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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-1β, 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.

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes. This study describes comprehensive synaptic engulfment by astrocytes, mediating synapse elimination in an activity-dependent manner; this elimination process involves the MEGF10 and MERTK phagocytic pathways and persists into adulthood, with mutant mice that lack these pathways in astrocytes exhibiting a failure to refine retinogeniculate connections during development. Astrocytes involved in synapse elimination Synapse elimination is an important aspect of brain development in which the number of synaptic contacts is reduced in an activity-dependent manner. Glial cells — non-neural cells that perform a variety of roles in the brain — were recently shown to have a role in synapse remodelling, with the phagocytic microglia responsible for a certain proportion of connection refinement, with little else known regarding the mechanisms underlying this. Here, Won-Suk Chung et al . describe comprehensive synaptic engulfment by astrocytes, mediating synapse elimination in an activity-dependent manner. This elimination process involved the MEGF10 and MERTK phagocytic pathways, with transgenic animals lacking these pathways in astrocytes exhibiting a failure to refine retinogeniculate connections during development. These mechanisms also extend into adulthood. This work has implications for our understanding of learning and memory as well as neurological disease processes.

Microglial phagocytosis is required for neurogenic niche maintenance and response to injury; the TAM kinases Mer and Axl are expressed by microglia in the adult CNS, and mediate the clearance of apoptotic cells from the niche. Mer and Axl regulate microglial physiology Microglial phagocytosis is required for neurogenic niche maintenance and response to central nervous system (CNS) injury. Here Greg Lemke and colleagues show that the TAM receptor kinases Mer and Axl are expressed by microglia and in the adult CNS, and mediate the clearance of apoptotic cells from the neurogenic niche. This work demonstrates that TAM receptors act as controllers of microglial physiology, and are potential targets for therapeutic intervention in CNS disease. Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells 1 . Here we show that the TAM receptor tyrosine kinases Mer and Axl 2 regulate these microglial functions. We find that adult mice deficient in microglial Mer and Axl exhibit a marked accumulation of apoptotic cells specifically in neurogenic regions of the CNS, and that microglial phagocytosis of the apoptotic cells generated during adult neurogenesis 3 , 4 is normally driven by both TAM receptor ligands Gas6 and protein S 5 . Using live two-photon imaging, we demonstrate that the microglial response to brain damage is also TAM-regulated, as TAM-deficient microglia display reduced process motility and delayed convergence to sites of injury. Finally, we show that microglial expression of Axl is prominently upregulated in the inflammatory environment that develops in a mouse model of Parkinson’s disease 6 . Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.

The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia–reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (MertkCR ). MertkCR mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.

Neuroblastoma: a genetic link to ALK Neuroblastoma is the most common childhood cancer. There is a strong familial association and it was predicted over 30 years ago that there was a genetic element to the disease. Four groups now report the identification of mutations in the tyrosine kinase receptor ALK (anaplastic lymphoma kinase) in neuroblastoma patients. ALK acts as a neuroblastoma predisposition gene, and somatic point mutations occur in sporadic neuroblastoma cases. These mutations promote ALK's kinase activity and can transform cells and display tumorigenic activity in vivo . ALK inhibitors decrease neuroblastoma cell proliferation, so have potential as anticancer drugs. This is one of four papers in this issue that identifies mutations in the tyrosine kinase receptor ALK in neuroblastoma, the most frequent childhood cancer. ALK is found to be a neuroblastoma predisposition gene and somatic points mutations were found in sporadic cases of neuroblastoma. These mutations lead the ALK kinase activation and are able to transform cells and display tumourigenic activity in vivo . ALK inhibitors decrease neuroblastoma cell proliferating and are potential anti-cancer drugs for the treatment of neuroblastoma. Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood 1 , 2 . It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene 1 , 2 , 3 , 4 . Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) 5 tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK , indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK -mutated cells, but also in cell lines overexpressing a wild-type ALK , led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments 6 , 7 .

Beating anti-fungal defences Immunocompromised individuals are at high risk from fungal infection, yet the molecular mechanisms that govern host defence against fungi are not well understood. Gross et al . now show that Candida albicans infection in mice activates the NALP3 inflammasome via a mechanism involving Sky-induced production of reactive oxygen induced by the tyrosine kinase Syk. Interleukin-1β (IL-1β) is a key pro-inflammatory factor in innate antifungal immunity, but the mechanism by which the mammalian immune system regulates IL-1β production after fungal recognition is unclear. Here it is demonstrated that the tyrosine kinase Syk controls both pro-IL-1β synthesis and Nlrp3 inflammasome activation after cell stimulation with Candida albicans . Fungal infections represent a serious threat, particularly in immunocompromised patients 1 . Interleukin-1β (IL-1β) is a key pro-inflammatory factor in innate antifungal immunity 2 . The mechanism by which the mammalian immune system regulates IL-1β production after fungal recognition is unclear. Two signals are generally required for IL-1β production: an NF-κB-dependent signal that induces the synthesis of pro-IL-1β (p35), and a second signal that triggers proteolytic pro-IL-1β processing to produce bioactive IL-1β (p17) via Caspase-1-containing multiprotein complexes called inflammasomes 3 . Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1β synthesis and inflammasome activation after cell stimulation with Candida albicans . Whereas Syk signalling for pro-IL-1β synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1β processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1β production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo .

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T cell receptor signaling, while its role in CAR signaling is unknown. By electroporation of CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing analyses revealed downregulation of exhaustion and upregulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T cell products for clinical use.

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase ( ALK ) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23–24 using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194 high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability, to be oncogenic drivers. Mutations resulted in constitutive phosphorylation, and targeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in all cell lines harbouring mutant or amplified ALK , as well as in two out of six wild-type cell lines for ALK . Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy. Neuroblastoma: a genetic link to ALK Neuroblastoma is the most common childhood cancer. There is a strong familial association and it was predicted over 30 years ago that there was a genetic element to the disease. Four groups now report the identification of mutations in the tyrosine kinase receptor ALK (anaplastic lymphoma kinase) in neuroblastoma patients. ALK acts as a neuroblastoma predisposition gene, and somatic point mutations occur in sporadic neuroblastoma cases. These mutations promote ALK's kinase activity and can transform cells and display tumorigenic activity in vivo . ALK inhibitors decrease neuroblastoma cell proliferation, so have potential as anticancer drugs. ALK is identified as a neuroblastoma predisposition gene. Germline mutations were found in ALK , a tryrosine kinase receptor, in affected families. In addition, somatic point mutations in ALK were found in sporadic cases of neuroblastomas. ALK mutations seem to lead to constitutive activation of its kinase activity and promote cell proliferation.

Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF’s receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

The cell death of inhibitory neurons, which originate far from the cortical areas to which they migrate during embryonic development, is determined autonomously rather than by competition for trophic signals from other cell types. Cell death in cortical interneurons It has long been known that apoptosis, a form of programmed cell death, eliminates young cells from developing tissues. In the field of neurobiology, it is widely believed that developmental neuronal-cell death results from cellular competition for environmentally derived survival signals that selects for an optimally sized and properly wired population of neurons. This study of developmental cell death in the mouse cortex in vivo, in vitro and after transplantation suggests that developmental neuronal-cell death is instead intrinsically determined for interneurons, the main inhibitory cells of the cerebral cortex. Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis 1 , 2 , 3 , 4 , 5 , is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo , in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system 6 , 7 , 8 . Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.