In the Eyes of the Beholder-New Mertk Knockout Mouse and Re-Evaluation of Phagocytosis versus Anti-Inflammatory Functions of MERTK.

Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke's scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

Introduction: The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina. Methods: We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods. Results and discussion: This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvß5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

Inflammation of the retinal pigment epithelium drives early-onset photoreceptor degeneration in Mertk-associated retinitis pigmentosa.

Severe, early-onset photoreceptor (PR) degeneration associated with MERTK mutations is thought to result from failed phagocytosis by retinal pigment epithelium (RPE). Notwithstanding, the severity and onset of PR degeneration in mouse models of Mertk ablation are determined by the hypomorphic expression or the loss of the Mertk paralog Tyro3. Here, we find that loss of Mertk and reduced expression/loss of Tyro3 led to RPE inflammation even before eye-opening. Incipient RPE inflammation cascaded to involve microglia activation and PR degeneration with monocyte infiltration. Inhibition of RPE inflammation with the JAK1/2 inhibitor ruxolitinib mitigated PR degeneration in Mertk-/- mice. Neither inflammation nor severe, early-onset PR degeneration was observed in mice with defective phagocytosis alone. Thus, inflammation drives severe, early-onset PR degeneration-associated with Mertk loss of function.

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the "eat me" signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.

Tissue-specific modifier alleles determine Mertk loss-of-function traits.

Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with Mertk KO mice. Mertk KO mice suffer from severe, early-onset retinal degeneration. MERTK, expressed in retinal pigment epithelia, is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed MERTK-mediated phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Here, we report that the loss of Mertk alone is not sufficient for retinal degeneration. The widely used Mertk KO mouse carries multiple coincidental changes in its genome that affect the expression of a number of genes, including the Mertk paralog Tyro3. Retinal degeneration manifests only when the function of Tyro3 is concomitantly lost. Furthermore, Mertk KO mice display improved anti-tumor immunity. MERTK is expressed in macrophages. Therefore, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose of cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. The resistance against two syngeneic mouse tumor models observed in Mertk KO mice is not, however, phenocopied by the loss of Mertk alone. Neither Tyro3 nor macrophage phagocytosis by alternate genetic redundancy accounts for the absence of anti-tumor immunity. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determines Mertk KO traits.

Cell culture models to study retinal pigment epithelium-related pathogenesis in age-related macular degeneration.

Age-related macular degeneration (AMD) is a disease that affects the macula - the central part of the retina. It is a leading cause of irreversible vision loss in the elderly. AMD onset is marked by the presence of lipid- and protein-rich extracellular deposits beneath the retinal pigment epithelium (RPE), a monolayer of polarized, pigmented epithelial cells located between the photoreceptors and the choroidal blood supply. Progression of AMD to the late nonexudative "dry" stage of AMD, also called geographic atrophy, is linked to progressive loss of areas of the RPE, photoreceptors, and underlying choriocapillaris leading to a severe decline in patients' vision. Differential susceptibility of macular RPE in AMD and the lack of an anatomical macula in most lab animal models has promoted the use of in vitro models of the RPE. In addition, the need for high throughput platforms to test potential therapies has driven the creation and characterization of in vitro model systems that recapitulate morphologic and functional abnormalities associated with human AMD. These models range from spontaneously formed cell line ARPE19, immortalized cell lines such as hTERT-RPE1, RPE-J, and D407, to primary human (fetal or adult) or animal (mouse and pig) RPE cells, and embryonic and induced pluripotent stem cell (iPSC) derived RPE. Hallmark RPE phenotypes, such as cobblestone morphology, pigmentation, and polarization, vary significantly betweendifferent models and culture conditions used in different labs, which would directly impact their usability for investigating different aspects of AMD biology. Here the AMD Disease Models task group of the Ryan Initiative for Macular Research (RIMR) provides a summary of several currently used in vitro RPE models, historical aspects of their development, RPE phenotypes that are attainable in these models, their ability to model different aspects of AMD pathophysiology, and pros/cons for their use in the RPE and AMD fields. In addition, due to the burgeoning use of iPSC derived RPE cells, the critical need for developing standards for differentiating and rigorously characterizing RPE cell appearance, morphology, and function are discussed.

Galectin-3 Promotes Müller Glia Clearance Phagocytosis via MERTK and Reduces Harmful Müller Glia Activation in Inherited and Induced Retinal Degeneration.

Clearance phagocytosis is a documented function of Müller glia in the retina. However, the molecular mechanisms of Müller glia phagocytosis remain largely undefined. Here, we show that extracellular galectin-3 and protein S promote clearance phagocytosis by immortalized human MIO-M1 Müller cells in an additive, saturable manner. Galectin-3 promotes phagocytosis by primary Müller glia from wild-type (WT) mice but not from mice that lack the engulfment receptor MERTK and therefore develop postnatal photoreceptor degeneration. Probing a possible functional link between Müller galectin-3 and MERTK, we discovered that mertk -/- Müller glia in situ show excess galectin-3 at postnatal day 20 (P20), an age prior to detectable photoreceptor degeneration. Moreover, double knockout (DKO) mice lacking both galectin-3 and MERTK show increased activation of Müller cells (but not of microglia) at P20 and more pronounced photoreceptor loss at P35 compared to mice lacking MERTK alone. Exploring the well-established sodium iodate injury model, we also found more severe activation specifically of Müller glia, and worse retinal damage in mice lacking galectin-3 compared to WT mice. Indeed, galectin-3 deficiency significantly increased sensitivity to injury, yielding Müller activation and retinal damage at a sodium iodate concentration that had no effect on the WT retina. Altogether, our results from both inherited and acutely induced models of retinal degeneration agree that eliminating galectin-3 exacerbates Müller cell activation and retinal degeneration. These data identify an important protective role for the MERTK ligand galectin-3 in the retina in restraining Müller glia activation.

Sex-specific multi-level 3D genome dynamics in the mouse brain.

The female mammalian brain exhibits sex hormone-driven plasticity during the reproductive period. Recent evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown. Here, we mapped the 3D genome of ventral hippocampal neurons across the oestrous cycle and by sex in mice. In females, we find cycle-driven dynamism in 3D chromatin organization, including in oestrogen response elements-enriched X chromosome compartments, autosomal CTCF loops, and enhancer-promoter interactions. With rising oestrogen levels, the female 3D genome becomes more similar to the male 3D genome. Cyclical enhancer-promoter interactions are partially associated with gene expression and enriched for brain disorder-relevant genes and pathways. Our study reveals unique 3D genome dynamics in the female brain relevant to female-specific gene regulation, neuroplasticity, and disease risk.

Probing Photoreceptor Outer Segment Phagocytosis by the RPE In Vivo: Models and Methodologies.

In the vertebrate retina, the light-sensitive photoreceptor rods and cones constantly undergo renewal by generating new portions of the outer segment and shedding their distal, spent tips. The neighboring RPE provides the critical function of engulfing the spent material by phagocytosis. RPE phagocytosis of shed rod outer segment fragments is a circadian process that occurs in a burst of activity shortly after daily light onset with low activity at other times, a rhythm that has been reported for many species and over 50 years. In this review, we compare studies on the rhythm and quantity of RPE phagocytosis using different in vivo model systems and assessment methods. We discuss how measurement methodology impacts the observation and analysis of RPE phagocytosis. Published studies on RPE phagocytosis investigating mice further suggest that differences in genetic background and housing conditions may affect results. Altogether, a comparison between RPE phagocytosis studies performed using differing methodology and strains of the same species is not as straightforward as previously thought.

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

Lack of the antioxidant enzyme methionine sulfoxide reductase A in mice impairs RPE phagocytosis and causes photoreceptor cone dysfunction.

Methionine sulfoxide reductase A (MsrA) is a widely expressed antioxidant enzyme that counteracts oxidative protein damage and contributes to protein regulation by reversing oxidation of protein methionine residues. In retinal pigment epithelial (RPE) cells in culture, MsrA overexpression increases phagocytic capacity by supporting mitochondrial ATP production. Here, we show elevated retinal protein carbonylation indicative of oxidation, decreased RPE mitochondrial membrane potential, and attenuated RPE phagocytosis in msra-/- mice. Moreover, electroretinogram recordings reveal decreased light responses specifically of cone photoreceptors despite normal expression and localization of cone opsins. Impairment in msra-/- cone-driven responses is similar from 6 weeks to 13 months of age. These functional changes match dramatic decreases in lectin-labeled cone sheaths and reduction in cone arrestin in msra-/- mice. Strikingly, cone defects in light response and in lectin-labeled cone sheath are completely prevented by dark rearing. Together, our data show that msra-/- mice provide a novel small animal model of preventable cone-specific photoreceptor dysfunction that may have future utility in analysis of cone dystrophy disease mechanisms and testing therapeutic approaches aiming to alleviate cone defects.

Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3.

Purpose: Galectin-3 (gal-3) is a soluble glycoprotein that has been associated with diverse forms of phagocytosis, including some mediated by the engulfment receptor MerTK. Retinal pigment epithelium (RPE) in vivo uses MerTK (or the related Tyro3) for phagocytosis of shed outer segment fragments during diurnal outer segment renewal. Here, we test if gal-3 plays a role in outer segment renewal in mice and if exogenous gal-3 can promote MerTK-dependent engulfment of isolated outer segment fragments by primary RPE cells in culture. Methods: We explored age- and strain-matched wild-type (wt), lgals3-/- and mertk-/- mice. Immunofluorescence and immunoblotting characterized gal-3 and RPE/retina protein expression, respectively. Outer segment renewal was investigated by live imaging of phosphatidylserine (PS) exposure on photoreceptor outer segment distal tips and by microscopy of rhodopsin-labeled RPE phagosomes in tissue sections. Retinal function was assessed by recording electroretinograms (ERGs). Phagocytosis assays feeding purified outer segment fragments (POS) were conducted with added recombinant proteins testing unpassaged primary mouse RPE. Results: Gal-3 localizes to neural retina and RPE in wt mice. The lgals3-/- photoreceptor outer segments display normal diurnal PS exposure at distal tips. The number of rhodopsin-positive phagosomes in wt and lgals3-/- RPE does not differ at peak or trough of diurnal phagocytosis activity. lgals3-/- mice show light responses like wt, and their eyes contain wt levels of retinal and RPE proteins. Unlike purified protein S, recombinant gal-3 fails to promote POS engulfment by mouse primary RPE in culture. Conclusions: Gal-3 has no essential role in MerTK-dependent outer segment renewal in mice.

Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in mertk, the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris. However, the early onset and very fast progression of degeneration in mutMerTK-RP remains unexplained. Here, we explored the role of microglia in the Royal College of Surgeons (RCS) rat model of mutMerTK-RP. We found elevated levels of inflammatory cytokines and CD68 microglia activation marker, and more ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia in RCS retina when compared to wild-type retina as early as postnatal day 14 (P14). Strikingly, renewal of photoreceptor outer segments in P14 wild-type rat retina is still immature with low levels of RPE phagocytosis implying that at this early age lack of this process in RCS rats is unlikely to distress photoreceptors. Although the total number of Iba-1 positive retinal microglia remains constant from P14 to P30, we observed increasing numbers of microglia in the outer retina from P20 implying migration to the outer retina before onset of photoreceptor cell death at ~P25. Iba-1 and CD68 levels also increase in the retina during this time period suggesting microglia activation. To determine whether microglia affect the degenerative process, we suppressed retinal microglia in vivo using tamoxifen or a combination of tamoxifen and liposomal clodronate. Treatments partly prevented elevation of Iba-1 and CD68 and relocalization of microglia. Moreover, treatments led to partial but significant retention of photoreceptor viability and photoreceptor function. We conclude that loss of the phagocytosis receptor MerTK causes microglia activation and relocalization in the retina before lack of RPE phagocytosis causes overt retinal degeneration, and that microglia activities accelerate loss of photoreceptors in mutMerTK-RP. These results suggest that therapies targeting microglia may delay onset and slow the progression of this blinding disease.

No Difference Between Age-Matched Male and Female C57BL/6J Mice in Photopic and Scotopic Electroretinogram a- and b-Wave Amplitudes or in Peak Diurnal Outer Segment Phagocytosis by the Retinal Pigment Epithelium.

Mice provide informative models of enormous utility for eye research. Sex as biological variable must be considered when conducting studies exploring mouse models. To determine if sex confounds neural retina or retinal pigment epithelium (RPE) activity in wild-type C57BL/6J mice, we compared male and female mice with respect to retinal light response and RPE phagocytosis. We tested 2-month-old mice at peak fertility and 12-month-old mice past fertility. Retinal function was assessed by quantifying a- and b-wave amplitudes of photopic and scotopic electroretinograms (ERGs). These experiments did not reveal differences between male and female mice at either age. As expected from earlier studies, 12-month-old mice showed reduced light responses compared to 2-month-old mice, but age-related decline was identical for male and female mice. RPE functionality was assessed by quantifying RPE phagosome content 1 h after light onset in mice 2 months of age, an age of maturity of the process of outer segment turnover that includes RPE phagocytosis. These experiments did not reveal differences in RPE phagocytosis between male and female mice. Altogether, male and female C57BL/6J mice do not differ in retinal light response and peak RPE phagocytic activity. Retinal activity is impaired with age to the same extent in male and female mice. Our results justify testing mixed-sex mouse cohorts in studies on outer segment renewal and RPE phagocytosis and illustrate the importance of careful consideration of cohort age.

Annexin A5 regulates surface αvß5 integrin for retinal clearance phagocytosis.

Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) is a conserved efferocytosis process whose binding step is mediated by αvß5 integrin receptors. Two related annexins, A5 (ANXA5) and A6 (ANXA6), share an αvß5 integrin-binding motif. Here, we report that ANXA5, but not ANXA6, regulates the binding capacity for spent photoreceptor outer segment fragments or apoptotic cells by fibroblasts and RPE. Similar to αvß5-deficient RPE, ANXA5-/- RPE in vivo lacks the diurnal burst of phagocytosis that follows photoreceptor shedding in wild-type retina. Increasing ANXA5 in cells lacking αvß5 or increasing αvß5 in cells lacking ANXA5 does not affect particle binding. Association of cytosolic ANXA5 and αvß5 integrin in RPE in culture and in vivo further supports their functional interdependence. Silencing ANXA5 is sufficient to reduce levels of αvß5 receptors at the apical phagocytic surface of RPE cells. The effect of ANXA5 on surface αvß5 and on particle binding requires the C-terminal ANXA5 annexin repeat but not its unique N-terminus. These results identify a novel role for ANXA5 specifically in the recognition and binding step of clearance phagocytosis, which is essential to retinal physiology.This article has an associated First Person interview with the first author of the paper.

Non-invasive in vivo fluorescence imaging of apoptotic retinal photoreceptors.

Phosphatidylserine externalization is an early molecular signature for apoptosis. In many retinal degenerative diseases, photoreceptor neurons die by apoptosis. Here, we report utility of the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in transiently labeling apoptotic photoreceptors in living pigmented or albino rats and mice with retinal degeneration. Applying PSVue-550 as eyedrop is non-toxic and eliminates need for intraocular injection. PSVue-550 fluorescence specifically and transiently labeling dying retinal photoreceptors is detectable in anesthetized animals using standard retinal or whole small animal imaging systems. Importantly, prior PSVue-550 eyedrop administration and imaging does not affect repeat testing. Altogether, our results establish PSVue-550 imaging as a completely non-invasive method that provides the opportunity to longitudinally monitor retinal photoreceptor cell death in preclinical studies.

Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture.

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we provide experimental protocols to synchronize the POS binding and engulfment steps of phagocytosis. Finally, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Expression and characterization of αvß5 integrin on intestinal macrophages.

Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvß5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvß5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvß5 is induced during the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF-like molecule 8. In the absence of αvß5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and IL 10. Mice lacking αvß5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvß5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.