Drugging the efferocytosis process: concepts and opportunities

Abstract

The daily removal of billions of apoptotic cells in the human body via the process of efferocytosis is essential for homeostasis. To allow for this continuous efferocytosis, rapid phenotypic changes occur in the phagocytes enabling them to engulf and digest the apoptotic cargo. In addition, efferocytosis is actively anti-inflammatory and promotes resolution. Owing to its ubiquitous nature and the sheer volume of cell turnover, efferocytosis is a point of vulnerability. Aberrations in efferocytosis are associated with numerous inflammatory pathologies, including atherosclerosis, cancer and infections. The recent exciting discoveries defining the molecular machinery involved in efferocytosis have opened many avenues for therapeutic intervention, with several agents now in clinical trials.

Fig. 1. Steps in efferocytosis and therapeutic opportunities.

Smell phase: clearance of apoptotic cells is a multistep process, initiated by secreted molecules released by the apoptotic corpse, inducing mobilization and migration of phagocytes towards dying cells. Pannexin 1 channel-mediated release of endogenous metabolic modulators (EMMis) from apoptotic cells induces pro-resolution and anti-inflammatory gene expression in phagocytes. Plausible methods for targeting this phase include administration of EMMis, altering activity of metabolic enzymes involved in generation of EMMi or enhancing pannexin 1 channel activity. Administration of donor apoptotic cells, for example via extracorporeal photopheresis (ECP), can reduce transplant rejection in patients and intravenous injections of apoptotic cells suppress anti-donor humoral immune response. In contrast, necroptotic cells elicit a strong immunogenic response and are used in targeting tumour cells. Engulfing phase: apoptotic cell phosphatidylserine (PS) recognition by phagocytes occurs either directly (for example, brain-specific angiogenesis inhibitor 1 (BAI1), T cell immunoglobulin mucin receptor TIM1) or by indirect PS recognition (for example, AXL, MerTK). Key downstream efferocytic signalling involves GTPase RAC1, which is activated via ELMO and DOCK proteins to enhance actin remodelling and formation of the phagocytic cup. Context-dependent modulation of PS receptor expression using gene editing technologies such as CRISPR and AAV-mediated gene delivery helps in disease models. Small-molecule inhibitors of PS receptor signalling are in advanced stages of clinical trials as cancer therapeutics (Table 1). CD47, the classical ‘do not eat-me’ signal, is often overexpressed in pathologies with defective apoptotic cell clearance and blocking CD47–SIRP1 interaction used in cancer treatments and atherosclerosis. Digestion phase: engulfment of multiple apoptotic cells leads to high metabolic burden on phagocytes. Complex signalling machinery enabling cargo digestion and successive cargo engulfment is being deciphered. Nuclear receptors such as PPARγ and liver-X receptor (LXR) enable cargo digestion. Metabolite transfer between intracellular compartments and from extracellular space during efferocytosis (via solute carriers (SLCs)) can improve anti-inflammatory gene expression and continuous efferocytosis. Further, specialized pro-resolving mediator (SPM) synthesis and secretion by phagocytes is another step in modulating anti-inflammatory function of efferocytosis. AMP, adenosine-5′-monophosphate; Dbl2, guanine nucleotide exchange factor (gene name mcf2); DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerol-3-phosphate; GMP, guanosine-5′-monophosphate; IMP, inosine-5′-monophosphate; PQLC2 (also known as SLC66A1), PQ loop repeat-containing protein 2; UDP-G, UDP-glucose.

Fig. 2. Opportunities to target efferocytosis steps in specific pathologies.

Systemic lupus erythmatosus (SLE): defects in multiple steps of efferocytosis have been recognized in SLE. High levels of autoantibodies against scavenger receptor class F member 1 (SCARF1) (a phosphatidylserine (PS) receptor acting in conjunction with C1q) are found in patients with SLE, and correlated with accumulation of apoptotic cells. Enhanced cleavage of PS receptors MerTK, Axl, Tyro and Tim by ADAM17/10 proteases, and reduced activity and bioavailability of DNASE1L3, further suppress efferocytosis, exacerbating inflammation. Reduction in scramblase Xkr1, responsible for PS exposure, is also seen in mouse models of SLE. Plausible therapeutic opportunities in SLE could include: targeting PS receptors to improve efferocytosis; targeting PPAR/liver-X receptor (LXR) to enhance efferocytosis and LC3-associated phagocytosis (US Patent US20190145961); and extending administration of nanosphere-coupled DNAse1 to SLE. Diabetes and obesity: in diabetes, defective efferocytosis has been recognized. Further, downregulation of miR-126 that suppresses ADAM9 leads to MerTK cleavage and reduced efferocytosis. Overexpression of miR-126 was shown to restore efferocytosis. Erythropoietin (EPO) and PPARγ-mediated expression of phagocytosis receptors/associated molecules (Mfge8, MerTK, CD-36, Gas6) is defective in obese mice and a potential point of intervention in diabetes. Further, administration of endogenous metabolic modulators (EMMis) may also be effective strategies for enhancing efferocytosis in diabetes and obesity. Atherosclerosis: defective efferocytosis contributes to accumulation of lipid-laden apoptotic cells, which eventually add to necrotic lesions. Causes for poor efferocytosis include ADAM17/10-dependent cleavage of MerTK and decrease in ABCA1 upregulation. Reduced cholesterol export from phagocytes further diminishes engulfment capacity of cells. Enhanced expression of CD47 on apoptotic cells further suppresses apoptotic cell engulfment in atherosclerotic plaques. Numerous clinical trials are underway, targeting CD47–SIRP1α interaction in cancers, and repurposing these for atherosclerosis can be of value. Turning specialized pro-resolving mediator (SPM) ratios in favour of resolution and efferocytosis may help delay atherogenesis. Induction of foam cell apoptosis and localized administration of EMMis in plaques may be a supplementary therapeutic strategy. EAE, experimental autoimmune encephalomyelitis; LRP1, low-density lipoprotein receptor-related protein 1; TIM, T cell immunoglobulin mucin receptor.

Fig. 3. The metabolic dynamics of efferocytosis.

The immense metabolic burden that apoptotic cells carry into a phagocyte requires phagocytes to rapidly change their immuno-metabolic landscape to enable immunologically silent clearance of apoptotic cells. Metabolites secreted by apoptotic cells before engulfment, apart from being potent chemokines, also drive transcriptional changes in phagocytes, including expression of anti-inflammatory and pro-resolution genes. Lysosomal degradation of apoptotic cargo releases free fatty acids, cholesterol, amino acids, sugars and nucleic acids into metabolic flux cycles of the phagocyte. There are multiple modes by which apoptotic cells alter the immunometabolism of the phagocyte. First, apoptotic cargo as signal transducers — apoptotic cell binding to phosphatidylserine (PS) receptors leads to activation of liver-X receptor (LXR) signalling and upregulation of ABCA1 transporters, leading to plasma membrane export of cholesterol. Second, degradation of apoptotic cells for continuous efferocytosis — mitochondrial oxidation of apoptotic cell-derived fatty acids alter NAD⁺/NADH balance, activating sirtuin 1 signalling, which then triggers expression of pro-resolving cytokine IL-10; apoptotic cargo digestion enhances mitochondrial fission, enabling successive cargo engulfment allowing phagolysosomal sealing and membrane recycling. Third, direct utilization of apoptotic cell metabolites — arginine derived from lysosomal degradation of apoptotic cargo is transported via PQLC2 lysosomal transporter into phagocyte cytosol; ornithine decarboxylase converts arginine into putrescine, which aids in Rac1 activation, driving successive rounds of apoptotic cell uptake. Fourth, reprogramming phagocyte solute balance via solute carrier (SLC) family reprogramming — binding of apoptotic cells to receptors that recognize PS leads to upregulation of glucose transporter 1 (GLUT1; also known as SLC2A1) and serum/glucocorticoid regulated kinase 1 (SGK1), which is required for GLUT1 transport to the plasma membrane. The increased glycolysis supported by enhanced glucose import is necessary for ATP generation and actin polymerization. The lactate, generated as a result of increased aerobic glycolysis, acts as a paracrine anti-inflammatory mediator after secretion through the upregulated lactate transporter SLC16a1. Along with adjusting metabolite compartmentalization and transport, efferocytosis also equilibrates ionic balance in engulfing cells. Plasma membrane chloride import via SLC12A2 channels was upregulated and activated after efferocytosis. Inhibiting transporter activity caused an increase in efferocytosis but the response to this corpse uptake was pro-inflammatory rather than anti-inflammatory.