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Head and neck squamous cell carcinoma (HNSCC) is diagnosed in more than 71,000 patients each year in the United States, with nearly 16,000 associated deaths. One significant hurdle in the treatment of HNSCC is acquired and intrinsic resistance to existing therapeutic agents. Over the past several decades, the University of Wisconsin has formed a multidisciplinary team to move basic scientific discovery along the translational spectrum to impact the lives of HNSCC patients. In this review, we outline key discoveries made throughout the years at the University of Wisconsin to deepen our understanding of therapeutic resistance in HNSCC and how a strong, interdisciplinary team can make significant advances toward improving the lives of these patients by combatting resistance to established therapeutic modalities. We are profoundly grateful to the many scientific teams worldwide whose groundbreaking discoveries, alongside evolving clinical paradigms in head and neck oncology, have been instrumental in making our work possible. [ABSTRACT FROM AUTHOR]

Background Hepatitis B virus–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 patients with HBV-ACLF. Methods Transcriptomics analysis evaluated MERTK expression and function during disease progression. The diagnostic and prognostic significance of MERTK for patients with HBV-ACLF were verified by enzyme-linked immunosorbent assay, area under the receiver operating characteristic curve (AUROC) analysis, and immunohistochemistry (IHC) of liver tissues. Results MERTK mRNA was highly expressed in patients with HBV-ACLF compared to those with liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NC). Elevated MERTK mRNA predicted poor prognosis for HBV-ACLF at 28 and 90 days (AUROC = 0.814 and 0.731, respectively). Functional analysis showed MERTK was significantly associated with toll-like receptor 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- and 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 cutoff value. Moreover, IHC staining showed increasing MERTK expression from NC, CHB, and LC to HBV-ACLF. Conclusions MERTK shows promise as a candidate biomarker for early diagnosis and prognosis of HBV-ACLF. [ABSTRACT FROM AUTHOR]

Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk−/− mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk−/− mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk−/− mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response. [ABSTRACT FROM AUTHOR]

Disruption of the blood-spinal cord barrier (BSCB) is a critical event in the secondary injury following spinal cord injury (SCI). Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics. However, the specific involvement of Mertk in BSCB remains elusive. Here, we demonstrated a distinct role of Mertk in the repair of BSCB. Mertk expression is decreased in endothelial cells following SCI. Overexpression of Mertk upregulated tight junction proteins (TJs), reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis. Ultimately, this led to enhanced neural regeneration and functional recovery. Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk. These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair. [ABSTRACT FROM AUTHOR]

Abstract Japanese encephalitis is an acute infectious disease of the central nervous system caused by neurotropic Japanese encephalitis virus (JEV). As a member of TAM (Tyro3, Axl and Mertk) family, Mertk has involved in multiple biological processes by engaging with its bridging ligands Gas6 and Protein S, including invasion of pathogens, phagocytosis of apoptotic cells, inflammatory response regulation, and the maintenance of blood brain barrier (BBB) integrity. However, its role in encephalitis caused by JEV infection has not been studied in detail. Here, we found that Mertk−/− mice exhibited higher mortality and more rapid disease progression than wild-type mice after JEV challenge. There were no significant differences in viral load and cytokines expression level in peripheral tissues between Wild type and Mertk−/− mice. Furthermore, the absence of Mertk had little effect on the inflammatory response and immunopathological damage while it can cause an increased viral load in the brain. For the in vitro model of BBB, Mertk was shown to maintain the integrity of the BBB. In vivo, Mertk−/− mice exhibited higher BBB permeability and lower BBB integrity. Taken together, our findings demonstrate that Mertk acts as a protective factor in the development of encephalitis induced by JEV infection, which is mainly associated with its beneficial effect on BBB integrity, rather than its regulation of inflammatory response.

Simple Summary: Ewing sarcoma family tumors are the second most common bone cancer affecting adolescents and young adults. Outcomes are poor in patients with metastatic or relapsed disease, and new treatments are urgently needed. MERTK is a protein found in leukemia, melanoma, lung cancer, and other cancer types where it promotes cancer cell survival and resistance to therapies. MRX-2843 is a new drug that targets MERTK and is currently in clinical trials. We show that MERTK is present on Ewing sarcoma cells and that MRX-2843 has therapeutic activity against these cells. MRX-2843 is even more effective against Ewing sarcoma cells when combined with other drugs, venetoclax or navitoclax, that target a protein called BCL-2. These results suggest that MRX-2843 could be useful to treat Ewing sarcoma in patients. Outcomes are poor in patients with advanced or relapsed Ewing sarcoma (EWS) and current treatments have significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. MER proto-oncogene tyrosine kinase (MERTK) promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies in a variety of cancers. MERTK was ubiquitously expressed in five EWS cell lines and five patient samples. Moreover, data from CRISPR-based library screens indicated that EWS cell lines are particularly dependent on MERTK. Treatment with MRX-2843, a first-in-class, MERTK-selective tyrosine kinase inhibitor currently in clinical trials, decreased the phosphorylation of MERTK and downstream signaling in a dose-dependent manner in A673 and TC106 cells and provided potent anti-tumor activity against all five EWS cell lines, with IC50 values ranging from 178 to 297 nM. Inhibition of MERTK correlated with anti-tumor activity, suggesting MERTK inhibition as a therapeutic mechanism of MRX-2843. Combined treatment with MRX-2843 and BCL-2 inhibitors venetoclax or navitoclax provided enhanced therapeutic activity compared to single agents. These data highlight MERTK as a promising therapeutic target in EWS and provide rationale for the development of MRX-2843 for the treatment of EWS, especially in combination with BCL-2 inhibitors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction Diabetic peripheral neuropathy (DPN) impacts patient quality of life. In such patients, increased expression of mer tyrosine kinase (MerTK) has been demonstrated; however, its mechanism of action remains unclear. In this study, type 2 diabetes mellitus (T2DM) and DPN models were established in Sprague Dawley rats via low-dose streptozotocin and a high-fat diet and the mode of action of MerTK was examined. Methods MerTK-specific inhibitors were administered by gavage once daily for 2 weeks. Sciatic nerve conduction velocity and nerve structure were measured. The levels of MerTK, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and relevant biochemical indexes were detected. Results The study revealed upregulation of MerTK expression in T2DM and more so in DPN groups. Inhibiting MerTK led to reduced nerve conduction velocity and further deterioration of sciatic nerve structure, as evidenced by structural morphology. Concurrently, serum levels of total cholesterol, glycated hemoglobin, and triglyceride significantly increased. Moreover, levels of NF-κB increased in both serum and nerve tissue, alongside a significant rise in TNF-α and IL-1β expressions. MerTK could bind to the inhibitor of kappa B kinase beta (Ikbkb) in Schwann cells, establishing Ikbkb as a precursor to NF-κB activation. Discussion Inhibition of MerTK exacerbates neuropathy, indicating its protective role in DPN by suppressing the NF-κB pathway, highlighting a potential new target for its diagnosis and treatment. [ABSTRACT FROM AUTHOR]

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 (TGFβ). In this brief review, we provide an overview of the potential role of MERTK as a targeted and valuable approach for treating organ fibrosis. [ABSTRACT FROM AUTHOR]

Simple Summary: We identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in Triple-negative breast cancer (TNBC). We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic. [ABSTRACT FROM AUTHOR]

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 ∼21 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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphic Abstract

Abstract 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. Graphical Abstract

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB-NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Adenosine A3 receptor (ADORA3) belongs to the adenosine receptor families and the role of ADORA3 in vascular dementia (VaD) is largely unexplored. The present study sought to determine the therapeutic role of ADORA3 antagonist in a mouse model of VaD. Methods: The GSE122063 dataset was selected to screen the differential expression genes and pathways between VaD patients and controls. A mouse model of bilateral carotid artery stenosis (BCAS) was established. The cognitive functions were examined by the novel object recognition test, Y maze test, and fear of conditioning test. The white matter injury (WMI) was examined by 9.4 T MRI, western blot, and immunofluorescence staining. The mechanisms of ADORA3‐regulated phagocytosis by microglia were examined using qPCR, western blot, dual immunofluorescence staining, and flow cytometry. Results: The expression of ADORA3 was elevated in brain tissues of VaD patients and ADORA3 was indicated as a key gene for VaD in the GSE122063. In BCAS mice, the expression of ADORA3 was predominantly elevated in microglia in the corpus callosum. ADORA3 antagonist promotes microglial phagocytosis to myelin debris by facilitating cAMP/PKA/p‐CREB pathway and thereby ameliorates WMI and cognitive impairment in BCAS mice. The therapeutic effect of ADORA3 antagonist was partially reversed by the inhibition of the cAMP/PKA pathway. Conclusions: ADORA3 antagonist alleviates chronic ischemic WMI by modulating myelin clearance of microglia, which may be a potential therapeutic target for the treatment of VaD. [ABSTRACT FROM AUTHOR]

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTKCR) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTKCR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTKCR and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTKCR RPE primary cultures phagocytosed less POS in vitro, while in vivo, the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Despite multiple therapeutic modalities, the overall survival of patients with gastric adenocarcinoma remains poor, especially for advanced tumor stages. Although the tyrosine kinase MerTK has shown therapeutic relevance in several tumor entities, its potential effects in gastric adenocarcinoma have not yet been sufficiently characterized. Methods: MerTK expression and its influence on patient survival were evaluated by immunohistochemistry in a cohort of 140 patients with gastric adenocarcinoma. CRISPR/Cas9 knockout and siRNA knockdown of MerTK in the gastric cancer cell lines SNU1, SNU5, and MKN45 was used to analyze protein expression, growth, migration, and invasion properties in vitro and in a murine xenograft model. MerTK was pharmacologically targeted with the small molecule inhibitor UNC2025 in vitro and in vivo. Results: In patients, high MerTK expression was associated with decreased overall survival (OS) and lymph node metastasis especially in patients without neoadjuvant therapy (p < 0.05). Knockout and knockdown of MerTK reduced cell proliferation and migration both in vitro and in vivo. UNC2025, a small‐molecule inhibitor of MerTK, exhibited a significant therapeutic response in vitro and in vivo. Additionally, MerTK expression attenuated the response to neoadjuvant treatment, and its inhibition sensitized tumor cells to 5‐Fluorouracil (5‐FU)‐based chemotherapy in vitro. Conclusions: Our findings demonstrate the potential value of MerTK as a prognostic biomarker for gastric adenocarcinoma. Targeting MerTK may become a new treatment option, especially for patients with advanced tumors, and may overcome resistance to established chemotherapies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-1β 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. [ABSTRACT FROM AUTHOR]

Following myocardial infarction (MI), blood neutrophils quickly and extensively infiltrate the heart, where they are temporally polarized into pro-inflammatory (N1) and anti-inflammatory (N2) subpopulations. Neutrophil transmigration is rapidly followed by the accrual of macrophages (MACs), which are believed to undergo local phenotypic transformations from pro-inflammatory to pro-healing MACs that mediate inflammation resolution. We hypothesized that N2 neutrophils can reprogram MACs toward a healing phenotype with increased efferocytosis capacity. To examine this, human neutrophils isolated from healthy subjects were polarized in N1 and N2 neutrophils, and their secretome was added to human MACs derived from THP monocytes. The impact of neutrophil factors on macrophages was investigated using qPCR, ELISA, Western blot, immunofluorescence, or an efferocytosis assay. The results show that the MACs exposed to N2 neutrophil secretome exhibited (i) increased expression of the anti-inflammatory molecules CD206, TGF-β, and IL-10 and the nuclear factors associated with reparatory macrophages (PPARγ, Nur77, and KLF4); (ii) enhanced expression of efferocytosis receptors (MerTK, CD36, CX3CR1, and integrins αv/β5) and of the bridge molecules Mfage8 and Gas6; and (iii) enhanced efferocytosis. In conclusion, factors released by N2 neutrophils induce a pro-healing phenotype of MACs by upregulating anti-inflammatory molecules and efferocytosis receptors and ensuing the efferocytosis capacity. The data suggest that molecular therapy to foster N2 polarization, which boosts macrophages' pro-healing phenotype, could be a promising strategy to speed up inflammation resolution and tissue repair. [ABSTRACT FROM AUTHOR]

Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs. [ABSTRACT FROM AUTHOR]

Previous works from our group show that Semaphorin3B (Sema3B) is reduced in RA and plays a protective role in a mouse arthritis model. In turn, MerTK plays a protective function in murine arthritis models, is expressed by synovial tissue macrophages and is linked to remission in patients with RA. In this study, we examined the role of Sema3B in the phenotypic characteristics of RA macrophages and the implication of MerTK. Peripheral blood monocytes from RA patients were differentiated into IFN-γ (RA MØIFN-γ) or M-CSF (RA MØM-CSF) macrophages and stimulated with LPS, Sema3B or their combination. Alternatively, RA fibroblast like synoviocytes (FLS) were stimulated with RA MØIFN-γ and RA MØM-CSF supernatants. Gene expression was determined by qPCR and protein expression and activation by flow cytometry, ELISA and western blot. Sema3B down-regulated the expression of pro-inflammatory mediators, in both RA MØIFN-γ and RA MØM-CSF. We observed a similar reduction in RA FLS stimulated with the supernatant of Sema3B-treated RA MØIFN-γ and RA MØM-CSF. Sema3B also modulated cell surface markers in macrophages towards an anti-inflammatory phenotype. Besides, MerTK expression and activation was up-regulated by Sema3B, just as GAS6 expression, Resolvin D1 secretion and the phagocytic activity of macrophages. Importantly, the inhibition of MerTK and neuropilins 1 and 2 abrogated the antiinflammatory effect of Sema3B. Our data demonstrate that Sema3B modulates the macrophage characteristics in RA, inducing a skewing towards an antiinflammatory/pro-resolving phenotype in a MerTK-dependant manner. Therefore, here we identify a new mechanism supporting the protective role of Sema3B in RA pathogenesis. [ABSTRACT FROM AUTHOR]

The TAM (TYRO3, MERTK, and AXL) family of receptor tyrosine kinases are pleiotropic regulators of adult tissue homeostasis maintaining organ integrity and self-renewal. Disruption of their homeostatic balance fosters pathological conditions like autoinflammatory or degenerative diseases including rheumatoid arthritis, lupus erythematodes, or liver fibrosis. Moreover, TAM receptors exhibit prominent cell-transforming properties, promoting tumor progression, metastasis, and therapy resistance in various cancer entities. Emerging evidence shows that TAM receptors are involved in bone homeostasis by regulating osteoblastic bone formation and osteoclastic bone resorption. Therefore, TAM receptors emerge as new key players of the regulatory cytokine network of osteoblasts and osteoclasts and represent accessible targets for pharmacologic therapy for a broad set of different bone diseases, including primary and metastatic bone tumors, rheumatoid arthritis, or osteoporosis. [ABSTRACT FROM AUTHOR]

Background and aimsMetabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition.MethodsMice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning.ResultsIn mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB− NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation.ConclusionOur findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Abstract Background Despite multiple therapeutic modalities, the overall survival of patients with gastric adenocarcinoma remains poor, especially for advanced tumor stages. Although the tyrosine kinase MerTK has shown therapeutic relevance in several tumor entities, its potential effects in gastric adenocarcinoma have not yet been sufficiently characterized. Methods MerTK expression and its influence on patient survival were evaluated by immunohistochemistry in a cohort of 140 patients with gastric adenocarcinoma. CRISPR/Cas9 knockout and siRNA knockdown of MerTK in the gastric cancer cell lines SNU1, SNU5, and MKN45 was used to analyze protein expression, growth, migration, and invasion properties in vitro and in a murine xenograft model. MerTK was pharmacologically targeted with the small molecule inhibitor UNC2025 in vitro and in vivo. Results In patients, high MerTK expression was associated with decreased overall survival (OS) and lymph node metastasis especially in patients without neoadjuvant therapy (p

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTKCR) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTKCR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTKCR and control mice between the ages of 3 and 18 months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTKCR RPE primary cultures phagocytosed less POS in vitro, while in vivo, the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina.

Rationale: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for the clearance of apoptotic cells (efferocytosis) and plays important roles in redox-related human diseases. We will explore MerTK biology in human cells, tissues, and diseases based on big data analytics. Methods: The human RNA-seq and scRNA-seq data about 42,700 samples were from NCBI Gene Expression Omnibus and analyzed by QIAGEN Ingenuity Pathway Analysis (IPA) with about 170,000 crossover analysis. MerTK expression was quantified as Log2 (FPKM + 0.1). Results: We found that, in human cells, MerTK is highly expressed in macrophages, monocytes, progenitor cells, alpha-beta T cells, plasma B cells, myeloid cells, and endothelial cells (ECs). In human tissues, MerTK has higher expression in plaque, blood vessels, heart, liver, sensory system, artificial tissue, bone, adrenal gland, central nervous system (CNS), and connective tissue. Compared to normal conditions, MerTK expression in related tissues is altered in many human diseases, including cardiovascular diseases, cancer, and brain disorders. Interestingly, MerTK expression also shows sex differences in many tissues, indicating that MerTK may have different impact on male and female. Finally, based on our proteomics from primary human aortic ECs, we validated the functions of MerTK in several human diseases, such as cancer, aging, kidney failure and heart failure. Conclusions: Our big data analytics suggest that MerTK may be a promising therapeutic target, but how it should be modulated depends on the disease types and sex differences. For example, MerTK inhibition emerges as a new strategy for cancer therapy due to it counteracts effect on anti-tumor immunity, while MerTK restoration represents a promising treatment for atherosclerosis and myocardial infarction as MerTK is cleaved in these disease conditions.

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.

Previous works from our group show that Semaphorin3B (Sema3B) is reduced in RA and plays a protective role in a mouse arthritis model. In turn, MerTK plays a protective function in murine arthritis models, is expressed by synovial tissue macrophages and is linked to remission in patients with RA. In this study, we examined the role of Sema3B in the phenotypic characteristics of RA macrophages and the implication of MerTK. Peripheral blood monocytes from RA patients were differentiated into IFN-γ (RA MØIFN-γ) or M-CSF (RA MØM-CSF) macrophages and stimulated with LPS, Sema3B or their combination. Alternatively, RA fibroblast like synoviocytes (FLS) were stimulated with RA MØIFN-γ and RA MØM-CSF supernatants. Gene expression was determined by qPCR and protein expression and activation by flow cytometry, ELISA and western blot. Sema3B down-regulated the expression of pro-inflammatory mediators, in both RA MØIFN-γ and RA MØM-CSF. We observed a similar reduction in RA FLS stimulated with the supernatant of Sema3B-treated RA MØIFN-γ and RA MØM-CSF. Sema3B also modulated cell surface markers in macrophages towards an anti-inflammatory phenotype. Besides, MerTK expression and activation was up-regulated by Sema3B, just as GAS6 expression, Resolvin D1 secretion and the phagocytic activity of macrophages. Importantly, the inhibition of MerTK and neuropilins 1 and 2 abrogated the anti-inflammatory effect of Sema3B. Our data demonstrate that Sema3B modulates the macrophage characteristics in RA, inducing a skewing towards an anti-inflammatory/pro-resolving phenotype in a MerTK-dependant manner. Therefore, here we identify a new mechanism supporting the protective role of Sema3B in RA pathogenesis.

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.

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.

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.

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.

Following myocardial infarction (MI), blood neutrophils quickly and extensively infiltrate the heart, where they are temporally polarized into pro-inflammatory (N1) and anti-inflammatory (N2) subpopulations. Neutrophil transmigration is rapidly followed by the accrual of macrophages (MACs), which are believed to undergo local phenotypic transformations from pro-inflammatory to pro-healing MACs that mediate inflammation resolution. We hypothesized that N2 neutrophils can reprogram MACs toward a healing phenotype with increased efferocytosis capacity. To examine this, human neutrophils isolated from healthy subjects were polarized in N1 and N2 neutrophils, and their secretome was added to human MACs derived from THP monocytes. The impact of neutrophil factors on macrophages was investigated using qPCR, ELISA, Western blot, immunofluorescence, or an efferocytosis assay. The results show that the MACs exposed to N2 neutrophil secretome exhibited (i) increased expression of the anti-inflammatory molecules CD206, TGF-β, and IL-10 and the nuclear factors associated with reparatory macrophages (PPARγ, Nur77, and KLF4); (ii) enhanced expression of efferocytosis receptors (MerTK, CD36, CX3CR1, and integrins αv/β5) and of the bridge molecules Mfage8 and Gas6; and (iii) enhanced efferocytosis. In conclusion, factors released by N2 neutrophils induce a pro-healing phenotype of MACs by upregulating anti-inflammatory molecules and efferocytosis receptors and ensuing the efferocytosis capacity. The data suggest that molecular therapy to foster N2 polarization, which boosts macrophages’ pro-healing phenotype, could be a promising strategy to speed up inflammation resolution and tissue repair.

Infectious diseases are a major cause of mortality in spite of existing public health, anti-microbial and vaccine interventions. We aimed to define plasma proteomic associates of infection mortality and then apply Mendelian randomisation (MR) to yield biomarkers that may be causally associated. We used UK Biobank plasma proteomic data to associate 2923 plasma proteins with infection mortality before 31st December 2019 (240 events in 52,520 participants). Since many plasma proteins also predict non-infection mortality, we focussed on those associated with >1.5-fold risk of infection mortality in an analysis excluding survivors. Protein quantitative trait scores (pQTS) were then used to identify whether genetically predicted protein levels also associated with infection mortality. To conduct Two Sample MR, we performed a genome-wide association study (GWAS) of infection mortality using UK Biobank participants without plasma proteomic data (n = 363,953 including 984 infection deaths). After adjusting for clinical risk factors, 1142 plasma proteins were associated with risk of infection mortality (false discovery rate <0.05). 259 proteins were associated with >1.5-fold increased risk of infection versus non-infection mortality. Of these, we identified genetically predicted increasing MERTK concentration was associated with increased risk of infection mortality. MR supported a causal association between increasing plasma MERTK protein and infection mortality (odds ratio 1.46 per unit; 95% CI 1.15- 1.85; p = 0.002). Plasma MERTK is causally associated with infection mortality and warrants exploration as a potential therapeutic target. [Display omitted] • We identified plasma proteins associated with long-term infection mortality. • 259 were more strongly associated with infection than non-infection mortality. • From these, genetically predicted MERTK also associated with infection mortality. • Mendelian randomisation supported this to be a causal association. • Understanding this association may lead to therapies that reduce fatal infection. [ABSTRACT FROM AUTHOR]

HuoXueTongFu Formula (HXTF) is a traditional Chinese herbal formula that has been used as a supplement and alternative therapy for intraperitoneal adhesion (IA). However, its specific mechanism of action has not been fully understood. In surgery, IA presents an inevitable challenge, significantly impacting patients' physical and mental well-being and increasing the financial burden. Our previous research has confirmed the preventive effects of HXTF on IA formation. However, the precise mechanism of its action still needs to be understood. In this study, the IA model was successfully established by using the Ischemic buttons and treated with HXTF for one week with or without Mer Tyrosine Kinase (MerTK) inhibitor. We evaluated the pharmacodynamic effect of HXTF on IA mice. The MerTK/phosphoinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-associated proteins were detected by Western blotting. Neutrophil extracellular traps (NETs) were detected by immunofluorescence. Macrophage phenotype was assessed by immunohistochemistry and flow cytometry. Inflammatory cytokines were detected by Real Time Quantitative PCR and Western blotting. HXTF reduced inflammatory response and alleviated IA. HXTF significantly enhanced MerTK expression, increased the number of M2c macrophages, and decreased the formation of NETs. In addition, the MerTK/PI3K/AKT pathway was significantly activated by HXTF. However, after using MerTK inhibitors, the role of HXTF in inducing M2c macrophage through activation of the PI3K/AKT pathway was suppressed and there was no inhibitory effect on NETs formation and inflammatory responses, resulting in diminished inhibition of adhesion. HXTF may improve IA by activating the MerTK/PI3K/AKT pathway to induce M2c polarization, which removes excess NETs and attenuates the inflammatory response. The caption of the abstract graphic: The HXTF improves IA by modulating the MerTK/PI3K/AKT pathway to induce M2c macrophages, increase phagocytosis of excess NETs, and reduce inflammation. [Display omitted] • HuoXueTongFu Formula (HXTF) ameliorated intraperitoneal adhesion (IA) in Mice. • HXTF induced M2c macrophage and removed excessive NETs. • HXTF regulated the MerTK/PI3K/AKT pathway to induce M2c macrophage and was verified by using MerTK inhibitor. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is a challenging autoimmune disease characterized by progressive fibrosis affecting the skin and internal organs. Despite the known infiltration of macrophages and neutrophils, their precise contributions to SSc pathogenesis remain elusive. In this study, we elucidated that CD206 hi MHCII lo M2-like macrophages constitute the predominant pathogenic immune cell population in the fibrotic skin of a bleomycin-induced SSc mouse model. These cells emerged as pivotal contributors to the profibrotic response by orchestrating the production of TGF-β1 through a MerTK signaling-dependent manner. Notably, we observed that neutrophil infiltration was a prerequisite for accumulation of M2-like macrophages. Strategies such as neutrophil depletion or inhibition of CXCR1/2 were proven effective in reducing M2-like macrophages, subsequently mitigating SSc progression. Detailed investigations revealed that in fibrotic skin, neutrophil-released neutrophil extracellular traps were responsible for the differentiation of M2-like macrophages. Our findings illuminate the significant involvement of the neutrophil-macrophage-fibrosis axis in SSc pathogenesis, offering critical information for the development of potential therapeutic strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune therapy using checkpoint inhibitors or adoptive cell transfer has revolutionized the treatment of several types of cancers. However, response to treatment is currently limited to a fraction of patients. Elucidation of immune modulatory mechanisms might optimize patient selection and present ways to modify anti-cancer immune responses. We recently discovered the expression and an important costimulatory role of TAM receptor MerTK signaling on activated human primary CD8+ T cells. Here we extend our study of the costimulatory role of MerTK expression in human CD8+ T cells. We uncover a clear link between MerTK expression and less differentiated Central Memory T cells based on an increased expression of CCR7, CD45RO, CD28, CD62L, and an altered metabolic profile. In addition, we observe an improved proliferative capacity and elevated expression of effector molecule IFNγ upon recall responses of MerTK-expressing cells in vitro. Finally, using gp100TCR-transduced T cells, we demonstrate how PROS1 treatment results in improved cytotoxicity and killing of tumors. Our findings describe a role of MerTK expression in T cells, which could be exploited in the search for improving immunotherapeutic approaches., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Exposure to fine particulate matter (PM 2.5) and polycyclic aromatic hydrocarbons (PAHs) is known to be associated with the polarization of pro-inflammatory macrophages and the development of various cardiovascular diseases. The pro-inflammatory polarization of resident cardiac macrophages (cMacs) enhances the cleavage of membrane-bound myeloid-epithelial-reproductive receptor tyrosine kinase (MerTK) and promotes the formation of soluble MerTK (solMER). This process influences the involvement of cMacs in cardiac repair, thus leading to an imbalance in cardiac homeostasis, myocardial injury, and reduced cardiac function. However, the relative impacts of PM 2.5 and PAHs on human cMacs have yet to be elucidated. In this study, we aimed to investigate the effects of PM 2.5 and PAH exposure on solMER in terms of myocardial injury and left ventricular (LV) systolic function in healthy children. A total of 258 children (aged three to six years) were recruited from Guiyu (an area exposed to e-waste) and Haojiang (a reference area). Mean daily PM 2.5 concentration data were collected to calculate the individual chronic daily intake (CDI) of PM 2.5. We determined concentrations of solMER and creatine kinase MB (CKMB) in plasma, and hydroxylated PAHs (OH-PAHs) in urine. LV systolic function was evaluated by stroke volume (SV). Higher CDI values and OH-PAH concentrations were detected in the exposed group. Plasma solMER and CKMB were higher in the exposed group and were associated with a reduced SV. Elevated CDI and 1-hydroxynaphthalene (1-OHNa) were associated with a higher solMER. Furthermore, increased solMER concentrations were associated with a lower SV and higher CKMB. CDI and 1-OHNa were positively associated with CKMB and mediated by solMER. In conclusion, exposure to PM 2.5 and PAHs may lead to the pro-inflammatory polarization of cMacs and increase the risk of myocardial injury and systolic function impairment in children. Furthermore, the pro-inflammatory polarization of cMacs may mediate cardiotoxicity caused by PM 2.5 and PAHs. [Display omitted] • PM 2.5 and PAH exposure was higher in e-waste-exposed children. • Elevated soluble form of MerTK (solMER) in exposed children was observed. • PM 2.5 and PAH exposure was related to reduced left ventricular systolic function. • PM 2.5 and PAH exposure was related to myocardial injury and mediated by solMER. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Microglia wait 44 days to phagocytose dying oligodendrocytes after single-cell DNA damage • Microglial engagement, phagocytosis, and clearance of dying oligodendrocytes occur in 1 day • CX3CR1 deletion delays the clearance of oligodendrocyte somas but not sheaths • MERTK deletion has no effect on the clearance of oligodendrocytes or myelin sheaths Olveda et al. use intravital imaging and single-cell demyelination to discover that microglia clear dying oligodendrocytes and their myelin sheaths through a stereotyped and rapid phagocytic sequence. This clearance occurs 44 days after oligodendrocyte DNA damage and is delayed by removal of CX3CR1 but not MERTK. [ABSTRACT FROM AUTHOR]

Exposure to fine particulate matter (PM 2.5 ) and polycyclic aromatic hydrocarbons (PAHs) is known to be associated with the polarization of pro-inflammatory macrophages and the development of various cardiovascular diseases. The pro-inflammatory polarization of resident cardiac macrophages (cMacs) enhances the cleavage of membrane-bound myeloid-epithelial-reproductive receptor tyrosine kinase (MerTK) and promotes the formation of soluble MerTK (solMER). This process influences the involvement of cMacs in cardiac repair, thus leading to an imbalance in cardiac homeostasis, myocardial injury, and reduced cardiac function. However, the relative impacts of PM 2.5 and PAHs on human cMacs have yet to be elucidated. In this study, we aimed to investigate the effects of PM 2.5 and PAH exposure on solMER in terms of myocardial injury and left ventricular (LV) systolic function in healthy children. A total of 258 children (aged three to six years) were recruited from Guiyu (an area exposed to e-waste) and Haojiang (a reference area). Mean daily PM 2.5 concentration data were collected to calculate the individual chronic daily intake (CDI) of PM 2.5 . We determined concentrations of solMER and creatine kinase MB (CKMB) in plasma, and hydroxylated PAHs (OH-PAHs) in urine. LV systolic function was evaluated by stroke volume (SV). Higher CDI values and OH-PAH concentrations were detected in the exposed group. Plasma solMER and CKMB were higher in the exposed group and were associated with a reduced SV. Elevated CDI and 1-hydroxynaphthalene (1-OHNa) were associated with a higher solMER. Furthermore, increased solMER concentrations were associated with a lower SV and higher CKMB. CDI and 1-OHNa were positively associated with CKMB and mediated by solMER. In conclusion, exposure to PM 2.5 and PAHs may lead to the pro-inflammatory polarization of cMacs and increase the risk of myocardial injury and systolic function impairment in children. Furthermore, the pro-inflammatory polarization of cMacs may mediate cardiotoxicity caused by PM 2.5 and PAHs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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., (© 2024. The Author(s).)

Rationale: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for the clearance of apoptotic cells (efferocytosis) and plays important roles in redox-related human diseases. We will explore MerTK biology in human cells, tissues, and diseases based on big data analytics., Methods: The human RNA-seq and scRNA-seq data about 42,700 samples were from NCBI Gene Expression Omnibus and analyzed by QIAGEN Ingenuity Pathway Analysis (IPA) with about 170,000 crossover analysis. MerTK expression was quantified as Log2 (FPKM + 0.1)., Results: We found that, in human cells, MerTK is highly expressed in macrophages, monocytes, progenitor cells, alpha-beta T cells, plasma B cells, myeloid cells, and endothelial cells (ECs). In human tissues, MerTK has higher expression in plaque, blood vessels, heart, liver, sensory system, artificial tissue, bone, adrenal gland, central nervous system (CNS), and connective tissue. Compared to normal conditions, MerTK expression in related tissues is altered in many human diseases, including cardiovascular diseases, cancer, and brain disorders. Interestingly, MerTK expression also shows sex differences in many tissues, indicating that MerTK may have different impact on male and female. Finally, based on our proteomics from primary human aortic ECs, we validated the functions of MerTK in several human diseases, such as cancer, aging, kidney failure and heart failure., Conclusions: Our big data analytics suggest that MerTK may be a promising therapeutic target, but how it should be modulated depends on the disease types and sex differences. For example, MerTK inhibition emerges as a new strategy for cancer therapy due to it counteracts effect on anti-tumor immunity, while MerTK restoration represents a promising treatment for atherosclerosis and myocardial infarction as MerTK is cleaved in these disease conditions., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)