MERTK tyrosine kinase receptor together with TIM4 phosphatidylserine receptor mediates distinct signal transduction pathways for efferocytosis and cell proliferation.

Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, leading to efferocytosis, i.e. their engulfment by resident macrophages that express the PtdSer receptor T cell immunoglobulin mucin receptor 4 (TIM4) and TAM family receptor tyrosine kinase receptors (MERTK, AXL, and TYRO3). TAM family receptors stimulate cell proliferation, and the many aspects of the growth signaling pathway downstream of TAM family receptors have been elucidated previously. However, the signaling cascade for TAM receptor-mediated efferocytosis has been elusive. Here we observed that efferocytosis by mouse-resident peritoneal macrophages was blocked by inhibitors against the MERTK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), AKT Ser/Thr kinase (AKT), focal adhesion kinase (FAK), or STAT6 pathway. Accordingly, apoptotic cells stimulated the phosphorylation of MERTK, ERK, AKT, FAK, and STAT6, but not of IκB or STAT5. A reconstituted efferocytosis system using MERTK- and TIM4-expressing NIH3T3-derived cells revealed that the juxtamembrane and C-terminal regions of MERTK have redundant roles in efferocytosis. The transformation of murine IL-3-dependent Ba/F3 cells (a pro-B cell line) with MERTK and TIM4 enabled them to proliferate in response to apoptotic cells in a PtdSer-dependent manner. This apoptotic cell-induced MERTK-mediated proliferation required both MERTK's juxtamembrane and C-terminal regions and was blocked by inhibitors of not only ERK, AKT, FAK, and STAT6 but also of NF-κB and STAT5 signaling. These results suggest that apoptotic cells stimulate distinct sets of signal transduction pathways via MERTK to induce either efferocytosis or proliferation.

ACK1 and BRK non-receptor tyrosine kinase deficiencies are associated with familial systemic lupus and involved in efferocytosis.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with systemic lupus erythematosus (SLE) we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, non-receptor tyrosine kinases (NRTKs) regulate activation, migration, and proliferation of immune cells. We found that the patients' ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced Pluripotent Stem Cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.

Depth elemental imaging of forensic samples by confocal micro-XRF method.

Micro-XRF is a significant tool for the analysis of small regions. A micro-X-ray beam can be created in the laboratory by various focusing X-ray optics. Previously, nondestructive 3D-XRF analysis had not been easy because of the high penetration of fluorescent X-rays emitted into the sample. A recently developed confocal micro-XRF technique combined with polycapillary X-ray lenses enables depth-selective analysis. In this paper, we applied a new tabletop confocal micro-XRF system to analyze several forensic samples, that is, multilayered automotive paint fragments and leather samples, for use in the criminaliztics. Elemental depth profiles and mapping images of forensic samples were successfully obtained by the confocal micro-XRF technique. Multilayered structures can be distinguished in forensic samples by their elemental depth profiles. However, it was found that some leather sheets exhibited heterogeneous distribution. To confirm the validity, the result of a conventional micro-XRF of the cross section was compared with that of the confocal micro-XRF. The results obtained by the confocal micro-XRF system were in approximate agreement with those obtained by the conventional micro-XRF. Elemental depth imaging was performed on the paint fragments and leather sheets to confirm the homogeneity of the respective layers of the sample. The depth images of the paint fragment showed homogeneous distribution in each layer expect for Fe and Zn. In contrast, several components in the leather sheets were predominantly localized.

Clearance of Apoptotic Cells and Pyrenocytes.

Apoptotic cells are engulfed and digested by macrophages to maintain homeostasis in animals. If dead cells are not engulfed swiftly, they undergo secondary necrosis and release intracellular components that activate the immune system. Apoptotic cells are efficiently cleared due to phosphatidylserine (PtdSer) exposed on the cell surface that acts as an "eat me" signal. PtdSer is exposed through the activation of phospholipid scramblase and the inactivation of phospholipid flippase, which are both caspase-mediated events. Macrophages express a variety of molecules to recognize PtdSer, and use a sophisticated mechanism to engulf apoptotic cells. In red blood cells, the nucleus is lost when it is extruded as a pyrenocyte during definitive erythropoiesis. These pyrenocytes (nuclei surrounded by plasma membrane) also expose PtdSer on their surface and are efficiently engulfed by macrophages in a PtdSer-dependent manner. Macrophages transfer the engulfed apoptotic cell or pyrenocyte into lysosomes, where the components of the dead cell or pyrenocyte are degraded. If lysosomes cannot digest the DNA from apoptotic cells or pyrenocytes, the undigested DNA accumulates in the lysosome and activates macrophages to produce type I interferon (IFN) via a STING-dependent pathway; in embryos, this causes severe anemia. Here, we discuss how macrophages clear apoptotic cells and pyrenocytes.

Tim4- and MerTK-mediated engulfment of apoptotic cells by mouse resident peritoneal macrophages.

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.