Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

Introduction: The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. Methods: In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. Results: At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS <3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2 = 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Conclusions: Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Phosphatidylserine and Tyro3-Axl-Mertk Receptor Tyrosine Kinase level detection in plasma and on plasma-derived extracellular vesicle surface in chronic lymphocytic leukemia.

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.

Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

Introduction: TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. Methods: We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 µL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-ß and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. Conclusion: This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

MERTK in the rat trigeminal system: a potential novel target for cluster headache?

The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.

Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

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.

Duodenal mucosa of untreated celiac disease patients has altered expression of the GAS6 and PROS1 and the negative regulator tyrosine kinase TAM receptors subfamily.

Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.

Discovery of Novel Macrocyclic MERTK/AXL Dual Inhibitors.

MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide antitumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as nonsmall cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated antitumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.

Disturbed flow impairs MerTK-mediated efferocytosis in aortic endothelial cells during atherosclerosis.

Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK-/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1ß, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22phox) and mitochondrial dysfunction. Interestingly, MerTK-/-induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis.

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-ß (TGFß) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFß signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFß-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFß expression and drove TGFß signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFß signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

CADHERIN-11 regulation of myeloid phagocytes and autoimmune inflammation in murine lupus.

BACKGROUND AND OBJECTIVE: Understanding the regulation of efferocytosis by myeloid phagocytes is important in identifying novel targets in systemic lupus erythematosus (SLE). Cadherin-11 (CDH11), a cell adhesion molecule, is implicated in inflammatory arthritis and fibrosis and recently been shown to regulate macrophage phagocytosis. The extent and mechanism of this regulation is unknown. Our objective was to examine the extent to which CDH11 regulates myeloid phagocytes and contributes to autoimmunity and tissue inflammation. METHODS: We analyzed efferocytosis in macrophages and dendritic cells (DCs) from WT and Cdh11-/- mice and investigated the mechanisms in vitro. We investigated the role of CDH11 in disease development in vivo using the pristane induced lupus model. To translate the clinical relevance of CDH11 in human disease, we measured serum CDH11 levels in two independent pediatric SLE (pSLE) cohorts and healthy controls. RESULTS: Using bone marrow derived macrophages (BMDMs) and DCs (BMDCs), we found impaired efferocytosis in phagocytes from Cdh11-/- mice, mediated by downregulated efferocytosis receptor expression and RhoGTPase activation. Specifically, loss of CDH11 downregulated Mertk expression and Rac1 activation in BMDMs, and integrin αVß3 expression and Cdc42 activation in BMDCs, highlighting distinct pathways. In vivo, Cdh11-/- mice displayed defective efferocytosis and increased accumulation of apoptotic debris in pristane-induced lupus. Further, Cdh11-/- mice had enhanced systemic inflammation and autoimmune inflammation with increased anti-dsDNA autoantibodies, splenomegaly, type I interferons, and inflammatory cytokines. Paradoxically, at the tissue level, Cdh11-/- mice were protected against glomerulonephritis, indicating a dual role in murine lupus. Finally, SLE patients had increased serum CDH11 compared to controls. CONCLUSION: This study highlights a novel role of CDH11 in regulating myeloid cells and efferocytosis and its potential as a contributor to development in autoimmunity murine lupus. Despite the increase in autoimmunity, Cdh11-/- mice developed decreased tissue inflammation and damage.

CX3CR1 deficiency promotes resolution of hepatic ischemia-reperfusion injury by regulating homeostatic function of liver infiltrating macrophages.

Hepatic ischemia-reperfusion injury(HIRI) remains to be an unsolved risk factor that contributes to organ failure after liver surgery. Our clinical retrospective study showed that lower donor liver CX3-C chemokine receptor-1(CX3CR1) mRNA expression level were correlated with upregulated pro-resolved macrophage receptor MERTK, as well as promoted restoration efficiency of allograft injury in liver transplant. To further characterize roles of CX3CR1 in regulating resolution of HIRI, we employed murine liver partial warm ischemia-reperfusion model by Wt & Cx3cr1-/- mice and the reperfusion time was prolonged from 6 h to 4-7 days. Kupffer cells(KCs) were depleted by clodronate liposome(CL) in advance to focus on infiltrating macrophages, and repopulation kinetics were determined by FACS, IF and RNA-Seq. CX3CR1 antagonist AZD8797 was injected i.p. to interrogate potential pharmacological therapeutic strategies. In vitro primary bone marrow macrophages(BMMs) culture by LXR agonist DMHCA, as well as molecular and functional studies, were undertaken to dissect roles of CX3CR1 in modulating macrophages cytobiological development and resolutive functions. We observed that deficiency or pharmacological inhibition of CX3CR1 facilitated HIRI resolution via promoted macrophages migration in CCR1/CCR5 manner, as well as enhanced MerTK-mediated efferocytosis. Our study demonstrated the critical roles of CX3CR1 in progression of HIRI and identified it as a potential therapeutic target in clinical liver transplantation.

The TAM Subfamily of Receptor Tyrosine Kinases: The Early Years.

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.

Nintedanib downregulates the profibrotic M2 phenotype in cultured monocyte-derived macrophages obtained from systemic sclerosis patients affected by interstitial lung disease.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs, including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs, and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFß1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients. METHODS: Fourteen SSc patients, fulfilling the 2013 ACR/EULAR criteria for SSc, 10 SSc patients affected by ILD (SSc-ILD pts), 4 SSc patients non affected by ILD (SSc pts no-ILD), and 5 voluntary healthy subjects (HSs), were recruited at the Division of Clinical Rheumatology-University of Genova, after obtaining Ethical Committee approval and patients' informed consent. Monocytes were isolated from peripheral blood, differentiated into MDMs, and then maintained in growth medium without any treatment (untreated cells), or treated with nintedanib (0.1 and 1µM) for 3, 16, and 24 h. Gene expression of macrophage scavenger receptors (CD204, CD163), mannose receptor-1 (CD206), Mer tyrosine kinase (MerTK), identifying M2 macrophages, together with TGFß1 and IL10, were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFß1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test. RESULTS: Cultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05), CD204, and MerTK (p < 0.01), together with a significant upregulation of the gene expression of MerTK and TGFß1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover, the protein synthesis of CD206 and MerTK and the gene expression of TGFß1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204, CD206, CD163 (p < 0.05), and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10, both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs, nintedanib 0.1 and 1µM significantly reduced the release of active TGFß1 after 24 h of treatment (p < 0.05 vs. untreated cells). CONCLUSIONS: In cultured MDMs from SSc-ILD pts, nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers, together with the significant reduction of TGFß1 release, and notably MerTK, a tyrosine kinase receptor involved in lung fibrosis.

Adjuvant Novel Nanocarrier-Based Targeted Therapy for Lung Cancer.

Lung cancer has the lowest survival rate due to its late-stage diagnosis, poor prognosis, and intra-tumoral heterogeneity. These factors decrease the effectiveness of treatment. They release chemokines and cytokines from the tumor microenvironment (TME). To improve the effectiveness of treatment, researchers emphasize personalized adjuvant therapies along with conventional ones. Targeted chemotherapeutic drug delivery systems and specific pathway-blocking agents using nanocarriers are a few of them. This study explored the nanocarrier roles and strategies to improve the treatment profile's effectiveness by striving for TME. A biofunctionalized nanocarrier stimulates biosystem interaction, cellular uptake, immune system escape, and vascular changes for penetration into the TME. Inorganic metal compounds scavenge reactive oxygen species (ROS) through their photothermal effect. Stroma, hypoxia, pH, and immunity-modulating agents conjugated or modified nanocarriers co-administered with pathway-blocking or condition-modulating agents can regulate extracellular matrix (ECM), Cancer-associated fibroblasts (CAF),Tyro3, Axl, and Mertk receptors (TAM) regulation, regulatory T-cell (Treg) inhibition, and myeloid-derived suppressor cells (MDSC) inhibition. Again, biomimetic conjugation or the surface modification of nanocarriers using ligands can enhance active targeting efficacy by bypassing the TME. A carrier system with biofunctionalized inorganic metal compounds and organic compound complex-loaded drugs is convenient for NSCLC-targeted therapy.

Axl and MerTK regulate synovial inflammation and are modulated by IL-6 inhibition in rheumatoid arthritis.

The TAM tyrosine kinases, Axl and MerTK, play an important role in rheumatoid arthritis (RA). Here, using a unique synovial tissue bioresource of patients with RA matched for disease stage and treatment exposure, we assessed how Axl and MerTK relate to synovial histopathology and disease activity, and their topographical expression and longitudinal modulation by targeted treatments. We show that in treatment-naive patients, high AXL levels are associated with pauci-immune histology and low disease activity and inversely correlate with the expression levels of pro-inflammatory genes. We define the location of Axl/MerTK in rheumatoid synovium using immunohistochemistry/fluorescence and digital spatial profiling and show that Axl is preferentially expressed in the lining layer. Moreover, its ectodomain, released in the synovial fluid, is associated with synovial histopathology. We also show that Toll-like-receptor 4-stimulated synovial fibroblasts from patients with RA modulate MerTK shedding by macrophages. Lastly, Axl/MerTK synovial expression is influenced by disease stage and therapeutic intervention, notably by IL-6 inhibition. These findings suggest that Axl/MerTK are a dynamic axis modulated by synovial cellular features, disease stage and treatment.

Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVß5, CD36, and TIM-3, whereas TIM-1, αVß3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

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.

GAS6/TAM Axis as Therapeutic Target in Liver Diseases.

TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor biology and autoimmune diseases. In contrast, TAM regulation of fibrogenesis and the inflammation mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and, ultimately, liver cancer has recently been revealed. GAS6 and PROS binding to phosphatidylserine exposed in outer membranes of apoptotic cells links TAMs, particularly MERTK, with hepatocellular damage. In addition, AXL and MERTK regulate the development of liver fibrosis and inflammation in chronic liver diseases. Acute hepatic injury is also mediated by the TAM system, as recent data regarding acetaminophen toxicity and acute-on-chronic liver failure have uncovered. Soluble TAM-related proteins, mainly released from activated macrophages and hepatic stellate cells after hepatic deterioration, are proposed as early serum markers for disease progression. In conclusion, the TAM system is becoming an interesting pharmacological target in liver pathology and a focus of future biomedical research in this field.

A single cell atlas of frozen shoulder capsule identifies features associated with inflammatory fibrosis resolution.

Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

Big data analytics for MerTK genomics reveals its double-edged sword functions in human diseases.

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.