TAM Receptors Research Articles

Diminished soluble levels of growth arrest specific protein 6 and tyrosine kinase receptor Axl in patients with rheumatoid arthritis.

Growth arrest specific protein 6 (Gas-6) and its tyrosine kinase receptor Axl plays an important role in apoptosis, and regulation of innate immune response, therefore, we investigated their plasma concentrations in Rheumatoid arthritis (RA) patients and correlated them to clinical, laboratory and radiological parameters of the disease. Plasma from 77 RA patients and 50 normal healthy subjects were assayed for plasma Gas6 and Axl levels. Demographic, clinical and serological data were prospectively assessed. Rheumatoid arthritis disease activity was assessed using 28-joint Disease Activity Score (DAS-28) and functional capacity by modified health assessment questionnaire (mHAQ). Standardized x-rays for hands and feet were done to all participants. The level of Gas6 and Axl were significantly decreased in the RA patients compared to those of the healthy control subjects. Levels of Gas6 correlated positively with Axl levels in both patients and healthy control. Gas6 levels were remarkably reduced in those patients with erosive RA than those without. Levels of Gas6 were found to be negatively correlated with the presence of erosive disease and positively correlated with DAS-28, ESR, Leucocytosis and IL6. The plasma concentrations of Gas6 and Axl are altered in RA patients and thus may have a role in RA pathogenesis. Further mechanistic studies on the involvement of all TAM receptors tyrosine kinases pathway in RA are needed to help in understanding the pathogenesis and possibly aid in diagnosis and future treatments of RA especially for patients with erosive disease.

TAM Receptor or CBL-B Inhibition Enables Tumor Rejection by NK Cells

Abstract CBL-B–dependent ubiquitination of TYRO3, AXL, and MER (TAM) receptors inhibits NK cell activation.

MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme

Glioblastoma is an aggressive tumor that occurs in both adult and pediatric patients and is known for its invasive quality and high rate of recurrence. Current therapies for glioblastoma result in high morbidity and dismal outcomes. The TAM subfamily of receptor tyrosine kinases includes Tyro3, Axl, and MerTK. Axl and MerTK exhibit little to no expression in normal brain but are highly expressed in glioblastoma and contribute to the critical malignant phenotypes of survival, chemosensitivity and migration. We have found that Foretinib, a RTK inhibitor currently in clinical trial, inhibited phosphorylation of TAM receptors, with highest efficacy against MerTK, and blocked downstream activation of Akt and Erk in adult and pediatric glioblastoma cell lines, findings that are previously unreported. Survival, proliferation, migration, and collagen invasion were hindered in vitro. Foretinib treatment in vivo abolished MerTK phosphorylation and reduced tumor growth 3-4 fold in a subcutaneous mouse model. MerTK targeted shRNA completely prevented intracranial and subcutaneous glioma growth further delineating the impact of MerTK inhibition on glioblastoma. Our findings provide additional target validation for MerTK inhibition in glioblastoma and demonstrate that robust MerTK inhibition can be achieved with the multi-kinase inhibitor Foretinib as an innovative and translational therapeutic approach to glioblastoma.

Erratum to: Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity

Erratum to: Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity

TAM Receptors Affect Adult Brain Neurogenesis by Negative Regulation of Microglial Cell Activation

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

EGFR signaling in the HGG-02 glioblastoma cell line with an unusual loss of EGFR gene copy

Epidermal growth factor receptor (EGFR) gene amplification and the overexpression of EGFR are described as common features of glioblastoma multiforme (GBM). Nevertheless, we previously reported the loss of EGFR gene copy in a GBM specimen from a patient with an unusually favorable course of the disease, and the HGG-02 cell line with this aberration was successfully derived from this tumor. Here, we present a detailed analysis of changes in gene expression and cell signaling in the HGG-02 cell line; the GM7 reference cell line with a standard EGFR gene copy number derived from a very aggressive GBM was used as a control. We confirmed the downregulation of EGFR expression and signaling in HGG-02 cells using different methods (RTK analysis, gene profiling and RT-PCR). Other changes that may have contributed to the non-aggressive phenotype of the primary tumor were identified, including the downregulated phosphorylation of the Axl and Trk receptors, as well as increased activity of JNK and p38 kinases. Notably, differences in PDGF signaling were detected in both of these cell lines; HGG-02 cells preferentially expressed and signaled through PDGFRα, and PDGFRβ was strongly overexpressed and phosphorylated in the GM7 reference cell line. Using expression profiling of cancer-related genes, we revealed the specific profile of HGG-02 cells that included upregulated tumor-suppressors as well as downregulated genes associated with the extracellular matrix. This study represents the first comprehensive analysis of gene expression and cell signaling in glioblastoma cells with lower EGFR gene dosage. As indicated by our results, the TAM receptors, Trk receptors and PDGFRs need to be investigated further since their regulation appears to be important for glioblastoma biological features as well as the clinical course of the disease.

Biology of the TAM Receptors

The TAM receptors--Tyro3, Axl, and Mer--comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands--Gas6 and Protein S--are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.

A Janus role for MerTK in the outcome of septic shock

Severe sepsis remains a clinical challenge today, even indeveloped countries, because of the increased age of thepatients and the high burden of antibiotic resistance [1]. Inaddition, in recent years, we have become increasinglyaware that, even in the early stages of sepsis, a high pro-portion of patients experience a severe dysfunction ofnative and adaptive immunity, which is closely related topatients’ outcome [2]. In this context, understanding thefunctionality of receptors able to modulate the immuneresponse, and in particular to downregulate inflammation,has great importance for designing new therapeutic strat-egies. Among these molecules, a main role is occupied bythe TAM family of receptor tyrosine kinases, which isformed by three members, i.e., Tyro3, AxI, and Mer, thathave two vitamin K-dependent ligands, i.e., the growtharrest-specific protein (Gas)6 and Protein S [3]. In theSeptember 2013 issue of Intensive Care Medicine, Gui-gnant et al. [4] explored by an elegant experimental studythe expression of TAM receptors in circulating immunecells in patients with septic shock and after trauma, and inhealthy volunteers. A significant early alteration of MerTKexpression on monocytes and neutrophils was observed inpatients with septic shock, and a persistent MerTK over-expression after septic shock was associated with adverseoutcome. The ex vivo analysis showed also a differentMerTK surface expression of healthy monocytes after48 h incubation with plasma from septic or traumapatients, supporting the difference in TAM expressionbetween infection-induced inflammation and sterileinflammation (i.e., trauma). The study provides newimportant data on the mechanisms of immune response insepsis, but the true reasons for the observed alterations inMerKT expression and their role in the complex patho-physiological context of sepsis remain to be clarified.TAM receptors are under the control of Toll-likereceptors (TLR), and are widely expressed by cells of theinnateimmunesystem[5].ActivationofTAMreceptorsbytheir ligands triggers a cascade of intracellular events thatculminate in the inhibition of nuclear factor-kappaB (NF-jB), a crucial proinflammatory molecule, and results indownregulation of inflammation [6]. When expressed bymonocytes and granulocytes, these receptors (and in par-ticular MerTK) can also mediate the phagocytosis ofapoptoticcells[7],whichisfundamentalinordertoremovedead cells without triggering inflammation. Several datanow exist that underline the importance of MerTK activa-tion in the elimination of potential autoantigens derived byapoptotic cells, in the inhibition of the production of pro-inflammatory cytokines, and in the prevention of theexpansion of autoreactive lymphocytes [8–10]. The func-tional impairment of MerTK promotes not onlyautoimmunity but also atherosclerosis, while its overex-pression is linked to poor prognosis in cancer [11].The strong relationships existing between TAMreceptors and a proinflammatory molecule such as tumornecrosis factor-alpha (TNF-a) are reinforced by theobservation that they can interact with a common protein,the matrix metalloproteinase TNF-a converting enzyme,also known as ‘‘a disintegrin and a metalloproteinase 17’’(ADAM17). In order to provide the mature, soluble,secretable form of TNF-a, ADAM17 cleaves the mem-brane form of this important proinflammatory cytokine[12]. Interestingly, ADAM17 is also able to cleave the

Viruses Play Dead to TAMe Interferon Responses

TAM receptors promote apoptotic cell uptake and function as inflammation suppressors. Many viruses mimic apoptotic cells, thus exploiting TAM receptors for attachment and entry. In this issue of Cell Host & Microbe, Bhattacharyya etal. show that TAM binding by enveloped viruses also induces receptor signaling to suppress cellular interferon responses.

Increased MerTK expression in circulating innate immune cells of patients with septic shock

A new pathway of three protein tyrosine kinase receptors, namely, the TAM receptor family [Tyro-3, Axl and Mer tyrosine kinase (MerTK)], has recently been described to negatively control immune responses. The objective of this prospective, observational, clinical study was to investigate the expression patterns of TAM receptors in circulating white blood cells collected from patients with septic shock. The expression of TAM receptors was measured by flow cytometry in circulating leukocytes from patients with septic shock sampled on days (D) 1-2 (n = 47) and D3-4 (n = 37) after the onset of shock, severe trauma patients at D1-2 after trauma (n = 51) and healthy individuals (n = 23). On D1-2 after injury, MerTK was overexpressed in monocytes and neutrophils collected from patients with septic shock in comparison with those collected from healthy volunteers and trauma patients. This phenomenon was also observed for mRNA. Conversely, the expression of Tyro-3 and Axl was higher in monocytes from trauma patients versus healthy volunteers or those in septic shock. MerTK expression between D1-2 and D3-4 remained elevated in patients suffering from septic shock who died or developed an intensive care unit-acquired infection, whereas it decreased in patients who recovered uneventfully. This in vivo observed expression pattern was reproduced ex vivo after the incubation of healthy volunteer cells with plasma from septic shock or trauma patients. MerTK expression in circulating innate immune cells is increased in patients with septic shock in comparison with healthy volunteers and trauma patients. Persistent MerTK overexpression after septic shock is associated with adverse outcome. The role of this family of receptors in the pathophysiology of injury-induced immune dysfunctions deserves to be specifically investigated.

T Cell-Derived Protein S Engages TAM Receptor Signaling in Dendritic Cells to Control the Magnitude of the Immune Response

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactiveimmune responses. Tcells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse Tcells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to Tcell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human Tcells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.

THU0178 Circulating free protein S levels may be linked to cardiovascular events and venous thrombosis in SLE

BackgroundSLE patients are at risk for diverse organ systems involvement, which increases the challenges for diagnosis and predictions for the development of specific clinical features. In recent studies we have...

TAM receptors and their role in the regulation of lung innate immunity (P3236)

Abstract Background: The role of TAM receptors are well characterised in autoimmunity and cancer biology. Their role in infectious diseases is now being elucidated. The goal of this study is to characterise TAM receptor expression profiles of various innate immune components at homeostasis as well as in various infection models. The hypothesis of this study is that up-regulation of TAM receptors, AXL and MerTK are responsible for the de-sensitisation of airway macrophages to bacteria following influenza infection. Furthermore, I hypothesise that TAM receptors may be involved in limiting innate immune activation at homoeostasis. Methods: Using a murine model, we characterised TAM receptor expression on various innate populations in the lung following viral (influenza A) and bacterial (Streptcoccus pneumoniae) infections. We also investigated the role of AXL in influenza/bacteria single and co-infection using AXL-/- mice. Results: AXL and MerTK are differentially expressed by innate populations in the lung depending on their location. Monocytes and neutrophils which infiltrate into the alveolar space during influenza infection significantly up-regulate TAM receptor expressions. Lack of AXL significantly potentiates innate immunity by upregulating the release of inflammatory cytokines in vitro. Conclusion: Up-regulation of AXL following a viral infection may explain the exacerbation of secondary bacterial complications as its upregulation limits the innate inflammatory responses.

Role of the TAM receptor Axl in differentiation and activation of B cells (P4058)

Abstract In dendritic cells (DCs), TLR activation upregulates TAM receptor expression and these TAM receptors initiated pathways subsequently shut down cytokine production. Moreover, TAM receptor deficient mice develop a severe SLE-like disease. TLR9 and TLR7 play a key role in the detection of autoantigens by both DCs and B cells in the context of systemic autoimmunity. To better understand the direct effects of TAM receptors on B cells, we have used BCR Tg mice (AM14) that recognize autologous IgG2a. AM14 B cells are activated by immune complexes (ICs) that incorporate either DNA or RNA through pathways dependent on TLR9 and TLR7, respectively. Contrary to DCs, expression of the TAM receptor Axl is not induced by TLR ligands but is dependent on crosslinking of the BCR per se. However, BCR/TLR9, but not BCR/TLR7, co-engagement inhibits Axl upregulation, pointing to a role for TLR9 in the regulation of Axl expression. Activation of AM14 B cells by RNA-containing ICs induces TLR7-dependent differentiation into plasma cells and these plasma cells express high levels of Axl. In contrast, BCR/TLR9-dependent activation of AM14 B cells by DNA-containing ICs blocks Axl expression and does not lead to plasma cell differentiation. These data identify unique outcomes of BCR/TLR7 and BCR/TLR9 co-engagement on B cell differentiation and Axl expression. Furthermore, these B cell intrinsic responses may account for the opposing roles of TLR7 and TLR9 in the regulation of systemic autoimmunity.

Determining the role of the Axl receptor tyrosine kinase in beta cell autoimmunity (P4089)

Abstract Type I diabetes (T1D) is an autoimmune disorder characterized by the T cell mediated destruction of the insulin-producing beta cells. Mechanisms involving the breakdown of self-tolerance in T1D are ill defined. We have previously shown that T1D progression is inhibited in NOD mice lacking expression of Mer, a member of the TAM family of receptor tyrosine kinases. Mer is expressed by dendritic cells (DC) and is critical for mediating the tolerogenic effects of apoptotic cells (AC) on DC. Axl, another TAM receptor is co-expressed by DC and binds AC. The relative role of Mer and Axl in mediating the tolerogenic effects of AC on DC, and regulating beta cell autoimmunity are unknown. Accordingly, we assessed the effects of AC on bone marrow-derived and splenic NOD DC lacking Axl expression. Pretreatment with AC of DC showed that Axl was required for AC-mediated inhibition of DC activation and maturation. Axl-deficient DC failed to activate c-Src and STAT3, which are key signaling molecules needed for AC-induced inhibition of DC. Furthermore, incidence of T1D in NOD mice lacking Axl was significantly reduced. Collectively, these findings indicate that Axl contributes to AC-induced inhibition of DC, and has a role in regulating T1D progression.

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity

IntroductionGrowth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population.MethodsFifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples.ResultsPlasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients.ConclusionsThe present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.

Genetic Dissection of TAM Receptor-Ligand Interaction in Retinal Pigment Epithelial Cell Phagocytosis

Although TAM receptor tyrosine kinases play key roles in immune regulation, cancer metastasis, and viral infection, the relative importance of the two TAM ligands-Gas6 and Protein S-has yet to be resolved in any setting invivo. We have now performed a genetic dissection of ligand function in the retina, where the TAM receptor Mer is required for the circadian phagocytosis of photoreceptor outer segments by retinal pigment epithelial cells. This process is severely attenuated in Mer mutant mice, which leads to photoreceptor death. We find that retinal deletion of either Gas6 or Protein S alone yields retinae with a normal number of photoreceptors. However, concerted deletion of both ligands fully reproduces the photoreceptor death seen in Mer mutants. These results demonstrate that Protein S and Gas6 function as independent, bona fide Mer ligands, and are, to a first approximation, interchangeable with respect to Mer-driven phagocytosis in the retina.

Gas6 and TAM Receptors in Mouse Platelets

Abstract 5199 Objective:Growth Arrest Specific-6 (Gas6) is a new vitamin K-dependent protein and is a ligand for TAM receptors, three receptors (Tyro, Axl and Mer) belonging to the receptor tyrosine kinase subfamily. The number of publications concerning the physiological importance of the new vitamin-K dependent proteins, which are not members of the coagulation cascade, is growing. Gas6 has a structural homology to natural anticoagulant of protein S, but has no anticoagulant activity. Some research groups have reported contradictory results concerning the presence of Gas6 and its receptors in mouse platelets. The purpose of this study is to investigate the presence of Gas6 and its receptors in mouse platelets as mRNA and protein levels. Material and Methods:25 specific pathogen-free, 8–10 week old, 25–30 g in weight, race of Balb-C male/female mice were anesthetized under light ether anesthesia and blood samples were taken from their hearts. Total RNAs were isolated from platelets, then mRNAs encoding Gas6 and TAM receptors were detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA. We were unable to measure the Mer protein level because of the absence of any commercial ELISA kit for mouse Mer. Results:RT-PCR results indicated the presence of mRNAs encoding to Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro. Receptor levels of Axl and Tyro were below the detection limits of the ELISA method. Plasma Gas6 concentration was found to be 26 ng/ml. Conclusion:We found the presence of mRNAs encoding Gas6 and only receptor Mer in mouse platelets but not Axl and Tyro. Protein Gas6, Axl and Tyro levels were below the detection limits of the ELISA methods. The presence of mRNA is not evidence of protein expression in platelets because they have no DNA. Further studies are required to clarify the presence of Gas6 and TAM receptors in platelets using Real Time PCR, and more sensitive immunological methods, and then, as studies of their effects on mechanisms. Original results from these further studies will provide reliable information as to whether Gas6 and its TAM receptors may be a pharmacological target in thrombosis, hemostasis, restenosis and atherosclerosis. Disclosures:No relevant conflicts of interest to declare.

The TIM and TAM Families of Phosphatidylserine Receptors Mediate Dengue Virus Entry

Dengue viruses (DVs) are responsible for the most medically relevant arboviral diseases. However, the molecular interactions mediating DV entry are poorly understood. We determined that TIM and TAM proteins, two receptor families that mediate the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, serve as DV entry factors. Cells poorly susceptible to DV are robustly infected after ectopic expression of TIM or TAM receptors. Conversely, DV infection of susceptible cells is inhibited by anti-TIM or anti-TAM antibodies or knockdown of TIM and TAM expression. TIM receptors facilitate DV entry by directly interacting with virion-associated PtdSer. TAM-mediated infection relies on indirect DV recognition, in which the TAM ligand Gas6 acts as a bridging molecule by binding to PtdSer within the virion. This dual mode of virus recognition by TIM and TAM receptors reveals how DVs usurp the apoptotic cell clearance pathway for infectious entry.

Growth arrest-specific gene 6 (Gas6) levels are elevated in patients with chronic renal failure

The TAM receptors (tyro3, axl and mer) and their ligands (vitamin K-dependent proteins-Gas6 and Protein S) are crucial modulators of inflammation, which may be relevant in chronic kidney disease (CKD). Gas6 and axl have multiple roles in mediating vascular atherosclerosis and injury, thrombosis and inflammation, yet nothing is known about the Gas6-axl pathway in humans with CKD. Given the prevalence of chronic inflammation and vascular disease in this population, we measured TAM ligands in patients with various levels of renal function. Gas6 and protein S were quantified in the plasma by ELISA in three patient groups: end-stage renal disease on chronic hemodialysis (HD), CKD and normal controls. Significantly increased levels of Gas6 and protein S were found in CKD patients compared with normal controls (P < 0.01 and <0.001, respectively). In HD patients, Gas6 levels were elevated compared with controls (P < 0.001) and positively associated with low albumin (r = 0.33; P = 0.01), dialysis vintage (r = 0.36; P = 0.008) and IV iron administration (r = 0.33; P = 0.01). The levels of Gas6 rose with CKD stage and were inversely associated with estimated GFR (P < 0.0001). Dysregulation of circulating Gas6 is associated with renal disease and inversely proportional to renal function. Low albumin and higher IV iron administration were associated with higher Gas6 levels, suggesting a possible connection between inflammation and oxidative stress mediated by iron. Protein S levels were also elevated in CKD patients, but the relevance of this finding needs to be further investigated.