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Douglas Vollrath - One of the best experts on this subject based on the ideXlab platform.
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gene therapy for MERTK associated retinal degenerations
Advances in Experimental Medicine and Biology, 2016Co-Authors: Matthew M Lavail, Wentao Deng, Douglas Yasumura, Michael T Matthes, Haidong Yang, William W Hauswirth, Douglas VollrathAbstract:MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in MERTK-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.
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Tyro3 Modulates MERTK-Associated Retinal Degeneration.
PLoS genetics, 2015Co-Authors: Douglas Vollrath, Douglas Yasumura, Michael T Matthes, Wei Feng, Gillie Benchorin, Natalie M. Nguyen, Cecilia D. Sedano, Melissa A. Calton, Matthew M LavailAbstract:Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of MERTK-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in MERTK-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. MERTK-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of MERTK. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in MERTK knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of MERTK knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates MERTK-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.
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MERTK drives myosin II redistribution during retinal pigment epithelial phagocytosis.
Investigative ophthalmology & visual science, 2008Co-Authors: David J. Strick, Wei Feng, Douglas VollrathAbstract:PURPOSE. MERTK is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, MERTK is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of MERTK function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which MERTK regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with MERTK during OS phagocytosis. METHODS. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with MERTK antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS. Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional MERTK. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS. MERTK mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of US.
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MERTK arginine-844-cysteine in a patient with severe rod-cone dystrophy: Loss of mutant protein function in transfected cells
Investigative ophthalmology & visual science, 2004Co-Authors: Christina L. Mchenry, Douglas Vollrath, Wei Feng, Yuhui Liu, Anita R. Nair, Kecia L. Feathers, Xiaoling Ding, Andreas Gal, Paul A. Sieving, Debra A. ThompsonAbstract:PURPOSE. Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod- cone degeneration in a young patient. METHODS. MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35 S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR. RESULTS. Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5° centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning. CONCLUSIONS. The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.
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MERTK Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells
Journal of Biological Chemistry, 2002Co-Authors: Wei Feng, Matthew M Lavail, Douglas Yasumura, Michael T Matthes, Douglas VollrathAbstract:Abstract The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning andin vivo genetic complementation to demonstrate thatMERTK is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of MERTK and to obtain functional evidence for a key role of MERTK in RPE phagocytosis. We found that MERTK protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat MERTK to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. MERTK protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated MERTK accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that MERTK likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.
Deborah Deryckere - One of the best experts on this subject based on the ideXlab platform.
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MERTK in cancer therapy: Targeting the receptor tyrosine kinase in tumor cells and the immune system.
Pharmacology & therapeutics, 2020Co-Authors: Justus M. Huelse, Deborah Deryckere, Diana M. Fridlyand, Shelton Earp, Douglas K. GrahamAbstract:The receptor tyrosine kinase MERTK is aberrantly expressed in numerous human malignancies, and is a novel target in cancer therapeutics. Physiologic roles of MERTK include regulation of tissue homeostasis and repair, innate immune control, and platelet aggregation. However, aberrant expression in a wide range of liquid and solid malignancies promotes neoplasia via growth factor independence, cell cycle progression, proliferation and tumor growth, resistance to apoptosis, and promotion of tumor metastases. Additionally, MERTK signaling contributes to an immunosuppressive tumor microenvironment via induction of an anti-inflammatory cytokine profile and regulation of the PD-1 axis, as well as regulation of macrophage, myeloid-derived suppressor cell, natural killer cell and T cell functions. Various MERTK-directed therapies are in preclinical development, and clinical trials are underway. In this review we discuss MERTK inhibition as an emerging strategy for cancer therapy, focusing on MERTK expression and function in neoplasia and its role in mediating resistance to cytotoxic and targeted therapies as well as in suppressing anti-tumor immunity. Additionally, we review preclinical and clinical pharmacological strategies to target MERTK.
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abstract a35 MERTK and bcl 2 as potential therapeutic targets in early t precursor acute lymphoblastic leukemia
Cancer Research, 2019Co-Authors: Ryan J Summers, Deborah Deryckere, Katherine Minson, Stephen V. Frye, Xiaodong Wang, Shelton H Earp, Douglas K. GrahamAbstract:Background: Early T-precursor acute lymphoblastic leukemia (ETP-ALL) is a subclass of T-cell ALL (T-ALL) accounting for 15% of pediatric T-ALL cases and characterized by an immature phenotype, resistance to therapy, and high rates of induction failure and relapse (Wood B et al., Blood 2009). MERTK receptor tyrosine kinase is not expressed in normal T cells but is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T-cell phenotype (Graham DK et al., Clin Cancer Res 2006), suggesting a role in ETP-ALL. One potential role is regulation of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2). BCL-2 is specifically expressed in double-negative T-cell precursors and is preferentially expressed in ETP-ALL compared to T-ALL (Chonghaile et al., Cancer Discovery 2014). Moreover, ETP-ALL cells are dependent on BCL-2 for survival. Our previous studies demonstrated regulation of BCL-2 and BCL-2 family members downstream of MERTK in B-ALL and acute myeloid leukemia cells (Linger RM et al., Blood 2013; Lee-Sherick AB et al., Oncotarget 2015). This interplay between MERTK and BCL-2 and their association with an immature T-ALL phenotype suggest that combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods: Publicly available mRNA expression data were used to assess MERTK and BCL-2 expression in T-ALL cell lines and patient samples. MERTK and BCL-2 protein expression were determined by immunoblot. ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 kinase inhibitor. MERTK protein was immunoprecipitated from cell lysates and phosphorylated and total proteins were assessed by immunoblot. Alternatively, cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Orthotopic xenografts were established in NSGS mice using an ETP-ALL patient sample, and leukemia burden in peripheral blood (%hCD45+) was monitored by flow cytometry. After engraftment (1.86 +/- 0.43% peripheral blasts), mice were treated once daily with 75 mg/kg MRX-2843 or saline vehicle administered orally. Mice were euthanized when symptoms of advanced leukemia were evident and median survival was determined by Kaplan-Meier analysis. Results: MERTK mRNA was expressed at significantly higher levels in ETP-ALL cell lines and patient samples relative to other T-ALLs. Similarly, MERTK protein was ubiquitously expressed in ETP-ALL cell lines (n=2), ETP-ALL patient samples (n=2), and a near-ETP-ALL patient sample (n=1). In contrast, only 60% of other T-ALL cell lines (n=5) expressed MERTK. BCL-2 mRNA was expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs and BCL-2 protein was expressed in 2 of 2 ETP-ALL cell lines and 2 of 3 ETP-ALL and near-ETP-ALL patient samples. Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK in ETP-ALL cells and induced dose-dependent cell death in the ETP-ALL cell lines PEER (43.2% vs 16% in vehicle-treated cultures, p Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843, a dual MERTK/FLT3 kinase inhibitor, has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. In particular, combination therapy targeting MERTK and BCL-2 may be an effective therapeutic option for ETP-ALL. Citation Format: Ryan J. Summers, Katherine A. Minson, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A35.
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MERTK Inhibition Promotes Anti-Leukemia Immunity By Reversing T Cell Suppression Via the PD-1 Axis
Blood, 2018Co-Authors: Alisa B. Lee-sherick, Deborah Deryckere, Stephen V. Frye, H. Shelton Earp, Curtis J. Henry, Rebecca E. Parker, Xiaodong Wang, Kristen Jacobsen, Douglas K. GrahamAbstract:Abstract The efferocytic receptor MERTK is expressed on monocytes/macrophages and facilitates tolerogenic T cell suppressive clearance of apoptotic cellular debris. Given the rapid cell turn-over in leukemia, we hypothesized that ongoing efferocytosis is leukemia-permissive and inhibition of MERTK on monocytes/macrophages will reverse T cell suppression leading to enhanced anti-leukemia immunity. To test this hypothesis, we inoculated mice with syngeneic murine BCR-ABL p185 acute lymphoblastic leukemia (ALL) cells, which do not express MERTK. The effects of MERTK inhibition were evaluated in wild-type (WT) mice treated daily with MRX-2843, a MERTK small molecule inhibitor, and in mice with a MERTK knockout mutation (MERTK-/-). Median survival was prolonged in WT mice treated with MRX-2843 (40 days) compared to vehicle treatment (20 days, p<0.01). In littermate MERTK-/- mice, all but one mouse survived without evidence of leukemia 150 days after inoculation (p<0.0001). To explore the T cell suppressive effects of MERTK inhibition on CD11b+ monocytes/macrophages, we used flow cytometry to study ex vivo mixed cell cultures using MERTK-/- or WT murine splenocytes +/- ALL cells treated +/- MRX-2843. Co-culture with ALL cells for 24 (36.0%), 48 (34.9%) and 72 (36.6%) hours resulted in a significant increase in expression of co-inhibitory ligands PD-L1 and PD-L2 on CD11b+ cells compared to cultures without ALL cells (2.03%, 3.2% and 7.5%, p<0.0001), or cultures with ALL cells treated with MRX-2843 (8.3%, 15.3% and 9.5%, p<0.0001). Similarly, PD-L1/PD-L2 co-expression on CD11b+ cells from MERTK-/- mice was not significantly altered in the presence (16.0%) or absence (7.0%) of ALL. Furthermore, there was no significant change in PD-L1/PD-L2 expression when MERTK-/- co-cultures were treated with MRX-2843 (12.6%). These findings were recapitulated in vivo. There was no change in PD-L1/PD-L2 expression on CD11b+ cells from MERTK-/- mice inoculated with ALL (4.8% spleen; 0.8% marrow) compared to uninoculated MERTK-/- mice (7.2% spleen; 1.2% marrow) or uninoculated WT littermates (2.0% spleen; 0.7% bone marrow), whereas CD11b+ cells from WT mice inoculated with ALL demonstrated a significant upregulation of PD-L1/PD-L2 (24.1% spleen, p<0.0001; and 4.2% bone marrow, p<0.001), which was downregulated upon treatment with MRX-2843 (1.9% spleen, p<0.01; 1.2% bone marrow, p<0.05) In these experiments, expression of PD-1, the co-inhibitory receptor which binds PD-L1/PD-L2, on T cells was also assessed. In WT mice inoculated with ALL, expression of PD-1 on T cells from spleens (74.2% CD4+; 49.2% CD8+) and bone marrow (65.9% CD4+; 73.0% CD8+) was significantly increased compared to spleens (10.0% CD4+; 4.1% CD8+) and bone marrow (36.4% CD4+; 8.5% CD8+) from uninoculated control mice (p<0.0001). However, expression of PD-1 was not significantly different in spleens (28.3% CD4+; 7.2% CD8+) and bone marrow (66.2% CD4+; 29.2% CD8+) from MERTK-/- mice inoculated with ALL compared with spleens (20.0% CD4+; 4.7% CD8+) and bone marrow (62.2% CD4+; 12.4% CD8+) from uninoculated MERTK-/- mice. To determine whether changes in PD-1 expression were indicative of T cell activation or exhaustion, T cells treated with α-CD3 beads were added to splenocyte and ALL cell co-cultures for 24 hours and intracellular IFN-ɣ and TNF-α levels were assessed using flow cytometry. The fraction of T cells producing IFN-ɣ/TNF-α significantly increased in co-cultures treated with MRX-2843 (44.2% CD4+; 51.1% CD8+) compared to vehicle-treated co-cultures (27.4% CD4+, p<0.0001; 36.8% CD8+, p<0.0001). Similarly, IFN-ɣ/TNF-α production increased in co-cultures with MERTK-/- splenocytes and ALL cells (48.3% CD4+, p<0.0001; 51.2% CD8+, p<0.0001) compared to co-cultures with WT splenocytes and activation was not further augmented by treatment with MRX-2843 (48.9% CD4+, 51.5% CD8+). Similar results were obtained when T cells from MERTK-/-mice were used, demonstrating that the effects of T cell activation were dependent on the presence of MERTK on CD11b+ cells, but not T cells. In conclusion, inhibition of MERTK on CD11b+ monocytes/macrophages reversed T cell suppression in response to leukemia via the PD-1 axis. MERTK inhibitors are in early phase clinical trials and these data demonstrate a potential immunotherapeutic use for MERTK inhibitors in the treatment of ALL. Disclosures Wang: Meryx: Equity Ownership, Patents & Royalties. Frye:Meryx: Equity Ownership, Patents & Royalties. Earp:Meryx: Equity Ownership. DeRyckere:Meryx: Equity Ownership. Graham:Meryx: Equity Ownership.
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MERTK inhibition in the leukemia microenvironment promotes a therapeutic immune response to acute lymphoblastic leukemia
Journal of Immunology, 2017Co-Authors: Kristen M. Jacobsen, Alisa B. Lee-sherick, Weihe Zhang, H. Shelton Earp, Madeline G. Huey, Lauren S. Page, Xiaodong Wang, Stephen V. Fyre, Craig T. Jordan, Deborah DeryckereAbstract:T cell immunotherapy is effective in some, but not all cancers, and targeting the immunosuppressive, innate immune tumor microenvironment is one key to further advancement. During efferocytosis, activation of MERTK tyrosine kinase receptor in innate immune cells suppresses proinflammatory cytokines, stimulates immunosuppressive cytokines and upregulates co-inhibitory ligands. MERTK has also been validated as a therapeutic target in the tumor microenvironment in solid tumor models. To investigate immunoregulatory roles for MERTK in leukemia, we utilized pharmacologic and genetic inhibition of MERTK in a MERTK-negative syngeneic murine model of acute lymphoblastic leukemia (ALL). Treatment with MRX-2843, a potent, orally bioavailable, small molecule MERTK inhibitor, significantly reduced leukemic burden and prolonged median survival in wildtype mice (38 days post-treatment vs. 24 days in vehicle-treated mice, p>0.01). Similarly, overall survival was increased more than four-fold in MERTK −/− mice transplanted with leukemia (>80 days vs. 21 days in wild-type mice, p>0.0001), suggesting that the observed therapeutic activity is immune-mediated. Indeed, treatment with MRX-2843 abrogated expression of the immune co-inhibitory ligands PD-L1 and PD-L2 on CD11b + monocytes/macrophages in the ALL microenvironment. Although T cells do not express MERTK, expression of the cognate PD-1 receptor was also reduced on CD8+ T cells and the frequency of FOXP3 + CD4 + regulatory T cells was decreased in the leukemia microenvironment following MRX-2843 treatment. These data demonstrate several mechanisms by which MERTK can suppress anti-leukemia immunity and validate MERTK as a novel immunotherapeutic target in ALL.
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Discovery of Macrocyclic Pyrimidines as MERTK‐Specific Inhibitors
ChemMedChem, 2017Co-Authors: Andrew L. Mciver, Weihe Zhang, Qingyang Liu, Xinpeng Jiang, Michael A. Stashko, James H. Nichols, Michael J. Miley, Jacqueline Norris-drouin, Mischa Machius, Deborah DeryckereAbstract:Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure-based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MERTK)-specific inhibitors. An enzyme-linked immunosorbent assay (ELISA) in 384-well format was employed to evaluate the inhibitory activity of macrocycles in a cell-based assay assessing tyrosine phosphorylation of MERTK. Through structure-activity relationship (SAR) studies, analogue 11 [UNC2541; (S)-7-amino-N-(4-fluorobenzyl)-8-oxo-2,9,16-triaza-1(2,4)-pyrimidinacyclohexadecaphane-1-carboxamide] was identified as a potent and MERTK-specific inhibitor that exhibits sub-micromolar inhibitory activity in the cell-based ELISA. In addition, an X-ray structure of MERTK protein in complex with 11 was resolved to show that these macrocycles bind in the MERTK ATP pocket.
C. Pitzalis - One of the best experts on this subject based on the ideXlab platform.
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THU0082 MERTK SYNOVIAL EXPRESSION CORRELATES WITH TREATMENT RESPONSE IN RHEUMATOID ARTHRITIS
Annals of the Rheumatic Diseases, 2020Co-Authors: Alessandra Nerviani, M. A. Boutet, G. M. Ghirardi, G. Lliso Ribera, Felice Rivellese, Myles Lewis, Michele Bombardieri, Frances Humby, C. PitzalisAbstract:Background: Despite substantial improvements in long-term clinical outcomes, a significant proportion of rheumatoid arthritis (RA) patients still fail to respond to treatment adequately, and early prognostic biomarkers of response are missing. Single-cell transcriptomic studies on RA synovial tissue (ST) have shown that MERTK is highly expressed in “anti-inflammatory” macrophages [1]. It has also been suggested that synovial macrophages isolated from RA patients in remission are characterised by a CD206+/MERTK+ signature [2]. Finally, monocyte-derived macrophages from RA patients treated with TNF-inhibitors (TNF-i) up-regulate MERTK. Objectives: To assess i) the modulation of synovial tissue MERTK+ macrophages upon treatment with conventional synthetic (cs) disease-modifying anti-rheumatic drugs (DMARDs) and ii) the relationship between baseline MERTK gene expression and response to TNFi. Methods: ST was obtained by US-guided synovial biopsies of an inflamed peripheral joint in patients with early ( Results: Before any treatment intervention, the percentage of MERTK+CD206+ macrophages was significantly higher in RA patients with low (DAS28 5.1) disease activity (24.5±20.1 versus 4.8±4.8, p Conclusion: Our whole-tissue protein expression data further support the hypothesis that a selective expansion of the MERTK+ macrophage subset characterise patients achieving remission. Moreover, the pre-treatment up-regulation of the MERTK gene in future responders to TNFi suggest that MERTK is implicated in modulating synovial inflammatory responses and may be exploited as a therapeutic target in RA. References: [1]F. Zhang et al, Nature Immunology, vol. 20, no. 7, pp. 928–942, 2019. [2]S. R. Finlay at al, Annals of the Rheumatic Diseases, vol. 77, Supplement 2, pp. 183–183, 2018. [3]Y. Degboe et al, Frontiers in Immunology, vol. 10, p. 3, 2019. Acknowledgments: Versus Arthritis Disclosure of Interests: None declared
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THU0033 A Functional Inhibitory Role for the GAS6/MERTK Axis in Modulating Synovial Inflammation in Rheumatoid Arthritis
Annals of the Rheumatic Diseases, 2015Co-Authors: Alessandra Nerviani, Michele Bombardieri, Frances Humby, Stephen Kelly, C. PitzalisAbstract:Background MERTK is an inhibitory tyrosine kinase receptor activated in several immune cells upon binding of its ligand Growth-Arrest-Specific-6 (Gas6). MERTK/Gas6 axis controls inflammation and immune system in animal models and its loss induces systemic chronic inflammation and autoimmunity. In Rheumatoid Arthritis (RA) patients Gas6 plasmatic level is decreased compared to healthy subjects 1 while the over-expression of MERTK agonists in collagen-induced arthritis ameliorates disease 2 . However, little is known about the MERTK/Gas6 system expression and functional role within the synovial tissue in RA patients. Objectives To characterize the expression of MERTK/Gas6 in human rheumatoid synovitis, investigating the mechanisms leading to activation/resistance of RA synovial cells to MERTK-mediated regulation of inflammation. Methods MERTK expression in synovial tissue was assessed by immunohistochemistry/immunofluorescence staining. MERTK/CD163 synovial tissue gene expression was analyzed in 30 early-RA patients treatment-naive. Mononuclear cells isolated from inflammatory synovial fluids were stimulated with rhGas6 (200 ng/ml) for 24 h and interleukin (IL) 10 production was measured in cells supernatant by ELISA. Synovial fibroblasts isolated from RA synovial tissue were stimulated with Toll-Like-Receptor (TLR) 3 and 4 ligands (respectively polyinosinic-polycytidylic acid and lipopolysaccharide); Gas6 secretion in stimulated and resting fibroblasts was quantified by ELISA at different time points. Results MERTK was strongly expressed by resident CD68+ synovial macrophages in both the lining and sub-lining; moreover, MERTK-expressing cells were detected within ectopic-lymphoid-structures. MERTK mRNA in treatment-naive early RA patients was positively correlated with CD163, a marker of anti-inflammatory-polarized-macrophages (r=0.58), but not with CD68. RA synovial fluid lympho-mononuclear cells up-regulated the expression of IL10 in the presence of rhGas6. Finally, RA synovial fibroblasts constitutively released Gas6 but at levels significantly lower compared to RA dermal fibroblast. Moreover, TLR-activation of RA synovial fibroblasts led to a significant decrease in Gas6 expression. Conclusions The MERTK/Gas6 inhibitory axis is expressed in RA synovitis. In vitro , exogenous Gas6 exerted an immune-modulatory role on synovial fluid mononuclear cells by increasing the release of anti-inflammatory cytokines. Conversely, the diminished availability of Gas6 in synovial stromal cells following pro-inflammatory stimuli may contribute to the chronic inflammation in the rheumatoid synovium. Our data suggest that the exploitation of the anti-inflammatory properties of the Gas6/MERTK axis might constitute a novel therapeutic approach in RA. References Bassyouni IH et al. Diminished soluble levels of growth arrest specific protein 6 and tyrosine kinase receptor Axl in patients with rheumatoid arthritis. Int J Rheum Dis. 2014 Apr van den Brand BT et al. Therapeutic efficacy of Tyro3, Axl, and Mer tyrosine kinase agonists in collagen-induced arthritis. Arthritis Rheum. 2013 Mar Disclosure of Interest None declared
Celia Parinot - One of the best experts on this subject based on the ideXlab platform.
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A Comprehensive Review of Mutations in the MERTK Proto-Oncogene.
Advances in experimental medicine and biology, 2015Co-Authors: Celia Parinot, Emeline F. NandrotAbstract:Phagocytosis and elimination of shed aged photoreceptor outer segments (POS) by retinal pigment epithelial cells is crucial for photoreceptor function and survival. Genetic studies on a natural animal model of recessive retinal degeneration allowed the identification of MERTK, the gene encoding the surface receptor required for POS internalization. Following this discovery, screenings of DNA samples from patients have revealed that MERTK mutations cause retinal degenerations in humans. MERTK patients present some of the classical symptoms of retinitis pigmentosa, but it is atypical in that the disease develops very early during childhood and the macula is also involved early on. Therefore, the phenotype ought to be qualified as a rod-cone dystrophy. Recently, MERTK has been implicated in various types of cancers and sclerosis. This review identifies the different MERTK mutations known so far and describes associated pathologies.
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Etude des mécanismes extracellulaires régulant la fonction du récepteur MERTK au cours de la phagocytose rétinienne
2015Co-Authors: Celia ParinotAbstract:Le récepteur MERTK est impliqué dans la phagocytose des segments externes des photorécepteurs (SEP) par l'épithélium pigmentaire rétinien (EPR), fonction cruciale pour la survie des photorécepteurs et la vision. Dans la rétine, ces deux tissus sont en contact permanent et la phagocytose ne survient qu'une fois par jour, cette fonction nécessite donc d'être contrôlée précisément. Le pic de phagocytose est lié à l'activation intracellulaire de MERTK via l'intégrine αvβ5. Ce projet a eu pour but d'étudier les mécanismes extracellulaires régulant la fonction de MERTK au cours de cette phagocytose.Nous avons montré que MERTK est clivé à la surface des cellules d'EPR in vivo avant et après le pic de phagocytose. Ceci permettrait d'éviter une phagocytose trop prononcée des SEP.Nous avons démontré le rôle opposé des ligands de MERTK, spécifique à l'EPR. Gas6 semble inhibiteur, il stimule le clivage de MERTK et inhibe la phagocytose in vitro, et son expression in vivo est faible au moment du pic de phagocytose. Au contraire, Protéine S, dont l'expression augmente in vivo au moment du pic, inhibe le clivage de MERTK et stimule la phagocytose in vitro, et pourrait ainsi potentialiser cette fonction.Parmi les protéases étudiées, l'inhibition d'ADAM17 in vitro engendre une diminution du clivage de MERTK corrélée à une augmentation de sa biodisponibilité à la surface cellulaire et de son activité. Cependant, cet effet n'étant pas total, l'implication d'une autre protéase n'est pas exclue.Ainsi, mes travaux de Doctorat permettent de mieux comprendre la régulation complexe de l'activité de MERTK dans la phagocytose rétinienne, essentielle pour le rythme circadien de cette fonction.
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cleavage of mer tyrosine kinase MERTK from the cell surface contributes to the regulation of retinal phagocytosis
Journal of Biological Chemistry, 2015Co-Authors: Ahlai Law, Celia Parinot, Jonathan Chatagnon, Basile GravezAbstract:Abstract Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins including MERTK receptors and associated Gas6 and Protein S ligands. In the retina, POS phagocytosis is rhythmic and MERTK is activated promptly after light onset via the alphavbeta5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a downregulation mechanism that limits its duration. Our previous data showed that MERTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MERTK (sMERTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MERTK ligands have an opposite effect on phagocytosis and sMERTK release, whilst the integrin ligand MFG-E8 markedly increases both phagocytosis and sMERTK levels. sMERTK acts as a decoy receptor blocking the effect of both MERTK ligands. Interestingly, stimulation of sMERTK release decreases POS binding. Conversely, blocking MERTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MERTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.
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cleavage of mer tyrosine kinase MERTK from the cell surface contributes to the regulation of retinal phagocytosis
Journal of Biological Chemistry, 2015Co-Authors: Ahlai Law, Celia Parinot, Jonathan Chatagnon, Basile GravezAbstract:Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MERTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MERTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MERTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MERTK (sMERTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MERTK ligands have an opposite effect on phagocytosis and sMERTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMERTK levels. sMERTK acts as a decoy receptor blocking the effect of both MERTK ligands. Interestingly, stimulation of sMERTK release decreases POS binding. Conversely, blocking MERTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MERTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.
Wei Feng - One of the best experts on this subject based on the ideXlab platform.
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Tyro3 Modulates MERTK-Associated Retinal Degeneration.
PLoS genetics, 2015Co-Authors: Douglas Vollrath, Douglas Yasumura, Michael T Matthes, Wei Feng, Gillie Benchorin, Natalie M. Nguyen, Cecilia D. Sedano, Melissa A. Calton, Matthew M LavailAbstract:Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of MERTK-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in MERTK-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. MERTK-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of MERTK. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in MERTK knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of MERTK knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates MERTK-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.
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MERTK drives myosin II redistribution during retinal pigment epithelial phagocytosis.
Investigative ophthalmology & visual science, 2008Co-Authors: David J. Strick, Wei Feng, Douglas VollrathAbstract:PURPOSE. MERTK is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, MERTK is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of MERTK function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which MERTK regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with MERTK during OS phagocytosis. METHODS. Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with MERTK antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS. Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional MERTK. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS. MERTK mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of US.
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MERTK arginine-844-cysteine in a patient with severe rod-cone dystrophy: Loss of mutant protein function in transfected cells
Investigative ophthalmology & visual science, 2004Co-Authors: Christina L. Mchenry, Douglas Vollrath, Wei Feng, Yuhui Liu, Anita R. Nair, Kecia L. Feathers, Xiaoling Ding, Andreas Gal, Paul A. Sieving, Debra A. ThompsonAbstract:PURPOSE. Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod- cone degeneration in a young patient. METHODS. MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse-chase studies of 35 S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR. RESULTS. Three MERTK sequence variants were identified in a patient with rod-cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5° centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull's-eye macular atrophy and widespread RPE thinning. CONCLUSIONS. The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.
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MERTK Triggers Uptake of Photoreceptor Outer Segments during Phagocytosis by Cultured Retinal Pigment Epithelial Cells
Journal of Biological Chemistry, 2002Co-Authors: Wei Feng, Matthew M Lavail, Douglas Yasumura, Michael T Matthes, Douglas VollrathAbstract:Abstract The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning andin vivo genetic complementation to demonstrate thatMERTK is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of MERTK and to obtain functional evidence for a key role of MERTK in RPE phagocytosis. We found that MERTK protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat MERTK to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. MERTK protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated MERTK accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that MERTK likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.