Deficiency of Axl aggravates pulmonary arterial hypertension via BMPR2.

Pulmonary arterial hypertension (PAH), is a fatal disease characterized by a pseudo-malignant phenotype. We investigated the expression and the role of the receptor tyrosine kinase Axl in experimental (i.e., monocrotaline and Su5416/hypoxia treated rats) and clinical PAH. In vitro Axl inhibition by R428 and Axl knock-down inhibited growth factor-driven proliferation and migration of non-PAH and PAH PASMCs. Conversely, Axl overexpression conferred a growth advantage. Axl declined in PAECs of PAH patients. Axl blockage inhibited BMP9 signaling and increased PAEC apoptosis, while BMP9 induced Axl phosphorylation. Gas6 induced SMAD1/5/8 phosphorylation and ID1/ID2 increase were blunted by BMP signaling obstruction. Axl association with BMPR2 was facilitated by Gas6/BMP9 stimulation and diminished by R428. In vivo R428 aggravated right ventricular hypertrophy and dysfunction, abrogated BMPR2 signaling, elevated pulmonary endothelial cell apoptosis and loss. Together, Axl is a key regulator of endothelial BMPR2 signaling and potential determinant of PAH.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

Pathogenic postzygotic mosaicism in the tyrosine receptor kinase pathway: potential unidentified human disease hidden away in a few cells.

Mutations occurring during embryonic development affect only a subset of cells resulting in two or more distinct cell populations that are present at different levels, also known as postzygotic mosaicism (PZM). Although PZM is a common biological phenomenon, it is often overlooked as a source of disease due to the challenges associated with its detection and characterization, especially for very low-frequency variants. Moreover, PZM can cause a different phenotype compared to constitutional mutations. Especially, lethal mutations in receptor tyrosine kinase (RTK) pathway genes, which exist only in a mosaic state, can have completely new clinical manifestations and can look very different from the associated monogenic disorder. However, some key questions are still not addressed, such as the level of mosaicism resulting in a pathogenic phenotype and how the clinical outcome changes with the development and age. Addressing these questions is not trivial as we require methods with the sensitivity to capture some of these variants hidden away in very few cells. Recent ultra-accurate deep-sequencing approaches can now identify these low-level mosaics and will be central to understand systemic and local effects of mosaicism in the RTK pathway. The main focus of this review is to highlight the importance of low-level mosaics and the need to include their detection in studies of genomic variation associated with disease.

Testosterone ameliorates vascular aging via the Gas6/Axl signaling pathway.

Low serum testosterone level is associated with aging-related vascular stiffness, but the underlying mechanism is unclear. The Growth arrest-specific protein 6 (Gas6) /Axl pathway has been proved to play important roles in cell senescence. In this study, we intend to explore whether Gas6/Axl is involved in the effect of testosterone on vascular aging amelioration. Vascular aging models of wild type and Axl-/- mice were established by natural aging. Mice of these two gene types were randomized into young group, aging group and testosterone undecanoate (TU) treatment group. Mice were treated with TU (37.9 mg/kg) in the TU group, which treated with solvent reagent served as control. The aging mice exhibited decreases in serum testosterone, Gas6 and Axl levels and an increase in cell senescence, manifested age-related vascular remodeling. Testosterone treatment induced testosterone and Gas6 levels in serum, and ameliorated cell senescence and vascular remodeling in aging mice. Furthermore, we uncover the underlying molecular mechanism and show that testosterone treatment restored the phosphorylation of Akt and FoxO1a. Axl knockout accelerated cell senescence and vascular remodeling, and resisted the anti-aging effect of testosterone. Testosterone might exert a protective effect on vascular aging by improving cell senescence and vascular remodeling through the Gas6/Axl pathway.

Duplication and divergence: New insights into AXR1 and AXL functions in DNA repair and meiosis.

Rubylation is a conserved regulatory pathway similar to ubiquitination and essential in the response to the plant hormone auxin. In Arabidopsis thaliana, AUXIN RESISTANT1 (AXR1) functions as the E1-ligase in the rubylation pathway. The gene AXR1-LIKE (AXL), generated by a relatively recent duplication event, can partially replace AXR1 in this pathway. We have analysed mutants deficient for both proteins and complementation lines (with the AXR1 promoter and either AXR1 or AXL coding sequences) to further study the extent of functional redundancy between both genes regarding two processes: meiosis and DNA repair. Here we report that whereas AXR1 is essential to ensure the obligatory chiasma, AXL seems to be dispensable during meiosis, although its absence slightly alters chiasma distribution. In addition, expression of key DNA repair and meiotic genes is altered when either AXR1 or AXL are absent. Furthermore, our results support a significant role for both genes in DNA repair that was not previously described. These findings highlight that AXR1 and AXL show a functional divergence in relation to their involvement in homologous recombination, exemplifying a duplicate retention model in which one copy tends to have more sub-functions than its paralog.

Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Tyro3/Axl/Mertk-deficient mice develop bone marrow edema which is an early pathological marker in rheumatoid arthritis.

Rheumatoid arthritis is an auto-immune disease of the synovial joints, hallmarked by chronic inflammation and subsequent progressive tissue destruction. TYRO3, AXL and MER (gene name Mertk) (TAM) receptors are part of a negative feedback signaling system in the immune reaction and mediate efferocytosis thereby tempering the inflammatory process. We have shown that Axl-/- and Mertk-/- mice develop more severe arthritis whereas activating these receptors by overexpressing their ligands Pros1 and Gas6 ameliorates arthritis. Mice genetically ablated for the three genes of the TAM receptor family Tyro3/Axl/Mertk (TAM triple knock-out or TKO) have been described to spontaneously develop macroscopic signs of arthritis. In this study we aimed to analyze arthritis development in TAM TKO mice histologically to determine the extent and sequence of pathological changes in the joint. Ankle joints of three different age groups, adolescence (14 weeks), mature adult (34 weeks) and middle-age (52 weeks), of TAM TKO or wild-type mice were examined macroscopically, histologically and immunohistochemically. Surprisingly, until the age of 52 weeks, none of the mice examined developed spontaneous macroscopic signs of arthritis. There was no synovial inflammation nor any signs of damage to the cartilage or bone. However, bone marrow edema was observed in TAM TKO mice in the two latter age groups. The infiltrate in the bone marrow was characterized by both myeloid cells and lymphocytes. This study showed that TAM TKO mice developed a pre-stage (pre-clinical phase) of arthritis marked by bone marrow edema.

Ron Receptor Signaling Ameliorates Hepatic Fibrosis in a Diet-Induced Nonalcoholic Steatohepatitis Mouse Model.

Liver fibrosis is commonly observed in the terminal stages of nonalcoholic steatohepatitis (NASH) and with no specific and effective antifibrotic therapies available, this disease is a major global health burden. The MSP/Ron receptor axis has been shown to have anti-inflammatory properties in a number of mouse models, due at least in part, to its ability to limit pro-inflammatory responses in tissue-resident macrophages and hepatocytes. In this study, we established the role of the Ron receptor in steatohepatitis-induced hepatic fibrosis using Ron ligand domain knockout mice on an apolipoprotein E knockout background (DKO). After 18 weeks of high-fat high-cholesterol feeding, loss of Ron activation resulted in exacerbated NASH-associated steatosis which is precedent to hepatocellular injury, inflammation and fibrosis. 1H nuclear magnetic resonance (NMR)-based metabolomics identified significant changes in serum metabolites that can modulate the intrahepatic lipid pool in hepatic steatosis. Serum from DKO mice had higher concentrations of lipids, VLDL/LDL and pyruvate, whereas glycine levels were reduced. Parallel to the aggravated steatohepatitis, increased accumulation of collagen, inflammatory immune cells and collagen producing-myofibroblasts were seen in the livers of DKO mice. Gene expression profiling revealed that DKO mice exhibited elevated expression of genes encoding Ron receptor ligand MSP, collagens, ECM remodeling proteins and pro-fibrogenic cytokines in the liver. Our results demonstrate the protective effects of Ron receptor activation on NASH-induced hepatic fibrosis.

The Long Non-Coding RNA MIR503HG Enhances Proliferation of Human ALK-Negative Anaplastic Large-Cell Lymphoma.

Anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma (ALCL) is a rare type of highly malignant, non-Hodgkin lymphoma. Currently, only a few gene rearrangements have been linked to ALK-negative ALCL progression. However, the specific molecular mechanisms underlying the growth of ALK-negative ALCL tumors remain unclear. Here, we investigated aberrantly expressed, long non-coding RNAs (lncRNAs) in ALK-negative ALCL and assessed their potential biological function. MIR503HG (miR-503 host gene) was highly expressed in ALK-negative cell lines and was significantly upregulated in tumors in mice formed from ALK-negative ALCL cell lines. Depletion of MIR503HG suppressed tumor cell proliferation in vivo and in vitro; conversely, its overexpression enhanced tumor cell growth. MIR503HG-induced proliferation was mediated by the induction of microRNA-503 (miR-503) and suppression of Smurf2, resulting in stabilization of the tumor growth factor-ß receptor (TGFBR) and enhanced tumor cell growth. Collectively, these findings support a potential role for MIR503HG in cancer cell proliferation through the miR-503/Smurf2/TGFBR axis and indicate that MIR503HG is a potential marker in ALK-negative ALCL.

Myasthenia gravis with antibodies to MuSK: an update.

Myasthenia gravis with antibodies to the muscle-specific tyrosine kinase (MuSK+ MG) is a rare disease with distinctive pathogenic mechanisms and clinical features. An acute onset and predominant bulbar muscle weakness are very common and highly suggestive of the disease. On the other hand, a more indolent course, atypical ocular presentation, and signs of cholinergic hyperactivity may complicate the diagnosis. Though MuSK+ MG is still a severe disease, over the years we have observed a steady reduction in the rate of respiratory crisis and a significant improvement in the clinical outcome, both likely related to earlier diagnosis and timely treatment. Despite the improved management, MuSK+ MG patients tend to remain dependent on long-term immunosuppressive treatment and may develop permanent disabling weakness. In uncontrolled studies, B cell depletion with rituximab proved effective in most patients with refractory disease, inducing prolonged clinical responses associated with a sustained reduction of serum antibody levels. Promising results from experimental studies and case reports suggest that both 3,4-diaminopyridine and albuterol may be effective as symptomatic agents.

High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization.

Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

MerTK Cleavage on Resident Cardiac Macrophages Compromises Repair After Myocardial Ischemia Reperfusion Injury.

RATIONALE: Clinical benefits of reperfusion after myocardial infarction are offset by maladaptive innate immune cell function, and therapeutic interventions are lacking. OBJECTIVE: We sought to test the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion. METHODS AND RESULTS: In humans, we discovered that clinical reperfusion after myocardial infarction led to significant elevation of the soluble form of MerTK (myeloid-epithelial-reproductive tyrosine kinase; ie, soluble MER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial ischemia reperfusion. More notably, Mertk(CR) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCIILOCCR2- (major histocompatibility complex II/C-C motif chemokine receptor type 2) macrophages expressed higher levels of MerTK and, when exposed to apoptotic cells, secreted proreparative cytokines, including transforming growth factor-ß. Mertk deficiency compromised the accumulation of MHCIILO phagocytes, and this was rescued in Mertk(CR) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced soluble MER levels post-ischemia reperfusion. CONCLUSIONS: Our data implicate monocyte-induced MerTK cleavage on proreparative MHCIILO cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury.

Phosphatidylserine Is the Signal for TAM Receptors and Their Ligands.

Nature repeatedly repurposes, in that molecules that serve as metabolites, energy depots, or polymer subunits are at the same time used to deliver signals within and between cells. The preeminent example of this repurposing is ATP, which functions as a building block for nucleic acids, an energy source for enzymatic reactions, a phosphate donor to regulate intracellular signaling, and a neurotransmitter to control the activity of neurons. A series of recent studies now consolidates the view that phosphatidylserine (PtdSer), a common phospholipid constituent of membrane bilayers, is similarly repurposed for use as a signal between cells and that the ligands and receptors of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases (RTKs) are prominent transducers of this signal.

Pharmacological inhibition of Anaplastic Lymphoma Kinase rescues spatial memory impairments in Neurofibromatosis 1 mutant mice.

Heterozygous Neurofibromatosis 1 (NF1) loss of function mutations are found in 90% of patients with neurofibromatosis, a syndrome associated with disabling cognitive impairment. Drosophila studies have demonstrated a genetic interaction between Anaplastic Lymphoma Kinase (Alk) and NF1 in cognitive performance. In addition, pharmacologic inhibition of Alk improves cognitive performance in heterozygous NF1 mutant flies. In this study, we tested whether pharmacological inhibition of Alk in heterozygous NF1 mutant mice attenuates or rescues cognitive impairments. Cognitive impairment of spatial memory retention observed in heterozygous NF1 mutant mice was rescued by the Alk inhibitor. These data support the hypothesis that inhibition of Alk may cognitively benefit patients with Neurofibromatosis 1.

DBA/2J Haplotype on Distal Chromosome 2 Reduces Mertk Expression, Restricts Efferocytosis, and Increases Susceptibility to Atherosclerosis.

OBJECTIVE: Arch atherosclerosis 4 (Aath4) is a quantitative trait locus for atherosclerotic plaque formation in the inner curve of the aortic arch previously identified in an F2 cross of Apoe-/- mice on DBA/2J and 129S6 backgrounds. C-mer proto-oncogene tyrosine kinase (Mertk), coding for a ligand-activated transmembrane tyrosine kinase, is a candidate gene within the same chromosomal region. Our objective was to determine whether strain differences in Mertk influence plaque formation. APPROACH AND RESULTS: To dissect the strain effects of Mertk on atherosclerosis, we first established a congenic mouse line (Aath4aDBA/DBA ) in which a 5' region of Aath4 of DBA/2J, including Mertk, was backcrossed onto a 129S6-Apoe-/- background. The resulting Aath4aDBA/DBA male mice developed significantly larger plaques compared with control mice (Aath4a129/129 ), proving that the DBA/2J allele of Aath4a is proatherogenic. Thioglycollate-elicited peritoneal macrophages from Aath4aDBA/DBA mice express less than 50% of Mertk mRNA and cell-surface MERTK protein compared with those from the control mice. Moreover, both large and small peritoneal Aath4aDBA/DBA macrophages showed reduced phagocytosis of apoptotic cells. When Mertk cDNAs from 129S6 and DBA/2J mice were overexpressed in HEK293T (human embryonic kidney 293T) cells, phagocytosis of apoptotic cells was equally enhanced in direct proportion to Mertk levels, indicating that phagocytosis is modulated by the amount of MERTK, but that it is not affected by MERTK amino acid differences between 129S6 and DBA/2J. CONCLUSIONS: Reduced transcription of Mertk, rather than differences in MERTK protein structure, determines the reduced efficiency of apoptotic cell clearance in the Aath4aDBA/DBA mice, which, in turn, contributes to their increased susceptibility to atherosclerosis.

Axl molecular targeting counteracts aggressiveness but not platinum-resistance of ovarian carcinoma cells.

Ovarian carcinoma, the most common gynaecological cancer, is characterized by high lethality mainly due to late diagnosis and treatment failure. The efficacy of platinum drug-based therapy in the disease is limited by the occurrence of drug resistance, a phenomenon often associated with increased metastatic potential. Because the Tyr-kinase receptor Axl can be deregulated in ovarian carcinoma and plays a pro-metastatic/anti-apoptotic role, the aim of this study was to examine if Axl inhibition modulates drug resistance and aggressive features of ovarian carcinoma cells, using various pairs of cisplatin-sensitive and -resistant cell lines. We found that mRNA and protein levels of Axl were increased in the platinum-resistant IGROV-1/Pt1 and IGROV-1/OHP cell lines compared to the parental IGROV-1 cells. IGROV-1/Pt1 cells displayed increased migratory and invasive capabilities. When Axl was silenced, these cells exhibited reduced growth and invasive/migratory capabilities compared to control siRNA-transfected cells, associated with decreased p38 and STAT3 phosphorylation. In keeping with this evidence, pharmacological inhibition of p38 and STAT3 decreased IGROV-1/Pt1 invasive capability. Molecular inhibition of Axl did not sensitize IGROV-1/Pt1 cells to cisplatin, but enhanced ErbB3 activation in IGROV-1/Pt1 cells and suppressed the clonogenic capability of various ovarian carcinoma cell lines. The combination of cisplatin and AZD8931, a small molecule which inhibits ErbB3, produced a synergistic effect in IGROV-1/Pt1 cells. Thus, Axl targeting per se reduces invasive capability of drug-resistant cells, but sensitization to cisplatin requires the concomitant inhibition of additional survival pathways.

Axl signaling induces development of natural killer cells in vitro and in vivo.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axl antibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rß (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment of Axl-Ig. Anti-Axl antibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axl antibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83 % by the administration of mNK cells treated with anti-Axl antibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

Deficiency of AXL in Bone Marrow-Derived Cells Does Not Affect Advanced Atherosclerotic Lesion Progression.

AXL, a member of the TAM (Tyro3, Axl, MerTK) family of receptors, plays important roles in cell survival, clearance of dead cells (efferocytosis), and suppression of inflammation, which are processes that critically influence atherosclerosis progression. Whereas MerTK deficiency promotes defective efferocytosis, inflammation, and plaque necrosis in advanced murine atherosclerosis, the role of Axl in advanced atherosclerosis progression is not known. Towards this end, bone marrow cells from Axl-/- or wild-type mice were transplanted into lethally irradiated Ldlr-/- mice. These chimeric mice were then fed the Western-type diet (WD) for 17 weeks. We demonstrate that lesional macrophages in WT mice express Axl but that Axl deficiency in bone marrow-derived cells does not affect lesion size, cellularity, necrosis, or inflammatory parameters in advanced atherosclerotic plaques. Moreover, apoptosis of lesional cells was unaffected, and we found no evidence of defective lesional efferocytosis. In contrast to previously reported findings with MerTK deficiency, hematopoietic cell-Axl deficiency in WD-fed Ldlr-/- mice does not affect the progression of advanced atherosclerosis or lesional processes associated with TAM receptor signaling. These findings suggest a heretofore unappreciated TAM receptor hierarchy in advanced atherosclerosis.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice.

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, ß) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl-/- mice show more severe symptoms than do wild-type (Axl+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.