RESUMO
In this issue honoring the contributions of Greg Lemke, the Earp and Graham lab teams discuss several threads in the discovery, action, signaling, and translational/clinical potential of MERTK, originally called c-mer, a member of the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases. The 30-year history of the TAM RTK family began slowly as all three members were orphan RTKs without known ligands and/or functions when discovered by three distinct alternate molecular cloning strategies in the pre-genome sequencing era. The pace of understanding their physiologic and pathophysiologic roles has accelerated over the last decade. The activation of ligands bridging externalized phosphatidylserine (PtdSer) has placed these RTKs in a myriad of processes including neurodevelopment, cancer, and autoimmunity. The field is ripe for further advancement and this article hopefully sets the stage for further understanding and therapeutic intervention. Our review will focus on progress made through the collaborations of the Earp and Graham labs over the past 30 years.
Assuntos
Neoplasias , c-Mer Tirosina Quinase , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/antagonistas & inibidores , c-Mer Tirosina Quinase/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Animais , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidoresRESUMO
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Fagocitose , Proteína S , c-Mer Tirosina Quinase , Animais , Camundongos , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligantes , Proteína S/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/citologiaRESUMO
Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Janus Quinase 1 , Receptores Proteína Tirosina Quinases , Sarcoma de Ewing , Transdução de Sinais , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Sarcoma de Ewing/genética , Humanos , Janus Quinase 1/metabolismo , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Linhagem Celular Tumoral , Animais , Transdução de Sinais/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Camundongos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fosforilação/efeitos dos fármacos , Feminino , Fator de Transcrição STAT6RESUMO
Oligodendrocyte death is common in aging and neurodegenerative disease. In these conditions, dying oligodendrocytes must be efficiently removed to allow remyelination and to prevent a feedforward degenerative cascade. Removal of this cellular debris is thought to primarily be carried out by resident microglia. To investigate the cellular dynamics underlying how microglia do this, we use a single-cell cortical demyelination model combined with longitudinal intravital imaging of dual-labeled transgenic mice. Following phagocytosis, single microglia clear the targeted oligodendrocyte and its myelin sheaths in one day via a precise, rapid, and stereotyped sequence. Deletion of the fractalkine receptor, CX3CR1, delays the microglial phagocytosis of the cell soma but has no effect on clearance of myelin sheaths. Unexpectedly, deletion of the phosphatidylserine receptor, MERTK, has no effect on oligodendrocyte or myelin sheath clearance. Thus, separate molecular signals are used to detect, engage, and clear distinct sub-compartments of dying oligodendrocytes to maintain tissue homeostasis.
Assuntos
Receptor 1 de Quimiocina CX3C , Microglia , Oligodendroglia , Fagocitose , c-Mer Tirosina Quinase , Animais , Oligodendroglia/metabolismo , Microglia/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Camundongos , Bainha de Mielina/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Morte CelularRESUMO
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.
Assuntos
Camundongos Knockout , Fagocitose , c-Mer Tirosina Quinase , Animais , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Camundongos , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Modelos Animais de Doenças , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Humanos , Inflamação/genética , Inflamação/metabolismoRESUMO
Introduction: TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. Methods: We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 µL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-ß and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. Conclusion: This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.
Assuntos
Receptor Tirosina Quinase Axl , Homeostase , Pulmão , Macrófagos Alveolares , Camundongos Knockout , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Silicose , c-Mer Tirosina Quinase , Animais , Camundongos , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Silicose/metabolismo , Silicose/imunologia , Silicose/patologia , MasculinoRESUMO
The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.
Assuntos
Cefaleia Histamínica , Gânglio Trigeminal , c-Mer Tirosina Quinase , Animais , Cefaleia Histamínica/metabolismo , Cefaleia Histamínica/sangue , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Gânglio Trigeminal/metabolismo , Humanos , Masculino , Ratos , Feminino , Proto-Oncogene Mas , Adulto , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Sanguíneas , GalectinasRESUMO
Organ fibrosis is a devastating medical challenge that is collectively responsible for an estimated 45% of all deaths in developed countries and poses a substantial health and economic burden. The process of fibrosis has common characteristics that can occur in various organs, such as the liver, kidney, lung, and skin. Currently, there is a paucity of effective treatments available for fibrosis. Therefore, it is crucial to identify new approaches to find potential therapeutic targets. Genetic studies have shown great promise in advancing the drug development process. Mer tyrosine kinase (MERTK) was recently identified as a crucial regulator of fibrosis that specifically controls the activity of transforming growth factor beta (TGFß). In this brief review, we provide an overview of the potential role of MERTK as a targeted and valuable approach for treating organ fibrosis.
Assuntos
Fibrose , c-Mer Tirosina Quinase , Humanos , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Animais , Fator de Crescimento Transformador beta/metabolismo , Terapia de Alvo MolecularRESUMO
Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.
Assuntos
Proliferação de Células , Neoplasias de Mama Triplo Negativas , c-Mer Tirosina Quinase , Humanos , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Feminino , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Endoglina/metabolismo , Endoglina/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Metástase Neoplásica , Transdução de Sinais , Apoptose/genéticaRESUMO
Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.
Assuntos
Receptor Tirosina Quinase Axl , Doença Celíaca , Duodeno , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal , Proteína S , Receptores Proteína Tirosina Quinases , c-Mer Tirosina Quinase , Humanos , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Doença Celíaca/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Masculino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/imunologia , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Adulto , Duodeno/metabolismo , Duodeno/imunologia , Duodeno/patologia , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Proteína S/metabolismo , Proteína S/genética , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Adulto Jovem , Transdução de Sinais , Adolescente , Interferons/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK-/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1ß, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22phox) and mitochondrial dysfunction. Interestingly, MerTK-/-induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis.
Assuntos
Aorta , Aterosclerose , Células Endoteliais , Fagocitose , c-Mer Tirosina Quinase , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Humanos , Células Endoteliais/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Camundongos , Apoptose , Proto-Oncogene Mas , Masculino , Camundongos Endogâmicos C57BL , Dieta Hiperlipídica , Células Cultivadas , EferocitoseRESUMO
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.
Assuntos
Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Proteólise , c-Mer Tirosina Quinase , Humanos , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células THP-1 , Macrófagos/metabolismo , Proteína S/metabolismo , Monócitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Hepatic ischemia-reperfusion injury(HIRI) remains to be an unsolved risk factor that contributes to organ failure after liver surgery. Our clinical retrospective study showed that lower donor liver CX3-C chemokine receptor-1(CX3CR1) mRNA expression level were correlated with upregulated pro-resolved macrophage receptor MERTK, as well as promoted restoration efficiency of allograft injury in liver transplant. To further characterize roles of CX3CR1 in regulating resolution of HIRI, we employed murine liver partial warm ischemia-reperfusion model by Wt & Cx3cr1-/- mice and the reperfusion time was prolonged from 6 h to 4-7 days. Kupffer cells(KCs) were depleted by clodronate liposome(CL) in advance to focus on infiltrating macrophages, and repopulation kinetics were determined by FACS, IF and RNA-Seq. CX3CR1 antagonist AZD8797 was injected i.p. to interrogate potential pharmacological therapeutic strategies. In vitro primary bone marrow macrophages(BMMs) culture by LXR agonist DMHCA, as well as molecular and functional studies, were undertaken to dissect roles of CX3CR1 in modulating macrophages cytobiological development and resolutive functions. We observed that deficiency or pharmacological inhibition of CX3CR1 facilitated HIRI resolution via promoted macrophages migration in CCR1/CCR5 manner, as well as enhanced MerTK-mediated efferocytosis. Our study demonstrated the critical roles of CX3CR1 in progression of HIRI and identified it as a potential therapeutic target in clinical liver transplantation.
Assuntos
Receptor 1 de Quimiocina CX3C , Fígado , Camundongos Knockout , Traumatismo por Reperfusão , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/genética , Camundongos , Fígado/metabolismo , Fígado/patologia , Masculino , Humanos , Células de Kupffer/metabolismo , Células de Kupffer/patologia , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Transplante de Fígado , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Homeostase , Modelos Animais de DoençasAssuntos
Macrófagos Alveolares , Fagocitose , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , c-Mer Tirosina Quinase , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/imunologia , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Animais , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Camundongos , Humanos , Apoptose , EferocitoseRESUMO
BACKGROUND AND OBJECTIVE: Understanding the regulation of efferocytosis by myeloid phagocytes is important in identifying novel targets in systemic lupus erythematosus (SLE). Cadherin-11 (CDH11), a cell adhesion molecule, is implicated in inflammatory arthritis and fibrosis and recently been shown to regulate macrophage phagocytosis. The extent and mechanism of this regulation is unknown. Our objective was to examine the extent to which CDH11 regulates myeloid phagocytes and contributes to autoimmunity and tissue inflammation. METHODS: We analyzed efferocytosis in macrophages and dendritic cells (DCs) from WT and Cdh11-/- mice and investigated the mechanisms in vitro. We investigated the role of CDH11 in disease development in vivo using the pristane induced lupus model. To translate the clinical relevance of CDH11 in human disease, we measured serum CDH11 levels in two independent pediatric SLE (pSLE) cohorts and healthy controls. RESULTS: Using bone marrow derived macrophages (BMDMs) and DCs (BMDCs), we found impaired efferocytosis in phagocytes from Cdh11-/- mice, mediated by downregulated efferocytosis receptor expression and RhoGTPase activation. Specifically, loss of CDH11 downregulated Mertk expression and Rac1 activation in BMDMs, and integrin αVß3 expression and Cdc42 activation in BMDCs, highlighting distinct pathways. In vivo, Cdh11-/- mice displayed defective efferocytosis and increased accumulation of apoptotic debris in pristane-induced lupus. Further, Cdh11-/- mice had enhanced systemic inflammation and autoimmune inflammation with increased anti-dsDNA autoantibodies, splenomegaly, type I interferons, and inflammatory cytokines. Paradoxically, at the tissue level, Cdh11-/- mice were protected against glomerulonephritis, indicating a dual role in murine lupus. Finally, SLE patients had increased serum CDH11 compared to controls. CONCLUSION: This study highlights a novel role of CDH11 in regulating myeloid cells and efferocytosis and its potential as a contributor to development in autoimmunity murine lupus. Despite the increase in autoimmunity, Cdh11-/- mice developed decreased tissue inflammation and damage.
Assuntos
Caderinas , Células Dendríticas , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico , Macrófagos , Fagocitose , Animais , Criança , Feminino , Humanos , Camundongos , Autoimunidade , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Caderinas/metabolismo , Caderinas/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Fagocitose/imunologia , TerpenosRESUMO
BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.
Assuntos
Oligonucleotídeos Antissenso , Radioimunoterapia , Feminino , Animais , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Linfócitos T CD8-Positivos , c-Mer Tirosina Quinase/genética , Proto-Oncogenes , Resultado do TratamentoRESUMO
The TAMs are a subfamily of receptor tyrosine kinases (RTKs) comprised of three members, Tyro3, Axl and Mer. Evidence in support of the existence of this subfamily emerged from a screen for novel RTKs performed in the laboratory of Dr. Greg Lemke in 1991. A PCR-based approach to selectively amplify tyrosine kinase-specific genes yielded 27 different tyrosine kinase genes, of which 13 were novel (the "Tyros"). Of these, Tyro3, 7 and 12 were more closely related to each other than to any other kinases and it was proposed that they constituted a novel subfamily of RTKs. Additional support for this hypothesis required determining the complete sequences for these receptor tyrosine kinases. By the end of 1991, full-length sequences for Tyro7 (Axl) revealed a unique extracellular domain organization that included two immunoglobulin-like domains and two fibronectin type III repeats. In 1994, the complete sequences for Tyro12 (Mer) and Tyro3 were shown to have an extracellular region domain structure similar to that of Axl. In 1995, Gas6 and Pros1 were reported as ligands for Tyro3 and Axl, setting the stage for functional studies. The Lemke lab and its many trainees have since played leading roles in elucidating the physiological relevance of the TAMs.
Assuntos
Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas , c-Mer Tirosina Quinase/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/química , Tamoxifeno , TirosinaRESUMO
The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.
Assuntos
Artrite Reumatoide , Receptores Proteína Tirosina Quinases , Humanos , Receptor Tirosina Quinase Axl , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Membrana Sinovial/metabolismoRESUMO
Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at â¼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.
Assuntos
Macrófagos Alveolares , Ozônio , Fagocitose , Proto-Oncogene Mas , c-Mer Tirosina Quinase , Ozônio/farmacologia , c-Mer Tirosina Quinase/metabolismo , c-Mer Tirosina Quinase/genética , Animais , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Humanos , Fagocitose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/patologia , Camundongos Knockout , Masculino , Inflamação/metabolismo , Inflamação/patologia , Inflamação/induzido quimicamente , Apoptose/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , EferocitoseRESUMO
Immune checkpoint inhibitors, particularly PD-1/PD-L1 blockades, have been approved for unresectable hepatocellular carcinoma (HCC). However, high resistance rates still limit their efficacy, highlighting the urgent need to understand the underlying mechanisms and develop strategies for overcoming the resistance. In this study, we demonstrate that HCC with high MER proto-oncogene tyrosine kinase (MerTK) expression exhibits anti-PD-1/PD-L1 resistance in two syngeneic mouse models and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, MerTK renders HCC resistant to anti-PD-1/PD-L1 by limiting ferroptosis with the upregulation of SLC7A11 via the ERK/SP1 pathway and facilitating the development of an immunosuppressive tumor microenvironment (TME) with the recruitment of myeloid-derived suppressor cells (MDSCs). Sitravatinib, an inhibitor of MerTK, sensitizes resistant HCC to anti-PD-L1 therapy by promoting tumor ferroptosis and decreasing MDSC infiltration into the TME. In conclusion, we find that MerTK could serve as a predictive biomarker for patient stratification and as a promising target to overcome anti-PD-1/PD-L1 resistance in HCC.