Autophagy in immunity

Abstract

Autophagy is now emerging as a spotlight in trafficking events that activate innate and adaptive immunity. It facilitates innate pathogen detection and antigen presentation, as well as pathogen clearance and lymphocyte homeostasis. In this review, we first summarize new insights into its functions in immunity, which underlie its associations with autoimmunity. As some lines of evidence are emerging to support its role in autoimmune and autoinflammatory diseases, we further discuss whether and how it affects autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus and multiple sclerosis, as well as autoinflammatory diseases, such as Crohn disease and vitiligo.

Keywords: :

• autoimmune disease
• autoinflammatory disease
• autophagy
• immunity

Introduction

Autoimmune diseases (AID) arise due to a breach in central and peripheral immune tolerance, amplification of the autoimmune response, and local processes in the target organ that facilitate end-organ disease. This may be restricted to certain organs (e.g., in autoimmune thyroiditis), certain tissues in different places [e.g., antiglomerular basement membrane (GBM) disease which may affect the basement membrane in both the lung and the kidney],¹ or may be systemic such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), in which the loss of immune tolerance is systemic and disease manifestations can occur at a variety of different organs or tissues in the body.² Although the precise mechanisms of autoimmune diseases are unclear, certain pathogenic mechanisms are ultimately triggered during the disease progression, and dysfunctions involving major cell signaling pathways, i.e., the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1/NFκB) pathway and the inflammatory responses, are consistent features in most AID.^3,4

Autophagy is now emerging as a spotlight in trafficking events that activate innate and adaptive immunity.⁵ As a central player in the immunological control of pathogen clearance, it may also deliver antigens to major histocompatibility complex (MHC) compartments, regulate lymphocyte survival and homeostasis, and mediate cytokine production.^6-9 Given the likely importance of autophagy in self-renovation for immunity, here we will review its immune functions and highlight its role in the pathogenesis of AID.

Mechanisms of Autophagy

Autophagy refers to a cellular degradation pathway that involves cytoplasmic content engulfment, delivery and degradation by the lysosome. At least three different forms termed macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been identified. Of these forms, macroautophagy has the best-known impact upon human diseases, which will be the main focus of this review. The process of autophagy is as shown in Figure 1. More details for this process are described extensively elsewhere.^10-21

Figure 1. Schematic diagram of autophagy. In macroautophagy, the phagophores engulf the cytoplasmic constituents including organelles, and the resulting autophagosomes fuse with lysosomes, resulting in complete degradation of the sequestered cytoplasmic components by lysosomal hydrolases. In sequence, the cellular events during autophagy follow distinct stages: vesicle nucleation (formation of the phagophore), vesicle elongation and completion (growth and closure), fusion of the double-membrane autophagosome with the lysosome to form an autolysosome, and lysis of the autophagosome inner membrane and breakdown of its contents inside the autolysosome. Microautophagy, on the other hand, happens when lysosomes directly engulf cytoplasm by invaginating, protrusion and/or septation of the lysosomal limiting membrane. In CMA, specific cytosolic proteins are transported into lysosomes via a molecular chaperone/receptor complex composed of HSPA8/HSC70 and LAMP2A (the CMA receptor lysosome-associated membrane protein type-2A).

In brief, autophagy is tightly regulated to prevent its unbalanced activation. It can be upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, endoplasmic reticulum (ER) stress, accumulation of unfolded proteins and pathogen infection. Autophagosomes can be formed from different membrane sources including the ER,^22,23 the Golgi complex,^24-26 the mitochondria,^27,28 and the plasma membrane.^14,19,29,30 To date, at least 35 autophagy-related (ATG) genes have been identified.³¹ Most of them contribute to autophagosome formation, and are conserved from yeast to human. In mammals, the “core” ATG proteins can be divided into five subgroups: the ATG13-RB1CC1/FIP200-ATG101-ULK1/ULK2 (equivalent of Atg1 in yeast) complex,¹¹PIK3C3/VPS34-BECN1(Atg6) class III PtdIns3-kinase complex (which can be divided into at least three types, the ATG14-PIK3C3-PIK3R4/VPS15-BECN1, UVRAG (UV radiation resistance-associated gene)-PIK3C3-PIK3R4-BECN1 and KIAA0226/RUBICON-UVRAG-PIK3C3-PIK3R4-BECN1 complexes),^32-34 ATG9-WIPI1 complex,^35-37 ATG12 (ATG12–ATG5-ATG16L1) conjugation system,³⁸ and LC3 (Atg8) conjugation system.^12,20,39-42 Each complex contributes to a different function during autophagy. Autophagy is impaired without any of these complexes or core gene products, indicating that a sequential reaction is indispensable for the autophagy process.⁴³ Post-translational modifications such as phosphorylation, ubiquitination and acetylation are thought to be important in regulating autophagy.

Physiological and Pathological Roles of Autophagy

The autophagy induced in response to nutrient deprivation is executed in a nonselective fashion, and its main purpose has long been recognized as a response to adapt to nutrient-poor conditions to maintain energy homeostasis. It preserves cellular bioenergetics by providing metabolic substrates (obtained through bulk cytoplasmic degradation), which maintains macromolecular synthesis and ATP production. Mice with systemic deletion of Atg3,⁴⁴ Atg5,⁴⁵ Atg7,⁴⁶ Atg9⁴⁷ or Atg16l1,⁴⁸ die immediately after birth and show reduced amino acid levels during the neonatal starvation period.^10,17,49 Although the exact reason for neonatal death in those knockout mice is still controversial, it demonstrates explicitly that amino acid supply through autophagy is important for infant survival. Later tissue-specific gene-targeting studies have revealed that autophagy functions with lineage specification.^10,17,49,50

Recent studies, however, have shed light on other indispensable roles for starvation-independent or constitutive autophagy in cellular homeostasis. Autophagy also occurs constitutively at low levels even under nutrient-rich conditions. One of the best-characterized substrates of selective autophagy is SQSTM1/p62 (sequestosome 1). SQSTM1 directly interacts with LC3 on the phagophore through the LC3-interacting region, and then is incorporated into the autophagosome and degraded. When autophagy is defective, accumulation of SQSTM1 will enhance its function as a scaffold protein in several signaling pathways, e.g., NFKB1 signaling.^10,51 Inclusion bodies with SQSTM1 can be observed in various neurodegenerative diseases and liver disorders. Other adaptive functions of autophagy are as follows: it works as a cellular housekeeper to eliminate defective proteins and organelles, to prevent abnormal protein aggregate accumulation, and to remove intracellular pathogens; it selectively delivers microbial genetic material and antigens to the innate and adaptive immune systems; it functions as a tumor suppressor in nontumor cells, but it favors tumor progression once tumors are established,^52,53 owing to an increased metabolic demand; it also interplays with life/death decisions of the cell by self-cannibalism. Accordingly, it protects humans against diverse pathologies, including obesity, diabetes, neurodegeneration, aging, heart disease, infections, cancer and AID.^10,16,17,54

Autophagy and Immunity

It is suggested autophagy has three principal types of contributions to the function of the immune system.⁵⁵ (1) Type I: Specialized immune processes. For example, by a process called xenophagy,⁵⁶ pathogens can be captured and eliminated. This will lead to pattern recognition receptors capture, and enhanced MHC presentation of antigens, including auto-antigens in some circumstances.⁵⁷ (2) Type II: Generic role in cellular homeostasis. This refers to the role of autophagy in controlling cellular homeostasis in all cell types. Intracellular organelles and energy will be normally controlled but with lineage specification as mentioned. For example, T cells are more dependent on autophagy whereas B2 cells are less sensitive. (3) Type III: Isolated ATG functions. This refers to gene-specific function instead of implementation of the entire autophagy pathway. For example, ATG12–ATG5 has been implicated in the inhibition of retinoic acid-inducible gene 1-like receptor (RLR) signaling, whereas ATG9 has been implicated in negatively regulating TBK1.

The many presently recognized roles of autophagy in innate and adaptive immunity have been steadily increasing in complexity (Fig. 2). There are complicated feedback loops between the autophagy pathway/proteins and immunity/inflammation; the autophagy proteins function in both the induction and suppression of immune and inflammatory responses, and, vice versa, immune and inflammatory signals function in both the induction and suppression of autophagy.^{5,7-9,55,57-62}

Figure 2. The multifaceted roles of autophagy in immunity. The many currently recognized roles of autophagy in innate and adaptive immunity have been steadily increasing in complexity. Normal autophagy function contributes to balanced immunity responses, resulting in protection against disease. Imbalanced autophagy results in maladaptive responses and more severe disease. Here “diseasome” stresses more on convergence of different diseases as “every road leads to Rome” in which autophagy may be a common pathway.

Autophagy as an Innate Immunity Mechanism

In keeping with its primary function as a cytoplasmic scavenger process, autophagy serves as a mechanism for the removal of intracellular pathogens, in a regulated manner. Autophagy proteins have diverse functions to benefit the host in the removal of invading pathogens, through xenophagy, phagolysosomal maturation, the recruitment of molecules that damage pathogen-derived membranes, and, possibly, many other as-yet undiscovered mechanisms.^63,64 Beyond targeting intracellular pathogens for degradation, other functions such as the control of downstream inflammatory signaling by an increase in NFKB1-dependent cytokine production, ROS production, production of antimicrobial peptides, cell-cell receptor-ligand interactions,⁶⁵ and necrotic cell death in autophagy-deficient cells⁶⁶ may have further beneficial effects in infected host cells. In signaling, diacylglycerol is beneficial to autophagy induction, whereas cAMP/PKA signaling may have the inverse effect.^67,68 In addition, the autophagy pathway has a crucial role in resistance to infection by intracellular pathogens. The genetic deletion, mutation or knockdown of autophagy genes prevents viral, fungal and bacterial infection. For example, in humans, recent studies support a link between autophagy and resistance to tuberculosis^69-71 and leprosy.^58,72,73

Although autophagy is a component of host defense, various pathogens have evolved several ways to adapt to host autophagy as reviewed elsewhere,^9,64,74 and even for their own self-serving purposes, e.g., viral replication.^75-77 Bacteria can inhibit lysosomal fusion or maturation by residing in phagosomes, or camouflage themselves to avoid autophagic recognition. Such disguise or escape may shed light on a fundamental aspect of autoimmunity, i.e., discrimination between self and nonself.

Recent studies revealed overlaps between autophagy and innate immune signaling via TBK1 and IκB family of kinases, underscoring the cooperation of pro-inflammatory, immune and physiological signaling pathways in the control of autophagy.^57,78-80 Other intersections between autophagy and innate immunity come from studies on pattern recognition receptors, including pathogen-associated molecular patterns: Toll-like receptors, Nod-like receptors, RLRs; DAMPs (damage-associated molecular patterns): self-DNA-containing complexes, HMGB1 (high mobility group box1), IL1B/IL-1β, defensins; SLRs (SQSTM1-like receptors): SQSTM1, NBR1, CALCOCO2/NDP52;^57,81immunity-related GTPases: immunity-related GTPase family, M (IRGM);⁸² and inflammasomes^83-85 (Fig. 2). The details of how pattern recognition receptors connect to autophagy have been elegantly reviewed elsewhere.^9,57,86 Pattern recognition receptors detect microbial components and induce innate immune responses; however, aberrant activation of immune responses often causes massive inflammation, leading to onset and/or promotion of autoimmune diseases.^87-90 Of interest, autophagy often has a dual function. That is, SLRs endow autophagic machinery with the capacity to find its microbial targets in the cytoplasm, whereas they can, via proinflammatory signaling, induce other innate immunity mechanisms of the cell (e.g., promoting NFKB1 induction and CASP8 aggregation, activation and cell death);⁹¹ DAMPs such as HMGB1 and IL1B can induce autophagy, whereas induction of autophagy can also promotes secretion of HMGB1 and IL1B.^92-94 Such forward loops may explain some of the reasons why inflammation or autoimmunity persist and amplify.

Neutrophil extracellular traps (NETs) composed of DNA, histones and neutrophil antimicrobial peptides can trap and kill various bacterial, fungal and protozoal pathogens, and their release is one of the first lines of defense against pathogens.⁹⁵ Recent data suggest NETosis, a cell death program also called NET cell death, is associated with the formation of autophagosomes. Autophagy inhibition blocks the intracellular chromatin decondensation, which is necessary for NET formation and NETosis.^96,97 NETs have recently been associated with infections, certain autoimmune diseases, such as SLE, anti-neutrophil antibody-associated small vessel vasculitis and autoinflammatory chronic granulomatous disease.^98-100 In addition, autophagy may be involved in neutrophil-mediated, antibody-dependent cellular cytotoxicity.¹⁰¹

Autophagy in Antigen Processing and Presentation

The major histocompatibility complex on human chromosome 6 represents one of the most important genetic loci for human autoimmune diseases. Specific alleles that differ from closely related alleles by only one or a few amino acids in the peptide binding groove are frequently strongly associated with disease susceptibility, raising the important question of which peptide presentation events are critical in disease initiation and progression.^102-104 Classically, MHC class I molecules present intracellular antigens to CD8⁺ T cells, and MHC class II molecules present extracellular antigens to CD4⁺ T cells. However, professional antigen-presenting cells, such as dendritic cells, can process extracellular antigens for MHC class I presentation by a pathway called cross-presentation.¹⁰⁵ Vice versa, some nuclear and cytosolic antigens can be presented by MHC class II after intracellular processing.^106,107

Autophagy has been implicated both in intracellular and extracellular antigen processing for MHC class II presentation to CD4⁺ T cells.¹⁰⁶ Autophagosomes fuse with lysosome to form autolysosomes, and can also fuse with late endosomes, such as multivesicular bodies, and MHC class II loading compartments, resulting in MHC class II presentation of the autophagic cargo.¹⁰⁸ In human B lymphoblastoid cell lines, mass spectrometry analysis of the MHC class II ligandome shows that 20% to 30% of self-class II epitopes are derived from cytosolic and nuclear proteins.¹⁰⁶ This self-antigen presentation via autophagy might be involved in autoimmune diseases.¹⁰⁹ Recent studies indicate self-derived,¹¹⁰ pathogen-derived,^111,112 and tumor antigens¹¹³ can be presented by autophagy. Autophagy might assist with extracellular antigen processing through pathogen associated molecular patterns, i.e., toll-like receptors¹¹⁴ and NOD-like receptors.¹¹⁵

The vast majority of peptides presented on MHC class I molecules are generated by the proteasome in the cytosol. However, some studies also suggest that autophagy might even assist MHC class I antigen presentation to CD8⁺ T cells.^116,117 It was suggested that antigens could be degraded by proteases in the lytic vacuole after autophagy. Peptides generated in the lytic vacuole might return to the cytosol and then enter the classical pathway after proteasome trimming, or alternatively, they could stay in the endovacuolar pathway and be loaded onto MHC class I molecules. This mechanism seems to be induced during viral infection, and requires more time and more antigen than the classical pathway involving the proteasome in the cytosol. Thus, this process might be limited to times of “cellular emergency.”¹¹⁸

Autophagy and T Cells

One major mechanism of tolerance induction is based on the elimination of autoreactive T cells in the thymus. The proposed model for how autophagy influences T cell repertoires accounts for the dual role of thymic epithelial cells, which play a role via endogenous antigen presentation in the MHC II-restricted fashion in both positive and negative selection.¹¹⁹ By transplantation of Atg5^−/− thymi into TCR transgenic or athymic recipient mice, positive selection of some but not all CD4⁺ T cell specificities and for all CD8⁺ T cell specificities is observed and Atg5 deficiency shapes the T cell repertoire toward multi-organ inflammation. Significantly, organs that appear particularly susceptible to autophagy-dependent autoimmunity show high baseline autophagy, which enhances endogenous MHC II antigen presentation, suggesting that an increase in MHC II-restricted presentation of self antigens has to be matched by autophagy-dependent tolerogenic selection mechanisms in the thymus.

Once T cells depart from the thymus, autophagy plays a role in their survival and homeostasis. A significant decrease in peripheral T and B lymphocyte numbers but with no alterations in the numbers of the myeloid lineage cells has been observed in Atg3-knockout, Atg5-knockout and Atg7-knockout mice.^120,121 Defective clearance of mitochondria,^122-126 and growth factor withdrawal¹²⁷ may be plausible explanations. In addition, although autophagic cell death is still a matter of debate, there is a report that T cells showing excessive autophagy undergo cell death with an autophagic component.¹²⁸

Autophagy and B Cells

Most B cells in peripheral lymphoid tissues are produced in adult bone marrow and are referred to as B-2 cells, which are generally considered to be mediators of adaptive immunity. A minor B cell subset called B-1 cells is part of the innate immune system.^129,130 It has been suggested that B-1a, B-1b and B-2 B cells may develop from distinct cell lineages.¹³¹ A study using B cell-specific deletion of Atg5 in mice has shown that autophagy plays a role in B cell development and homeostasis.¹³² The data indicate that ATG5 is required for the maintenance of B-1a B cells, which normally have self-renewal capacity, but not B-1b or B-2 B cells, although sufficient pre-B cells survive to populate the periphery. The reason might be due to a role for ATG5 in cytokine-driven differentiation or cell survival after growth factor withdrawal.

A few studies also suggest that autoantibodies can stimulate autophagy. For example, sera from type 2 diabetic patients with neuropathy or a subpopulation of patients with neurogenic chronic intestinal pseudo-obstruction,^133-137 are associated with increased levels of autophagosomes involving the FAS receptor complex. Similar autophagy activation was also observed in SH-SY5Y neurons treated by sera from active lupus patients.¹³³ These data might allow the hypothesis that autoimmune diseases correlate with unbalanced autophagy. But, to date, no clear link and precise mechanisms have been resolved with regard to autoantibodies from classical autoimmune disease in mediating autophagy.

Autophagy and Cytokines

Autophagy is regulated by immunologically relevant cytokines and cell surface receptors, with activation by IFNG/IFNγ, TNF/TNFα, NFKB, monocyte chemoattractant protein 1(CCL2),¹³⁸ Fcγ receptors, CD46, CD40 and inhibition by IL4, IL13, CLCF1, LIF, insulin-like growth factor-1 (IGF1), FGF2, CXCL12/SDF1, NFKB, STAT3 and BCL2.⁹ As IFNG, a major Th1 cytokine, induces autophagy, whereas the Th2 cytokines IL4 and IL13 inhibit autophagy, it may demonstrate that autophagy is an effector of Th1/Th2 polarization, explaining why Th1 cytokines are protective and Th2 cytokines permissive when it comes to controlling intracellular pathogens.⁶¹ Lines of evidence supporting the role of inflammatory cytokines in the regulation of autophagy may even indicate that the pro-inflammatory cytokine patterns usually observed in chronic autoimmune disorders ultimately disrupt the balance of autophagy in the affected organ.⁶² Also, autophagy has positive contributions to the biogenesis and secretion of the proinflammatory cytokines such as IL1B,⁹² IL18, adipocytokines,^139,140 type I IFN,¹⁴¹ TNF,¹⁴² IL7Rα chain,¹⁴³etc., which may be a possible explanation for the vicious pathogenic cytokine milieu often observed in infectious and autoimmune diseases.

Autophagy in Autoimmune and Autoinflammatory Diseases

The multitiered connections between autophagy and immunity uncovered to date strongly supports the idea that autophagy may exert both beneficial and aggravating effects on immunity and inflammation. Such a switch between the dual functions of autophagy in disease has become a priority when considering disease pathogenesis, and further its potential therapeutic implications. Of utmost importance, an imbalanced function of autophagy has recently been linked to the causation and prevention of autoimmune and/or autoinflammatory diseases, which strongly supports a role of autophagy at the heart of immunity rather than as a bystander.

Example of an autoinflammatory disorder: Crohn disease

Crohn disease (CD) has widely been described as an autoimmune disease, and a newer view describes it as a disease of immune deficiency.^144,145 This suggests an autoinflammatory rather than autoimmune origin of inflammation in Crohn disease.¹⁴⁶ Autoinflammatory disease is a brand new category of diseases that have several common characteristics of autoimmunity without involving autoantigen, marked by abnormally increased inflammation, mediated predominantly by the cells and molecules of the innate immune system, with a significant host predisposition. Both autoinflammatory diseases and autoimmune diseases result in flares of inflammation.^147,148 A combination of environmental factors and genetic predisposition is believed to cause CD. And plenty of genetic risk factors have been discovered.^149-151 Malfunctions in the innate immune system and Th17 system have been addressed. However, the discovery of the autophagy genes ATG16L1 and IRGM as risk factors for CD put autophagy in the spotlight in its pathogenesis.^152-169

A single-nucleotide polymorphism (SNP) of ATG16L1 (rs2241880, threonine300 is replaced by alanine, T300A) has been associated with CD with evidence from several northern Caucasian CD cohorts.^{150,151,166,167} However, its role in the pathogenesis of CD disease is not simple; the association was not observed in Asian populations.¹⁷⁰ For specific CD subphenotypes, it appears to be associated only with disease of the small bowel.¹⁷¹ So far, very little information is currently available about the functions of the human ATG16L1 protein, and even less about the consequences of its variations. Threonine 300 is located in the C-terminal WD repeat domain in ATG16L1, suggesting T300A would modify Atg16L1 conformation and interaction and be sufficient for conventional autophagy. However, T300A variants are fully competent in the formation of autophagosomes in both constitutive and starvation-induced conditions in ATG16L1^−/−fibroblasts.¹⁷²One study suggested that the ATG16L1T300A mutant is defective in autophagic sequestration of intracellular bacteria but not in canonical autophagy.¹⁷² Another study, however, suggested that this mutant has intact activity in both canonical autophagy and antibacterial autophagy.¹⁷³ This subtle consequence of the T300A variation might be critically dependent on the cell system examined: human gut epithelial Caco2 cells¹⁷² vs. murine fibroblasts.¹⁷³ ATG16L1-deficient mice die within the first day of delivery, however, ATG16L1 hypomorphic mice, which express a very low level of Atg16L1, are viable and grossly normal but exhibit structurally abnormal Paneth cells with cytoplasmic lysozyme staining. These data suggest ATG16L1 has a role in maintaining the normal function of Paneth cells.^139,174 ATG16L1-deficient Paneth cells exhibit not only defective granule exocytosis but also an unexpected gain of function including increased expression of genes involved in peroxisome proliferator-activated receptor signaling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Similarly, CD patients homozygous for the ATG16L1 CD risk alleles display comparable Paneth cell granule defects and increased leptin levels.¹³⁹ Further study suggests that CD can be determined by a virus-plus-susceptibility gene interaction in combination with additional environmental factors and commensal bacteria, showing a possible reason why the disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes.¹⁷⁴ In addition to these cell-intrinsic mechanisms, a link between autophagy and the immune system can also contribute to the pathogenesis of CD. This link occurs through a direct interaction between ATG16L1 and NOD2/CARD15 (which has the strongest association with Crohn disease) in the bacterial sensing pathway,^175,176 the regulation of inflammatory cytokine production^48,177 and antigen presentation.¹¹⁵

Mice that are null for Irgm1 develop normally, have no apparent phenotype and, yet, they are extremely susceptible to bacterial infection, all dying at 11–16 weeks postinfection. Previously, two polymorphisms of IRGM have been highly correlated with CD risk: a tag-SNP variation within the coding region (rs10065172, C313T);¹⁷⁸ and a 20-kb deletion upstream of the IRGM gene.¹⁷⁹ They are in perfect linkage disequilibrium (r² = 1.0). Follow-up genetic studies of IRGM have revealed unexpected complexity. Resequencing of the IRGM open reading frames did not reveal any disease-associated coding variants, suggesting that the causal variants are regulatory rather than structural.^179,180 A recent study shows rs10065172 alters a binding site for MIR196, which is strongly increased in colonic epithelial cells of individuals with CD compared with controls.¹⁸¹ Downregulation of IRGM seen in colonic epithelial cells of individuals homozygous for the protective allele (rs10065172 C) is mediated by MIR196, but that is not seen in carriers of the risk variant (rs10065172 T) because the T allele disrupts the target site. Hypothetically, increased IRGM expression in colonic mucosa should be protective. However, IRGM overexpression actually reduces the efficacy of xenophagy, as an increase in the number of surviving intracellular bacteria is observed. Effective xenophagy may thus require optimal IRGM expression levels, both overexpression and underexpression being potentially harmful.¹⁸² Alternatively, overexpression of specific IRGM isoforms induces cell death,⁸² which could be the main inflammatory trigger. It is possible that the rs10065172 T allele contributes to the maintenance (rather than initiation) of chronic inflammation in the gut by disrupting a MIR196-dependent autoregulatory loop controlling IRGM expression, thereby promoting epithelial apoptosis.¹⁸³ Further genetic evidence, combined with functional data, is needed, as the association can also be observed with susceptibility to tuberculosis in African Americans,¹⁸⁴ but cannot be detected in Asian CD patients,¹⁸⁰ which may reflect gene-by-environment or gene-by-gene interactions.

Along these lines, it would be critical to explore further genetic associations given that autophagy is a pathway, and interactions between the environment and the autophagic risk factors. A recent study thus suggests an association between a tSNP (rs12303764) in ULK1 and risk of CD.¹⁶³ However, no confirmed epistasis has been derived to date, in spite of a weak interaction with respect to ATG16L1 and NOD2 susceptibility variants.¹⁶⁷ In addition, the lack of association of these variants with CD in the Asian populations is intriguing.

Example of an autoimmune disease: Systemic lupus erythematosus

The pathogenic mechanisms of SLE—a prototypic systemic autoimmune disease— are incompletely understood, but are postulated to involve multiple diverse aspects in the dysregulated immune response system. Recent genome-wide association studies have improved our understanding of the genetic basis of SLE, especially since they have identified and robustly replicated several novel loci, which implicate newer layers of immunological disturbances or pathways involved in disease pathogenesis. Among the many convincingly established genetic associations, genetic variants within or near ATG5—a key autophagy gene required for the formation of autophagosomes—have been associated with SLE both in Caucasian and Chinese populations,^185-188 which suggested that autophagy may also be involved in SLE pathogenesis.

However, both the genetic data and the pathogenesis mechanisms are insufficient. The PRDM1 (PR domain containing 1, with ZNF domain, also known as BLIMP1) -ATG5 gene region is in low linkage disequilibrium and associated patterns are diversified between Caucasians and Chinese. Thus, it seems that multiple independent signals are associated with SLE, including rs573775 (ATG5 intron 1),¹⁸⁵ rs2245214 (ATG5 intron 6)¹⁸⁶ and intergenic region of PRDM1-ATG5 (rs548234, rs6937876 and rs6568431).^185-188 Although replication studies confirmed the association of ATG5 gene variants with SLE from Caucasians,^185,186 the consistent association signal in both Caucasians and Chinese is from the intergenic region of PRDM1-ATG5,¹⁸⁸ which may obscure the role of ATG5 in SLE. In spite of this, eQTL analysis both in immortalized lymphoblasts and in lymphocytes from Chinese lupus patients as well as healthy controls, supported the idea that polymorphisms within the intergenic region of PRDM1-ATG5 may be through upregulating ATG5 gene expression, giving clues that ATG5 is a candidate gene of SLE.¹⁸⁸ Genotypes of intergenic gene polymorphisms correlated with ATG5 (cis-QTL effect) and its downstream gene expressions (trans-QTL effect), including many autophagy genes, type I IFN-related genes, apoptosis-related genes and NFKB-related genes, suggesting ATG5 has a multifaceted function in lupus pathogenesis (Fig. 2).¹⁸⁸ Anyhow, the genetic mechanism by which intergenic gene polymorphisms affects gene expression is lacking and a further fine mapping of ATG5 is urgent. From the viewpoint that autophagy is a pathway, the study also suggests that ATG7 and IRGM are susceptibility genes to SLE, and positive gene-gene interactions occur among ATG5, ATG7 and IRGM.¹⁸⁸ The association between IRGM gene polymorphisms and SLE can be also derived from a meta-analysis conducted between SLE and 16 autoimmune diseases, which aims to detect shared and disease-specific genetic loci.¹⁸⁹ All the above strongly indicate that autophagy is involved in SLE pathogenesis. The upregulation of ATG5 is consistent with the observations that complement-inactivated sera from patients of SLE can significantly activate autophagy¹³³ and that ATG5 expression elevates in autoimmune demyelination and multiple sclerosis (MS).¹⁹⁰

ATG5 may participate in the pathogenesis of SLE at multiple levels (Fig. 3).¹⁹¹ It may be involved in pathogen clearance, ultraviolet (UV) irradiation resistance, and many other environmental trigger protections. For example, Atg5^−/− fibroblasts are resistant to death from UV light.¹⁹² This protein may be involved in the activation of innate immunity by delivering viral nucleic acids to endosomal compartments containing toll-like receptor 7, which signals the induction of type 1 IFN production—thus playing a central role in SLE pathogenesis.^193-195 Plasmacytoid dendritic cells from Atg5 knockout mice produce less IFNA/IFNα in response to viral infections, which may underscore a role for viral and bacterial triggers in SLE.¹⁴¹ Many other PPRs like HMGB1,¹⁹⁶ and DEFENSIN,¹⁹⁷ whose expression are higher in lupus patients, can activate autophagy as well.⁹³ The lack of efficient handling of apoptosis, secondary necrosis, and exposure of chromatin fragments may overcome tolerance to self-antigens and lead to SLE. Mice with dendritic cell-conditional deletion in Atg5, show a pronounced defect of processing and presentation of phagocytosed antigens containing toll-like receptor stimuli for MHC class II.¹¹² A more recent study directly investigating autophagy in T lymphocytes from both lupus-prone mouse models and human lupus patients by LC3 conversion assays and electron microscopy experiments indicated autophagy increased in lupus compared with that in normal controls and nonlupus autoimmune diseases. The data further support the idea that autophagy is genetically deregulated in lupus, promoting survival of autoimmune T cells.¹⁹⁸ Persistence of autoreactive T and B cell clones and their imbalanced crosstalk can also be interfered with by ATG5.^{124,132,191,199-201} Excessive inflammation and tissue damage may further be sustained by production of inflammatory cytokines,²⁰² the recruitment of immune cells,^203-205 and enhanced oxidative stress.^206,207 In addition, nonimmune mechanisms may also contribute to end-stage organ damage.²⁰⁸ For example, podocyte-specific deletion of Atg5 leads to a glomerulopathy, whereas human renal biopsies of patients with acquired proteinuric diseases often show elevated ATG3 expression, suggesting autophagy as a key homeostatic mechanism to maintain glomerular injury.²⁰⁹ As lupus nephritis is a common characteristic of SLE, further organ or tissue intrinsic mechanisms involving autophagy are also needed.

Figure 3. The possible role of ATG5 in systemic lupus erythematosus (SLE). An overview of the many possible mechanisms by which ATG5 gene polymorphisms may contribute to the pathogenesis of a type of autoimmune disease.

Of note, as discussed above, ATG5 may have its own autophagy-independent functions/Type III functions. It is suggested that autophagy-independent functions include: being a pro-apoptotic molecule translocating to mitochondria to trigger mitochondrial outer membrane permeabilization;^210,211 to function as a suppressor of innate immune signaling in the form of the ATG12–ATG5 conjugate by interactions with the caspase recruitment domain of retinoic acid-inducible gene-1 (DDX58/RIG1) and interferon-β promoter stimulator (MAVS/IPS-1), contributing to RNA virus replication,²¹² but to mediate intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.²¹³ In spite of its possibly specific function in such cellular processes, it would be cautious to assume that its association with SLE is due to its nonautophagy characteristics, as current understanding of the autophagy network is incomplete. Also, the above “nonautophagy” functions relate more with innate immunity rather than perturbations of adaptive immunity, which nevertheless laid the foundation of autoimmunity. In any case, systemic evaluations of ATG5 in SLE will be necessary and critical.

In addition, recent genetic studies also associate allelic variants of the HSPA/HSP70 genes with SLE in Caucasians, and correlate with the presence of autoantibodies to CALR/Ro and SSB/La.²¹⁴ This finding may indicate that CMA is also defective in SLE patients.^215,216

Other autoimmune/autoinflammatory diseases

A few pieces of evidence are emerging to support the role of autophagy in other autoimmune disease, such as rheumatoid arthritis, diabetes mellitus, MS and other autoinflammatory diseases such as vitiligo.

Anti-citrullinated protein antibodies, which are the most specific autoantibody indicators in patients with RA, are present before clinical onset, which may indicate that the initial loss of immune tolerance to citrullinated proteins occurs as a consequence of an inflammatory event outside the joint. A recent study suggests the presentation of the citrullinated peptides but not of the unmodified peptides was associated with autophagy. The presentation was reduced by 3-methyladenine or by reduction in ATG5 expression.¹¹⁰ Thus, ATG5 may also be involved in the pathogenesis of RA. Of interest, a recent large-scale replication study on RA also associated disease susceptibility with the PRDM1-ATG5 intergenic region, and this may further suggest a common mechanism of ATG5 in autoimmunity.²¹⁷ However, such genetic association is lacking in Asian populations, either with disease susceptibility or anticyclic citrullinated peptides antibody.²¹⁷

The majority of type 1 diabetes is an inflammatory autoimmune disease of the pancreas, resulting in a lack of insulin. Type 2 diabetes mellitus is characterized by insulin resistance and failure of pancreatic β-cells producing insulin. Deficiency of insulin by β-cells, or the insensitivity of its receptors plays a central role in all forms of diabetes mellitus. Recent studies strongly suggest that basal autophagy is important for maintenance of β-cell volume and function.^218,219 Impaired autophagy has been linked with diabetes and acute pancreatitis.^219-221 This process is considered to be an adaptive response, i.e., autophagy induction contributes to the beneficial metabolic effects of exercise.²²² However, a study also indicates autophagy can be stimulated by complement-inactivated sera from type 2 diabetic patients with neuropathy, which may suggest an autoantibody-mediated autophagy pathway in the pathogenesis of demise of neuronal tissue in type 2 diabetes.

Multiple sclerosis is currently believed to be an autoimmune-mediated disorder characterized by damage of myelin and axons by focal lymphocytic infiltration.²²³ In MS, prolonged T cell survival and increased T cell proliferation have been linked to disease relapse and progression. In blood samples of experimental autoimmune encephalomyelitis mice—a widely used model of MS—elevated expression of ATG5 and IRGM1 have been observed.^190,224 ATG5 is also higher in T cells as well as brain samples from active relapsing-remitting MS patients than that in nondiseased controls. IRGM may affect the survival of mature effector CD4⁺ T cells by protecting them from IFNG-inflicted apoptotic cell death, thus delaying a homeostatic mechanism that otherwise limits the duration and strength of the cell-mediated immunity response.^55,224,225 Thus, increased T cell expression of ATG5 and IRGM1 may contribute to inflammatory demyelination in MS.

As discussed above, UVRAG activates the BECN1-PIK3C3 complex, promoting autophagy. A small nonsegmental vitiligo cohort study from Korea suggests a possible association between UVRAG (rs1458836, rs7933235) and disease susceptibility, expanding the number of current disease associated autophagy genes.²²⁶ As UV is strongly associated with SLE disease occurrence, association between UVRAG gene polymorphisms and SLE susceptibility will be of interest.

Conclusion

Recent studies highlight the role of autophagy in positively shaping the landscapes of innate and adaptive immunity. One key issue is to understand whether and how it affects immunity-related diseases. Plenty of evidence is emerging to support its role in autoimmune and autoinflammatory diseases. The most telling lines of evidence are from an unbiased method, by genome-wide association studies in human populations. Key findings include associations between polymorphisms in ATG16L1 and IRGM with autoinflammatory Crohn disease, and polymorphisms in/near ATG5 with autoimmune SLE. Clues are also surfacing to link autophagy with other autoimmune diseases, such as RA, diabetes mellitus, MS and other autoinflammatory diseases such as vitiligo. These findings will bolster the significance of the immunological role of autophagy in human disease, but also will broaden our vision and develop a scientific research foundation for the pathogenesis of autoimmune and autoinflammatory diseases.

Abbreviations:
AID	=	autoimmune diseases
ATG16L1	=	autophagy-related gene 16-like 1
CD	=	Crohn disease
CMA	=	chaperone-mediated autophagy
DAMPs	=	damage-associated molecular patterns
ER	=	endoplasmic reticulum
HMGB1	=	high mobility group box 1
IGF1	=	insulin-like growth factor-1
IRGM	=	immunity-related GTPase family M
MHC	=	major histocompatibility complex
MS	=	multiple sclerosis
NETs	=	neutrophil extracellular traps
NFKB1	=	nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
RA	=	rheumatoid arthritis
RLR	=	retinoic acid-inducible gene 1-like receptor
SLE	=	systemic lupus erythematosus
SNP	=	single-nucleotide polymorphism
TNF	=	tumor necrosis factor
UV	=	ultraviolet

Acknowledgments

This work was supported by grants from the Major State Basic Research Development Program of China (973 program, No.2012CB517700), National Natural Science Foundation of China (No. 30825021) and Natural Science Fund of China to the Innovation Research Group (81021004). We apologize to those authors whose publications could not be quoted in this review due to space limitations.