Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Heit, Bryan

Abstract

Macrophages are key mediators of efferocytosis – the phagocytic engulfment and removal of apoptotic cells. During engulfment, the coordinated activity of efferocytic receptors induces the remodeling of the actin cytoskeleton, which facilitates the envelopment of the cell by the plasma membrane. Mer receptor tyrosine kinase (MERTK) is a crucial efferocytic receptor, but its role during actin remodeling is not well understood. Previously, our lab showed that MERTK is an activator of β2 integrins – which are comprised of receptors known to induce the actin polymerization that is required for engulfment. We hypothesized that MERTK is an indirect stimulator of actin polymerization via the stimulation of the actin-remodeling β2 integrins. Herein, the biomechanics of efferocytosis were investigated by fluorescence imaging of the efferocytic synapse. In a frustrated efferocytosis model – where macrophages attempted to engulf a planar substrate that mimicked apoptotic cells, MERTK formed discrete clusters within the centre of the efferocytic synapse, where the periphery of the synapse was delineated by an expanding ring of actin-associated β2 integrins. This suggests that integrins, not MERTK, are local directors of actin polymerization. Single particle tracking revealed that MERTK was less restricted in its free diffusion compared to the highly confined β2 integrins, indicating these receptors play unique biomechanical roles during engulfment. Finally, we began the development of a traction force microscopy assay to characterize the biophysics of MERTK-dependent engulfment. Preliminary data revealed that frustrated efferocytosis on opsonized hydrogels required the simultaneous activity of MERTK and efferocytic integrin receptors. We expect that integrins transduce the forces required for engulfment via the actin cytoskeleton, whereas MERTK stimulates the initial activation of the integrins. Further elucidation of the mechanism of MERTK-mediated engulfment will be critical in our understanding of pathologies where efferocytosis is defective, such as systemic lupus erythematosus and atherosclerosis.

Summary for Lay Audience

Macrophages are a type of immune cell that engulf and destroy dying cells, a process known as efferocytosis. This is required to prevent the accumulation of dead cells that would otherwise cause autoimmune disease or inflammation. MERTK is a receptor that is required for the recognition and engulfment of dying cells, how this works is not understood. Our laboratory previously showed that MERTK activates another group of receptors called the β2 integrins. Integrins are known to directly manipulate the structure of the actin cytoskeleton, a network of protein fibers that are responsible for altering the shape of cells. By reshaping the cell, reorganization of actin allows a macrophage to extend its membrane around a target, thus engulfing material from their surrounding environment. We predicted that MERTK activates the β2 integrins to induce the actin remodeling required for the engulfment of dying cells. In other words, we expect that the MERTK indirectly induces actin restructuring by working through integrins. To test this, we used fluorescence microscopy to observe the structures formed by macrophages during efferocytosis. In this project, we found that macrophages formed structured interactions with the dying cell being engulfed. β2 integrins were located at the outer edges of the extending membrane, where extensive actin remodeling also occurred. MERTK failed to localize at these sites, and instead was found in the centre of the macrophage. Analysis of the movement of MERTK and β2 integrins showed that the integrins were immobilized by their direct linkage with the actin cytoskeleton, while MERTK was not. Finally, we started the development of an experiment that will measure the forces induced by the actin cytoskeleton during efferocytosis. We expect that integrins are responsible for direct force production whereas MERTK plays a passive role by stimulating the initial activation of the integrins. Together, this work has revealed key insights into how MERTK and integrins coordinate the clearance of dying cells. This may represent a targetable pathway to treat conditions where MERTK’s activity is defective, such as heart attack, stroke, lupus, arthritis, or other severe inflammatory disorders.

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