A guide to membrane atg8ylation and autophagy with reflections on immunity

Deretic and Lazarou conceptualize atg8ylation as a membrane stress and remodeling response, with autophagy and nonautophagic processes as its outputs.


Introduction
Canonical autophagy in mammalian cells is a multitasking process engaged in cytoplasmic quality control (Levine and Kroemer, 2019;Morishita and Mizushima, 2019), metabolism (Lahiri et al., 2019), and innate and adaptive immunity (Clarke and Simon, 2019;Deretic, 2021). The immune, metabolic, and quality control aspects of autophagy are intertwined in many of its physiological functions (Deretic and Kroemer, 2022). Autophagy is responsive to diverse inputs, and while the outputs may appear different, the molecular machineries involved are shared and redundantly utilized, creating sometimes unanticipated but biologically effectual overlaps. When autophagy fails, cellular, tissue, and organismal manifestations often present as dysregulated inflammation and other abnormalities. These contribute to a wide spectrum of diseases and pathological conditions reflected in animal models and human populations (Mizushima and Levine, 2020).
The term "autophagy" is often encountered in the literature as a conflation of diverse lysosomal degradative processes, including macroautophagy (herein "canonical autophagy"; Morishita and Mizushima, 2019), microautophagy (Schuck, 2020), chaperonemediated autophagy (Bourdenx et al., 2021;Dong et al., 2021), and other systems such as formation of intralumenal vesicles and related processes in endosomes and lysosomes Loi et al., 2019;Mejlvang et al., 2018). They all have a common purpose of importing substrates into membranous organelles where the sequestered substrates are typically digested. This contributes to turnover of proteins, membranes, and whole organelles; destruction of microbes or their products; and generation of metabolic intermediates during starvation (Deretic and Kroemer, 2022;Gonzalez et al., 2020;Morishita and Mizushima, 2019). There are also nondegradative outcomes of autophagy, such as secretory autophagy (Ponpuak et al., 2015), which contributes to unconventional secretion of leaderless cytosolic proteins and excretion of bulkier material from the cytoplasm.
"Noncanonical autophagy" encompasses an assortment of autophagy-related processes akin to but different from canonical autophagy, in several cases, reflecting noncanonical activities of autophagy proteins that do not involve lysosomal degradation. These phenomena include LC3-associated phagocytosis (LAP; Sanjuan et al., 2007) and its variations (Galluzzi and Green, 2019;Ulferts et al., 2021) as well as a growing collection of diverse emerging manifestations (Goodwin et al., 2021;Guo et al., 2017a;Kumar et al., 2020;Lee et al., 2020;Leidal et al., 2020;Loi et al., 2019;Nakamura et al., 2020). A feature frequently shared by canonical and noncanonical autophagy is the engagement of mammalian ATG8 proteins (mATG8s). The mATG8 LC3B is traditionally used as an autophagy marker (Kabeya et al., 2000), although it cannot differentiate between canonical and noncanonical forms. Thus, it is necessary to reconsider what mATG8 lipidation and appearance of intracellular mATG8 puncta represent, and how we interpret them. A recent proposal is that the atg8ylation of membranes by mATG8s (Kumar et al., 2021b) represents a general membrane stress and remodeling response analogous to ubiquitylation of proteins. Under this concept, canonical autophagy (macroautophagy), LAP, and other noncanonical processes represent manifestations of the role of atg8ylation in membrane homeostasis (Kumar et al., 2021b). Here, we revisit the standard model of autophagy and related processes through the conceptual lens of atg8ylation as a general membrane stress and remodeling response (Fig. 1).
The known processes downstream of membrane atg8ylation (Fig. 2) are individually addressed in the next section. As listed in the Fig. 2 boxes, we herein often use immunity examples to illustrate the physiological impact of atg8ylation, which broadly affects diverse aspects of metabolism, quality control, and inflammation. Figure 2. Atg8ylation and its cell biological manifestations including canonical autophagy, noncanonical autophagy, and related nonautophagic processes. (A) Canonical autophagy as a classic output of atg8ylation and the process of double-membrane autophagosome formation with atg8ylationindependent and atg8ylation-dependent stages. ATG8-negative prophagophore (HyPAS) is defined by fusion of FIP200 + early-secretory pathway/cis-Golgiderived membrane with ATG16L1 + endosomal membarnes. HyPAS converts to ATG8 + (usually referred as LC3 + ) phagophore, which sequesters the cargo and, upon ESCRT-catalyzed membrane closure, fuses with lysosomes leading to cargo degradation. (B and C) Noncanonical autophagy-related processes that do not involve formation of double-membrane autophagosomes and instead rely on atg8ylation of single-membrane organelles induced in response to membrane stress or other signals requiring membrane remodeling. (D) Processes utilizing atg8ylation that do not include canonical or noncanonical autophagy-related processes. Boxes indicate immunological processes associated with particular atg8ylation outputs. Details in the text.
Autophagy and related processes as manifestations of atg8ylation Canonical autophagy. The hallmark of canonical autophagy is the emergence of double-membrane autophagosomes in the cytoplasm (Morishita and Mizushima, 2019). Canonical autophagosomes (Morishita and Mizushima, 2019;Fig. 2 A) can capture diverse cytoplasmic cargoes that are recognized through a range of autophagy receptors (Lamark and Johansen, 2021). The autophagosomes are decorated by ATG8 family proteins, with LC3B being the most ubiquitously used marker of autophagy (Kabeya et al., 2000). Many mATG8-associated processes are termed using an LC3-based nomenclature; however, this does not exclude roles for other mATG8 family members including GABARAPs. Indeed, GABARAPs have been linked to atg8ylation-mediated activation of transcription factor EB (TFEB; Goodwin et al., 2021). We therefore note that, despite the LC3 nomenclature that is used in the field, and within this perspective, exploration into the role of each mATG8 family member is both warranted and necessary to gain a full understanding of atg8ylation-related processes. Given that studies do not always specify the LC3 subfamily member used, one may assume that it is typically LC3B. Therefore, when we refer to a study that reports a process as being decorated with or associated with LC3, be mindful that LC3B is the most likely member being discussed.
The HyPAS prophagophore is at first LC3B-negative (Kumar et al., 2021a), and it may be free of all mATG8s. While the latter notion remains to be established, it is consistent with the independence of the initial stages of autophagy from the atg8ylation system (Dalle Pezze et al., 2021;Itakura et al., 2012;Nguyen et al., 2016;Zachari et al., 2019). Atg8ylation is uncoupled from FIP200 and autophagosome formation, as observed early on with bacterial phagosomes (Kageyama et al., 2011). The LC3-negative prophagophore can in principle make initial contacts with autophagic cargo receptors, since a significant number of these receptors directly bind to FIP200 to recruit initiation complexes (Ohnstad et al., 2020;Ravenhill et al., 2019;Smith et al., 2018;Turco et al., 2019;Vargas et al., 2019). These initial encounters between the cargo and the forming autophagosomes are subsequently augmented by atg8ylation and autophagy receptor-mATG8 interactions (Padman et al., 2019;Turco et al., 2019), which coincide with conversion of the prophagophore into an atg8ylated phagophore (Kumar et al., 2021a). The phagophore enlarges via a variety of mechanisms including direct delivery of phospholipids through lipid-transfer proteins such as ATG2 (Valverde et al., 2019) as well as lipid scramblases that relax lipid asymmetry between membrane leaflets to enable lipid flow into the growing phagophore (Maeda et al., 2020;Matoba et al., 2020). The enlarged phagophore enwrapping the cargo then closes with the help of the endosomal sorting complexes required for transport (ESCRT) machinery (Takahashi et al., 2018). Closed autophagosomes deliver the captured cargo to lysosomes for degradation via autophagosomelysosome fusion (Zhao and Zhang, 2019). Fig. 2 A also lists the principal immunological outputs of canonical autophagy. As recently reviewed (Deretic, 2021), this includes direct microbial elimination (xenophagy), antiinflammatory action by removing pathogen-and damageassociated molecular patterns (PAMPs and DAMPs), and development and differentiation of immune cells as well as their polarization and function. There are unique connections with immunometabolism (Deretic, 2021) because of the tight integration of canonical autophagy with the nutrient and energy sensors mammalian target of rapamycin (mTOR) and AMPactivated protein kinase (AMPK), which together determine the "posture" of key immune cells, with their populations assuming either a robust inflammatory phenotype associated with proliferation or tissue repair characteristics associated with quiescence (O'Neill et al., 2016;Saravia et al., 2020).
Noncanonical autophagy-related processes A number of noncanonical autophagy-related processes have been reported, and a diverse selection of newly described noncanonical trends involving mATG8s continue to emerge. The key feature of these phenomena is that they share certain but not all components with canonical autophagy and involve single instead of double membranes (Galluzzi and Green, 2019). The archetypal process in this category is LAP Sanjuan et al., 2007). Its close variations (LAP-related processes) on endosomal/phagosomal vesicles are LC3-associated endocytosis (LANDO; Heckmann et al., 2019) and LC3-associated micropinocytosis (LAM; Sonder et al., 2021;Fig. 2 B). They all exhibit the eponymous LC3 labeling but, unlike canonical autophagosomes, involve organelles with single delimiting membranes. We suggest that one may best grasp the relationship to canonical autophagy by viewing LAP and LAP-related processes as one half of the HyPAS prophagophore formation, engaging only the endosomal organelles where ATG16L1 E3 ligase resides but not the membranes coming from the early secretory pathway with FIP200 and its associated components. Consequently, FIP200 is not needed for LAP-related processes, whereas it is critical for canonical autophagy.
LAP-related processes can be induced by various stressors and membrane-damaging agents, including pharmacological agents or microbial products perturbing phagosomes and endosomes (Durgan et al., 2021;Fletcher et al., 2018;Florey et al., 2015a;Florey et al., 2011;Jacquin et al., 2017;Ulferts et al., 2021;Xu et al., 2019). The immunological roles of LAP and LAP-related processes are listed in the box in Fig. 2 B. They include antiinflammatory activity preventing autoimmunity and lupus , antimicrobial action (Kageyama et al., 2011;Martinez et al., 2015), and orderly removal of dead or dying cells and cell fragments by efferocytosis, entosis, and phagocytosis (Boada-Romero et al., 2020;Florey et al., 2011;Kim et al., 2013;Martinez et al., 2011). The downregulation of stimulator of interferon genes (STING), normally transmitting signals of ectopic presence of viral or mitochondrial dsDNA in the cytosol, via a LAP-related process (Fischer et al., 2020), has the potential to directly limit type I IFN responses, but this remains to be established. LAP may favor immune tolerance in cancer microenvironments (Cunha et al., 2018), albeit it promotes Toll-like receptor (TLR) signaling through IRF7-stimulating type I IFN response (Henault et al., 2012). In the context of neuroinflammatory diseases, LANDO clears β-amyloid aggregates and suppresses microglia activation (Heckmann et al., 2019). Of note, not all endosomes and phagosomes undergo atg8ylation, which requires a specific stress or danger signal such as described above, as well as TLR signaling upon encounter of fungal, bacterial, and microbial products known as PAMPs (Delgado et al., 2008;Sanjuan et al., 2007).
A number of other processes unrelated to LAP are manifestations of membrane atg8ylation (Fig. 2, C and D). These diverse phenomena include selective microautophagy of mammalian lysosomal membranes in response to osmotic stress or glucose starvation , selective removal of excess ER during recovery from ER stress via piecemeal micro-ER-phagy (Loi et al., 2019), and unconventional secretion via extracellular vesicles and secretory autophagy. Atg8ylation participates in the formation of exosomes (Guo et al., 2017a) and secretion of specific cytosolic cargo by extracellular vesicles (Leidal et al., 2020). Among the reported innate immunity functions for exosomes impacted by atg8ylation is neutralization of bacterial toxins before they can attack the host cells (Keller et al., 2020).
Atg8ylation furthermore plays a role in a type of unconventional secretion of leaderless cytosolic proteins, or excretion/ extrusion of cytoplasmic material, referred to as secretory autophagy (Gerstenmaier et al., 2015;Ponpuak et al., 2015). Secretory autophagy has been implicated in the export of a key proinflammatory cytokine, IL-1β, and of the alarmin HMGB1 (Dupont et al., 2011;Karmakar et al., 2020;Kimura et al., 2017;Razani et al., 2012;Thorburn et al., 2009), albeit IL-1β exit from cells uses multiple routes (Zhang et al., 2015;Zhang et al., 2020b), including prominently a passive release of IL-1β from dying cells through gasdermin pores on the plasma membrane during pyroptosis (Broz et al., 2020;Evavold et al., 2018). Atg8ylation does not always protect the host: for example, it has been coopted by influenza A to promote its filamentous mode of budding from the plasma membrane (Beale et al., 2014).
In summary, atg8ylation participates in phenomena historically referred to as noncanonical autophagy but functionally representing a range of membrane stress responses and membrane remodeling processes. This diversity is mirrored by the equally diverse immunological and anti-inflammatory outputs of atg8ylation further discussed below. Despite atg8ylation being linked to noncanonical processes, how exactly mATG8s are functioning during many of these processes remains to be fully explored. It is likely that atg8ylation is involved in the formation/expansion of membrane vesicles, but mATG8s may also recruit factors through LC3 interaction region (LIR)/GABARAP interaction motif (GIM), thereby acting as scaffolds for protein complexes that play roles in signaling. In addition, atg8ylation may play an adaptor role to recruit factors within the lumen of vesicles. In the next section, we cover some of the recent advances in the signaling roles of membrane atg8ylation.

Atg8ylation and signaling
Membrane atg8ylation can affect multiple signaling systems and therefore functions beyond the proposed direct physical role in membrane remodeling during the formation of canonical autophagosomes (Maruyama et al., 2021) and noncanonical LAP-like structures (Galluzzi and Green, 2019). The atg8ylation that takes place during canonical autophagy is the most understood (Kabeya et al., 2000). During PINK1/Parkin mitophagy, atg8ylation can serve as an amplification signal by increasing the concentration of autophagy machineries on phagophores (Padman et al., 2019). Although PINK1/Parkin mitophagy is largely independent of mTOR and AMPK signaling (Vargas et al., 2019), most forms of canonical autophagy depend on these principal regulators of cellular metabolism (Deretic and Kroemer, 2022). Localized at least in part to lysosomes (Fig. 3), mTOR inhibits (whereas AMPK activates) protein complexes controlling autophagosome biogenesis. Lysosomes are also the site where these systems, along with phosphatases such as calcineurin, control TFEB, a key regulator of lysosomal biogenesis, with functional lysosomes being essential for the completion of canonical autophagy and other roles (Ballabio and Bonifacino, 2020).
How is membrane atg8ylation induced on endolysosomal and phagosomal vesicles? Agents that deacidify/increase lumenal pH in endolysosomal vesicles  stimulate recruitment of the V 1 subunit of v-ATPase, which in turn recruits ATG16L1 E3 ligase (Xu et al., 2019;Fig. 3, box 4). This leads to direct membrane atg8ylation without formation of autophagosomes (Fischer et al., 2020;Hooper and Florey, 2021 Preprint;Ulferts et al., 2021). Of relevance for the immunological subplot of this article, many of the known physiological examples of membrane atg8ylation involve interactions of host cells with microbes. This includes Salmonella (Xu et al., 2019) and viruses such as influenza , response to microbial or ectopic dsDNA in the cytosol (Fischer et al., 2020), and activation of NADPH oxidase (Hooper and Florey, 2021 Preprint) by phagocytic cells as they kill microbes (Nauseef, 2019).
In addition to lysosomal membrane atg8ylation, extensive physical damage of these membranes eventually elicits canonical autophagy to remove excessively damaged lysosomes by lysophagy. Damage of endolysosomal organelles occurs physiologically, e.g., during exposure to exogenous and endogenous agents including biologically active crystals of silica, monosodium urate, Figure 3. Specific examples and circuitry of how atg8ylation controls different signaling and stress response processes at the lysosome. Note that both mTOR and AMPK as well as their regulatory elements are localized at the lysosome. AMPK positively regulates canonical autophagy, whereas mTOR negatively regulates this atg8ylation-associated process. Boxes 1-4 describe four of the expanding list of autophagy-independent atg8ylation-dependent processes. This includes control of AMPK, mTOR, and TFEB by mATG8s and atg8ylation (Boxes 1-3). Box 4-associated schematic depicts how increase in lumenal pH (phagosomes, organelles of the endolysosomal network) by the action of the influenza viroporin M2 that acts as an H + channel or proton scavenging during superoxide production induce membrane atg8ylation (LC3 shown as an example of LAP and LAP-related processes). This occurs due to the increased recruitment of ATG16L1 via its direct binding to the V1 subunit of vacuolar H + -ATPase, upon elevated V 1 V 0 assembly on membranes in response to neutralization of the lumenal pH. Further details in the text. and cholesterol (Maejima et al., 2013;Razani et al., 2012;Schroder and Tschopp, 2010), proteopathic fibrils or amyloid (Heneka et al., 2013;Masters et al., 2010;Papadopoulos et al., 2017;Parry et al., 2015), TRAIL-signaling induced lysosomal permeabilization (Werneburg et al., 2007), microbes including bacteria Salmonella and Mycobacterium tuberculosis (Chauhan et al., 2016;Fujita et al., 2013;Jia et al., 2018;Thurston et al., 2012;Watson et al., 2012), and coronaviruses including SARS-CoV-2 (Ghosh et al., 2020;Yue et al., 2018). A breach in lysosomal membrane integrity is subject to repair by ESCRTs (Jia et al., 2020b;Radulovic et al., 2018;Skowyra et al., 2018) or removal by canonical autophagy Jia et al., 2018;Jia et al., 2020b;Jia et al., 2020c;Maejima et al., 2013). Atg8ylation is a part of the canonical autophagy of excessively damaged lysosomes (lysophagy) set in motion by the inactivation of mTOR and the activation of AMPK (Fig. 3). The latter stages of lysophagy are aided by systems enabling selective autophagy of damaged lysosomes: the recognition of exposed lumenal glycans by ubiquitin E3 ligases and protein ubiquitination (Yoshida et al., 2017), ubiquitin remodeling (Papadopoulos et al., 2017), atg8ylation of autophagosomal membrane (Maejima et al., 2013), and engagement of selective autophagy receptors possessing LIRs (Lamark and Johansen, 2021) and ubiquitin-binding or galectin-binding capabilities, including p62/SQSTM1 (Papadopoulos et al., 2017), TRIM16 (Chauhan et al., 2016), and TAX1BP1, along with its interactors TBK1 and FIP200 (Eapen et al., 2021).
In conclusion, membrane atg8ylation, whether occurring on a single membrane delimiting an organelle of the endocytic pathway or on the double membrane of the canonical autophagosome, plays a significant role in upstream signaling and downstream effector processes.
IRGM. Human IRGM (Fig. 4) and its murine paralogs interact with mATG8s (Kumar et al., 2020), leading to multiple effector outputs. These include recruitment of STX17  as a component of the HyPAS fusion apparatus  leading to the formation of autophagic prophagophores (Kumar et al., 2021a), as well as inhibition of mTOR (Kumar et al., 2020) and stabilization of AMPK (Chauhan et al., 2015a), which regulate the FIP200 complex en route to canonical autophagosomes (Kumar et al., 2021a). IRGM and its paralogs bind to both ATG16L1 and mATG8s and may present mATG8s to the ATG16L1 E3 ligase, leading to atg8ylation. Among the IRGMdependent immunological effects is the protection of cells from excessive cGAS-STING and RIG-I-MAVS signaling (Jena et al., 2020), from excessive activation of the canonical (Mehto et al., 2019) and noncanonical inflammasomes, and from ensuing pyroptosis during bacterial invasion (Eren et al., 2020;Finethy et al., 2020).
SLRs and TRIMs interact with components from both halves of the autophagic apparatus defined by FIP200 and ATG16L1 (Fig. 4, components A and B).
AMPK and mTOR functionally interact with the v-ATPase at the lysosome ( Fig. 3; Eaton et al., 2021). This is important for components of the canonical autophagy pathway, and for atg8ylation processes in general via the ATG16L1 recruitment to and association with the V 1 subunit of the v-ATPase (Hooper and Florey, 2021 Preprint;Ulferts et al., 2021;Xu et al., 2019; Fig. 3, bottom left). The v-ATPase is a potent hub for integration of signals leading to canonical autophagy and other atg8ylation manifestations.

Anti-inflammatory functions of atg8ylation and autophagy
Deficiencies in canonical autophagy and noncanonical autophagy-related processes, along with the emerging concept of membrane atg8ylation, correlate with inflammatory or autoimmune disorders as detailed in recent reviews (Deretic, 2021;Galluzzi and Green, 2019;Youle, 2019). This includes (Fig. 5 A, box I) autoimmune diseases and conditions with systemic inflammation such as Crohn's disease (CD), lupus, asthma, rheumatoid arthritis, celiac disease, type 1 diabetes mellitus, and familial Mediterranean fever. These relationships extend to neurological disorders with inflammatory components, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. We refer the reader to a recent review for details (Deretic, 2021).
Some of the earliest genomewide association studies of any human disease (Consortium, 2007) revealed connections between polymorphisms in the genes encoding ATG16L1 and IRGM with the increased risk for CD. ATG16L1 and IRGM form a tripartite complex with NOD2 (Chauhan et al., 2015b), a familial CD predisposition locus (Horowitz et al., 2021;Fig. 5 A, box II). This complex participates in anti-inflammatory processes via several mechanisms that involve canonical or noncanonical autophagy (Jena et al., 2018;Jena et al., 2020;Mehto et al., 2019;Singh et al., 2006), both of which are downstream outputs of atg8ylation.
Metabolic inputs and innate immunity systems may integrate with autophagic and other atg8ylation processes in the context of neurodegenerative diseases (Fig. 5 A, box II;Youle, 2019). TBK1 is a frequent site of mutations in ALS (Ahmad et al., 2016). TBK1 can be controlled by AMPK and ULK1, whereas TBK1 directly phosphorylates mTOR and activates both mTORC1 and mTORC2 in response to growth factors or PAMP-PRR signaling (Bodur et al., 2018;Tooley et al., 2021). LRRK2, a protein kinase affecting autophagy (Gomez-Suaga et al., 2012) and axonal transport of autophagosomes (Boecker et al., 2021), is commonly associated with familial PD as well as CD, leprosy, and certain cancers. LRRK2 positively regulates AMPK (Usmani et al., 2021). Metabolic changes have been observed in astrocytes derived from PD patients carrying a common LRRK2 mutation (Sonninen et al., 2020). It is worth noting that LRRK2 operates, among other compartments, within the endolysosomal system where various manifestations of atg8ylation take place. PD has been associated with inflammation, including elevated cytokines in both serum and cerebrospinal fluid (Dzamko et al., 2015). In mouse models, the regulators of mitophagy PINK1 and Parkin (Youle, 2019) have been difficult to connect with PD, likely owing to the stress-responsive nature of PINK1 and Parkin and the lack of stress exposure to lab mice. However, under conditions promoting mitochondrial stress and inflammation, manifestations of parkinsonism could be detected (Sliter et al., 2018), including during an extended aging period (Noda et al., 2020). The PD phenotype could be rescued by manipulating STING (I199N missense mutant allele of the murine Sting gene, Tmem173) in Prkn −/− or Pink1 −/− mice (Sliter et al., 2018). These findings help connect the dots between failure in mitophagy (which includes atg8ylation), mitochondrial DNA release, cytosolic DNA sensing systems, and inflammation (Borsche et al., 2020;McArthur et al., 2018;Sliter et al., 2018;White et al., 2014). Although mitochondria are the major metabolic factories of the cell, here they are implicated as a source of DNA acting as a DAMP (Borsche et al., 2020), stimulating STING-TBK1 signaling to elicit a type I IFN response (Sliter et al., 2018).
Protein complexes that stimulate type I IFN production are affected by atg8ylation components or the whole autophagy pathway as one of the most studied downstream outputs of atg8ylation (Fig. 5 A, box II). The ATG5-ATG12 conjugate, a key part of the atg8ylation E3 ligase ATG16L1 complex, inhibits RIG-I (Jounai et al., 2007). ATG9A negatively controls trafficking of the ER-associated STING and inhibits activation of TBK1 (Saitoh et al., 2009). In the absence of effective mitophagy, RIG-I-like-MAVS signaling is increased due to high MAVS levels and ROS emanating from depolarized mitochondria (Tal et al., 2009). TRIM21 targets both IKKβ (Niida et al., 2010) and IRF3  for degradation ascribed to autophagy. cGAS , RIG-I, and TLR3 (Jena et al., 2020), as well as STING (Prabakaran et al., 2018), seem to be attenuated through autophagy to reduce type I IFN signaling. Targeting by mATG8s and downregulation of immune signaling proteins extends to TAB1/TAB2-TAK1, with effects on downstream IKK complexes and NFkB, as reported in Drosophila (Tsapras et al., 2022).
There are clear examples of nonautophagic processes where atg8ylation plays anti-inflammatory roles. LAP, a manifestation of atg8ylation, has important roles in inflammation and is required for elimination of pathogens (Martinez et al., 2015). LAP deficiency results in development of autoinflammatory, lupuslike syndrome . LAP is activated in patients with cirrhosis and protects against hepatic inflammation and fibrosis in mice (Wan et al., 2020). LAP provides immunity against Streptococcus pneumoniae, which diminishes as LAP declines with aging (Inomata et al., 2020). In addition to its role in

Deretic and Lazarou
Journal of Cell Biology inflammation and related complications, LAP has roles in cancer progression. LAP regulates the production of anti-inflammatory cytokines in tumor-associated macrophages, which promotes immune tolerance in the cancer microenvironment by suppressing T lymphocytes (Cunha et al., 2018). In summary, atg8ylation, in the context of autophagy or other autophagy-related and unrelated processes, is associated with nearly all key innate immunity systems and, with few exceptions, reduces inflammation. The systems and activities involved in anti-inflammatory activities of atg8ylation and canonical autophagy as one of its outputs engage both regulators of metabolism and key metabolic factories, i.e., mitochondria, with effects on immunometabolism that will be explored in the section on immune cells.

Antimicrobial functions of atg8ylation and autophagy
Xenophagy (Levine, 2005) is a term often encountered in describing autophagic elimination of intracellular microbes (Gomes and Dikic, 2014;Randow and Youle, 2014). Canonical autophagy or noncanonical atg8ylation processes can protect host cells against a variety of pathogens (Fig. 5 B, box I) including bacteria, viruses, and fungal pathogens. Mechanistic studies in vitro have established that selective autophagy receptors, in particular SLRs, recognize intracellular pathogens or vacuoles containing them when tagged by ubiquitin or galectins and execute their elimination by delivering the pathogens to degradative compartments (Gomes and Dikic, 2014;Randow and Youle, 2014). Some of the microbes studied in depth (Table S1) in this context are Salmonella, Streptococcus, and Mycobacterium tuberculosis. Additional notable examples of bacteria affected by autophagy or noncanonical atg8ylation processes include Shigella flexneri and Listeria. Besides bacteria, autophagy-related processes affect other microbes including viruses, fungal pathogens, and protozoa such as Toxoplasma gondii (Table S1).
The professional intracellular pathogens, such as intracellular bacteria (Engstrom et al., 2019;Laopanupong et al., 2021;Mitchell et al., 2018;Strong et al., 2020;Tan et al., 2018;Zhang et al., 2019b), viruses (Chang et al., 2019;Kumar et al., 2021a;Kyei et al., 2009;Orvedahl et al., 2007;Tiwari et al., 2020), and protozoa (Lopez Corcino et al., 2019), have evolved a multitude of ways to counter or disarm autophagy or components of atg8ylation (Xu et al., 2022;Xu et al., 2019;Fig. 5 B, box II). In extreme cases, highly adapted intracellular pathogens can use autophagy or factors contributing to atg8ylation and autophagyrelated processes to support their own growth (Fig. 5 B, box III). This includes Anaplasma phagocytophilum (formerly known as Ehrlichia, from order Rickettsiales; Niu et al., 2012), hepatitis C virus (Lee and Ou, 2021;Twu et al., 2021), influenza A virus (Beale et al., 2014), and SARS-CoV-2 Schneider et al., 2021;Twu et al., 2021). Probably the most elegant examples of bacterial defenses come from studies of Legionella pneumophila and Salmonella (Fig. 5 B, box II). L. pneumophila encodes a factor, RavZ, that irreversibly counters atg8ylation by proteolytically removing mATG8s' C-terminal glycines, thus precluding lipidation (Choy et al., 2012). Salmonella SopF acts as an ADP-ribosylating enzyme modifying v-ATPase to block its recruitment of ATG16L1 and inhibit atg8ylation (Xu et al., 2022;Xu et al., 2019). It is important to keep in mind that the majority of bacteria are not equipped to combat autophagy as one of the outputs of atg8ylation, and only a handful of highly adapted intracellular bacteria can persist in host cell's cytosol. Considering that mitochondria evolved from Rickettsia-like endosymbionts, this presents us with a clear example of a terminal bacterial-host adaptation in coevolution with autophagy (Deretic, 2010;Randow and Youle, 2014;Youle, 2019).
There is a relative dearth of studies of atg8ylation in animal models compared with the wealth of ex vivo studies using cellular models of microbial invasion. The available murine models suggest that xenophagy, even when it can be documented in vivo, is accompanied by an equal or more important action of canonical and noncanonical autophagy processes as atg8ylation outputs in protecting against excessive inflammation and tissue damage (Deretic, 2021;Deretic and Levine, 2018). A hyperinflammatory state can be artificially generated in murine models when genes contributing to autophagy or atg8ylation are inactivated. This paradoxically presents itself as protective against infections in experimental situations (Fig. 5 A, box III). For example, inactivation of key atg8ylation genes and/or components of HyPAS in myeloid cells elevates respiratory tract inflammation and confers resistance to influenza in mice . Similar genetic maneuvers can be protective against murine herpesvirus reactivation . Likewise, disrupting tissue macrophage quiescence by inactivating genes contributing to HyPAS but not atg8ylation, which normally maintains the anti-inflammatory state, confers resistance to Listeria in mice . Autophagy or autophagy-related processes are important to prevent cytotoxicity upon stimulation with PAMPs (Levy et al., 2020) or IFN-γ (Orvedahl et al., 2019). Two conclusions can be drawn from these explorations: First, processes associated with autophagylike phenomena and atg8ylation in principle play antiinflammatory roles. Second, not all components of what has been classically considered canonical autophagy are needed.
SARS-CoV-2 additionally interferes with other aspects of autophagosomal and lysosomal pathways. ORF3a affects autophagosome-lysosome fusion (Hayn et al., 2021;Miao et al., 2021), whereas SARS-CoV-2 ORF7 interferes with autolysosomal acidification (Hayn et al., 2021). On the flip side, autophagy factors counter actions of SARS-CoV-2 proteins: ATG9A repairs plasma membrane damage induced by SARS-CoV-2 ORF3a (Claude-Taupin et al., 2021). Adding to these antagonistic relationships between the virus and the host, atg8ylation phenomena may play a role in countering COVID-19 pathogenesis. Cells release exosomes dubbed decoy exosomes or "defensosomes" (Fig. 2 D) that can soak up microbial toxins so that they do not attack cells (Keller et al., 2020). This happens in an ATG16L1dependent fashion (Keller et al., 2020), and has been extended to the release of ACE2 + defensosomes that bind and block SARS-CoV-2 entry (Ching et al., 2021 Preprint). Presence of ACE2 + exosomes in bronchioalveolar lavages of critically ill COVID-19 patients was reported as being associated with reduced hospitalization times (Ching et al., 2021 Preprint). These findings suggest that atg8ylation and variants of secretory autophagy may contribute to antiviral defenses, albeit they can help viral egress in other cases, such as atg8ylation-dependent filamentous influenza A budding from the plasma membrane (Beale et al., 2014).

Immune cells, atg8ylation, and autophagy
The functions of ATG genes in immune cells have been extensively reviewed (Clarke and Simon, 2019;Deretic, 2021;Deretic et al., 2013;Macian, 2019;Riffelmacher et al., 2018). Since canonical autophagy (a classical downstream effector of atg8ylation) is a metabolic process, autophagy in immune cells manifests itself as an immunometabolic phenomenon (O'Neill et al., 2016), tracking with the known roles of mTOR and AMPK (Fig. 5 C). This is reflected in the reported beneficial effects of metformin, a clinically used AMPK activator, which improves immune responses by enhancing survival of memory CD8 + T cells (Bohme et al., 2020) and moderates Th17 responses by CD + T cells (Bharath et al., 2020) in infection, diabetes, and "inflammaging" contexts. Canonical autophagy is believed to support self-renewal and quiescence in immune stem cells, as well as cytoplasmic remodeling during immune cell differentiation . Atg8ylation components support neutrophils (Riffelmacher et al., 2017), B1a cells (Clarke et al., 2018;Miller et al., 2008), invariant NKT cells (Salio et al., 2014) and NKT cells (Zhu et al., 2018). They are required for T cell survival and function (Jia et al., 2011;Li et al., 2006;Pua et al., 2009) and for preservation of effector and memory CD8 + T cells Xu et al., 2014). Autophagy plays a role in the metabolism of CD8 + T cells, affecting their antitumor immunity (DeVorkin et al., 2019). These processes counter T cell anergy (Mocholi et al., 2018), whereas tissue-resident memory CD8 + cells and mucosa-associated invariant T cells display high autophagy levels (Swadling et al., 2020). Much of the role of atg8ylation in lymphocyte function is to enhance clearance of virus-infected and cancerous cells while minimizing excessive inflammation and tissue pathology.
The role of atg8ylation processes other than canonical autophagy in immune cells or in cells that are targets of cellmediated immunity are only beginning to be investigated. This often includes modulation of cell surface receptors. Plasma membrane presence of the cystine transporter SLC7A11 is diminished in pancreatic ductal adenocarcinoma cells knocked down for ATG5 or ATG7 (Mukhopadhyay et al., 2021). A further example of receptor regulation (Heckmann et al., 2019;Ulland et al., 2017) by atg8ylation processes and intersections between atg8ylation, autophagy, inflammation, and metabolism, is TREM2, a surface receptor required for microglial responses and a risk factor in sporadic Alzheimer's disease (Shi and Holtzman, 2018). An atg8ylation process LANDO promotes recycling of the TREM2 which allows more efficient clearance of Aβ by the microglia and mitigates inflammatory microglial activation (Heckmann et al., 2019;Fig. 5 A, box III). However, TREM2 also plays a role in immunometabolism through control of mTOR and canonical autophagy and maintains microglial metabolic fitness, which fails in TREM2 hypomorphs and is correctable by cyclocreatine supplementation (Ulland et al., 2017). MHC-I downregulation contributes to pancreatic cancer being immunologically cold and recalcitrant to check point inhibitors immunotherapy Fig. 5 A, box IV). This downregulation depends on FIP200 and other components of the FIP200 complex, whereas Rubicon seems not to affect it , thus ruling out LAP-like processes that depend on Rubicon (Heckmann et al., 2019;Martinez et al., 2015). Until recently, it has been very difficult to envision how a single membrane endosome formed during MHC-I endocytosis might result in degradation by canonical autophagy. However, the latest findings (Kumar et al., 2021a) demonstrating that canonical autophagosomes are generated through fusion between endosomes and FIP200 + cis-Golgi membranes thus forming the HyPAS pro-phagophore can explain how endocytosed proteins/ receptors end up being degraded in canonical autolysosomes. Atg8ylation, in the form of LAP, by tumor-associated macrophages is immunosuppressive, as found in a murine model of melanoma where LAP reduces STING-mediated type I IFN response normally important to spur infiltrating antitumor T cells (Cunha et al., 2018). This aspect seems to be of general import, as enhanced STING-mediated type I IFN responses due to inhibition of atg8ylation or autophagy are beneficial in cancer or cancer treatment models (Poillet-Perez et al., 2020;Yamazaki et al., 2020;Fig. 5 A, box IV). Thus, various proteins at the surface of immune cells or of cells that are their targets are affected by atg8ylation systems with important immunological consequences.
Mitochondrial abundance and their functionality depend on atg8ylation through mitophagy, which is essential for proper function of immune cells, and therefore their participation in clearance of infection or cancer and tissue repair and homeostasis Nakahira et al., 2011;Sliter et al., 2018). Developmental mitophagy is immunologically important in nonimmune cells because retention of erythroid mitochondria can trigger autoimmune diseases such as lupus (Caielli et al., 2021). Given the evolutionary origins of mitochondria from cytosol invading bacteria, it is not surprising that mitochondria to this day serve as signaling platforms for activation of protective innate immunity in response to bacterial and viral PAMPs. Under pathological conditions, mitochondria themselves become sources of endogenous DAMPs (oxidized mtDNA, dsDNA, dsRNA, cardiolipin, ROS), which stimulate a variety of PRRs and contribute to degenerative and inflammatory states (Youle, 2019). Mitochondria are furthermore the principal contributors to immunometabolism (Mills et al., 2017). This is of particular significance in immune cells whereby switching between oxidative phosphorylation (OXPHOS) and glycolysis has to match or even determines activation levels of T cells, macrophages, dendritic cells (Mills et al., 2017), and as recently shown, neutrophils (Willson et al., 2022;Fig. 5 C). It is well known that glycolysis dominates in proinflammatory classically activated macrophages whereas OXPHOS predominates in alternatively activated macrophages involved in tissue repair (Mills et al., 2017). Even in neutrophils, where mitochondrial electron transport complexes are traditionally believed to have very limited expression, recent studies show that they nevertheless contribute to ROS formation and stabilization of HIF-1α under hypoxic conditions in inflammatory sites (Willson et al., 2022), as during pulmonary complications in covid patients (Ackermann et al., 2021). Mitophagy has been shown to play an important role in immune cell metabolic switch from glycolysis to OXPHOS.
In conclusion, the common themes regarding the role of atg8ylation and autophagy as its thus far best studied output in immune cells include effects on immunometabolism, surface receptor expression, cytoplasmic immune signaling complexes, mitochondrial abundance, and quality control, all with consequences for proper function of immune cells and systems in various tissues and in diverse disease contexts.

Conclusions and outlook
The expanded concept of atg8ylation as a membrane stress response has many physiological effects based on quality control, metabolism, and roles in immunity. Experimental analyses have revealed that many mammalian ATG genes, including mATG8s, function in processes other than canonical autophagy. The atg8ylation concept-a covalent modification of stressed or remodeling membranes-mirrors ubiquitin modification of proteins. The crossovers between ubiquitylation and atg8ylation exist, as evident from examples of the ubiquitylation of glycolipids such as bacterial LPS when eroding into the mammalian cell cytoplasm (Otten et al., 2021). Conversely, protein atg8ylation can occur (Agrotis et al., 2019;Nguyen et al., 2021).
Atg8ylation participates in an expanding list of processes and includes but is not limited to canonical and noncanonical autophagy as its manifestations. In this view, the noncanonical membrane modifications and remodeling processes such as LAP, LANDO, selective microautophagy, EV/exosome secretion, and secretory autophagy, are easier to systematically organize and understand. The concept of atg8ylation also offers a new perspective on drug development by differentiating atg8ylation from canonical autophagy as targets of interest in different diseases.
The immune functions of canonical and noncanonical autophagy, and the emerging concept of atg8ylation as a membrane stress response, have been summarized herein. They manifest themselves in interactions with microbial pathogens as well as in cancer and as containment of endogenous sources of inflammation. Classically, autophagy is based on digestion of self at times of starvation, organellar malfunction, or elimination of incoming microbes during infection. These functions are conflated in mitophagy, as a product of an evolutionary "truce" between the host and an endosymbiont of bacterial ancestry. Autophagy is likely tirelessly at work against microbial wouldbe invaders, and, as emphasized here, in protection against endogenous sources of inflammation. Harnessing the full potential of autophagy and atg8ylation in separate and more granular ways may help in protection against current and future pandemics, as well as in treatments of cancer, neurodegenerative, and other diseases.
Online supplemental material Table S1 shows examples of intracellular microbes studied as targets for elimination by autophagy.