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      Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency

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          Abstract

          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.

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          Most cited references44

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          Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine.

          Liposomes can be used as vehicles for intracellular delivery of drugs into phagocytic cells. Clodronate and propamidine, delivered into macrophages in this way, will kill these cells as a result of intracellular accumulation and irreversible metabolic damage. The so-called liposome-mediated macrophage 'suicide' approach, which is based on this principle, is now frequently applied in studies aimed at unravelling macrophage function. In the present study, the mechanism of phagocytic cell death induced by liposome encapsulated drugs was investigated 'in vitro'. Peritoneal macrophages and macrophages of the RAW 264 cell line were cultured in the presence of the liposome encapsulated drugs clodronate, propamidine and several forms of ethylenediaminetetraacetic acid (EDTA). The results obtained suggest that apoptotic death is induced in phagocytic cells both by liposomally delivered clodronate and by liposomally delivered propamidine. Although intracellular EDTA did induce apoptosis in a minority of the experiments, the results support earlier findings that EDTA does not deplete macrophages as effectively as clodronate and propamidine.
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            INHERITED RETINAL DYSTROPHY IN THE RAT

            Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.
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              Inducible Cre mice.

              The Cre/lox site-specific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. A major technical advance in terms of in vivo inducibility was the development of ligand-dependent Cre recombinases that can be activated by administration of tamoxifen to the animal. Here we describe how tamoxifen-dependent Cre recombinases, so-called CreER recombinases, work and how they can be used to generate time- and tissue-specific mouse mutants. The focus will be on the CreER(T2) recombinase, which is currently the most successful CreER version. We will give an overview of available CreER(T2) transgenic mouse lines and present protocols that detail the generation of experimental mice for inducible gene knockout studies, the induction of recombination by tamoxifen treatment, and the analysis of the quality and quantity of recombination by reporter gene and target gene studies. Most of the protocols can also be used as general guidelines for the generation and characterization of Cre/lox-mediated genome modifications in mice.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                16 July 2020
                2020
                : 11
                : 1463
                Affiliations
                Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University , Bronx, NY, United States
                Author notes

                Edited by: Carlos Rosales, National Autonomous University of Mexico, Mexico

                Reviewed by: Lian Zhao, National Eye Institute (NEI), United States; Hemant Khanna, University of Massachusetts Medical School, United States

                *Correspondence: Silvia C. Finnemann finnemann@ 123456fordham.edu

                This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

                †These authors have contributed equally to this work

                Article
                10.3389/fimmu.2020.01463
                7381113
                32765507
                e49703f6-ef8e-4ecb-9647-3aae67c653f2
                Copyright © 2020 Lew, Mazzoni and Finnemann.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 01 February 2020
                : 05 June 2020
                Page count
                Figures: 7, Tables: 1, Equations: 0, References: 66, Pages: 15, Words: 9907
                Funding
                Funded by: National Eye Institute 10.13039/100000053
                Categories
                Immunology
                Original Research

                Immunology
                retinitis pigmentosa,mertk,phagocytosis,microglia,retinal degeneration
                Immunology
                retinitis pigmentosa, mertk, phagocytosis, microglia, retinal degeneration

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