LC3-Associated Phagocytosis and Inflammation

doi:10.1016/j.jmb.2017.08.012

Journal of Molecular Biology

Volume 429, Issue 23, 24 November 2017, Pages 3561-3576

https://doi.org/10.1016/j.jmb.2017.08.012 Get rights and content

Highlights

•
Comprehensive overview of LC3-associated phagocytosis (LAP).
•
LAP bridges the phagocytic and autophagic pathways.
•
Signaling to LAP and regulation of LAP activation by the canonical autophagy machinery.
•
LAP quenches inflammation and shapes the immune response towards anti-inflammation.
•
LAP promotes the immunosilent clearance of dying cells and prevents autoimmunity.

Abstract

LC3-associated phagocytosis (LAP) is a novel form of non-canonical autophagy where LC3 (microtubule-associated protein 1A/1B–light chain 3) is conjugated to phagosome membranes using a portion of the canonical autophagy machinery. The impact of LAP to immune regulation is best characterized in professional phagocytes, in particular macrophages, where LAP has instrumental roles in the clearance of extracellular particles including apoptotic cells and pathogens. Binding of dead cells via receptors present on the macrophage surface results in the translocation of the autophagy machinery to the phagosome and ultimately LC3 conjugation. These events promote a rapid form of phagocytosis that produces an “immunologically silent” clearance of the apoptotic cells. Consequences of LAP deficiency include a decreased capacity to clear dying cells and the establishment of a lupus-like autoimmune disease in mice. The ability of LAP to attenuate autoimmunity likely occurs through the dampening of pro-inflammatory signals upon engulfment of dying cells and prevention of autoantigen presentation to other immune cells. However, it remains unclear how LAP shapes both the activation and outcome of the immune response at the molecular level. Herein, we provide a detailed review of LAP and its known roles in the immune response and provide further speculation on the putative mechanisms by which LAP may regulate immune function, perhaps through the metabolic reprogramming and polarization of macrophages.

Graphical abstract

Introduction

Traditional macro-autophagy (autophagy hereafter) has long been implicated in regulating multiple facets of the immune response including pathogen capture, metabolic regulation, and cellular homeostasis. Autophagy is best described as “self-eating” and is a critical mechanism that cells utilize under conditions of nutrient deprivation and stress to compensate and preserve homeostasis. Activation of the autophagic pathway leads to both the en masse and selective capture and degradation of cytosolic components in a signaling-dependent fashion [1], [2], resulting in energy production derived from the recycling of organelles, proteins, amino acids, and other macromolecules. The ability of the autophagic pathway to govern metabolic status is of particular interest for cells of the immune system. Immune cells including lymphocytes and phagocytic cells such as macrophages require a fluidity in their energy architecture as they respond to activation signals [3], [4]. While both quiescent and activated immune cells require ATP, the transition in response to immune perturbation necessitates the appropriation of nutrients into varying pathways to support or direct functional alterations [3]. As expected, major energetic pathways including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid oxidation, fatty acid synthesis, and amino acid metabolism play vital roles in sculpting the energetic profile of individual immune cells [5]. Autophagy also plays a prominent role in facilitating these primary energetic pathways through the production of metabolic substrates and/or the quality control of organelles. While the autophagy pathway is the only known mechanism to dispose of and recycle intracellular organelles, multiple unique forms of autophagy exist. These divergent forms, deemed non-canonical autophagy, differ in their requirements for specific autophagy proteins (ATGs) required for the canonical autophagy pathway. These studies reveal limitations in our knowledge of how individual components function in either canonical or non-canonical autophagy. Perhaps it should not be surprising that components of a molecular pathway that has existed for eons might acquire new roles in related processes.

One such form of non-canonical autophagy is present in phagocytic cells, including macrophages, epithelial cells, and endothelial cells. This novel pathway of non-canonical autophagy utilizes components of the canonical autophagy machinery including a subset of the ATGs to conjugate the microtubule-associated protein 1A/1B–light chain 3 (LC3) to phagosome membranes [6]. As such, this process of non-canonical autophagy is called LC3-associated phagocytosis (LAP). Unlike other forms of non-canonical autophagy, LAP is unique in that it is an autophagosome-independent process that results in the formation of LC3-positive phagosomes called LAPosomes. Animals deficient in the LAP pathway have a propensity to develop autoimmunity, particularly systemic lupus erythrematosus (SLE) as discussed below [7]. In addition, LAP has been shown to have an >obligatory function in the efficient clearance of dying cells by macrophages and likely serves in dampening pro-inflammatory responses. Importantly, this ability for LAP to quench inflammatory responses results in a shaping of the immune response toward anti-inflammation.

In this review, we not only provide an overview of the LAP pathway, signaling to LAP, and the role of LAP in the clearance of dying cells and other phagocytic substrates, but further speculate on the potential for LAP to be a mechanism for the metabolic reprogramming of immune cells including macrophages in a variety of both physiological and pathological scenarios.

Section snippets

LAP bridges phagocytosis and autophagy

To understand LAP, we must first distinguish how cells, in particular phagocytes, identify potential cargo to be internalized. Surface receptors at the plasma membrane of phagocytic cells, including macrophages, can be engaged by ligands that are present on target particles. This binding event can result in the activation of the traditional phagocytic pathway, characterized by cytoskeletal rearrangements that mechanically pull the particle into the cell, leading to the formation of a phagosome

PI3K and Rubicon are required for LAP

Following the recognition of phagocytic cargo and after phagosome formation, the phosphotidylinositol 3-kinase complex (PI3KC3) is the first multi-protein complex involved in LAP regulation (Fig. 1, upper inset). As such, it is the most upstream complex shared between LAP and the canonical autophagic pathway. The core components of the PI3KC3 complex are VPS34, VPS15, and Beclin1 [19]. VPS34 is the catalytic subunit in the complex that phosphorylates the inositol ring of phosphotidylinositides

NOX2, ROS, and LC3 lipidation in LAP

In addition to PI3P, we likewise have eluded to the importance of ROS in LAP activation. The nicotamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX2) generates ROS during phagosome maturation in macrophages [57], [58], and both NOX2 function and ROS are indispensable for LAP [17], [42], [59]. NOX2 is a multiprotein complex comprised of both membrane-bound and cytosolic subunits (Fig. 1, middle inset). The membrane-bound cytochrome b-245 is a heterodimer of two transmembrane proteins:

LAP requires the autophagy conjugation system

While it remains unclear how the conjugation machinery is recruited to the phagosome membrane, the two ubiquitin-like (UB-like) conjugation systems required for LC3 conjugation during canonical autophagy [73] are also involved in LAP (Fig. 1, lower inset) [6], [10], [13], [17]. Briefly, in the first UB-like conjugation system, the exposed terminal glycine in ATG12 is covalently linked to an internal lysine in ATG5 through isopeptide bonding [74]. ATG7 and ATG10 function as the activating (E1)

Timing is everything

It is important to bear in mind, as exemplified above, the differences in the timing of lipidated LC3 production between LAP and canonical autophagy, and the functions derived from this divergence. LC3 is lipidated when the phagosome is fully sealed [10], and the lipidation machinery is most likely attached to the membrane after phagosome closure. Conversely, LC3 is conjugated during autophagosome membrane elongation [78], [79] and the ATG16L complex is released from autophagosome precursors

LAP promotes differential outcomes during immune activation

Upon fusion of the phagosome with the lysosome, luminal acidic hydrolases degrade the engulfed material and transmembrane pumps in the lysosomal membranes aid in the recycling of sugars, amino acids, lipids, and nucleotides to replenish cellular stores. These surplus molecules generated from extracellular material are reminiscent of amoeboid nutrition and can potentially be used as building blocks and energy sources within the phagocyte. Therefore, while LAP most likely impacts cellular

A role for LAP in antigen presentation and pathogen clearance

Activation of the C-type lectin receptor Dectin-1 or TLR2 leads to recruitment of LC3 to phagosomal membranes [37], [90], [91]. It has been well characterized that activation of Dectin-1 occurs in response to fungal infections and fungal-derived antigens. This mechanism of LAP activation promotes the sustained presentation of antigen by MHC-II [37]. Further evidence supporting a function for LAP in antigen presentation stems from work performed in human macrophages and dendritic cells, showing

LAP in efferocytosis

The connection of LAP with immune regulation is even more evident in the elimination of apoptotic cells by macrophages, a process known as efferocytosis. At any given time, professional phagocytes, specifically macrophages, efficiently patrol and eliminate dead cells to prevent the release of inflammatory signals by cell corpses, and thus protect tissue homeostasis. Although efferocytosis is generally defined as an “immunologically silent” process, the engulfment of apoptotic cells orchestrates

Metabolic reprograming in macrophage functional polarization

The link between LAP and clearance of apoptotic cells by efferocytosis hints at a deeper involvement for LAP in the regulation of macrophage metabolism and polarization. Macrophage functions are defined in response to microenvironmental cues, such as recognition of dying cells, that drive the acquisition of a spectrum of polarization states that find their extremes in either a pro-inflammatory, pathogen eliminating direction (M1, classically activated), or anti-inflammatory, immunosuppressive

Implications of LAP in macrophage immunometabolism

Upon engulfment of apoptotic cells, macrophages upregulate fatty acid oxidation, raising the possibility that metabolic changes contribute to the immune response to efferocytosis described above [112]. Whether LAP-dependent immunoregulation in macrophages upon engulfment and degradation of apoptotic cells is ultimately driven by metabolic reprogramming is still a matter of speculation, but some interesting studies suggest a possible relationship. Efferocytosis induces IL-4 production in

LAP as a potential regulator of vascular inflammation

It is well established that macrophages constitute a large portion of immune cells found in atherosclerotic plaques. In addition, macrophage specific depletion of Atg5 in an atherosclerotic mouse model increases atherosclerotic lesion size, at least in part due to an increase in infammasome activation, a defect in cholesterol efflux, an increase in ER/oxidative stress, and a defect in efferocytosis [123], [124], [125]. Since Atg5 is a known regulator of both LAP and canonical autophagy, we can

LAP in adipose tissue macrophages and insulin resistance

SLE patients in particular are at a high propensity to have increased visceral adiposity and epicardial fat accumulation [147]. Since LAP promotes anti-inflammation, it is possible that adipose-infiltrating macrophages, which display profound pro-inflammatory characteristics, have either a restriction or deficiency in LAP. As is the case with generalized obesity, establishment of this inflammatory milieu in the adipose tissue has been directly linked with insulin resistance and type 2 diabetes

Conclusions and outlook

The functions of LAP in inflammation and autoimmunity, taken in combination with the important role of LAP in mediating bacterial clearance and antifungal host defense, reinforce the broad importance of this form of non-canonical autophagy to not only cell but also organismal homeostasis. As shown in Fig. 2, the capacity for cells to engage LAP is a critical determinant of autoimmunity in response to apoptotic cells. In this model, a perturbation of LAP is likely a contributing factor to the

Acknowledgments

E.B.R. is a recipient of an EMBO Long-Term Fellowship (ALTF 1526-2016). D.R.G. is supported by funding from the National Institutes of Health and the Lupus Research Alliance. We would also like to thank Dr. Bart Tummers for his input and helpful discussions.

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Journal of Molecular Biology

ReviewLC3-Associated Phagocytosis and Inflammation

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

LAP bridges phagocytosis and autophagy

PI3K and Rubicon are required for LAP

NOX2, ROS, and LC3 lipidation in LAP

LAP requires the autophagy conjugation system

Timing is everything

LAP promotes differential outcomes during immune activation

A role for LAP in antigen presentation and pathogen clearance

LAP in efferocytosis

Metabolic reprograming in macrophage functional polarization

Implications of LAP in macrophage immunometabolism

LAP as a potential regulator of vascular inflammation

LAP in adipose tissue macrophages and insulin resistance

Conclusions and outlook

Acknowledgments

To be or not to be? How selective autophagy and cell death govern cell fate

Cell

The relationship between metabolism and the autophagy machinery during the innate immune response

Cell Metab.

Metabolic pathways in immune cell activation and quiescence

Immunity

Metabolic reprogramming and metabolic dependency in T cells

Immunol. Rev.

Changing the energy of an immune response

Am. J. Clin. Exp. Immunol.

Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells

Proc. Natl. Acad. Sci. U. S. A.

Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells

Nature

Phagocytosis: receptors, signal integration, and the cytoskeleton

Immunol. Rev.

Phagosome maturation: going through the acid test

Nat. Rev. Mol. Cell Biol.

Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis

Nature

Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling

J. Immunol.

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages

J Infect Dis

Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes

Immunity

An apoptotic ‘Eat Me’ signal: phosphatidylserine exposure

Trends Cell Biol.

Do not let death do us part: ‘find-me’ signals in communication between dying cells and the phagocytes

Cell Death Differ.

Noncanonical fungal autophagy inhibits inflammation in response to IFN-gamma via DAPK1

Cell Host Microbe

Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins

Nat. Cell Biol.

Autophagy proteins are not universally required for phagosome maturation

Autophagy

The intricate regulation and complex functions of the class III phosphoinositide 3-kinase Vps34

Biochem. J.

Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells

J. Biol. Chem.

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p–Vps15p protein sorting system

EMBO J.

Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae

J. Biol. Chem.

Induction of autophagy and inhibition of tumorigenesis by Beclin 1

Nature

Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages

Nat. Cell Biol.

Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1–phosphatidylinositol-3-kinase complex

Nat. Cell Biol.

ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy

J. Biol. Chem.

Nutrient-dependent mTORC1 association with the ULK1–Atg13–FIP200 complex required for autophagy

Mol. Biol. Cell

ULK–Atg13–FIP200 complexes mediate mTOR signaling to the autophagy machinery

Mol. Biol. Cell

Noncanonical autophagy promotes the visual cycle

Cell

Dynamin 2 is required for phagocytosis in macrophages

Review
LC3-Associated Phagocytosis and Inflammation