Identification of Tim-4 binding proteins and small molecules modulating efferocytosis

Abstract

Phagocytosis of apoptotic cells, referred to as efferocytosis, is essential cellular process for tissue homeostasis and immune regulation and during development. Defects of efferocytosis are known to cause autoimmune diseases such as rheumatoid arthritis and lupus due to generation of autoantibodies against intracellular contents released from uncleared apoptotic cells. In efferocytosis, Tim-4 is considered as a pivotal protein because it makes phagocytes distinguish apoptotic cells from normal cells through binding to phosphatidylserine specifically exposed on apoptotic cells. However, although efferocytosis is an essential cellular process and Tim-4 is a key molecule in efferocytosis, it has been incompletely explored that proteins interacting with Tim-4 and small molecules are able to modulate efferocytosis. Here, I report new Tim-4 interacting proteins and small molecules which are able to enhance the ability of phagocytes in efferocytosis. In first and second part, I report candidate proteins, Mertk, EphA2 and Cdon which have a possibility of interaction with Tim-4 for enhancing efferocytosis. Especially, at first, I report that Tim-4 cooperates with Mertk for efficient efferocytosis through a biochemical interaction. I observed that Tim-4 and Mertk localized close to each other for interaction by immunofluorescence and proximity ligation assay. Biochemical interaction between Tim-4 and Mertk, mediated by an interaction between IgV domain of Tim-4 and FnIII domain of Mertk, was addressed by immunoprecipitation. Furthermore, the cooperation effect of Mertk on Tim-4 mediated efferocytosis was abrogated by MertkFnIII, purified soluble form of FnIII domain of Mertk which can inhibit interaction between Tim-4 and Mertk. To sum up, these data suggest that biochemical interaction between Tim-4 and Mertk is essential for Mertk to cooperate with Tim-4 on efferocytosis. Secondly, I report other candidate proteins interacting with Tim-4, EphA2 and Cdon. I hypothesized that transmembrane protein containing a FnIII domain and expressed in a tissue, in which Tim-4 is also expressed, can interact and cooperate with Tim-4 to modulate efferocytosis. EphA2 biochemically interacted with Tim-4 but not Cdon. I observed close proximity between EphA2 and Tim-4 on plasma membrane. Especially, this interaction was directly mediated by the IgV domain of Tim-4 and the FnIII domain of EphA2. Nevertheless, I found that EphA2 failed to promote Tim-4-mediated efferocytosis. Taken together, the findings suggest that a new Tim-4 binding protein, EphA2 may intervene Tim-4-mediated cellular event including T cell proliferation, except efferocytosis or phagocytosis. Next, I report a new drug enhancing effeocytosis in part III. I developed a high throughput screening system to search for a small molecule promoting or inhibiting efferocytosis. Using the system, 18 candidate small molecules promoting efferocytosis more than 20% compared to the control were found and finally 3 candidate drugs were selected. Among these drugs, only two drugs reproducibly enhanced efferocytosis and both candidates increased the number of apoptotic cells binding to phagocytes. In sum, these candidates can be used a drug to treated diseases caused by defects of efferocytosis.