RESUMO
The TAM receptor tyrosine kinases (RTKs)-TYRO3, AXL, and MERTK-together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease.
Assuntos
Homeostase , Imunidade/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Suscetibilidade a Doenças , Humanos , Ligantes , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genéticaRESUMO
Childhood neglect and/or abuse can induce mental health conditions with unknown mechanisms. Here, we identified stress hormones as strong inducers of astrocyte-mediated synapse phagocytosis. Using in vitro, in vivo, and human brain organoid experiments, we showed that stress hormones increased the expression of the Mertk phagocytic receptor in astrocytes through glucocorticoid receptor (GR). In post-natal mice, exposure to early social deprivation (ESD) specifically activated the GR-MERTK pathway in astrocytes, but not in microglia. The excitatory post-synaptic density in cortical regions was reduced in ESD mice, and there was an increase in the astrocytic engulfment of these synapses. The loss of excitatory synapses, abnormal neuronal network activities, and behavioral abnormalities in ESD mice were largely prevented by ablating GR or MERTK in astrocytes. Our work reveals the critical roles of astrocytic GR-MERTK activation in evoking stress-induced abnormal behaviors in mice, suggesting GR-MERTK signaling as a therapeutic target for stress-induced mental health conditions.
Assuntos
Astrócitos , Fagocitose , Estresse Psicológico , Animais , Criança , Humanos , Camundongos , Astrócitos/metabolismo , c-Mer Tirosina Quinase/genética , Hormônios/metabolismo , Sinapses/metabolismo , Estresse Psicológico/metabolismoRESUMO
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Assuntos
Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Nucleotídeos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , c-Mer Tirosina Quinase/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Antígeno B7-H1/imunologia , Células Cultivadas , Feminino , Imunidade Inata , Imunoterapia , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , Fagocitose , Receptor de Morte Celular Programada 1/imunologia , Receptores Purinérgicos P2X7/deficiência , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase/genéticaRESUMO
Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat-me" signal that initiates glia-mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal-specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well-known "eat-me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post-synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post-synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, we found that phosphatidylserine is used for microglia-mediated pruning of inhibitory post-synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat-me" signal for inhibitory post-synapse elimination.
Assuntos
Microglia/metabolismo , Fosfatidilserinas/metabolismo , Convulsões/fisiopatologia , Sinapses/fisiologia , c-Mer Tirosina Quinase/metabolismo , Animais , Encéfalo/fisiopatologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fagocitose/fisiologia , Convulsões/genética , c-Mer Tirosina Quinase/genéticaRESUMO
Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1ß secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain.
Assuntos
Doença por Corpos de Lewy , Doença de Parkinson , Sinucleinopatias , Humanos , alfa-Sinucleína/metabolismo , c-Mer Tirosina Quinase/metabolismo , Doença por Corpos de Lewy/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Proteínas Tirosina Quinases , Sinucleinopatias/metabolismoRESUMO
Recurrent seizure is a common comorbidity in early-stage Alzheimer's disease (AD) and may contribute to AD pathogenesis and cognitive decline. Similarly, many mouse models of Alzheimer's disease that overproduce amyloid beta are prone to epileptiform seizures that may result in early sudden death. We studied one such model, designated APP/PS1, and found that mutation of the TAM receptor tyrosine kinase (RTK) Mer or its ligand Gas6 greatly exacerbated early death. Lethality was tied to violent seizures that appeared to initiate in the dentate gyrus (DG) of the hippocampus, where Mer plays an essential role in the microglial phagocytosis of both apoptotic and newborn cells normally generated during adult neurogenesis. We found that newborn DG neurons and excitatory synapses between the DG and the cornu ammonis field 3 (CA3) field of the hippocampus were increased in TAM-deficient mice, and that premature death and adult neurogenesis in these mice were coincident. In contrast, the incidence of lethal seizures and the deposition of dense-core amyloid plaques were strongly anticorrelated. Together, these results argue that TAM-mediated phagocytosis sculpts synaptic connectivity in the hippocampus, and that seizure-inducing amyloid beta polymers are present prior to the formation of dense-core plaques.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Convulsões , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Animais , Giro Denteado , Modelos Animais de Doenças , Hipocampo/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Neurogênese , Fagocitose , Placa Amiloide/patologia , Polímeros , Proteínas Tirosina Quinases , Convulsões/genética , Convulsões/patologiaRESUMO
BACKGROUND: Hepatitis B-related acute-on-chronic liver failure (HBV-ACLF) has a high short-term mortality. This study aimed to determine the diagnostic and prognostic role of MER tyrosine kinase (MERTK) in HBV-ACLF patients. METHODS: Transcriptomics analysis evaluated MERTK expression and function during disease progression. The diagnostic and prognostic significance of MERTK for HBV-ACLF patients were verified by ELISA, the area under the receiver operating characteristic curve (AUROC) analysis, and immunohistochemistry (IHC) of liver tissues. RESULTS: MERTK mRNA was highly expressed in the HBV-ACLF compared to the liver cirrhosis (LC), chronic hepatitis B (CHB) and normal controls (NC) groups. Elevated MERTK mRNA predicted poor prognosis for HBV-ACLF at 28/90 days (AUROCs=0.814/0.731). Functional analysis showed MERTK was significantly associated with TLR and inflammatory signaling, and several key biological processes. External validation with 285 plasma subjects confirmed the high diagnostic accuracy of plasma MERTK for HBV-ACLF (AUROC=0.859) and potential prognostic value for 28/90-day mortality rates (AUROC=0.673 and 0.644, respectively). Risk stratification analysis indicated higher mortality risk for patients with plasma MERTK level above the cut-off value. Moreover, IHC staining showed increasing MERTK expression from NC, CHB and LC to HBV-ACLF patients. CONCLUSIONS: MERTK shows promise as a candidate biomarker for early diagnosis and prognosis of HBV-ACLF.
RESUMO
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
Acute-on-chronic liver failure (ACLF) is a severe disease with a high mortality. Macrophage-related inflammation plays a crucial role in ACLF development. Mesenchymal stem cells (MSCs) treatment was demonstrated to be beneficial in ACLF in our previous study; however, the underlying mechanisms remain unknown. Therefore, mouse bone marrow-derived MSCs were used to treat an ACLF mouse model or cocultured with RAW264.7/J774A.1 macrophages that were stimulated with LPS. Histological and serological parameters and survival were analyzed to evaluate efficacy. We detected changes of Mer tyrosine kinase (Mertk), JAK1/STAT6, inflammatory cytokines, and markers of macrophage polarization in vitro and in vivo. In ACLF mice, MSCs improved liver function and 48-h survival of ACLF mice and alleviated inflammatory injury by promoting M2 macrophage polarization and elevated Mertk expression levels in macrophages. This is significant, as Mertk regulates M2 macrophage polarization via the JAK1/STAT6 signaling pathway.
Assuntos
Insuficiência Hepática Crônica Agudizada , Células-Tronco Mesenquimais , Camundongos , Animais , Insuficiência Hepática Crônica Agudizada/metabolismo , Proteínas Tirosina Quinases/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Células-Tronco Mesenquimais/metabolismo , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies.
Assuntos
Vesículas Extracelulares , Leucemia Linfocítica Crônica de Células B , Fosfatidilserinas , Receptores Proteína Tirosina Quinases , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Feminino , Fosfatidilserinas/metabolismo , Fosfatidilserinas/sangue , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/sangue , Masculino , Pessoa de Meia-Idade , Idoso , Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/metabolismo , Adulto , c-Mer Tirosina Quinase/metabolismo , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: Clearance of apoptotic cells by efferocytosis is crucial for prevention of atherosclerosis progress, and impaired efferocytosis contributes to the aggravated atherosclerosis. RESULTS: In this study, we found that diabetic ApoE-/- mice showed aggravated atherosclerosis as hyperglycemia damaged the efferocytosis capacity at least partially due to decreased expression of Mer tyrosine kinase (MerTK) on macrophages. To locally restore MerTK in the macrophages in the plaque, hybrid membrane nanovesicles (HMNVs) were thus developed. Briefly, cell membrane from MerTK overexpressing RAW264.7 cell and transferrin receptor (TfR) overexpressing HEK293T cell were mixed with DOPE polymers to produce nanovesicles designated as HMNVs. HMNVs could fuse with the recipient cell membrane and thus increased MerTK in diabetic macrophages, which in turn restored the efferocytosis capacity. Upon intravenous administration into diabetic ApoE-/- mice, superparamagnetic iron oxide nanoparticles (SMN) decorated HMNVs accumulated at the aorta site significantly under magnetic navigation, where the recipient macrophages cleared the apoptotic cells efficiently and thus decreased the inflammation. CONCLUSIONS: Our study indicates that MerTK decrease in macrophages contributes to the aggravated atherosclerosis in diabetic ApoE-/- mice and regional restoration of MerTK in macrophages of the plaque via HMNVs could be a promising therapeutic approach.
Assuntos
Aterosclerose , Diabetes Mellitus , Humanos , Animais , Camundongos , Eferocitose , Células HEK293 , Membrana Celular , Proteínas Tirosina Quinases , Apolipoproteínas E/genética , Nanopartículas Magnéticas de Óxido de FerroRESUMO
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
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
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
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
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
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
BACKGROUND & AIMS: Macrophages are key elements in the pathogenesis of cholestatic liver diseases. Arid3a plays a prominent role in the biologic properties of hematopoietic stem cells, B lymphocytes and tumor cells, but its ability to modulate macrophage function during cholestasis remains unknown. METHODS: Gene and protein expression and cellular localization were assessed by q-PCR, immunohistochemistry, immunofluorescence staining and flow cytometry. We generated myeloid-specific Arid3a knockout mice and established three cholestatic murine models. The transcriptome was analyzed by RNA-seq. A specific inhibitor of the Mertk receptor was used in vitro and in vivo. Promoter activity was determined by chromatin immunoprecipitation-seq against Arid3a and a luciferase reporter assay. RESULTS: In cholestatic murine models, myeloid-specific deletion of Arid3a alleviated cholestatic liver injury (accompanied by decreased accumulation of macrophages). Arid3a-deficient macrophages manifested a more reparative phenotype, which was eliminated by in vitro treatment with UNC2025, a specific inhibitor of the efferocytosis receptor Mertk. Efferocytosis of apoptotic cholangiocytes was enhanced in Arid3a-deficient macrophages via upregulation of Mertk. Arid3a negatively regulated Mertk transcription by directly binding to its promoter. Targeting Mertk in vivo effectively reversed the protective phenotype of Arid3a deficiency in macrophages. Arid3a was upregulated in hepatic macrophages and circulating monocytes in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Mertk was correspondingly upregulated and negatively correlated with Arid3a expression in PBC and PSC. Mertk+ cells were located in close proximity to cholangiocytes, while Arid3a+ cells were scattered among immune cells with greater spatial distances to hyperplastic cholangiocytes in PBC and PSC. CONCLUSIONS: Arid3a promotes cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes by macrophages during cholestasis. The Arid3a-Mertk axis is a promising novel therapeutic target for cholestatic liver diseases. IMPACT AND IMPLICATIONS: Macrophages play an important role in the pathogenesis of cholestatic liver diseases. This study reveals that macrophages with Arid3a upregulation manifest a pro-inflammatory phenotype and promote cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes during cholestasis. Although we now offer a new paradigm to explain how efferocytosis is regulated in a myeloid cell autonomous manner, the regulatory effects of Arid3a on chronic liver diseases remain to be further elucidated.
Assuntos
Colestase , Proteínas de Ligação a DNA , Hepatopatias , Fatores de Transcrição , c-Mer Tirosina Quinase , Animais , Camundongos , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Colestase/metabolismo , Hepatopatias/metabolismo , Macrófagos/metabolismo , Camundongos Knockout , Fagocitose/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening disease characterised by high-grade inflammation and immunoparesis, which is associated with a high incidence of death from sepsis. Herein, we aimed to describe the metabolic dysregulation in ALF and determine whether systemic immune responses are modulated via the lysophosphatidylcholine (LPC)-autotaxin (ATX)-lysophosphatidylcholinic acid (LPA) pathway. METHODS: Ninety-six individuals with ALF, 104 with cirrhosis, 31 with sepsis and 71 healthy controls (HCs) were recruited. Pathways of interest were identified by multivariate statistical analysis of proton nuclear magnetic resonance spectroscopy and untargeted ultraperformance liquid chromatography-mass spectrometry-based lipidomics. A targeted metabolomics panel was used for validation. Peripheral blood mononuclear cells were cultured with LPA 16:0, 18:0, 18:1, and their immune checkpoint surface expression was assessed by flow cytometry. Transcript-level expression of the LPA receptor (LPAR) in monocytes was investigated and the effect of LPAR antagonism was also examined in vitro. RESULTS: LPC 16:0 was highly discriminant between ALF and HC. There was an increase in ATX and LPA in individuals with ALF compared to HCs and those with sepsis. LPCs 16:0, 18:0 and 18:1 were reduced in individuals with ALF and were associated with a poor prognosis. Treatment of monocytes with LPA 16:0 increased their PD-L1 expression and reduced CD155, CD163, MerTK levels, without affecting immune checkpoints on T and NK/CD56+T cells. LPAR1 and 3 antagonism in culture reversed the effect of LPA on monocyte expression of MerTK and CD163. MerTK and CD163, but not LPAR genes, were differentially expressed and upregulated in monocytes from individuals with ALF compared to controls. CONCLUSION: Reduced LPC levels are biomarkers of poor prognosis in individuals with ALF. The LPC-ATX-LPA axis appears to modulate innate immune response in ALF via LPAR1 and LPAR3. Further investigations are required to identify novel therapeutic agents targeting these receptors. IMPACT AND IMPLICATIONS: We identified a metabolic signature of acute liver failure (ALF) and investigated the immunometabolic role of the lysophosphatidylcholine-autotaxin-lysophosphatidylcholinic acid pathway, with the aim of finding a mechanistic explanation for monocyte behaviour and identifying possible therapeutic targets (to modulate the systemic immune response in ALF). At present, no selective immune-based therapies exist. We were able to modulate the phenotype of monocytes in vitro and aim to extend these findings to murine models of ALF as a next step. Future therapies may be based on metabolic modulation; thus, the role of specific lipids in this pathway require elucidation and the relative merits of autotaxin inhibition, lysophosphatidylcholinic acid receptor blockade or lipid-based therapies need to be determined. Our findings begin to bridge this knowledge gap and the methods used herein could be useful in identifying therapeutic targets as part of an experimental medicine approach.
Assuntos
Falência Hepática Aguda , Sepse , Animais , Camundongos , Lisofosfatidilcolinas , Monócitos , Leucócitos Mononucleares/metabolismo , c-Mer Tirosina Quinase/metabolismo , Falência Hepática Aguda/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Imunidade Inata , Sepse/metabolismo , Lisofosfolipídeos/metabolismoRESUMO
BACKGROUND: Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined. METHODS: We used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages. RESULTS: Single-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP+ monocyte/macrophages and resident microglia engulfing NeuN+ or TUNEL+ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway. CONCLUSIONS: Our findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.