Efferocytosis meets endocytosis

ABSTRACT
Apoptosis happens continuously along with the active clearance of apoptotic cells (ACs) by phagocytes or
efferocytes, termed “efferocytosis”, to maintain tissue homeostasis. When efferocytosis becomes defective,
uncleared ACs undergo post-apoptotic necrosis and release immunogenic epitopes and pro-inflammatory
mediators, which leads to chronic inflammatory diseases. Our recent studies revealed a novel role of
efferocytosis in accelerating tissue repair as it promotes inflammation resolution by inducing the biosynthesis of
specialized pro-resolving mediators (SPMs) that stop inflammatory responses. Therefore, understanding how
efferocytosis is successfully carried out is of paramount importance. Much has been learned about the
mechanisms of AC recognition and uptake, but how efferocytes degrade ACs and process the metabolic cargo,
e.g., cholesterol released from AC digestion, is incompletely understood. Moreover, although efferocytosis and
endocytosis share common features, such as involving cytoskeleton rearrangement and intracellular transport
of vesicular membrane-bound cargoes, whether efferocytes hijack the endocytic machinery to process AC-
derived cargo remains uncertain. In our unpublished results, we found that resolvin D1 (RvD1), a
docosahexaenoic acid (DHA)–derived SPM, enhanced the acidification of the AC-containing compartments
(efferosomes) and LC3-II lipidation, key features in LC3-associated phagocytosis (LAP)-mediated corpse
degradation. As our recent study showed that the activation of MerTK, the efferocytosis receptor, was required
for RvD1 biosynthesis, these results indicate a novel role of MerTK-RvD1 signaling in LAP-mediated AC
degradation. To study whether the key endocytic regulators—the C-terminal Eps15 Homology Domain (EHD)
proteins comprising EHD1, EHD2, EHD3, and EHD4—are involved in efferocytosis-related events, we analyzed
a single-cell RNA-sequencing (scRNA-seq) dataset from atherosclerotic lesions where a lot of cells undergo
apoptosis and found that EHD proteins had heterogeneous expression with high expression of EHD1 and EHD4
in macrophages, the professional efferocytes. We further found that EHD1 enhanced the cell surface levels of
the cholesterol efflux transport protein ABCA1 in macrophages during efferocytosis, which indicates that EHD1-
mediated endocytic trafficking of ABCA1 may play a role in removing the excess free cholesterol released from
digested ACs. Here, we propose to combine approaches in cell biology, biochemistry, mouse genetics, and
functional genomics to determine the function and mechanisms of MerTK-RvD1 signaling in LAP and EHD
proteins in efferocytosis-related events including maintaining cellular cholesterol homeostasis and controlling
endocytic trafficking of MerTK. We will also perform unbiased genome-wide CRISPR screening to identify novel
regulators of MerTK levels on the cell surface where macrophages receive ACs. Taken together, understanding
these aspects of efferocytosis will shed light on key physiological and pathophysiological processes and suggest
novel therapeutic strategies for diseases driven by defective efferocytosis.

Grant Number: 7R35GM147269-04
NIH Institute/Center: NIH
Principal Investigator: Bishuang Cai