Carla V. Rothlin

TAM Receptors Are Pleiotropic Inhibitors of the Innate Immune Response

The activation of Toll-like receptors (TLRs) in dendritic cells (DCs) triggers a rapid inflammatory response to pathogens. However, this response must be tightly regulated because unrestrained TLR signaling generates a chronic inflammatory milieu that often leads to autoimmunity. We have found that the TAM receptor tyrosine kinases-Tyro3, Axl, and Mer-broadly inhibit both TLR and TLR-induced cytokine-receptor cascades. Remarkably, TAM inhibition of inflammation is transduced through an essential stimulator of inflammation-the type I interferon receptor (IFNAR)-and its associated transcription factor STAT1. TLR induction of IFNAR-STAT1 signaling upregulates the TAM system, which in turn usurps the IFNAR-STAT1 cassette to induce the cytokine and TLR suppressors SOCS1 and SOCS3. These results illuminate a self-regulating cycle of inflammation, in which the obligatory, cytokine-dependent activation of TAM signaling hijacks a proinflammatory pathway to provide an intrinsic feedback inhibitor of both TLR- and cytokine-driven immune responses.

Immunobiology of the TAM receptors

Recent studies have revealed that the TAM receptor protein tyrosine kinases — TYRO3, AXL and MER — have pivotal roles in innate immunity. They inhibit inflammation in dendritic cells and macrophages, promote the phagocytosis of apoptotic cells and membranous organelles, and stimulate the maturation of natural killer cells. Each of these phenomena may depend on a cooperative interaction between TAM receptor and cytokine receptor signalling systems. Although its importance was previously unrecognized, TAM signalling promises to have an increasingly prominent role in studies of innate immune regulation.

TAM receptor function in the retinal pigment epithelium.

The TAM receptor tyrosine kinase Mer is expressed by cells of the retinal pigment epithelium (RPE), and genetic studies have demonstrated that Mer is essential for RPE function. RPE cells that lack Mer exhibit a severely compromised ability to phagocytose the distal ends of photoreceptor (PR) outer segments, which leads to the complete postnatal degeneration of photoreceptors and to blindness. Although in vitro experiments have implicated Gas6 as the critical TAM ligand for this process, we find that Gas6 mutant mice have a histologically intact retina with no photoreceptor degeneration. We further find that, in addition to Mer, RPE cells also express another TAM receptor--Tyro 3--and that both of these receptors are instead activated independently by the Gas6-related ligand Protein S. This protein is also expressed by RPE cells. Finally, we demonstrate that loss of Mer function is accompanied by a substantial down-regulation in Tyro 3 as well. These observations indicate that both Mer and Tyro 3 act in mouse RPE cells and suggest that their biologically relevant ligand in these cells is Protein S.

Twist mediates suppression of inflammation by type I IFNs and Axl

Type I interferons (IFNs) are pleiotropic cytokines with antiviral and immunomodulatory properties. The immunosuppressive actions of type I IFNs are poorly understood, but IFN-mediated suppression of TNFα production has been implicated in the regulation of inflammation and contributes to the effectiveness of type I IFNs in the treatment of certain autoimmune and inflammatory diseases. In this study, we investigated mechanisms by which type I IFNs suppress induction of TNFα production by immune complexes, Fc receptors, and Toll-like receptors. Suppression of TNFα production was mediated by induction and activation of the Axl receptor tyrosine kinase and downstream induction of Twist transcriptional repressors that bind to E box elements in the TNF promoter and suppress NF-κB–dependent transcription. Twist expression was activated by the Axl ligand Gas6 and by protein S and apoptotic cells. These results implicate Twist proteins in regulation of TNFα production by antiinflammatory factors and pathways, and provide a mechanism by which type I IFNs and Axl receptors suppress inflammatory cytokine production.

Enveloped Viruses Disable Innate Immune Responses in Dendritic Cells by Direct Activation of TAM Receptors

Upon activation by the ligands Gas6 and Protein S, Tyro3/Axl/Mer (TAM) receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection. Upon virus challenge, TAM-deficient DCs display type I IFN responses that are elevated in comparison to wild-type cells. As a consequence, TAM-deficient DCs are relatively resistant to infection by flaviviruses and pseudotyped retroviruses, but infection can be restored with neutralizing type I IFN antibodies. Correspondingly, a TAM kinase inhibitor antagonizes the infection of wild-type DCs. Thus, TAM receptors are engaged by viruses in order to attenuate type I IFN signaling and represent potential therapeutic targets.

TAM receptors regulate multiple features of microglial physiology

Microglia are damage sensors for the central nervous system (CNS), and the phagocytes responsible for routine non-inflammatory clearance of dead brain cells. Here we show that the TAM receptor tyrosine kinases Mer and Axl 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 is normally driven by both TAM receptor ligands Gas6 and protein S. 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. Together, these results establish TAM receptors as both controllers of microglial physiology and potential targets for therapeutic intervention in CNS disease.

TAM receptor signaling in immune homeostasis.

The TAM receptor tyrosine kinases (RTKs)-TYRO3, AXL, and MERTK-together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease.

Paradoxical role of the proto-oncogene Axl and Mer receptor tyrosine kinases in colon cancer

The receptor tyrosine kinases Axl and Mer, belonging to the Tyro3, Axl and Mer (TAM) receptor family, are expressed in a number of tumor cells and have well-characterized oncogenic roles. The therapeutic targeting of these kinases is considered an anticancer strategy, and various inhibitors are currently under development. At the same time, Axl and Mer are expressed in dendritic cells and macrophages and have an essential function in limiting inflammation. Inflammation is an enabling characteristic of multiple cancer hallmarks. These contrasting oncogenic and anti-inflammatory functions of Axl and Mer posit a potential paradox in terms of anticancer therapy. Here we demonstrate that azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced inflammation-associated cancer is exacerbated in mice lacking Axl and Mer. Ablation of Axl and Mer signaling is associated with increased production of proinflammatory cytokines and failure to clear apoptotic neutrophils in the intestinal lamina propria, thereby favoring a tumor-promoting environment. Interestingly, loss of these genes in the hematopoietic compartment is not associated with increased colitis. Axl and Mer are expressed in radioresistant intestinal macrophages, and the loss of these genes is associated with an increased inflammatory signature in this compartment. Our results raise the possibility of potential adverse effects of systemic anticancer therapies with Axl and Mer inhibitors, and underscore the importance of understanding their tissue and cell type-specific functions in cancer.

Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells

Local macrophage clean-up Infection, especially by helminths or bacteria, can cause tissue damage (see the Perspective by Bouchery and Harris). Minutti et al. studied mouse models of helminth infection and fibrosis. They expressed surfactant protein A (a member of the complement component C1q family) in the lung, which enhanced interleukin-4 (IL-4)-mediated proliferation and activation of alveolar macrophages. This activation accelerated helminth clearance and reduced lung injury. In the peritoneum, C1q boosted macrophage activation for liver repair after bacterial infection. By a different approach, Bosurgi et al. discovered that after wounding caused by migrating helminths in the lung or during inflammation in the gut of mice, IL-4 and IL-13 act only in the presence of apoptotic cells to promote tissue repair by local macrophages. Science, this issue p. 1076, p. 1072; see also p. 1014 Just as infection needs to be limited, so must healing responses be contained to reduce scarring and allergy. Tissue repair is a subset of a broad repertoire of interleukin-4 (IL-4)– and IL-13–dependent host responses during helminth infection. Here we show that IL-4 or IL-13 alone was not sufficient, but IL-4 or IL-13 together with apoptotic cells induced the tissue repair program in macrophages. Genetic ablation of sensors of apoptotic cells impaired the proliferation of tissue-resident macrophages and the induction of anti-inflammatory and tissue repair genes in the lungs after helminth infection or in the gut after induction of colitis. By contrast, the recognition of apoptotic cells was dispensable for cytokine-dependent induction of pattern recognition receptor, cell adhesion, or chemotaxis genes in macrophages. Detection of apoptotic cells can therefore spatially compartmentalize or prevent premature or ectopic activity of pleiotropic, soluble cytokines such as IL-4 or IL-13.

Phagocytosis imprints heterogeneity in tissue-resident macrophages

Tissue-resident macrophages display varying phenotypic and functional properties that are largely specified by their local environment. One of these functions, phagocytosis, mediates the natural disposal of billions of cells, but its mechanisms and consequences within living tissues are poorly defined. Using a parabiosis-based strategy, we identified and isolated macrophages from multiple tissues as they phagocytosed blood-borne cellular material. Phagocytosis was circadianally regulated and mediated by distinct repertoires of receptors, opsonins, and transcription factors in macrophages from each tissue. Although the tissue of residence defined the core signature of macrophages, phagocytosis imprinted a distinct antiinflammatory profile. Phagocytic macrophages expressed CD206, displayed blunted expression of Il1b, and supported tissue homeostasis. Thus, phagocytosis is a source of macrophage heterogeneity that acts together with tissue-derived factors to preserve homeostasis.