at P45 and at P60. This way, we collected data on cone survival to be paired with data on inflammation levels at the same time points. This analysis was combined with Western blots for an inflammatory mediator (CCL2) and a cone specific protein (cone arrestin). Furthermore, we investigated cone morphology in control mice and Dexamethasone-administered animals

c) Behavioral assessment (Prusky water maze) of the photopic vision measuring visual acuity at P45 and at P60. This study evaluated if eventual effects of anti-inflammatory treatment on cone survival and morphology translated also in better-preserved cone functionality.

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ix 2. The second part of this thesis illustrates our attempt to gain deeper insight in the nature of the inflammation in rd10 mice, focusing on some characteristics of this response. In particular:

i. The presence of different classes of MPs (i.e. microglial cells and monocyte-derived macrophages) in the infiltrate of inflammatory cells in the outer retina was checked with cell-specific staining.

ii. Possible mechanism(s) that may lead to changes in the blood-retina barrier (BRB) permeability, responsible for the entrance of monocyte-derived macrophages into the diseased retinas, were discussed. Particular attention was payed to the outer BRB constituted by the RPE layer because of the vicinity with the site of PRs degeneration.

iii. The level of CCL2 expression in the retina, as an indication of the strength of the inflammatory response but also, specifically, of the intensity of the chemotactic signaling operating in the recruitment of monocyte-derived macrophages, was analyzed.

As a last point, we carried on morphological observations of the RPE, comparing the tissue of control and Dexamethasone-treated rd10 mice. The occurrence of RPE changes associated with the rd10 retinal pathology was investigated through the analysis of basic morphological aspects of the RPE cells.

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1. INTRODUCTION

In humans, vision is of paramount importance for quality of life and impairment of sight represents a highly incapacitating condition. Dysfunction or complete loss of visual ability may be caused by obstruction in the light path to the neural retina or inability of the retina to detect and/or transmit light-triggered signals to the brain (Veleri et al., 2015). The latter can be the result of several disease conditions.

Inherited Retinal Degenerations (IRDs) are a family of dystrophies characterized by the progressive loss of retinal function largely responsible for incurable blindness. In IRDs, mutations occur in PR or pigment epithelium-specific genes leading to PR dysfunction and death (Bessant et al., 2001, Wright et al., 2010). Dozen different types of diseases are included in this family. Retinitis Pigmentosa accounts for approximately one-half of cases (Daiger et al., 2013).

1.1. Retinitis pigmentosa

1.1.1. Disease clinical overview

By definition, the term Retinitis Pigmentosa refers to a group of inherited progressive, degenerative diseases of the retina, in which a genetic mutation triggers the primary degeneration of rods, later followed by the secondary loss of cones, leading to profound loss of vision and often culminating into blindness (Daiger et al., 2014).

Clinically, during the course of RP patients undergo through three stages of the disease: a) an early stage, typically during adolescence, characterized by night blindness and “tunnel vision”, caused by rod degeneration, but no visual defects in daylight (Figure 1); b) a mid-stage, in which patients have difficulty performing most night-time activities, perceive a loss of peripheral visual field even in daylight and may become photophobic. This is the stage when usually patients are clinically diagnosed with RP: fundus examination shows the presence of bone spicule-shaped pigment deposits and RPE atrophy. Functionally, the electroretinogram (ERG) indicates complete loss of scotopic visual response, which reflects the loss of the majority of rods, and an attenuated visual response in cone cells; c) a late stage, when clinical findings that occur in the mid-stage of the disease worsen and central

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- 2 - visual field is lost, due to secondary cone death; at that time RP patients lose their ability to perform daily tasks and may be irreversibly blind.

From the clinically manifested stage, ophthalmic examination usually displays the three clinical features considered the hallmarks of RP: bone spicule pigmentation, attenuation of retinal vessels, and a waxy pallor of the optic nerve. Pigmentation is caused by RPE cells that detach from Bruch membrane following PRs degeneration and migrate to intra-retinal perivascular sites, where they form melanin pigment deposits (Verbakel et al., 2018). Precisely what triggers RPE migration is unknown but it is likely related to the perturbation of the fine, interdependence link between RPE and PRs and likely reflecting also tissue suffering.

Several other secondary ocular conditions can be associated with RP primary symptoms. Among them: nystagmus, cystoid macular edema (CME) and epithelial membrane formation. In particular, CME has been shown to occur in up to 50% of patients with RP, likely due to breakdown of the BRB or impaired function of the RPE pumping mechanisms, leading to subretinal fluid accumulation (Strong et al., 2017).

Figure 1. Representation of “Tunnel vision” experienced by RP patients in the first clinical stage (from

WebMD).

1.1.2. Etiology and incidence

RP represents the most common cause of hereditary blindness worldwide, affecting approximately 1:3,000/1:4,000 people (Wert et al., 2014). RP is known to be highly heterogeneous, with hundreds of mutations in more than 70 different genes and 61 loci presently known to cause the non-syndromic forms of the disease (Retnet,

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heterogeneity concerns the various patterns of inheritance, which include autosomal dominant (20-30% of cases), autosomal recessive (20–25% of cases), and X-linked (10–15%) of the disease. The remaining cases are currently of unclassified inheritance and designated as simplex RP cases believed to be autosomal recessive (Figure 2) (Wang et al., 2005, Hamel, 2006, Hartong et al., 2006). As anticipated, there are syndromic forms of RP in which vision loss is associated with other systemic abnormalities. Genetic variability is a hallmark also of syndromic RP: mutations in 12 genes cause Usher syndrome (RP associated with deafness) and 17 genes are associated with Bardet-Biedl syndrome (RP associated variably to obesity, polydactyly, mental retardation, and infertility) (Daiger et al., 2014, Wert et al., 2014).

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Figure 2. Genes and their relative contribution to RP

Causal genes and their contributions to (A) autosomal-recessive disease, (B) autosomal-dominant disease, and (C) X-linked disease. (From Hartong et al., 2006)

Heterogeneity of RP goes beyond genetic and allelic variability and includes phenotypic heterogeneity – where mutations in the same gene cause different diseases, and clinical heterogeneity – insofar clinical consequences of a certain mutation differ in different individuals (Daiger et al., 2013). These represent confounding factors in diagnosis and obstacles in finding therapeutic approaches with a broad efficacy.

1.1.3. Animal models of RP

The use of model organisms can facilitate the elucidation of cellular mechanisms underlying human diseases and various natural or genetically altered animals have been largely employed in RP studies. Large mammals, particularly those primates that have the fovea, would be better suited for understanding retinal degeneration also occurring in humans and particularly cone death; however, lack of naturally occurring primate models of RP, ethical concerns and difficulties in the management and genetic manipulation pose limitations in the use of these animals. In general, only a limited number of spontaneously arising models of IRDs have been identified in large mammals. Conversely, rodent models, and particularly mice, have become the most widely used models, as multiple mutations associated to retinal diseases have long been recognized or can be generated relatively easily (Veleri et al., 2015) in these small, readily manageable laboratory animals. Among them, rats and mice with defects in the rhodopsin gene are among the most used, also because of the high frequency mutations in this gene responsible for the human disease. As an example, the P23H mutation in rhodopsin gene has been transgenically introduced in rats, mice and swine models (Naash et al., 1996, Ross et al., 2012).

RP resulting from mutations in the beta subunit of the rod phosphodiesterase gene (Pde6b) is one of the earliest onset and most aggressive form of this disease, accounting for up to 5% of autosomal RP in humans (Han et al., 2013). PDE is an essential part of the phototrasduction cascade, playing a role in hydrolysing the cGMP second messenger and resulting in the cationic channel closure in response to light. Thus, mutations in Pde gene lead to severe retinal dysfunction.

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Multiple mouse and dog models exist with spontaneously occurring mutations in Pde6b gene. Among these, rd1 and rd10 mouse lines are commonly used as models for the recessive form of the human RP disease (Figure 3).

In rd1 mice, the Pde6b gene bears a non-sense mutation that leads to a phenotype characterized by severe, early onset, rapid retinal degeneration. By post-natal day 17 (P17) only about 2% of the rods remain in the rd1 retina and virtually none by P36 (Han et al., 2013).

The rd10 mice was described for the first time by Chang et al in 2002; it carries a missense mutation in exon 13 of the Pde6b gene that leads to a relatively slower onset of retinal degeneration with respect to rd1 mice. It has been shown that PR degeneration in this model starts from the central retina at around P16 and spreads to the peripheral retina by P20. Rapid degeneration occurs between P18 and P25, and by P60 only one row of PR, mainly aberrant cones, remains, albeit no ERG can be recorded (Chang et al., 2007, Gargini et al., 2007).

Numerous missense mutations in human Pde6b result in partial loss of function and reduced PDE6B enzymatic activity, as seen in rd10 mice (Mclaughlin et al., 1995). Because of human analogy and milder progression of the degeneration, rd10 mice represents a good model to study RP pathogenesis and for developing strategies for treating humans with recessive RP (Han et al., 2013).

Figure 3. Schematic representation of the mouse Pde6b gene and protein, and the localization of spontaneous mutations in animal models. (From Han et al., 2013)

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1.2. Pathogenesis of RP

Despite a great research effort in this field, the pathophysiological mechanism of retinal degeneration in RP is still under investigation.

Most mutations in genes responsible for RP in humans and animal models primarily affect rods, leading to their death (Figure 4). Genetic defects targeting PR-specific functions are only marginally more numerous than mutations affecting more general functions (Wright et al., 2010). Common mutated proteins include classical components of the phototrasduction cascade (i.e. rhodopsin in first place, but also PDE etc.), transcription factors (i.e. CRX, NRL), disc membrane-associated glycoproteins (peripherin), ionic channels (the light sensitive cationic channel), structural proteins of the cilium etc. (Daiger et al., 2013).

Figure 4. A broad classification of proteins associated with IRDs according to their localization or function

in rod and RPE cell. (From Veleri et al., 2015)

The reason why defects in many different genes cause the same outcome likely resides in the unique physiology and biochemistry of PRs, underpinning their vulnerability to cell death. Rods appear to live in balance on a “knife-edge” separating functioning and survival

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from dysfunction and death: almost any abnormality is capable of tipping them toward cell death (Wright et al., 2010).

Later along the disease progression, when rods death approaches the peak, cones also progressively lose light sensitivity and degenerate, even though some of their cell bodies persist for extended periods before eventually disappearing (Hartong et al., 2006) (Figure

5).

Figure 5. PRs death in RP.

Simplified time course of rod and cone death kinetics during degeneration. (From Punzo et al., 2012)

1.2.1. Primary rod death in RP

As mentioned before, rod death represents the primary event in typical RP. However, besides the general notion that rods degenerate due to a primary genetic defect, the biological mechanisms linking the primary mutation to the lethal cellular damage remain elusive.

Apoptosis has been considered the main modality of PR demise (Cottet and Schorderet, 2009, Cuenca et al., 2014) but recent studies on various animal models demonstrated that necrosis and autophagy are equally involved (Sancho-Pelluz et al., 2008, Arango-Gonzalez et al., 2014). Moreover, it has been shown that multiple different apoptotic pathways are active in PRs at the same time and that apoptosis can be either caspase-independent or – dependent (Marigo, 2007, Kroemer et al., 2009).

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- 8 - To explain why single rods, affected by the same mutations, degenerate almost synchronously but independently, Wright et al. proposed a model mechanism based on exponential kinetic, almost universally applicable to neurodegenerative diseases of the brain and the retina. The model states that individual rods have a probability of death predicted to vary around a mean value for a given genotype and species; this probability is increased further by any RP-causing mutation. This happens because mitochondrial signalling pathways, which exert dominant control over the propensity of a cell to undergo apoptosis, integrate the diverse cellular stresses associated with different inherited defects (Wright et al., 2010). Light exposure, triggering reactive oxygen and nitrogen species (ROS/RNS), lipid oxidation, ciliary transport defects, endoplasmic reticulum stress, dysregulated survival signalling, metabolic stress and altered bioenergetics functions, are all likely involved as sources of cellular stress, finally leading to cell death activation.

1.2.2. Secondary cone death in RP

In typical RP, cones do not bear mutations and die because of a non-cell autonomous process. For reasons that remain unclear, they cannot survive indefinitely in the absence of the normal microenvironment dominated by rods (Lin et al., 2009).

Cone death follows that of rods but is a critical step in RP pathogenesis.

Although cones represent less than 5% of all PRs in the retina of most mammals, their role in human vision is crucial and their degeneration leads to the most severe clinical sign of RP, finally resulting in irreversible vision loss.

In rd1 and rd10 RP mouse models, the pattern of cone death has been studied extensively; cone degeneration begins centrally and proceeds toward the periphery. By P35 in the rd1 and P50 in the rd10, the majority of cones have been eliminated (Carter-Dawson et al., 1978, Chang et al., 2002).

In humans with RP, as in animal models described above, cones tend to be preserved until most of the rods have died. Initial visual field defects occur in the mid-periphery of the retina, then enlarge and coalesce into a ring, known as ring scotoma. Ring scotoma tends to spread in both directions reducing peripheral and central fields, finally leaving just a central island of residual visual acuity (Lin et al., 2009, Campochiaro and Mir, 2018).

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Preservation of even a fraction of cones or rescue of their sensitivity would ensure relatively normal lives to RP patients: this option represents a major goal of ongoing therapeutic strategies (Piano et al., 2013).

The biological mechanisms linking the death of rods and the bystander demise of cones remains also elusive. Among the proposed mechanism, there are decreased survival factors, dysregulation of glucose metabolism and oxidative stress (Murakami et al., 2015) (Figure 6).

Figure 6. Rod and cone cell death in RP

Possible biological mechanisms linking rod and cone death in RP. (From Murakami et al., 2015)

The first line of thinking postulated that cone cell death is directly caused by rod demise as an effect of a) release of toxic substances from dying rods or b) decrement in levels of survival factors produced by rods and needed by cones. Along this line of thinking, Ripps, proposed a bystander hypothesis for cone death, meaning that rod-cone coupling allows flux of noxious effectors through the gap junctions between dying rods and mutation-free cones, leading to the degeneration of the latter. Nucleotides, ions and small molecules are among the proposed death-inducing signals (Ripps, 2002). However, this possibility is unlikely, considering that deletion of connexin 36, an important component of PR gap junctions, does not affect the pattern or timing of cone death (Kranz et al., 2013).

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- 10 - Another line of research lead to the discovery of the Rod-derived Cone Viability Factor (RdCVF). RdCVF is an alternative splice variant of the nucleoredoxin-like 1 (Nxnl1) gene, whose other longer splice product is RdCVFL, an active thioredoxin. Ait-Ali et al. demonstrated that RdCVF, but not RdCVFL, protects cone function in several genetically distinct models of RP (Leveillard et al., 2004, Yang et al., 2009b, Ait-Ali et al., 2015, Byrne et al., 2015). These authors demonstrated that RdCVF acts through binding to Basigin 1 (BSG1), a transmembrane receptor protein expressed specifically in PRs. The following interaction between BSG1 and the glucose transporter GLUT1 results in increased glucose entry into cones and stimulation of the aerobic glycolytic metabolism (Cepko and Punzo, 2015) (Figure 7).

Figure 7. Glucose uptake in cone cells

a, Rod and cone PRs receive glucose from choroidal blood vessels, through RPE cells. Glucose uptake is

mediated by the transporter protein GLUT1; b, In RP, where rods die, cones seem to be unable to take up enough glucose to fuel their metabolism and support their survival; c, RdCVF associates with BSG1 that interacts with GLUT1 to improve glucose uptake. (From Cepko and Punzo, 2015)

The understanding of the role of RdCVF and its mechanism of action provided support for a previous hypothesis to explains cone degeneration in RP, postulated by Punzo et al. who claimed that cone degeneration in RP occurs secondarily to nutrient deficiency leading to

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cone starvation (Punzo et al., 2012). Indeed, this study demonstrated that genes involved in cellular metabolism, and especially those belonging to the mechanistic target of rapamycin (mTOR) signalling pathway, were downregulated in numerous mouse models of RP. mTOR actions are related to the energy status of the cell: under normal condition, when glucose levels is high, mTOR activity is positively affected by the ATP produced and the amino acid availability, and it allows energy consuming processes, such as translation, and prevents autophagy (Punzo et al., 2009, Takei and Nawa, 2014) (Figure 8). On the other hand, under stress conditions (i.e. diminished nutrient availability or hypoxia) the balance is shifted toward catabolism. Indeed, starvation negatively regulates mTOR and removes the inhibition to autophagy catabolic processes (Punzo et al., 2009).

Figure 8. Schematic representation of mTOR signalling through mTORC1.

Representative substrates and cellular responses mentioned in the text are shown (From Takei N et al., 2014).

These studies indicate that reduced levels of active mTOR in RP reflect poor nutrient conditions, leading to activation of autophagy within cones, finally resulting in cell death. Indeed, constitutive activation of mTORC1 (a complex consisting of mTOR and other proteins) in cones maintains cone function and promotes long-term survival of these cells (Venkatesh et al., 2015).

The same studies and further research support the notion that rod death affects the communication with the adjacent RPE, responsible of facilitating delivery of nutrients and

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- 12 - oxygen to PRs. When rod loss exceeds a critical threshold, the contact between RPE and cone outer segments is also irreversibly disrupted, compromising the uptake of nutrients from cones. Thus, both the theories proposed by (Punzo et al., 2009) and (Ait-Ali et al., 2015) support the idea that in RP cones ultimately die from starvation via a mechanism that involves compromised glucose uptake (Narayan et al., 2016). “Feeding” cones may be the key strategy to protecting them.

RdCVF relevance in cone survival is limited by the observation that the knockout for the Nxnl1 gene (Nxnl1-/-_{) results in only a 17% reduction in cone density with respect to the}

Nxnl1 wild type (WT), suggesting that loss of RdCVF is not the primary cause of cone cell death in RP (Campochiaro and Mir, 2018). Likely, this factor plays a contributing role, demonstrated also by the increased cone susceptibility to oxidative stress after loss of RdCVF (Elachouri et al., 2015).

Oxidative stress, an imbalance between the production and elimination of ROS, is one of the main causative factors contributing to PR cell death in the progression of a number of retinal diseases and it has also been suggested to play a role in cone degeneration in RP as well (Figure 9) (Shen et al., 2005). RP patients show a significant decrease in the ratio of reduced -to oxidize- glutathione and a significant increase in protein carbonyl content in aqueous humour (Campochiaro, 2015).

Campochiaro et al. investigated oxidative damage influence on cone cell death in RP using mouse models (rd1, rd10, P23H) and Pro347Leu pig models. Degeneration of rods, metabolically active cells constituting 95% of the outer nuclear layer (ONL), reduces dramatically oxygen consumption in the retina. Because choroidal vessels are not sensitive to oxygen consumption, the resultant condition of hyperoxia in the outer retina causes worsening of nitrosative and oxidative damage to lipids, proteins and DNA predominantly in surviving cells, and namely cones. Campochiaro et al. showed that reduction of oxidative stress following administration of a mixture of four antioxidants promotes cone survival and function in rd1 and rd10 mice (Campochiaro and Mir, 2018).

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Figure 9. Suggested mechanism to explain the role of oxidative stress on cone death in RP. (From Narayan

et al., 2016)

Although all the above-mentioned biological processes have been demonstrated to be active during rod degeneration and likely participate in cone demise, none of them is able to justify per se the whole pathophysiology of cone death during RP. Other mechanisms are likely to be implicated.

An intriguing hypothesis arises from recent studies implicating inflammatory processes in neurodegenerative diseases of the CNS. The recent notion that inflammatory and immune responses show activation/dysregulation in CNS in diseases not primarily inflammatory in their etiology indicates that these processes might be directly involved in neuronal death (Fakhoury, 2016, Zhang et al., 2017).

Considering the mounting evidence of a sustained immune response in RP, the implication of inflammatory reaction in cone loss deserves to be addressed.

1.3. Inflammation in the CNS

Efficient immune responses are needed in any organ to limit endogenous damage and/or the entry of exogenous agents, but they might come at the price of harming local tissue. Therefore, the type and strength of the local immune response reflect a compromise

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- 14 - between severity of the threat and capability of a given tissue to tolerate potential collateral damage, possibly regenerating after the response has been resolved (Deczkowska et al., 2018).

The CNS consists of a series of vulnerable neural networks with very limited capacity for regeneration and has evolved as an immune-privileged site, where immune activities are dampened and tightly regulated (Galea et al., 2007).

Of note, the CNS is endowed with a well organized, resident immune system that protects it from exaggerated responses, and constituted primarily by endogenous microglial cells that occupy the brain and retinal parenchyma since embryonic stages. This special immune status requires multiple checkpoint mechanisms encompassing numerous levels of regulation to restrain microglia activity in homeostatic condition (Figure 10).

Figure 10. Microglial checkpoints mechanisms.

Four major types of checkpoint mechanisms restrain microglia immune activity under physiological conditions. a, Physical barriers. b, Soluble factors. c, Cell–cell interactions d, Microglia-specific transcription factors and chromatin regulators. (From Deczkowska et al., 2018)

In first place, the CNS is physically secluded from the general circulation by the presence of the BBB, the BRB as well as by blood-cerebrospinal fluid and meningeal barriers. These

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immunologically active interfaces provide a highly selective gateway toward leukocyte entry and blood-derived factors access to the CNS (Harrison-Brown et al., 2016). Breakdown of these barriers represents the first cause of disruption of the immune homeostasis of the nervous tissue and often exacerbates pathological condition (Goldmann and Prinz, 2013).

Several soluble factors and cell-cell interactions also participate in the maintenance of the CNS immune-privilege. Secreted anti-inflammatory factors include cytokines (i.e. interleukin IL-4, IL-13), transforming growth factor-β (TGF-β) and IL-10, as well as neurotrophic factors, such as Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and neurotrophin-3 (NT-3) (Neumann and Wekerle, 1998).

Immune activity is restrained also by direct cell-cell interaction of microglial cells with neighbouring neurons in virtue of precise, inhibitory signals confined in space. It has been demonstrated that binding of the fractalkine receptor (CX3CR1), expressed ubiquitously on microglial cells, to its neurons-specific ligand, CX3CL1, attenuates microglia activation. Similarly, the interaction between microglial CD200 receptor (CD200R) and CD200L expressed on neurons, astrocytes and endothelial cells confines the immune response. These represent just two examples of negative interactions continuously involved in the maintenance of the CNS immune privilege (Biber et al., 2014, Deczkowska et al., 2018) and CNS inflammation often results from the breakdown of one or more checkpoints contemporarily (Lee and Landreth, 2010). However, the exact mechanisms that link the inflammatory response to neuronal death is still a matter of investigation.

In this context, it has been shown recently that inflammatory cells and their mediators may trigger several basic mechanisms commonly activated during cell death in neurodegenerative diseases, such as apoptosis, necroptosis, neuronal autophagy, retrograde degeneration, demyelination and astrogliopathy (Soto and Estrada, 2008, Chitnis and Weiner, 2017).

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Figure 11. The neuro-inflammatory process.

Exposure of astrocytes and microglia to damage signals promotes secretion of pro-inflammatory factors, inducing mutual activation of microglial cells and astrocytes, along with other neuro-inflammatory process eventually leading to neuronal death. (From Morales et al., 2014)

It is likely that multiple CNS degenerative pathologies are characterized by an early acute phase of microglial activation needed for the effective removal of toxic agents, misfolded proteins, dead cells etc. (Block et al., 2007). Defective responses and/or incomplete removal of threats might results in an escalating, vicious cycle of local cytotoxic inflammation (Figure 11) (Morales et al., 2014, Schwartz and Baruch, 2014). This stage, likely coincident with chronicization of the disease, is characterized by an inflammatory response with negative traits, to which cells other than endogenous microglia also contribute as explained below.

1.3.1. Immune cells and major signalling involved in neuroinflammation

Inflammation-mediated neurodegeneration in the CNS may result from the participation and behaviour of endogenous or exogenous immune cells. Endogenous cells are mainly microglia with minor populations of CNS phagocytes, constituted by meningeal, choroid plexus and perivascular macrophages, the latter colonizing the perivascular spaces of the CNS parenchyma (Ransohoff and Engelhardt, 2012). In addition, macroglial cells (i.e.

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astrocytes and retinal Müller cells) can participate in driving inflammation, despite their non-primarily immune nature.

It has been shown that most brain lesions contain a heterogeneous population of MPs that include resident immune cells and a majority of infiltrating macrophages derived from monocytes, normally residing in the blood stream, and absent in healthy CNS parenchyma. Despite similarities between microglia and monocytes, both endowed with phagocytic activity, it is becoming clear that these cells are not identical, especially for their roles in inflammation response.

- Microglia

The term microglia was first coined by Pio del Rio-Hortega to describe the neural, non-astrocytic “third element of the nervous system”, distinct from neuroectodermal oligodendroglia and oligodendrocytes (Sevenich, 2018).

Microglia represent the innate immune cells of the CNS and the first responders to environmental changes. They constitute the largest population of myeloid cells in the CNS and are localized throughout the brain parenchyma. Their ontogeny is now clear: microglial cells derive from erythro-myeloid progenitors from the yolk sac (Gomez Perdiguero et al., 2015) and their development is completely dependent on colony-stimulating factor 1 receptor (CSF1R) signalling (Ginhoux et al., 2010). Microglial cells were detected as early as embryonic day 11 in the foetal mouse and rat brains and they are believed to be maintained through an organism lifetime independently of circulating precursors (Waisman et al., 2015).

The term “double-edged sword” has been often used to describe the beneficial and/or detrimental effects of microglial cells (Block et al., 2007). Indeed, microglia produce potentially toxic molecules as inflammatory cytokines (es. Interleukin 1 beta (IL-1β), Tumor Necrosis Factor alpha (TNF-α), Interleukin 6 (IL-6)), superoxide, nitric oxide and excitatory amino acid, as well as neuroprotective neurotrophins, including BDNF, Glial cell-Derived Neurotrophic Factor (GDNF), and NGF.

Microglial cells forms a stable cellular network within the CNS. The peculiar localization and the highly ramified morphology of microglial cells perfectly fit their role in steady state condition. Indeed, microglial cells are anything but “resting”: they perform continuous immune surveillance extending and retracting their processes to monitor the brain parenchyma; they provide trophic support to neurons, eliminate dysfunctional synapses,

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- 18 - apoptotic debris, and other waste products (Nimmerjahn et al., 2005, Deczkowska et al., 2018).

When an immune stimulus manifests, microglia put in action a defence program characterized by the acquisition of a pro-inflammatory phenotype, with potentiated phagocytic activity and increased expression/secretion of receptors, cytokines and chemokines. These inflammatory-related molecules have the role to monitor and propagate the alert state to other cells but may easily became a source of tissue damage if the response is not ceased out shortly (Wolf et al., 2017). This process is usually referred as microglia activation.

Molecular and transcriptional changes that determine microglia activation are accompanied by a morphological transition from a ramified to a more amoeboid cell shape, with only a few branched processes (Karlstetter et al., 2010). This amoeboid morphology likely favours microglial migration to the site of damage, as typically observed during brain or retinal diseases (Karlstetter et al., 2010, Karlstetter et al., 2015, Wolf et al., 2017). The exact triggers involved in microglial activation are not completely understood but the rapidity of the process can be attributed to their responsiveness to subtle, sudden imbalances in ionic homeostasis, alterations in signalling molecules like ATP, acetylcholine and noradrenaline occurring in the local microenvironment (Lewis et al., 2005, Vessey et al., 2012).

Growing evidence started with studies in vitro demonstrates that activated microglia can be neurotoxic and it is now accepted that it can be implicated in neuronal death in degenerative diseases of the CNS.

- Macroglia

Microglia are the first but not the solely cell type implicated in CNS inflammation. Macroglial cells (i.e. astrocytes and Müller cells in the retina) undergo a pronounced transformation called “reactive gliosis” in response to almost any kind of CNS injury and disease, whereby they upregulate many genes and change their overall behaviour (Liddelow et al., 2017). Roles and gene expression changes in macroglial cells during inflammation are heterogeneous and strongly dependent on surrounding clues. For instance, it has been shown that astrocytes with two distinct phenotypes, named A1 and A2, can be found in different pathological conditions: A1 astrocytes predominate in

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inflammation-induced CNS injuries, while astrocyte with A2 phenotype are predominant in ischemia-induced CNS lesions. A1-phenotype induction is dependent on IL-1α, TNF-α and complement C1q in combination (Liddelow et al., 2017).

The relation between micro-and macroglia during inflammation is bidirectional: on one hand, microglial-derived pro-inflammatory molecules can stimulate astrocytes transition to an “A1-like” (potentially harmful phenotype); in turn, macroglia can produce pro-inflammatory cytokines and chemokines that could stimulate a positive loop in the inflammatory process. A meaningful example is shown by the ability of A1 astrocytes to secrete CCL2, a chemokine with a fundamental role in recruiting monocytes from the blood stream. Hence, macroglia represents a connection between endogenous and exogenous activation of immune cells in CNS overall immune response.

- Monocyte-derived inflammatory macrophages

Monocytes are bone-marrow-derived myeloid cells normally residing in the blood stream. They invade the inflamed tissues differentiating to inflammatory phagocytes and becoming a potent source of free radicals and cytokines. As already mentioned, monocytes-derived macrophages are absent in the healthy brain and retina where they can penetrate only if the BBB/BRB is damaged or modified, as it happens during inflammation (Waisman et al., 2015).

Monocytes attraction and recruitment to the injured site are strongly dependent on chemokine signalling. By definition chemokines (or, chemotactic cytokines), are characterized by their ability to induce directional migration and activation of leukocyte subsets into inflammatory sites. It has been demonstrated by several studies that the major chemokine involved in monocytes attraction is CCL2, also known as Monocytes Chemotactic Protein-1 (MCP-1), the first characterized human chemokine (Yoshimura et al., 1989, Rollins et al., 1996).

Short-lived circulating monocytes strongly express the CCL2 receptor, CCR2 (Geissmann et al., 2003), Mice deficient in CCL2 are unable to recruit monocytes in response to an inflammatory stimulus, despite normal number of circulating leukocytes (Lu et al., 1998). In the brain and the retina, the main sources of CCL2 are astrocytes (or Müller glia) and resident microglia, and to a lesser extent, endothelial cells (Barna et al., 1994, Berman et al., 1996, Harkness et al., 2003). CCL2 is also released by infiltrating monocytes upon their

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- 20 - migration into the brain parenchyma, indicating the presence of autocrine regulation that perpetuates cell recruitment and activation (Gourmala et al., 1997, Gunn et al., 1997, Peterson et al., 1997)

In healthy conditions, CCL2 is present at low levels in the CNS while its expression is strongly upregulated concomitantly with most of the inflammatory threats of the brain and the retina. A wide range of stimuli can trigger CCL2 production and release. Treatment with lipopolysaccharide, interferon, IL-1β, CSF-1, TGF-β and TNF-α can all induce CCL2 expression in different cell types either in vivo or in vitro (Huang et al., 2000, Thibeault et al., 2001, Harkness et al., 2003). In contrast, retinoic acid, glucocorticoids, and estrogens reportedly inhibit CCL2 production (Melgarejo et al., 2009). Once CCL2 binds its receptor, downstream targets of CCR2 signalling are activated. These include phosphatidylinositol-3 kinase, mitogen-activated protein kinases (MAPK), and protein kinase C, indicating the wide spectrum of intracellular pathways involved in cellular responses elicited by CCL2 and resulting, at first, in increased migratory abilities and cytokine production by monocyte-derived macrophages (Wain et al., 2002, Stamatovic et al., 2005).

Recently, it has been shown that also monocyte-derived macrophages can be directly neurotoxic. Using a mouse model of status epilepticus, Tian et al., 2017 demonstrated that signalling between CCL2 and its receptor on infiltrated monocytes induces the production of IL-1β, mediating neuronal cell death (Figure 12) (Tian et al., 2017).

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Figure 12. Schematic representation of CCL2–CCR2 signalling in causing neuronal cell death via STAT3 activation and IL-1β production.

CCL2 is upregulated aiding in CCR2-positive monocyte infiltration. CCL2/CCR2 interaction leads to the activation of pSTAT3 resulting in IL-1β upregulation and release from activated macrophages. IL-1β induces neuronal cell death. Consistently, both CCL2−/− mice and CCR2−/− mice show a phenotype of decreased neuronal death when used as models of KA-induced status epilepticus. (From Tian et al., 2017)

In addition to its chemotactic properties and neurotoxic potential , it has been shown that CCL2 has direct effects on BBB/BRB permeability (Song and Pachter, 2004, Dzenko et al., 2005), Changes in the barrier properties are among the first events observed during CNS inflammation and play a great role in its outcome.

Stamatovic et al. showed that exposure of astrocytes and brain microvascular endothelial cells to CCL2 in vitro induced changes in actin cytoskeletal structure, as well as redistribution of tight junction (TJs) protein expression, rendering the BBB more porous and facilitating the trans-endothelial migration of blood-born leukocytes into the brain. These effects were attenuated in CCL2-treated animals previously depleted of peripheral macrophages, indicating that this chemokine acts both directly on endothelial cells of the BBB; and indirectly, through the recruitment of macrophages and subsequent changes in BBB permeability (Stamatovic et al., 2005).

1.3.2. Distinguishing between resident and recruited MPs in the CNS

Existing data indicate a specific role of resident microglia and monocyte-derived macrophages in the normal function and in pathological conditions of the brain and of the retina. An emphasis, in the case of macrophages, has been posed to alterations in the BBB/BRB given the fact that they are normally secluded from brain and retina tissues. Indeed, while microglia serve a variety of functions to ensure neuronal homeostasis in health and disease, infiltrating macrophages intervene only in threatening situations (Yamasaki et al., 2014). In addition, recruited monocyte-derived macrophages are not self-regulated, meaning that they are not under restrain mechanisms as microglia do. Signalling threshold for cytokines production is lower in monocyte-derived macrophages, resulting in more pronounced toxicity respect to endogenous immune cells.

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- 22 - Most likely, monocyte-derived macrophages participate in a second wave of inflammatory activation, aimed at restoring the initial conditions by removing the initial trigger. This response often comes at the price of tissue damage due to secondary side effects.

Obviously, one needs to avoid generalization by speaking indifferently of MPs in the CNS. However, the lack of experimental systems that distinguish between recruited and brain-resident myeloid cells has previously halted analysis of cell- type-specific functions in CNS inflammation. The development of new methodologies provides unprecedented opportunities for comprehensive in-depth analyses of the immune landscape of the CNS under steady state and pathological conditions. Recently it has been demonstrated that Tmem119 is expressed exclusively on microglia with ramified and amoeboid morphologies, whereas infiltrating macrophages do not express this marker (Satoh et al., 2016). Conversely, there is evidence that CCR2 is absent in microglial cells, both in the healthy and diseased brain (Mizutani et al., 2012, Olah et al., 2012, Hickman and El Khoury, 2013) while peripheral monocytes require CCR2 to invade the diseased CNS (Mildner et al., 2007, Schilling et al., 2009, Prinz and Mildner, 2011).

Thus, CCR2 in the brain and retina can be used as a selective marker of monocyte-derived macrophages.

1.3.3. Prototypic neuro-inflammatory diseases as examples of the complexity of the inflammatory response

Increasing appreciation for the role of inflammation in neurodegenerative diseases of the CNS, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and the prototypic neuro-inflammatory disease multiple sclerosis (MS), has identified differential immune responses involving adaptive versus innate immune systems at various stages of these pathological conditions (Chitnis and Weiner, 2017).

It is now understood that the disease etiology has great impact on the nature of the local inflammatory response it evokes (Sospedra and Martin, 2005, Venken et al., 2008, He and Balling, 2013). Thus, although obvious difficulties associated to the extremely complex scenario of “neuro-inflammation”, a concept of the existence of an inflammatory response in CNS diseases can be generalized.

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For example, ALS and MS are autoimmune inflammatory disorders that lead to irreversible axonal damage and progressive neurological disability. In these cases, infiltration of adaptive immune cells (T- and B-lymphocytes) is causative. In MS, demyelinating white matter lesions are accompanied by infiltration of innate immune cells (i.e., monocytes) (Dendrou et al., 2015, Hemmer et al., 2015). Using a mouse model of MS, Yamasaki et al. demonstrated that monocyte-derived macrophages are the primary cause of demyelination, whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-derived macrophages are highly phagocytic and inflammatory, whereas microglial cells demonstrate an unexpected signature of remarkable down-regulation of their metabolism at the nuclear, cytoplasmic and cytoskeletal levels at disease onset (Yamasaki et al., 2014). Thus, the occurrence of specific roles for different classes of CNS MPs is confirmed.

AD represents the most common form of dementia and considered as a prototypic example of CNS chronic neurodegenerative disease. The contribution of inflammatory reaction to its progression has been controversial for a long time. Present knowledge on AD indicates association of this disease and innate immune reactions insofar infiltration of lymphocytes only occurs at late disease stages when integrity of the BBB is lost. Actually, AD provides a fundamental example of context-specific regulation of microglial function, as explained below.

Some studies have proposed a protective role of microglia in AD (Simard et al., 2006, Jay et al., 2015), while others have shown that in disease conditions, microglia acquires pro-inflammatory properties associated with disease acceleration. These conflicting data are probably due to a high phenotypic and functional heterogeneity of the disease, which includes multiple stages.

Two recent studies used single-cell RNA-Seq analysis to characterize the immune landscape at different stages of AD using the 5XFAD mouse model. Both studies described the occurrence of two distinct microglia subpopulations during disease progression. The earlier represented a transient, intermediate activation state, part of the reprogramming of homeostatic microglia in response to neurodegeneration. Late-response microglia, instead, were characterized by increased expression levels of many genes known as common risk factors for AD, including Apoe, Ctsd, Lpl, Tyrobp and Trem2. These distinct microglia states

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- 24 - likely represent intermediate stages of a continuum of microglial reprogramming that ultimately converts protective/beneficial functions into neurotoxic effects (Keren-Shaul et al., 2017).

Microglia responds to the Amyloid beta (Aβ) peptides and promotes their clearance through the release of cytotoxic factors, which promote the phagocytosis of these peptides. Therefore, if, on one hand, phagocytosis of amyloid- peptides could improve the disease course, on the other the release of pro-inflammatory mediators reinforces disease progression.

Recent works underlined a role of infiltrating monocytes in AD. Macrophages have been observed in the brain tissue of the APP/PS1 mouse model of AD (Kelly et al., 2013, McManus et al., 2014, Minogue et al., 2014) along with an increase in BBB permeability reported in models of the disease (Farrall and Wardlaw, 2009, Starr et al., 2009). In addition, Khoury showed that the deletion of CCR2 in an AD mouse model resulted in a substantial reduction of MPs accumulation around amyloid plaques and in increased deposition of Aβ, thus demonstrating that the chemokine CCR2 mediates the recruitment of inflammatory peripherals monocytes in Alzheimer brains (Khoury, 2008, Theriault et al., 2015).

In conclusion, the nature of the inflammatory response in CNS diseases is highly dynamic and involves different cellular players and signalling cascades. It is likely that neuro-inflammation is the results of a prolonged struggle between a pathological process unique to a given disease, and the attempts of the immune system to restore local conditions, in a process reminiscent of the ‘competition’ between tumors and immune cells (Ziv and Schwartz, 2008). Similarly to the triumph of a cancer and its rapid propagation when the tumor manages to escape from immune control, the damaging potential of inflammation in neurodegenerative diseases arises from the chronic immune cells activation, accompanied by overproduction of pro-inflammatory cytokines overriding the initial beneficial effect of this response (Block et al., 2007, Hanisch and Kettenmann, 2007)

1.3.4. The eye like the brain: inflammatory response in retinal diseases

As part of the CNS, the retina is endowed with immune-privilege. The underlying mechanisms of this condition are similar to those discussed for the brain, including the occurrence of physical constrains and the nature of the molecular signals.

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Figure 13. Blood–retina barrier.

TJs at two sites create the BRB: between endothelial cells of the retinal vessels that supply the retina from inside, organized in three plexa in mice and humans; and junctional complexes between RPE cells, which filter blood from the fenestrated, leaky choroidal vessels. Breakdown of the BRB can occur at the level of retinal vessels, at the RPE layer or both. (From Forrester and Xu, 2012)

Unlike the BBB, however, the Blood Retinal Barrier (BRB) consists of both inner and outer components (Figure 13) (Forrester et al., 2010, Forrester and Xu, 2012), both essential for retinal homeostasis (Campbell et al., 2012). The inner BRB, similar to the BBB is located in the inner retinal microvasculature and comprises the microvascular endothelium, which line these vessels, and whose cells are connected by TJs, highly selective for diffusion of molecules from the blood to the retina. The outer is formed at the RPE cell layer and regulates the movement of solutes and nutrients from fenestrated choroidal vessels to the subretinal space. Both barriers are active in monitoring the passage of immune cells toward the retina and become more permissive as a result of inflammatory reactions. In particular, during inflammation, leukocytes (i.e. monocyte-derived macrophages, natural killer cells and lymphocytes), can access the retinal parenchyma from both inner retina vessels and choroidal capillaries (Figure 14), in this case crossing the RPE gateway (Crane and Liversidge, 2008).

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Figure 14. Mechanisms of leukocyte trafficking across the BRB.

Chemokine and cytokine signals and gradients trigger the steps involved in leukocytes entry into the retina, from the initial interactions with the endothelium to the diapedesis. These signals act also in inducing alterations in the regulatory molecules of the TJ that control the interaction between the membranous component and the cytoskeleton of the endothelial cell, leading to breakdown of the BRB. (From Crane and Liversidge, 2008)

In IRDs, which primarily involve PRs, the contribution of MPs coming from the choroidal circulation is expected to be substantial (Sennlaub et al., 2013).

Indeed, it has been demonstrated that inflammatory mediators might signal to RPE cells triggering changes in components of TJ complexes and resulting in a reduction in the strength of cell-cell adhesion (Ma et al., 2009).

Pigment epithelial cells per se play an immunomodulatory role through both the secretion of soluble immunosuppressive factors and by means of contact-dependent mechanisms (Whitcup et al., 2013). Alterations in RPE physiology and function might results in changes in the contribution this layer provides to retinal immune privilege.

Under normal physiological conditions, the posterior segment of the eye contains several types of MPs. As already illustrated, quiescent microglial cells are normally present as perivascular cells in the retina layers, forming three plexa at the level of the ganglion cell layer (GCL), inner plexiform layer (IPL) and outer plexiform layer (OPL), respectively

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(Penfold et al., 2001). The PR cell layer and the subretinal space (located between the RPE and the PR outer segments) are physiologically devoid of resident and infiltrating MPs (Gupta et al., 2003, Combadiere et al., 2007b, Sennlaub et al., 2013, Levy et al., 2015, Eandi et al., 2016).

Resident macrophages are found also in the choroid and along the major retinal vessels and are generally named choroidal and perivascular macrophages. Dendritic cells are rare in the retina (Streilein, 2003, Forrester et al., 2010, Kumar et al., 2014).

The retinal immune response is obviously adaptive and oriented to restore homeostasis. As for the brain, however, acute injures, infections, aging, metabolic abnormalities, altered vascular perfusion, or degenerative genetic conditions may initiate inflammatory cascades, which are themselves pathogenic or lead to vision threatening complications (Whitcup et al., 2013).

Inflammatory processes have classically been implicated in the pathogenesis and sequelae of infectious and non-infectious uveitis and the development of macular edema (Johnson and Morrison, 2009). Recent evidence has demonstrated a prominent role for inflammation in the pathogenesis of age-related macular degeneration (AMD) (Buschini et al., 2011b), diabetic retinopathy (DR) (Joussen et al., 2004), retinal vein occlusion (RVO) (Ehlers and Fekrat, 2011), and RP (Yoshida et al., 2013b, a), all leading to retinal degeneration.

Despite the different primary triggers, retinal cell death is major component of degenerative process in all these pathologies. Thus, inflammatory responses in retinal diseases share most of the leading aspects with inflammation-associated brain disorders discussed before.

The concept that microglia is a “double-edged sword”, able to protect or to destruct the tissue that inhabit, has been proved true for the retina too. For example, there are works highlighting the role of microglia, in particular of CX3CR1 signalling, in postnatal development of PRs within the mouse retina (Jobling et al., 2018). On the other hand, it has been demonstrated that media taken from LPS-stimulated microglial cell cultures are able to kill PRs (Roque et al., 1999, Lewis et al., 2005).

In the active, effector phase, amoeboid microglia migrates toward the site of retinal damage where it may interact with infiltrating blood cells (Figure 15). Short-lived circulating

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- 28 - monocytes are recruited and locally differentiate into inflammatory macrophages (Geissmann et al., 2003). This event is favoured under conditions of increased vascular permeability, pronounced RPE atrophy, breakdown of Bruch's membrane and choroidal neovascularization, often associate with retinal degenerative diseases (Xu and Forrester, 2009, Tang and Kern, 2011, Ardeljan and Chan, 2013).

Hence, the immune privilege of the eye is far from absolute.

Figure 15. Schematic representation of microglial activity in the retina.

A) In the normal retina, resident microglia mainly populates the plexiform layers. B) Different insults leading to abnormal cell functions or degeneration rapidly alert microglia. C) Resident microglia activates and migrates to the lesion sites. (Modified from Karlstetter et al. 2010)

 The case of Age-related macular degeneration

AMD is a common, complex disease that results from interplay of aging, genetic and environmental risk factors. It is characterized by pigmentary abnormalities and accumulation of membranous, lipo-proteinaceous debris located between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane of the central retina. In AMD the death of PRs by apoptosis is documented and results in loss of central visual acuity, leading to severe/ permanent visual impairment and irreversible vision loss (Curcio, 2001, Dunaief et al., 2002). Late AMD represents the third cause of irreversible vision loss

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worldwide, with an incidence of 6-8% among people older than 60 years (Joachim et al., 2015, Mitchell et al., 2018).

Late-stage AMD can be classified into either geographic atrophy coinciding with RPE and retinal atrophy in the macula or “wet” AMD with neovascular growth from the choroid into the macula causing subretinal edema, haemorrhage and RPE detachment (Jager et al., 2008).

Ample genetic and biochemical evidence supports the notion that AMD is related to a dysregulation of the innate immune system, mainly involving complement factors and reactive macrophages and microglia (Penfold et al., 2001, Fritsche et al., 2014). For the genetic component of AMD etiology, a strong associations of particular mutations in the complement factor H (CFH) protein, a key regulatory component of the complement alternative pathway, and AMD has been identified (Haines et al., 2005).

Evidence of active inflammation includes the fact that bloated phagocytes are closely associated with drusen of patients with early AMD and that human drusen contain inflammatory molecules capable of activating the NLRP3 inflammasome (a multiprotein oligomer implicated in activation of inflammatory responses in MPs), inducing the secretion of active IL-1β (Doyle et al., 2012).

IL-1β signalling stimulates expression and secretion of chemokines, as IL-8 and CCL2, in RPE cells. The consequent loss of TJ proteins leads to a breakage of BRB and the recruitment of leukocytes and macrophages, triggering an auto-amplifying inflammatory vicious circle (Ardeljan et al., 2014).

Importantly, increased intra-ocular levels of CCL2 are observed in human neovascular (Kramer et al., 2012, Fauser et al., 2015, Rezar-Dreindl et al., 2016) and atrophic-AMD (Newman et al., 2012, Sennlaub et al., 2013). CCR2 immunostaining have demonstrated that subretinal inflammatory infiltrate observed in human sections of GA patients consists, at least in part, of blood-derived CCR2-positive monocyte-derived macrophages (Sennlaub et al., 2013). Altogether, this evidence indicates a prominent role of the exogenous component in inflammation associated with AMD pathogenesis.

Laboratory studies confirm this fact. In experimental animal models of non-neovascular AMD, the inhibition of CCR2+_{monocyte recruitment nearly completely prevents the}