REVIEWS

Autophagy in infection, inflammation

and immunity
Vojo Deretic1, Tatsuya Saitoh2 and Shizuo Akira2
Abstract | Autophagy is a fundamental eukaryotic pathway that has multiple
effects on immunity. Autophagy is induced by pattern recognition receptors and,
through autophagic adaptors, it provides a mechanism for the elimination of
intracellular microorganisms. Autophagy controls inflammation through regulatory
interactions with innate immune signalling pathways, by removing endogenous
inflammasome agonists and through effects on the secretion of immune mediators.
Moreover, autophagy contributes to antigen presentation and to T cell homeostasis,
and it affects T cell repertoires and polarization. Thus, as we discuss in this Review,
autophagy has multitiered immunological functions that influence infection,
inflammation and immunity.

Macroautophagy
The autophagy-related protein
(ATG)-dependent pathway,
sequestering cargo into
autophagosomes that fuse
with lysosomes, in which
the cargo is degraded.
Proteins, organelles (such as
mitochondria) and invading
microorganisms are selectively
degraded by macroautophagy.
Bulk cytoplasmic autophagy
occurs as a starvation
response.
1
Department of Molecular
Genetics and Microbiology,
University of New Mexico
Health Sciences Center, 915
Camino de Salud, North East,
Albuquerque, New Mexico
87131, USA.
2
Laboratory of Host Defense,
World Premier International
Immunology Frontier
Research Center, Osaka
University, 3–1 Yamada-oka,
Suita, Osaka 565–0871,
Japan.
Correspondence to V.D.
e‑mail:
vderetic@salud.unm.edu
doi:10.1038/nri3532
The term autophagy (from the Greek words auto
meaning ‘self ’ and phagein meaning ‘to eat’) refers
to a collection of diverse processes — including
macroautophagy, microautophagy, chaperone-mediated
autophagy1 and non-canonical autophagy2 — that enable
cells to digest their cytoplasmic contents in lysosomes.
Macroautophagy, which is autophagy in its strictest
form and which is the focus of this Review, depends
on specialized autophagy-related proteins (ATGs)1
(BOX 1) and is distinct from other cytoplasmic diges-
tive processes, including proteasomal degradation, as
a result of its ability to capture and to eliminate large
targets such as toxic protein aggregates, defunct or dis-
used organelles and invading microorganisms. Several
autophagy-related factors have also been recognized to
have autophagy-independent immune functions3, but
these are not discussed in this Review.
Autophagy may be the primordial form of eukary-
otic innate immunity against invading microorganisms.
Our understanding of the immunological functions
of autophagy has dramatically increased in recent
years4, and it is now appreciated that, in mammals,
these primordial functions of autophagy have evolved
and have been incorporated into multiple innate and
adaptive immune pathways. In this Review, we dis-
cuss the four main roles of autophagy in immunity:
the direct elimination of microorganisms, the control
of inflammation, the control of adaptive immunity
through the regulation of antigen presentation and
lymphocyte homeostasis, and the secretion of immune
mediators (FIG. 1).
The autophagy pathway
ATG-dependent autophagy. The principal morphologi-
cal feature of autophagy is the formation of endomem-
branous organelles, which are known as autophagosomes
(BOX 1). An ensemble of ATG factors drives the formation
in the cytoplasm of the autophagic isolation membrane
(also known as the phagophore)1 (BOX 1). Beclin 1 (the
mammalian orthologue of the yeast protein ATG-6), the
serine/threonine protein kinase ULK1 (the mammalian
paralogue of the yeast protein ATG-1), autophagy-
related LC3 proteins and γ-aminobutyric acid recep-
tor-associated proteins (GABARAPs; the mammalian
paralogues of the yeast ATG-8 protein) are key regula-
tors of phagophore formation1 (BOX 1). A phagophore,
which is often depicted in models as an isolated crescent
in the cytoplasm, is transiently connected to and derived
from phosphatidylinositol-3‑phosphate (PtdIns3P)-
positive domains of the endoplasmic reticulum (ER),
which are known as omegasomes 1. Other cellular
compartments, such as the Golgi apparatus, the mito-
chondria and the plasma membrane-derived endocytic
organelles, also contribute to phagophore formation1.
Recent studies suggest that the ER‑derived autophago-
somes form at ER–mitochondria contact sites5. A pha-
gophore sequesters the captured cytoplasmic cargo
that is destined for autophagic disposal and, following
elongation and closure, an autophagosome is formed.
At this stage, the corresponding endomembranes are
commonly visualized as double membrane structures1.
The degradation of the captured cargo begins when the
double membrane autophagosome matures into a single

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Box 1 | The autophagy pathway

Autophagy is controlled by the regulatory serine/threonine protein kinases ULK1 and ULK2 (which are the mammalian homologues of the yeast
autophagy-related protein 1 (ATG‑1)) and by the lipid kinase activity of the class III phosphatidylinositol-3‑phosphate kinase (PtdIns3K) VPS34 (VPS‑34 in
yeast), which forms a complex with beclin 1 (which is the mammalian orthologue of yeast ATG‑6) and ATG14‑like protein (ATG14L). ULK1, ULK2 and the
VSP34–beclin 1–ATG14L complex integrate upstream signals. These factors, which are engaged in the activation of autophagy during nutritional and
immune responses, induce the downstream ATG conjugation cascade1. This involves the association of the ATG5–ATG12 conjugate with ATG16L1. The
ATG5–ATG12–ATG16L1 complex facilitates the addition of a phosphatidylethanolamine group to the carboxyl terminus of the mammalian paralogues
of ATG-8: LC3A, LC3B (which has been used as a marker for the identification
of autophagosomes in mammalian cells), LC3C, γ-aminobutyric acid Donor membrane
receptor-associated protein (GABARAP), GABARAP-like 1 (GABARAPL1) • ER omegasome
and GABARAPL2 (REF. 1). Lipidated mammalian paralogues of ATG-8 function • Golgi apparatus
together with other factors to assemble, elongate and lead to the closure • ER–mitochondria contact sites
• Endosomes
of nascent autophagic organelles1 (see the figure). The lipid kinase VPS34, • Plasma membrane
through its production of PtdIns3 phosphate (PtdIns3P), has a dual function in
autophagy1. During initiation, VPS34‑derived PtdIns P is recognized by the Initiation
3
PtdIns3P-binding factors WD repeat domain phosphoinositide-interacting Isolation
ULK1 ATG16L1
protein 1 (WIPI1)–WIPI4 (which are the mammalian paralogues of yeast membrane
ATG‑18), and these factors cooperate with ATG2 and ATG9 to form the Beclin 1 VPS34 ATG5 ATG12
phagophore1. During the final maturation of autophagosomes into LC3
ATG14L
autolysosomes, the attachment of a tail-anchored SNARE, syntaxin 17 LC3A GABARAP
(REF. 6), to the autophagosomal membrane enables fusion with lysosomes.
WIPI ATG2 Elongation LC3B GABARAPL1
At this stage, the VPS34–beclin 1 complex contains ultraviolet radiation
ATG9 and closure
resistance‑associated gene protein (UVRAG) instead of ATG14L1. LC3C GABARAPL2
Autophagy requires the rapid remodelling of endomembranes in the
cytoplasm, which involves the formation of transient PtdIns3P-positive
structures (known as omegasomes) on the endoplasmic reticulum (ER), Autophagosome
the ER–mitochondria contact sites, the Golgi apparatus and the plasma Lysosome
membrane-derived endosomal organelles. This happens independently of
transcriptional changes. However, transcriptional adjustments are necessary Syntaxin 17
for sustained autophagy, for instance, to replenish ‘consumables’ such as LC3 Fusion
and autophagic adaptors including sequestosome 1 (also known as p62), which Maturation
are degraded along with the captured cargo in autolysosomes. Transcriptional
adjustments are accomplished by transcription-positive regulators (for Beclin 1 VPS34
example, transcription factor EB (TFEB), forkhead box protein O3A (FOXO3A) UVRAG
Autolysosome
and NFE2‑related factor 2 (NRF2)) and transcription-negative regulators (for HOPS
example, zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3; also
known as ZNF306), which coordinate autophagy with lysosomal, proteasomal,
lipolytic and oxidative stress response systems138,139.
Degradation
HOPS, homotypic fusion and vacuole protein sorting.
Nature Reviews | Immunology

membrane-delimited autolysosome. Autophagosomal The ancient defence mechanism of autophagy has

Microautophagy
Lysosomal import and
degradation of small
portions of the cytoplasm
that is independent of
autophagy-related proteins
(ATGs), and that is often
manifested as invaginations of
the lysososomal membrane
into the lysosomal lumen.
Chaperone-mediated
autophagy
The import and degradation of
soluble cytoplasmic proteins
by chaperone-dependent
direct translocation across
the lysosomal membrane.
maturation is induced by the translocation of the SNARE
protein syntaxin 17 (REF. 6) to the outer membrane of
the completed autophagosome. This facilitates fusion
of the autophagosome with lysosomes5,6 and results
in the acidification of the autophagosome lumen, the
acquisition of lysosomal hydrolases and the degradation
of the cargo as well as the inner of the two membranes1.
Immune signalling and autophagy. A deficiency of
intracellular nutrients because of competition from
invading microorganisms is likely to have been one
of the primordial danger signals that was available
to eukaryotic cells to detect microbial invasion and to
eliminate pathogens through autophagy. Consistent
with this idea, metabolic signalling downstream of
amino acid starvation has been associated with anti-
microbial autophagy in response to bacterial7 and viral
infection8 (FIG. 1a). Metabolic regulation by mammalian
target of rapamycin (mTOR), which inhibits autophagy,
and AMP-activated protein kinase (AMPK), which acti-
vates autophagy, has been covered in detail elsewhere1.
been well integrated with other immune-sensing sys-
tems. Pattern recognition receptor (PRR) signalling
that is induced following the recognition of pathogen-
associated molecular patterns (PAMPs) and damage-
associated molecular patterns (DAMPs) can activate
autophagy (BOX 2); for example, Toll-like receptor 4
(TLR4) signalling leads to ubiquitylation of the ATG
beclin 1 by the E3 ligase TNF receptor-associated fac-
tor 6 (TRAF6)9. Ubiquitylated beclin 1 is released from
its inhibitor B cell lymphoma 2 (BCL‑2) and becomes
active9. TRAF6 also activates ULK1 through ubiquityla-
tion10 and thereby controls both key pathways that lead
to autophagy (BOX 1). TRAF6, ubiquitylated ULK1 and
the IκB kinase (IKK) regulators TAK1‑binding protein 2
(TAB2) and TAB3 also have roles in starvation-induced
autophagy (BOX 2), which supports the idea that a simi-
lar circuitry, with some variations, becomes induced
by nutritional and innate immune signals. In BOX 2,
we propose a model by which immune and nutritional
triggers converge in common signalling pathways to
activate autophagy.

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a Elimination of microorganisms b Control of pro-inflammatory signalling

Bacteria Virus Virus

Ubiquitin DAMPs ATG12

K+ efflux RIG-I
IPS1 ATG5
Amino acid TLR
starvation
Galectin Damaged
SLR
mitochondria

mTOR TRAF6 NOX2 NOD2 Viral

ssRNA
ROS ATG16L1 Leak of
lysosomal
protease ROS mtDNA
LC3 SLR

NLRP3 AIM2
inflammasome inflammasome
Endosome
Auto-
phago- Delivery of
somes PAMPs to PRRs IL-1β
IL-18
Autophagy LAP Xenophagy TLR7 or SLR
TLR8 Failing
autophagy

TRAF6 Lysosome
Lysosome

Pro-inflammatory
mediators Autolysosome
Autolysosome

c Adaptive immunity d Secretion of immune mediators

Regulated Constitutive Unconventional
Particulate
secretion secretion secretion
antigen
Cathepsin K
B cell effects (in osteoclasts) and DAMPs (e.g. IL-1β,
Conventional • HSC self renewal lysozyme (in Paneth cells) IL-6 and IL-8 IL-18 and HMGB1)
MHC class I • B1 cell development
presentation • Plasma cell survival
and IgG secretion

Cytosolic T cell effects

proteins • Thymic selection of
T cell repertoire ATGs
DICER • T cell maturation
AGO (clearance of
Citrullinated mitochondria and ER) ?
antigen • T cell survival after TCR
activation
miRNA
• Immunological synapse
destabilization
Autophagy NOD2 LAP mRNA
Secretory
Innate immune cell lysosomes
effects
Proteins • Control of activation ATGs
and release of IL-1α and
IL-1β (which influences
T cell polarization to
TH17 cell phenotype)
Proteasome Golgi apparatus
MHC
class II ER

ER
Unconventional
MHC MHC class I
class I presentation

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Non-canonical autophagy DAMPs, such as DNA complexes11, ATP12 and high-
Macroautophagy that mobility group box 1 protein (HMGB1)13, also activate
has been reported to occur autophagy. HMGB1 derepresses beclin 1 by displacing
independently of one or its negative regulator BCL‑2 (BOX 2), and can also extra-
more components of the
autophagy-related protein
cellularly activate autophagy by interacting with its cell
(ATG) system. It should not be surface receptor RAGE (receptor for advanced glycation
confused with non-canonical end products).
functions of ATGs, which Inflammatory cytokines are also involved in the
refers to the participation
activation of autophagy (BOX  2). Indeed, the induc-
of individual ATG factors
in processes other than
tion of autophagy by the pro-inflammatory cytokine
autophagy. interleukin‑1β (IL‑1β) is crucial for the control of
Mycobacterium tuberculosis in infected macrophages14.
IL‑1β signals through the IL‑1 receptor (IL‑1R)14 and
◀ Figure 1 | Four principal roles of autophagy in immunity.  a | The role of autophagy
in the elimination of microorganisms is shown. An incoming microorganism can induce
autophagy by competing for nutrients or by stimulating innate immune receptors,
such as Toll-like receptors (TLRs). When the microorganism is taken up by phagocytosis
and remains in an intact vacuole, an autophagic process termed LC3‑associated
phagocytosis (LAP) can promote the maturation of autophagosomes into autolysosomes.
Xenophagy of pathogens that enter the cytosol can be initiated by sequestosome 1‑like
receptors (SLRs) or other mechanisms, including nucleotide-binding oligomerization
domain-containing protein 2 (NOD2)–autophagy-related protein 16‑like 1 (ATG16L1)
interactions. b | Several examples of the role of autophagy in the control of
pro-inflammatory signalling (see also FIG. 3) are shown. Failure to remove SLRs by
autophagy can increase the levels of these receptors and the levels of pro-inflammatory
signalling. Autophagy can deliver cytoplasmic pathogen-associated molecular patterns
(PAMPs) to endocytic TLRs and can stimulate their activity. NOD-like receptors (NLRs;
such as NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3)) and RIG‑I‑like
receptors (RLRs; such as absent in melanoma 2 (AIM2)) show complex positive and
negative co‑regulation with autophagy: the ATG5–ATG12 complex inhibits retinoic
acid-inducible gene I (RIG‑I) signalling, and autophagy limits inflammasome activation
by removing damaged mitochondria, which then release the inflammasome activators
reactive oxygen species (ROS) and mitochondrial DNA. c | The role of autophagy in
adaptive immunity is shown. Autophagy can increase the MHC class II presentation
of cytoplasmic antigens, including self or viral antigens, as well as promoting the
citrullination of antigens. LAP can enhance the processing of particulate antigens
for MHC class II presentation. NOD2 enhances autophagic antigen presentation.
Autophagy may directly or indirectly affect MHC class I presentation by competing with
the proteasome for substrates, by influencing the peptidome pools through the control
of levels of components of microRNA (miRNA) machinery (for example, argonaute (AGO)
and DICER), or by supporting unconventional MHC class I presentation. In addition,
autophagy affects the self-renewal of haematopoietic stem cells (HSCs), B1 cell
development, plasma cell survival and IgG secretion. Autophagy affects T cell survival
following T cell receptor (TCR) activation, and it destabilizes the immunological synapse.
It also controls innate immune cell (such as macrophage) signalling through the release
of interleukin‑1α (IL‑1α) and IL‑1β, which influence the polarization of T cells into
T helper 17 (TH17) cells. Autophagy also affects naive T cell repertoire selection in the
thymus and the survival and function of maturing T cells by removing the mitochondria
and endoplasmic reticulum (ER), thus ensuring calcium homeostasis. d | The role of
autophagy in the secretion of immune mediators is shown. Autophagy affects the quality
of regulated secretion from pre-stored granules. Autophagy affects the quality and the
quantity of the output of the constitutive secretory pathway (which is the conventional
pathway of protein secretion via the ER, the Golgi apparatus and the plasma membrane).
Autophagy supports a form of unconventional secretion that captures cytoplasmic
proteins for extracellular release. Note that secretory protein cargo in the regulated
and constitutive secretory pathways contains conventional leader peptides for
co‑translational import into the ER lumen, whereas protein cargo that enters the
unconventional secretory pathway lacks leader peptides and does not enter the ER.
Dashed arrow indicates that this pathway remains to be defined. DAMPs, damage-
associated molecular patterns; HMGB1, high-mobility group box 1 protein; IPS1, IFNβ
promoter stimulator protein 1; mtDNA, mitochondrial DNA; mTOR, mammalian target
of rapamycin; NOX2, NADPH oxidase 2; PRRs, pattern recognition receptors;
ssRNA, single-stranded RNA; TRAF6, TNF receptor-associated factor 6.
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REVIEWS
probably through the downstream recruitment of
TRAF6 and the subsequent TRAF6‑dependent ubiqui-
tylation of beclin 1 (REF. 9). T helper 1 (TH1) cell-derived
cytokines, such as interferon‑γ (IFNγ), also induce
autophagy in effector cells; for example, these cytokines
induce autophagy in macrophages to enable them to
resist mycobacteria infection15–17. IFNγ might activate
autophagy both through the function of immunity-
related GTPases15–17 and through the phosphorylation
of beclin 1 on Thr119 of its BH3 domain by death-
associated protein kinase 1 (DAPK1). This results in
the dissociation of beclin 1 from its inhibitor BCL‑2
(REF.18) and, together with other processes (BOX 2), leads
to beclin 1 activation. In human macrophages, but not
in mouse macrophages, IFNγ cooperates with 1,25‑dihy-
droxyvitamin D3 (also known as calcitriol) to induce
antimycobacterial autophagic activities19. Similarly,
tumour necrosis factor (TNF) has been shown to stimu-
late autophagy to restrict intracellular bacteria such as
Shigella spp. and Listeria spp20.
By contrast, TH2 cell-associated cytokines, such as
IL‑4 and IL‑13, inhibit autophagy 16. The signalling path-
ways that lead to the inhibition of autophagy by IL‑4
and IL‑13 are context dependent: they occur through
AKT signalling when autophagy is induced by starva-
tion, and are AKT independent and signal transducer
and activator of transcription 6 (STAT6) dependent
when autophagy is induced by IFNγ16. IL‑10 can also
inhibit autophagy through AKT signalling 21. Moreover,
STAT3, which transduces signals downstream of vari-
ous signals including IL‑6, can inhibit autophagy 22. This
inhibitory pathway does not involve transcriptional
signalling, but instead involves the binding of cyto­
plasmic STAT3 to protein kinase R (PKR; also known
as IFN-induced double-stranded RNA-activated protein
kinase)22. STAT3 interacts through its SH2 domain with
PKR and inhibits it. This prevents PKR from facilitating
autophagy through the hyperphosphorylation of eukary-
otic translation initiation factor 2α (EIF2α; also known
as EIF2S1) and the subsequent inhibition of cellular and
viral protein synthesis22.
Reactive oxygen species (ROS) are classical anti­
microbial effectors, which have an important role in
immune signalling. ROS, and the oxidases that generate
them, affect autophagy 23–27 (BOX 2; FIG. 1a). NADPH oxi-
dase and the autophagic machinery are connected via an
autophagy regulatory protein known as RUBICON (run
domain beclin 1‑interacting and cysteine-rich-containing
protein), which interacts with beclin 1 and the PtdIns3
kinase catalytic subunit VPS34. By physically disassociat-
ing from the autophagy inhibitory complex and associating
with the NADPH oxidase activating complex, RUBICON
activates two bactericidal mechanisms (that is, autophagy
and ROS production), although it is not known whether
the activation of these two mechanisms occurs simulta-
neously 26. Nitric oxide inhibits autophagy by inactivating
Jun N‑terminal kinase 1 (JNK1) and IKKβ through direct
S‑nitrosylation27. This prevents JNK1‑dependent BCL‑2
phosphorylation27 and BCL‑2 dissociation from beclin
128,29 and inhibits IKKβ-associated AMPK-dependent
autophagy initiation27,30.
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SNARE
(Soluble NSF attachment
protein receptor). A member
of a class of proteins that
catalyse membrane fusion
and thus regulate organelle
identity and vesicular
trafficking. The membrane
fusion occurs through the
formation of a cognate Qa-,
Qb-, Qc- and R‑SNARE
four-helix bundle, which
consists of SNAREs on donor
and acceptor membranes.
Mammalian target of
rapamycin
(mTOR). A serine/threonine
protein kinase that regulates
cell growth and metabolism.
mTOR is stimulated by growth
factor receptor and phosphati-
dylinositol‑3,4,5‑phosphate-
dependent signalling. It
responds to the availability of
nutrients (for example, amino
acids). Active mTOR inhibits
autophagy via the serine/
threonine protein kinase ULK1.
AMP-activated protein
kinase
(AMPK). A sensor of cellular
energetic state. It is activated
by increased AMP levels which
indicate decreased energy
status following hypoxia or
nutrient deprivation.
Xenophagy
The selective degradation
of intracellular pathogens
(such as bacteria or viruses)
through macroautophagy.
LC3‑associated
phagocytosis
(LAP). A shared pathway that
involves conventional
phagocytosis and autophagy
at the maturation stage that is
mediated by the recruitment
of the autophagy protein LC3
(which is the mammalian
homologue of yeast ATG‑8).
LAP results in a more robust
phagolysosome, which can
also function as a specialized
signalling compartment or
an antigen-presentation
compartment.
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Box 2 | Parallels between metabolic and immune signalling in autophagy activation
The model (see the figure) proposes that immune and nutritional signals converge to activate a similar cascade. Nutritional
triggers, such as starvation, are transduced via mammalian target of rapamycin (mTOR) and AMP-activated protein kinase
(AMPK). Starvation inhibits mTOR and activates AMPK, which in turn activates the serine/threonine protein kinase
ULK1 to phosphorylate beclin 1 (REF. 140), activating molecule in BECN1‑regulated autophagy protein 1 (AMBRA1)10
and TAK1‑binding protein 2 (TAB2)141. In the resting state, TAB2 and TAB3 bind beclin 1 and repress its activity30,140.
TAB2 activates TGFβ-activated kinase 1 (TAK1)30 to further enhance AMPK activity. AMBRA1 (REF. 10), along with
beclin 1 (REF. 107), recruits TNF receptor-associated factor 6 (TRAF6). TRAF6 functions as an E3 ubiquitin ligase, generating
polyubiquitin chains that in turn stabilize and activate ULK1 (REF. 10) and beclin 1 (REF. 107), and that probably activate
the TAB2 and TAB3–TAK1 complexes. Once fully amplified, the concomitant activation of ULK1 and beclin 1 leads to
autophagy through both branches
of autophagy regulatory kinases —
that is, the protein kinase ULK1 (via AMPK P TAK1
Starvation
AMPK) and the lipid kinase VPS34
(via beclin 1) (not shown)107.
P
Immune signals activate
RAPTOR
autophagy and engage at least
some components of the mTOR
pathways, including ULK1
P
(REF. 108), beclin 1 (REF. 142) and
TRAF6 (REF. 107). Downstream of NOD2 RIPK2 ULK1 P TAB2 and
TAB3
the TAB2 and TAB3–TAK1 Ub
IFNγ DAPK1 Ub
complexes, IκB kinases (IKKs)
affect autophagy independently of Ub
HMGB1 JNK1
nuclear factor-κB143. TANK-binding Ub
P Ub
kinase 1 (TBK1) also controls
NLRP3,
autophagy14,77 and promotes the NLRP4 and Beclin 1
elimination of intracellular NLRP10 Ub
microorganisms independently of Ub
its role in type I interferon (IFN) Cardiolipin IRGM P AMBRA1
P
activation (not shown)144. TBK1
controls both the capture of TRAF6 BCL-2
RUBICON
autophagic cargo (for example,
NOX2 ROS
microorganisms) via specialized
adaptor proteins14,35,36,45 and the
TLR4
maturation of autophagosomal
organelles into degradative
compartments14,77. TBK1 may also IL-1β
contribute to autophagosome
formation at stages earlier than CD40 Autophagy
autophagosome maturation77.
Nature
BCL‑2, B cell lymphoma 2; DAPK1, death-associated protein kinase 1; HMGB1, high-mobility group box 1 Reviews
protein; IL‑1β,| Immunology
interleukin‑1β; IRGM, immunity-related GTPase family M protein; JNK1, Jun N-terminal kinase 1; NLRP, NOD-, LRR- and pyrin
domain-containing protein; NOD2, nucleotide-binding oligomerization domain-containing protein 2; NOX2, NADPH oxidase 2;
P, phosphorylation; RAPTOR, regulatory-associated protein of mTOR; RIPK2, receptor-interacting serine/threonine-protein kinase 2;
ROS, reactive oxygen species; RUBICON, run domain beclin 1-interacting and cysteine-rich-containing protein; TLR4, Toll-like
receptor 4; Ub, ubiquitylation.
The connections between autophagy and immune confined in the nascent and presumably intact phago-
signalling seem to be surprisingly complex. From an some24,31,32 (BOX 3). Finally, a group of autophagic adap-
evolutionary perspective, these connections reflect the tors33, known as sequestosome 1‑like receptors (SLRs), are
integration of autophagy with several immune regulatory involved in eliminating microorganisms from the cyto-
systems. plasm. SLRs recognize molecular tags (such as ubiqui-
tin, galectin and membrane phospholipid modifications)
Direct elimination of microorganisms present on invading microorganisms or on damaged
Autophagy intercepts pathogen invasion. The antimi- host membranes that are associated with the pathogen
crobial functions of autophagy provide a series of barri- and that physically recruit the autophagic machinery 34–38
ers against invading microorganisms (FIG. 1a). The first (FIG. 2). The importance of autophagy in protecting the
antimicrobial function is xenophagy, which is the uptake cytoplasm from microbial invasion is highlighted by the
of intracellular microorganisms into double-membrane microbial countermeasures and adaptations that have
autophagosomes4, and the second is LC3‑associated evolved to inhibit, to block specific stages or to com-
phagocytosis (LAP), which involves the engagement mandeer autophagy 39 (see BOX 4 for recently discovered
of the autophagic machinery while the bacterium is examples).
© 2013 Macmillan Publishers Limited. All rights reserved

Box 3 | LAP: a shared maturation step for autophagy and phagocytosis
Autophagy is often morphologically defined by the formation of double-membrane
autophagosomes in the cytoplasm. This is the geometrical consequence of the
formation of a phagosome that is derived from a pre-existing membranous intracellular
compartment, such as the endoplasmic reticulum (ER), rather than the consequence of
a functional requirement for a double membrane. An exception to this rule is the
formation of standard phagolysosomes by the autophagy-related protein (ATG) LC3,
through a process known as LC3‑associated phagocytosis (LAP)24,31,32,53. LAP occurs
following the uptake of various extracellular targets: particles coated with Toll-like
receptor (TLR) agonists, phagocytosed dead cells, live epithelial cells that are
engulfed via entosis by neighbouring cells, and the Fcγ receptor-dependent uptake
of immune complexes. When a microorganism or a TLR ligand is taken up by
conventional phagocytosis31,53, the autophagic machinery enhances the maturation
of the conventional phagosomes through the same maturation pathway that is used
in internally formed autophagosomes. Thus, LAP uses beclin 1–VPS34 complexes and
LC3 conjugation systems. LAP is independent of the serine/threonine protein kinase
ULK1 (REF. 32) — ULK1 is only required to generate autophagosomes from internal
ER membranes during starvation.
To initiate autophagy, mammalian cells detect the
presence, location and extent of the cytoplasmic inva-
sion by a pathogen. Conventional PRRs can elicit
autophagic responses at different stages of the host–
pathogen encounter 40. First, TLRs and NOD-like recep-
tors (NLRs) detect released microbial products (that is,
PAMPs) very early following infection and this stimu-
lates autophagy. Second, autophagy can be initiated
during adhesion- and pathogen-induced uptake of bac-
teria by the host cell, or during active phagocytosis of
bacteria by macro­phages31,41,42. Third, at stages after bac-
terial uptake, autophagy is induced following pathogen-
inflicted damage to the newly formed parasitophorous
vacuoles35,36,38,43–45 and on the escape of bacteria into the
cytoplasm34,46,47. Similarly, the initiation of autophagy
following virus infection occurs at various stages of the
virus life cycle25,48–57 (FIG. 1a).
TLRs and autophagy cooperate in the response to
PAMPs. The early induction of autophagy downstream of
TLR stimulation ensures the prompt upregulation
of antimicrobial activities, including the upregulation of
autophagy systems in advance of microbial invasion58,59
Sequestosome 1‑like (FIG.  1a) . This and other triggers, such as IFNγ and
receptors 1,25‑dihydroxyvitamin D3, may promote the expres-
(SLRs). These are autophagic
sion and the early delivery of antimicrobial peptides
adaptors that recognize
microbial targets and that link to the parasitophorous vacuoles19,60–62, or they may
them to autophagy machinery direct the autophagic response to the points of micro-
by binding to mammalian bial entry, as occurs during LAP31 (FIG. 1a). In addition,
autophagy-related protein 8 autophagic membranes deliver cytoplasmic PAMPs,
(ATG-8) proteins (for example,
autophagy related LC3
such as single-stranded viral RNA, to the endosomal
proteins and γ-aminobutyric lumen where they can make contact with the luminal
acid receptor-associated portion of endosomal TLRs, such as TLR7, to stimulate
proteins (GABARAPs)). other responses, including the type I IFN response 50
(FIG. 1b). Autophagy that is stimulated by TLRs, in con-
Crohn’s disease
A form of chronic inflammatory junction with LAP53,63, enhances antigen presentation
bowel disease that can affect by dendritic cells (DCs) (FIG. 1c). LAP also contributes
the entire gastrointestinal to the trafficking of TLR9 into specialized IFN signal-
tract, but is most common ling compartments in plasmacytoid DCs (pDCs) (FIG. 3).
in the terminal ileum. It is
characterized by transmural
Thus, TLRs and autophagy influence each other, which
inflammation, strictures and amplifies the outputs of both systems in response to
granuloma formation. microbial invasion.
NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 727
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
NLRs interact with ATGs to localize autophagy. The
cooperation between NLRs and autophagy in antimi-
crobial defence is conserved from flies64 to humans41,42.
NOD1 (nucleotide-binding oligomerization domain-
containing protein 1) and NOD2 detect muramyl pep-
tides in the cytoplasm and they direct the autophagic
machinery by recruiting ATG16‑like 1 (ATG16L1) to
the plasma membrane at the site of bacterial entry 41
(FIG. 1a). The NOD2‑assisted localization of ATG16L1
at the plasma membrane is consistent with the role of
ATG16L1 in the formation of a portion of autophagic
precursors from the plasma membrane65 (BOX 1). Of
note, polymorphisms at the ATG16L1 and NOD2
loci have been associated with an increased risk of
Crohn’s disease66. Cells from donors who are homozy-
gous for the Crohn’s disease risk allele ATG16L1*300T
have a decreased capacity for autophagy induction in
response to the NOD2 agonist muramyl dipeptide.
The truncation of NOD2 that occurs in patients with
Crohn’s disease renders it cytoplasmic, which results in
the retention of ATG16L1 in the cytoplasm. This pre-
cludes ATG16L1 recruitment to bacterial entry sites
and thereby prevents timely and site-specific control of
autophagy.
Other NLRs also interact with the autophagic machin-
ery. NLRX1 and its interacting partner mitochondrial
Tu elongation factor (TUFM), which associates with
ATG5–ATG12 complexes and with ATG16L1, promote
autophagy 67 (FIG. 3b). NLRC4 (NOD-, LRR- and CARD-
containing protein 4), NLRP3 (NOD-, LRR- and pyrin
domain-containing protein 3), NLRP4 and NLRP10
interact with beclin 1 (REF. 68) (BOX 2). The recruitment
of NLPR4 to the plasma membrane during the phago-
cytosis of group A streptococci leads to its transient dis-
sociation from beclin 1. This enables the initiation of
beclin 1‑mediated autophagic responses68. Collectively,
NLRs may gather autophagy factors in the vicinity of the
incoming microorganism (or resident mitochondria),
which leads to the activation of autophagy.
Nucleic acid sensors and autophagy. RIG‑I‑like receptors
(RLRs), including retinoic acid-inducible gene I (RIG‑I),
melanoma differentiation-associated protein 5 (MDA5;
also known as IFIH1) and LGP2 (also known as DHX58),
recognize viral RNA to induce the production of type I
IFNs and other pro-inflammatory cytokines. Unlike other
PRRs, RLRs seem to negatively regulate autophagy67,69.
However, downstream molecules that are engaged by
RLRs may have an RLR-independent (and possibly
competing) function in the induction of autophagy; for
example, downstream of nucleic acid sensing by RLRs,
the cytoplasmic adaptor protein stimulator of IFN genes
protein (STING) activates TANK-binding kinase 1
(TBK1) signalling and type I IFN production, but can
also induce autophagy, possibly by the direct recogni-
tion of DNA or second messengers that are associated
with the presence of cytoplasmic DNA. Indeed, infec-
tion with double-stranded DNA viruses, such as herpes
simplex virus 1 (HSV1) or human cytomegalovirus,
induces autophagy that is dependent on STING70,71.
Similarly, bacterial DNA that is released during infection
REVIEWS

can induce autophagy via STING45 (FIG. 3e). Such bacte-
rial DNA can escape from M. tuberculosis-containing
phagosomes through pores that are introduced by the
bacterial secretion system ESX1 (REF. 45). It has recently
become evident that, in response to cytoplasmic DNA,
host cell systems generate a second messenger, cyclic
GMP–AMP (cGAMP)72 (FIG. 3e). This cyclic dinucleotide
is generated by host cGAMP synthase and activates the
type I IFN pathway via STING72. It remains to be shown
whether cGAMP production in response to bacterial and

a Sequestosome 1-like receptors b

NLS NES UBA Bacteria
Sequestosome 1 PB1 ZZ Parasitophorous
PAMP vacuole
TRAF6-interacting region LIR KIR
332 342 β-galactoside Galectin

X/(D,E,S,T)-X/(D,E,S,T)-X/(D,E,S,T)-W/F/Y-X/(D,E,S,T)-X-L/I/V
LC3C NDP52
NBR1 PB1 ZZ CC FW CC
Isolation
LIR UBA
membrane
NDP52 (human) SKICH CC GIR UBZ LRSAM1
(E3 ligase)
CLIR TBK1
NDP52 (mouse) SKICH RAB8B
Optineurin

TAX1BP1 SKICH CC CC CC UBZ UBZ

Optineurin CC CC CC UBAN ZnF

LIR
Figure 2 | Autophagy-mediated clearance of intracellular pathogens.  a | Protein domains
of sequestosome 1‑like receptors (SLRs) are shown. The LC3‑interacting region (LIR) motif
of an SLR binds to autophagy-related LC3 proteins through its consensus sequence at
amino acids 332–342 in sequestosome 1 (also known as p62). The conserved residues are Ub
shown. X/(D,E,S,T) indicates that any amino acid (X) is allowed but that acidic (D,E) or Ub
Ub
phosphorylatable amino acids (S,T) are often present (usually at least one or more within Ub Ub
Ub
the entire consensus sequence). The core LIR motif residues are aromatic pocket-filling W
(or F or Y) residues and aliphatic pocket-filling L (or I or V). They form an intermolecular Ubiquitylation
parallel β-sheet with LC3 proteins or γ-aminobutyric acid receptor-associated proteins
(GABARAPs). The CLIR motif, which is a LIR motif that is specific for LC3C, lacks the aromatic
residue found in the LIR motif and, instead, uses hydrophobic contacts provided by
additional aliphatic residues located between the W and L position anchors to stabilize
interactions with LC3C. All human SLRs also contain a ubiquitin-binding domain (UBD):
UBA (as found in sequestosome 1 and NBR1) is a three-helix bundle UBD that has affinity TBK1
for monoubiquitin and K63 ubiquitin linkages; UBAN (as found in optineurin) is a parallel RAB8B
coiled-coiled dimer UBD that has specificity for linear ubiquitin chains; and UBZ
(as found in nuclear dot protein 52 (NDP52)) is a zinc finger ββα-fold UBD that binds to
monoubiquitin and polyubiquitin. b | A model of cooperative action between different
SLRs and E3 ligases in bacterial targeting for xenophagy is shown. The schematic shows a
parasitophorous vacuole with glycosylated molecules (in this case β‑galactosides) facing
the lumen of the vacuole that contains a bacterium and that is experiencing membrane
damage. This membrane tear exposes β‑galactosides to galectins (for example, galectin 8)
which in turn bind to the galectin-interacting region (GIR) motif of NDP52. NDP52 also LC3 or
directly interacts with the E3 ligase LRSAM1 and indirectly with the serine/threonine protein GABARAP
P TBK1
kinase TANK-binding kinase 1 (TBK1), which interacts with optineurin. The CLIR motif of Ub
NDP52 binds to LC3C, which is a proposed initiator in the LC3 and GABARAP cascade Ub
Sequestosome 1 Ub
during bacterial xenophagy. LRSAM1 or other E3 ubiquitin ligases polymerize ubiquitin at multimer Ub
molecular targets that are yet to be identified. The hypothetical model includes the putative Ub
Ub
recognition of bacterial pathogen-associated molecular patterns (PAMPs) by LRSAM1
through its leucine-rich repeat domain. Ubiquitin tags are recognized by UBDs of NDP52, Ub
optineurin and sequestosome 1. The LIR motif of optineurin is phosphorylated by TBK1 and Ub
Ub
this improves LC3 and GABARAP binding. The UBA of sequestosome 1 is also phosphorylated Ub
by TBK1 and this improves ubiquitin chain binding. As a consequence, the autophagic Ub Ub
P
isolation membrane initiates at the appropriate location and grows to capture the bacterium Ub
and to eliminate it through autophagy. CC, coiled-coil domain; FW, four W domain (also Ub
Ub
known as the NBR1 domain); KIR, KEAP1 interacting region; NES, nuclear export signal; NLS, Ub
nuclear localization signal; P, phosphorylation; PB1, protein-binding domain 1; SKICH, skeletal
muscle and kidney enriched inositol phosphatase carboxyl homology domain; TAX1BP1,
TAX1-binding protein 1; Ub, ubiquitylation; ZnF, zinc finger domain; ZZ, ZZ‑type ZnF domain.

728 | O CTOBER 2013 | VOLUME 13 www.nature.com/reviews/immunol

© 2013 Macmillan Publishers Limited. All rights reserved Nature Reviews | Immunology

Ubiquitin-binding domains
(UBDs). Domains that have
different specificities for
ubiquitin chains: UBA, which
is found in sequestosome 1,
favours K63 polyubiquitin
chains; UBZ, which is found
in nuclear dot protein 52
(NDP52), binds to
monoubiquitin and
polyubiquitin; and UBAN,
which is found in optineurin,
has specificity for linear
ubiquitin chains.
LC3‑interacting region motif
(LIR motif). Canonical LIR
motif, which is X/(D,E,S,T)-X/
(D,E,S,T)-X/(D,E,S,T)-W/F/Y-X/
(D,E,S,T)-X-L/I/V, where X/
(D,E,S,T) indicates that any
amino acid (X) is allowed
but that acidic (D,E) or
phosphorylatable amino
acids (S,T) are often present.
Aromatic residues (W/F/Y)
occupy the aromatic pocket
and aliphatic side chains (L/I/V)
occupy an aliphatic pocket
present in all autophagy-
related protein 8 (ATG-8)
homologues. The LIR motif
forms an intermolecular
β‑sheet with ATG-8 homologues,
not discriminating between
mammalian paralogues of
yeast ATG-8.
CLIR motif
A variant of the LC3‑interacting
region (LIR) motif (L-V-V),
which is found in nuclear
dot protein 52 (NDP52).
The CLIR motif binds to the
LIR-interacting region in LC3C.
The CLIR motif lacks the
signature aromatic residue of
the LIR motif. Instead, it makes
compensatory hydrophobic
contacts with LC3C.
NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 729
REVIEWS
host-derived cytoplasmic DNA can induce autophagy. SLRs contain one or more cargo recognition domains
Analogous cyclic dinucleotides that are secreted by (CRDs) that recognize ubiquitin-tagged 20,34–36,45,46,73
bacteria (for example, cyclic di‑GMP or di‑AMP) bind or galectin-tagged38,79 microbial or microorganism-
to STING and can activate autophagy when they are associated targets. Ubiquitin-binding domains (UBDs)
introduced into the host cell cytoplasm45 (FIG. 3e). that are specific for different ubiquitin chains (UBA,
In conclusion, conventional PRRs recognize microbial UBZ or UBAN)80 are present in all known SLRs. SLR
products at different stages of an infection and initiate CRDs that recognize tags other than ubiquitin include
an autophagic response that contributes to the efficient a hook-like CRD (which is the galectin-interacting
elimination of the invading microorganism. However, region (GIR) motif) that enables NDP52 to interact
in some instances, autophagy can suppress type I IFN with galectin 8 (REF. 79) (FIG. 2a). Galectin 8 links NDP52
responses, notably by inhibiting RLR signalling 67,69. This to cytoplasmically exposed β‑galactoside glycans on
relationship paradoxically promotes viral replication, and pathogen-damaged host membranes38 (FIG. 2b).
may explain why autophagy can have a pro-infective role. Moreover, all SLRs have an LC3‑interacting region
motif (LIR motif), which is X/(D,E,S,T)-X/(D,E,S,T)-X/
SLRs clear microorganisms from the cytoplasm. If a path- (D,E,S,T)-W/F/Y-X/(D,E,S,T)-X-L/I/V, where X/
ogen escapes the autophagy barriers that are controlled (D,E,S,T) indicates that any amino acid (X) is allowed
by conventional PRRs, it can still be captured in the cyto- but that acidic (D,E) or phosphorylatable amino acids
plasm or even in the cytosol. The autophagy-mediated (S,T) are often present (at least one or more within the
elimination of cytoplasmic pathogens depends on special- entire motif)33. A modified LIR, known as a CLIR motif
ized adaptors known as SLRs20,34–36,46,73,74 (FIG. 2a). SLRs, (L-V-V) has been identified in NDP52 (REF. 81) and pos-
which are named after the archetypical protein seques- sibly in TAX1BP1 (REF. 77) (FIG. 2a). Phosphorylation of
tosome 1 (also known as p62) include sequestosome 1 the LIR motif and of the CRDs of SLRs modulates their
(REF. 75), NBR1 (REF. 76), optineurin36, nuclear dot protein autophagic activities14,36,82 (FIG. 2b). The number of SLRs
52 (NDP52; also known as CALCOCO2; it is full size and the type of unique or repetitive structures that are
in humans but truncated in mice)35 and an NDP52‑like recognized as tags may increase as more research is
receptor TAX1-binding protein 1 (TAX1BP1; also known carried out.
as CALCOCO3)77 (FIG. 2a). SLRs have been shown to The process of autophagic clearance of microorgan-
affect inflammation in several ways34,74,78 and, as they can isms from the cytoplasm involves the sensing of DAMPs
be consumed during autophagy 33, it is possible that SLR and PAMPs, and often the function of multiple SLRs. An
accumulation (following autophagy inhibition) or SLR example of autophagy-inducing DAMPs are the glycans
depletion (following autophagy activation) may modulate that become exposed following damage to host mem-
inflammatory processes (FIG. 1b). branes, as in the case of infection with Salmonella spp.38,79.
Box 4 | Microbial countermeasures against autophagy
Microorganisms use a wide range of mechanisms to prevent, to counteract or to commandeer autophagy39.
These inhibitory mechanisms involve the targeting of beclin 1 (REFS 49,145), the inhibition of autophagosomal
maturation51,57, the perforation of autophagosomal membranes to prevent acidification44, the proteolytic cleavage
of autophagy-related protein 8 (ATG8) to irreversibly remove carboxy-terminal lipid modifications146, and the
masking of epitopes or tags that are recognized by sequestosome 1‑like receptors (SLRs)46,147. Autophagy may
even be activated to generate nutrients for invading microorganisms148.
With regard to bacteria, Listeria spp. proteins AktA46 and InlK147 interfere with recognition via host ubiquitin tags,
and Shigella spp. protein IcsB masks bacterial epitopes149, which are recognized by the autophagic machinery, either
directly or by recruiting cytoplasmic proteins. The Salmonella spp. deubiquitinase SseL removes ubiquitin tags88.
The Legionella spp. effector protein RavZ is injected into the host cytoplasm through the bacterial type IV secretion
system and inhibits autophagy through irreversible deconjugation of mammalian homologues of ATG-8. This involves
the proteolysis of the C‑terminal glycine in ATG-8 homologues, which prevents future phosphatidylethanolamine
modification146. Listeria spp. block autophagosome acidification through the pore-forming toxin listeriolysin O44.
Bacterial actin-based intracytoplasmic motility may be another factor in evading capture by autophagosomes.
This is supported by the role of septin scaffolds in enabling autophagy of cytoplasmic Shigella spp. as they start
to polymerize actin20.
Viruses also interfere with autophagy; for example, herpes simplex virus 1 infected cell protein 34.5 (ICP34.5)49,
influenza virus M2 protein52 and HIV protein Nef51,57, all target beclin 1 to either completely block autophagy or
to inhibit autophagosomal maturation. Nef binds to the evolutionarily conserved domain of beclin 1 at the
same region as the endogenous inhibitor GAPR1 (Golgi-associated plant pathogenesis-related protein 1)57.
Mouse herpesvirus 68‑encoded protein M11 (which is the viral homologue of B cell lymphoma 2) inhibits beclin 1
through its BH3 domain145, whereas Kaposi sarcoma-associated virus (KSHV) FLICE-like inhibitory protein (FLIP)
blocks ATG3 in the LC3 lipidation cascade127. HIV Nef, hepatitis C virus NS3 and measles virus Mev3 interact with
autophagy factor immunity-related GTPase family M protein (IRGM), which has consequences that are yet to be
fully understood150.
Finally, several microorganisms can harness autophagy for their own benefit, including using autophagy for
replication39; for example, the obligatory intracellular pathogen Anaplasma phagocytophilum uses its type IV
secretion effector Ats1 to activate autophagy to supply nutrients148.
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS

a DNA-containing b c
antigen
RIG-I Dectin RIG-I
B cell pDC
BCR ATG12
↑ Autophagy RUBICON
ATG5 BCL-10
Beclin 1
IPS1 MALT1 CARD9 ↓ Autophagy
LAP VPS34
TUFM
TLR9 NF-κB
NLRX1
Type I
IFN BCL-10
MALT1
CARD9 RUBICON
B cell activation Type I IFN
Degradation
BCL-10 Ub of BCL-10 by
autophagy
Autoimmune plasma cells Sequestosome 1

d Autophagy e
Virus DNA cGAMP
synthase

ROS ↑ RLR signalling Mitochondria cGAMP

GMP AMP
Depolarized Bacteria
mitochondria Inflammasome Calpain Type I IFNs
Cyclic di-AMP STING ATG9 ↑ Autophagy
Cyclic di-GMP
IL-1β IL-1α

Autophagy TBK1

Type I IFN
Figure 3 | Autophagy controls inflammatory processes.  a | Autophagy promotes Toll-like receptor 9 (TLR9)
Nature Reviews
signalling in B cells and type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs). | Immunology
b | The autophagy
protein complex autophagy-related protein 5 (ATG5)–ATG12 inhibits RIG‑I‑like receptor (RLR) signalling by binding
to the caspase recruitment domains of retinoic acid-inducible gene I (RIG‑I) and IFNβ promoter stimulator protein 1
(IPS1), which is the mitochondrial adaptor of RIG‑I signalling. The NOD-like receptor X1 (NLRX1)-interacting partner
mitochondrial Tu elongation factor (TUFM) associates with the ATG5–ATG12 complex to promote autophagy while
inhibiting RLR-dependent type I IFN activation. c | Autophagy factors negatively regulate the caspase recruitment
domain-containing protein 9 (CARD9)–B cell lymphoma 10 (BCL‑10)– mucosa-associated lymphoid tissue lymphoma
translocation protein 1 (MALT1) complex. RUBICON (run domain beclin 1-interacting and cysteine-rich-containing
protein), which is a binding partner and a negative regulator of beclin 1, inhibits CARD9, whereas sequestosome 1
leads to the degradation of BCL‑10. d | Excessive production of reactive oxygen species (ROS) by depolarized
mitochondria that are not cleared by autophagy enhance RLR signalling. e | Viral, mitochondrial or bacterial DNA lead
to the activation of stimulator of IFN genes protein (STING), probably through cGAMP synthase and cyclic GMP–AMP
(cGAMP) production, which increases the type I IFN response. Autophagy removes sources of agonists that stimulate
STING, whereas autophagic factors (for example, ATG9) inhibit the activation of STING by affecting its cytoplasmic
translocation. Bacterial cyclic dinucleotides (di‑AMP and di‑GMP) can activate autophagy, thereby functioning as a
regulatory loop that amplifies the removal of infectious or endogenous irritants. BCR, B cell receptor; IL, interleukin;
LAP, LC3‑associated phagocytosis; NK-κB, nuclear factor-κB; TBK1, TANK-binding kinase 1; Ub, ubiquitylation.

Leucine-rich repeat
(LRR). A domain that is often
found in pattern recognition
receptors and that is involved
in pathogen-associated
molecular pattern recognition.
LRRs are repeats of L-X-X-L-X-
L-X-X-N-X-L or L-X-X-X-L-X-L-X-
X-C-X-X-L motifs (where X
represents any amino acid),
which form a horseshoe or
solenoid tertiary structure.
Moreover, microbial polymers, such as DNA, that are
present in the cytoplasm might function as autophagy-
inducing PAMPs, as occurs during M. tuberculosis infec-
tion45. In the cytoplasm, microorganisms may become
coated with ubiquitin20,34–36,45,46,73,which may involve
specialized E3 ubiquitin ligases, such as LRSAM1, which
recognize Salmonella spp. through their leucine-rich
repeat (LRR) domains83. Following auto-ubiquitylation,
LRSAM1 ubiquitylates microbial targets that are yet to be
defined and possibly host molecules that are associated
with microorganisms. Any of these ubiquitin tags alone
or in combination may lead to the recruitment of SLRs
through their UBDs (FIG. 2b).
There is further cooperation (FIG. 2b) between the
recognition of the initial tears in microorganism-
harbouring vacuoles and ubiquitylation, as exemplified
in the case of Salmonella spp. infection. In this exam-
ple, LRSAM1 directly interacts with NDP52 (REF. 83). In
human cells, NDP52 seems to function as an important
hub, as it can recognize LRSAM1 (REF. 83), galectin 8
bound to β‑galactoside glycans38,79, ubiquitin tags35 and
LC3C (also known as MAP1LC3C), which is required
for the acquisition of other LC3 proteins to mediate
autophagy 81. The action of NDP52 is non-redundantly
reinforced by other SLRs that may be recruited to
Salmonella spp., such as optineurin (which localizes with

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© 2013 Macmillan Publishers Limited. All rights reserved


Mitophagy
A special form of autophagy
by which mitochondria (in a
damaged or depolarized
state) are engulfed by
autophagosomes and
degraded.
NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 731
NDP52 in microdomains surrounding Salmonella spp.)
and sequestosome 1 (REF. 36). These SLRs bind to ubiqui-
tin through their UBDs that have varied specificities for
ubiquitin chains80, and, through their LIR motifs, they
recruit the remaining LC3 proteins and GABARAPs to
mediate autophagosome formation. The affinity of SLRs
for LC3 and ubiquitin is further enhanced by phospho-
rylation of the LIR motif in optineurin and the UBD
in sequestosome 1 by TBK1 (FIG. 2b). TBK1 is recruited
to these sites via protein complexes containing NDP52
(REF. 35), optineurin36 and RAB8B14, which is a small
GTPase that regulates membrane trafficking. The cargo
capture and autophagy is coordinated with maturation
into autolysosomes by RAB8B14. Notably, the NDP52
product that is encoded by the corresponding mouse
gene Calcoco2 is carboxy‑terminally truncated (and lacks
motifs for ubiquitin and galectin binding) (FIG. 2a) and
may be primarily expressed in embryonic tissues (see the
expressed sequence tags NCBI database). This suggests
that, in mice, the function of CALCOCO2 may be pri-
marily conferred through the recruitment of LRSAM1,
TBK1 and LC3, as the binding sites for these proteins are
retained in the truncated mouse protein. Alternatively,
NDP52 activities in mice may be compensated for by
the NDP52‑like receptor CALCOCO3.
Other pathways to target cytoplasmic microorgan-
isms for autophagy. Autophagy-associated factors have
been shown to directly target microbial proteins 47;
for example, ATG5 binds directly to a Shigella spp.
surface protein, VirG. The ATG5–VirG interaction
only occurs when the VirG recognition epitope is not
masked by another Shigella spp. protein, IcsB (BOX 4),
and requires the ATG5‑binding partner TECPR1 (tec-
tonin β-propeller repeat-containing protein 1) which
interacts with WIPI2 (WD repeat domain phospho-
inositide-interacting protein 2)47. WIPI2 is one of the
four mammalian PtdIns3P-binding ATG18 paralogues
and has a role in phagophore formation (BOX 1). Of note,
the control of icsB-mutant Shigella spp. also requires
NOD1 activity to induce autophagy and to limit intra-
cellular growth41. Moreover, these mechanisms against
Shigella spp. are further complemented by ubiquitylation
and SLR activity 20,34. Taken together, these findings sup-
port the idea that autophagic defences are multilayered
and cooperative in nature.
Other antibacterial pathways include the autophagy
factors beclin 1 (REF. 84) and immunity-related GTPase
family M protein (IRGM) which directly bind to cardio­
lipin, a lipid that is only present in bacteria and mito-
chondria17. IRGM is necessary for the optimal induction
of autophagy 17, is partially localized in mitochondria
(hence its affinity for cardiolipin)17 and is a genetic
predisposition factor for Crohn’s disease66,85,86. The
E3 ubiquitin-protein ligase SMURF1 is required for anti-
viral responses to Sindbis virus and to HSV1 (REF. 87).
SMURF1 is involved in mitophagy87, which in some
respects resembles bacterial xenophagy. Surprisingly, the
HECT (homologous to the E6‑AP C terminus) domain
of SMURF1 (and hence the region that has its E3 ligase
activity) is dispensable for mitophagy and, instead, its
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
phospholipid-binding domain C2 is required. In keep-
ing with the idea that membrane phospholipids or their
derivatives are potential tags or signalling intermedi-
ates, diacylglycerol has also been implicated in bacterial
autophagy 37. Thus, the presence of an E3 ligase domain
in an autophagy-targeting factor does not necessarily
indicate that it ubiquitylates the cargo as a requirement
for selective autophagy.
In summary, we now understand in greater detail
how autophagy is activated in response to microbial
presence. Autophagy is controlled by nearly all classes
of PRRs and is also regulated by cytokines and recep-
tors that modulate innate and adaptive immunity.
Integration of the immune triggers is an obvious area
for continuing study, and it is already clear that the
antimicrobial functions of autophagy can be promoted
(for example, by TH1 cell-associated cytokines and
IL‑1β) or inhibited (for example, by TH2 cell-associated
cytokines) by immune mediators. At the intracellular
level, an idea has emerged of a graded, multitiered sys-
tem of autophagic responses that are commensurate
with the extent of microbial penetration. An interesting
recent development was the discovery of the hierarchi-
cal cooperation of several proteins in controlling anti-
microbial autophagy. However, gaps in our knowledge
remain, especially with regard to alternative, that is, not
ubiquitin-based, modes of target recognition. At pre-
sent, recognition of ubiquitin tags seems to be crucial
for autophagy, and this is further supported by evidence
of bacterial countermeasures that involve deubiquity-
lation88. Moreover, ubiquitin tags might only activate
autophagy after more specialized mechanisms of recog-
nition have failed. It remains to be determined whether
overly strong activation of antimicrobial autophagy
comes at a price, as exemplified by its interference with
type I IFN signalling. Finally, although autophagy is a
mode of self defence that is available, in principle, to
any eukaryotic cell, the magnitude of its contribution
in complex organisms is cell type dependent, as shown
by the fact that it has an important role in protecting
neurons against HSV1 infection whereas it is absent
in keratinocytes of the vaginal mucosa56. Thus, at the
organismal level, tissue- and cell-specific engagement
of antimicrobial autophagy may be a key factor.
Control of inflammation
Genetic links with immune disorders. The role of
autophagy in inflammatory diseases was initially
established through genome-wide association stud-
ies (GWASs)66. Polymorphisms in autophagy-asso-
ciated genes, such as ATG16L1 and IRGM, are linked
to Crohn’s disease66. In addition to single nucleotide
polymorphisms, IRGM is an example of a gene dosage
(through copy number variants) correlation with a pre-
disposition to Crohn’s disease in human populations85.
One of the common IRGM polymorphisms in Crohn’s
disease leads to an escape from the negative regula-
tion of IRGM expression by a microRNA (miRNA)86.
A recent GWAS analysis of 75,000 individuals indicates
an overlap between susceptibility loci for inflammatory
bowel disease and mycobacterial infections89. Moreover,
REVIEWS
a link has been reported between Crohn’s disease and
the autophagy-targeting factor SMURF1 (REF.  89) .
Furthermore, polymorphisms in ULK1 have also been
linked with Crohn’s disease90.
In addition, polymorphisms in autophagy-associated
genes have been associated with autoimmune dis-
orders. IRGM polymorphisms may be a risk factor
in systemic lupus erythematosus (SLE)91. Moreover,
GWASs have linked ATG5 variants with SLE92–94 and
asthma95. Rheumatoid arthritis has been associated
with variations in the PR domain-containing gene 1
(PRDM1)–ATG5 intergenic region96. A new human
autophagy locus, which was first functionally identified
in Caenorhabditis elegans screens as epg5 and which was
shown in mice to be required for autophagosomal matu-
ration97, has been linked to the complex Vici syndrome
that includes immunodeficiency 98. Thus, autophagy
shows clinical relevance as a result of genetic links with
immunological and inflammatory disorders.
Autophagy affects PRR-mediated type I IFN signalling.
The autophagic machinery can amplify TLR signalling;
for example, autophagy enhances the delivery of cyto-
plasmic PAMPs to endosomal TLR7, which enables the
recognition of cytoplasmic viral replication intermedi-
ates and IFNα production by pDCs50. However, the role
of autophagy in PAMP–PRR amplification loops can
lead to aberrations and auto­immunity. In the case of
TLR9, which normally responds to microbial unmethyl-
ated CpG DNA, the autophagic machinery can enhance
aberrant self DNA reactivity. This occurs via intracellular
trafficking events following B cell receptor (BCR) stim-
ulation by self DNA-containing antigens11. Autophagy
promotes the fusion of DNA-containing antigen-loaded
BCR compartments and TLR9‑containing endosomes
and leads to B  cell hyper­responsiveness 11 (FIG.  3a) .
Similarly, in pDCs the autophagic machinery enhances
the trafficking of particulate self DNA-containing
immune complexes and of TLR9 to signalling-
competent compartments, which results in increased
IFNα production32 (FIG. 3a). It is possible that these aber-
rant autophagic activities in B cells and pDCs could
cooperate and lead to the generation of autoimmune
plasma cells (FIG. 3a).
By contrast, there are examples of autophagy-related
factors that directly inhibit the formation or that sup-
press the activation of pro-inflammatory protein com-
plexes. The ATG5–ATG12 complex negatively regulates
RLR signalling by directly binding to the caspase recruit-
ment domains (CARDs) of RIG‑I and IFNβ promoter
stimulator protein 1 (IPS1; also known as MAVS, VISA
or CARDIF; it is a mitochondrial adaptor of RIG‑I sig-
nalling)69 (FIG. 3b). RUBICON inhibits CARD9–BCL‑10–
MALT1 (mucosa-associated lymphoid tissue lymphoma
translocation protein 1) signalling complexes by binding
to CARD9, which thereby terminates pro-inflammatory
signalling downstream of RIG‑I or dectin 1 (REF. 99)
(FIG. 3c). The absence of autophagy amplifies RLR sig-
nalling through increased IPS1 levels as a result of an
accumulation of mitochondria, and increased pools of
depolarized mitochondria in the absence of mitophagy
732 | O CTOBER 2013 | VOLUME 13 www.nature.com/reviews/immunol
© 2013 Macmillan Publishers Limited. All rights reserved
are a source of ROS that also enhance RLR effects25
(FIG. 3d). NLRX1 promotes autophagy and inhibits the
RLR-dependent induction of type I IFN signalling and
inflammation (FIG. 3b); indeed, the two processes are
reciprocally affected by NLRX1 (REF. 67). Finally, ATG9
negatively controls the trafficking of STING and sup-
presses the activation of TBK1 in type I IFN signalling
in response to double-stranded DNA100 (FIG. 3e).
Collectively, these phenomena may represent feed-
back loops by which autophagy downregulates type I
IFN responses following a period of productive induc-
tion or to increase the threshold for activation of type I
IFN signalling. Alternatively, autophagic interference
with RLR signalling could reflect competition for limited
resources, such as TBK1, that are shared between type I
IFN signalling and autophagy pathways14,36,45,101. These
phenomena, in addition to other factors that are not dis-
cussed here, could be an explanation for why autophagy
paradoxically enhances the replication of certain viruses.
Autophagy suppresses inflammasome activation. The
recognition that autophagy has an anti-inflammatory
function stems from the observation that the production
of IL‑1β and IL‑18 is increased in the absence of func-
tional ATG16L1 in a mouse model of Crohn’s disease102.
Inflammasomes are cytoplasmic complexes that respond
to PAMPs and DAMPs by inducing the proteolytic pro-
cessing and secretion of IL‑1β and IL‑18 (REF. 103). An
inflammasome consists of pro-caspase 1, the adaptor
protein ASC and a sensor protein from the NLR family
(such as NLRP1, NLRP3, NLRC4, NLRP6 or NLRP12)
or from the PYHIN (pyrin and HIN domain-containing
protein) family (which includes absent in melanoma 2
(AIM2) and IFNγ-inducible protein 16 (IFI16)) 103.
Several convergent reports show that autophagy has a
negative role in inflammasome activation104–108. Under
sterile conditions, autophagy clears the cytoplasm of
debris, protein aggregates and defective organelles that
can function as endogenous inflammasome agonists.
Studies suggest that basal levels of autophagy control the
set point for inflammasome activation104,105. If autophagy
is blocked, this leads to an accumulation of depolarized
mitochondria that leak endogenous inflammasome ago-
nists, such as mitochondrial DNA (which is detected,
at least partly, by AIM2) and ROS (which activate the
NLRP3 inflammasome)104,105 (FIG. 1b).
During infection, the removal of damaged mitochon-
dria may not be a passive process; for example, during
infection with influenza A virus, a NOD2–receptor-
interacting serine/threonine protein kinase 2 (RIPK2)
pathway activates the autophagy factor ULK1 to main-
tain or to increase mitophagy and to thereby reduce
inflammasome activation108. This RIPK2‑dependent
regulation of autophagy may complement the
RIPK2‑independent action of NOD2 following the
recruitment of ATG16L1 to microorganisms41 and both
processes may contribute to inflammatory syndromes
such as Crohn’s disease. Importantly, autophagy may
downregulate prolonged inflammasome activity by
removing aggregated inflammasome components107.
This depends on the recruitment of sequestosome 1 to

Exocyst
An evolutionarily conserved
protein complex that consists
of eight subunits and that is
best known for targeting
exocytic vesicles to sites of
docking and fusion at the
plasma membrane. It also
functions as a protein complex
assembly platform.
NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 733
K63‑ubiquitylated ASC107. A role for exocyst has been
implicated in the overall process107, but the process could
be self-starting, as sequestosome 1 associates with the
E3 ligase TRAF6 (REF. 78) that ubiquitylates beclin 1 to
initiate autophagy 9; however, this has not been inves-
tigated. In summary, basal autophagy protects cells
from inadvertent inflammasome activation initiating
damaging sterile inflammation, whereas a deficiency in
autophagy causes increased IL‑1β levels (FIG. 1b).
Autophagy suppresses calpain-dependent IL‑1α activa-
tion. Similarly to IL‑1β, IL‑1α is synthesized as a cyto-
plasmic pro-form, and is processed by calpain or other
proteases before being actively secreted from cells or
passively released following cell death109. The activation
and the secretion of IL‑1α involve caspase 1‑dependent
and caspase 1‑independent processes109. Autophagy-
defective (Atg5fl/fl LysM–Cre+) macrophages secrete
high levels of IL‑1α through a calpain-dependent but
inflammasome-independent pathway 109. This pheno-
type has been observed both in vitro and in vivo 45,109.
The processing of pro‑IL‑1α by calpain is induced by
ROS that have been released from accumulated depo-
larized mitochondria in autophagy-deficient cells109.
Thus, ROS in autophagy-defective cells activate both the
inflammasome and the calpain pathways that lead to the
excess production of IL‑1β and IL‑1α, respectively 104,109.
IL‑1α that has been released from ATG5‑defective mac-
rophages has important consequences in vivo, as it leads
to enhanced and prolonged TH17 cell responses in the
context of M. tuberculosis infection, which contributes to
lung tissue damage in a mouse model of tuberculosis109.
Autophagy and degradation of pro-inflamma-
tory signalling factors. Autophagy factors inhibit
BCL‑10‑containing complexes99, and autophagy degrades
BCL‑10 to reduce nuclear factor‑κB (NF‑κB) activation,
as shown in antigen-activated T cells110. This occurs via
sequestosome 1 association with K63‑polyubiquitylated
BCL‑10 (FIG. 3c). As mentioned above, BCL‑10‑containing
complexes are also inhibited by the binding of RUBICON
to CARD9 (REF. 99). As RUBICON is a negative regulator
of autophagy, its translocation to BCL‑10 complexes leads
to the direct inhibition of CARD9–BCL‑10–MALT1
signalling and may also enhance the autophagic deg-
radation of these complexes (FIG. 3c). NF‑κB signalling
may also be downregulated by autophagy via NSFL1C
cofactor p47, which is a protein that has a ubiquitin-
binding UBA domain and its orthologue in yeast binds
to ATG‑8. This potential adaptor functions as a negative
regulator of IKK through the lysosomal (and presum-
ably autophagic) degradation of polyubiquitylated NF‑κB
essential modulator (NEMO)111. The targeting of NEMO
for autophagic degradation is specifically induced by the
murine cytomegalo­virus protein M45; this suppresses
antiviral processes during infection112.
In summary, genetic and functional studies indicate
that autophagy is an anti-inflammatory mechanism
that affects numerous pathways. The most prominent
examples are the dampening of inflammasome acti-
vation and type I IFN signalling; however, broader
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
targeting of other inflammatory signalling pathways is
also becoming apparent. This is consistent with the idea
that autophagy eliminates microorganisms (and micro-
bial products or corpses — an area that deserves more
investigation) and endogenous irritants to prevent exces-
sive inflammation. When autophagic clearance fails,
inflammation ensues as the body’s response to persistent
danger. This may be an explanation for the selection of
genetic alleles in autophagy-related factors that promote
inflammation at certain anatomical sites. This has been
recently shown in the case of the Crohn’s disease risk
factor ATG16L1, whereby a defect in autophagy may
paradoxically confer protection against uropathogenic
Escherichia coli (UPEC) by increasing the production
of pro-inflammatory cytokines and by promoting the
recruitment of innate immune cells to UPEC-infected
bladders in ATG16L1‑hypomorphic mice113.
Autophagy in adaptive immunity
Autophagy in antigen presentation and T cell responses.
Autophagy functions as a bulk ‘topological inverter’ by
transporting proteins from the cytoplasm into the lumen
of antigen-processing compartments54,114,115 (FIG. 1c). This
property can be artificially exploited by fusing antigens
(such as influenza virus matrix protein 1) to LC3 to
enhance MHC class II‑mediated antigen presentation
to CD4+ T cells116. The topological inversion principle
is more generally applicable as it supplies cytoplasmic
ligands to endosomal receptors; for example, autophagy
transfers viral RNA replication intermediates to lumenal
TLR7, as discussed above.
The contributions of autophagy to MHC class II
presentation are not restricted to cytoplasmic pro-
teins and extend through LAP to exogenous antigens.
Autophagy augments the MHC class II‑dependent pro-
cessing and presentation of phagocytosed extracellular
particulate antigens, as shown using PRR-stimulated
LAP of ovalbumin-coated latex beads in DCs53. PRR-
mediated enhancement of MHC class II presenta-
tion also occurs through the induction of autophagy
following NOD2 stimulation with bacterial muramyl
dipeptides42 (FIG. 1c).
Autophagy-enhanced MHC class II presentation
possibly has a role in the selection of naive T cell rep-
ertoires in the thymus. In autophagy-deficient mice,
both positive and negative selection of CD4 + T cells
but not of CD8+ T cells were affected, and this had
consequences for the selection of appropriate T cell
receptor (TCR) repertoires and for the elimination
of autoreactive CD4 + T  cells 115. Transplantation of
ATG5‑deficient thymi into wild-type mice caused an
infiltration of autoreactive CD4+ T cells into multiple
organs and induced autoimmune colitis that was similar
to Crohn’s disease-related phenotypes115. These effects
have been attributed to the defective presentation of
cytoplasmic antigens by autophagy-deficient thymic
epithelial cells115. Other studies investigating DC func-
tion indicate that autophagy-dependent antigen pres-
entation is defective in DCs from patients with Crohn’s
disease that express the ATG16L1 or NOD2 disease risk
variants42. The contributions of autophagy-dependent
REVIEWS
Citrullinated antigens
Citrullin is enzymatically
generated from arginine
residues by peptidylarginine
deaminase, which is an
enzyme that may be enriched
in autophagosomes. The
presence of autoantibodies in
patients with rheumatoid
arthritis correlates with the
levels of citrullinated antigens
(such as vimentin).
Epithelial-to‑mesenchymal
transition
A reversal of the mesenchymal-
to‑epithelial transition that
occurs during development.
Epithelial-to‑mesenchymal
transition or the
dedifferentiation of epithelial
cells can have normal
physiological roles (such as
in wound healing) or can be
associated with fibrotic
pathologies and cancer.
miRNA response elements
Sequences on RNA
transcripts that have partial
complementarity to
microRNAs (miRNAs). miRNA
binding to the response
elements typically leads to
repression of target gene
expression.
734 | O CTOBER 2013 | VOLUME 13 www.nature.com/reviews/immunol
MHC class II antigen presentation in antimicrobial
defence are experimentally detectable in the context
of adaptive immunity against viral54,114 and bacterial63
infections. As a result of the enrichment of peptidy-
larginine deaminase in autophagosomes117, autophagy
promotes the presentation of citrullinated antigens that
may be relevant to autoreactivity (FIG. 1c). This process
is constitutive in DCs and macrophages, but in B cells it
only occurs following BCR stimulation117.
In contrast to this augmentation of MHC class II
antigen processing by autophagy, autophagic degrada-
tion promotes the disassembly of immunological syn-
apses118. Inhibition of ATG16L1 and IRGM expression
in DCs leads to hyperstable interactions with T cells and
increases T cell activation118. This activation might be
further enhanced in the T cells by the lack of autophagic
degradation of BCL‑10 (REF. 110) (FIG. 3c). Therefore,
although autophagy initially promotes MHC class II
antigen processing, at later stages it may help to down-
regulate the response. Consistent with this idea, during
cancer cell invasion the epithelial-to‑mesenchymal transi-
tion is associated with an attenuation of immunological
synapses between target cells and cytotoxic lympho-
cytes in a process that is dependent on autophagy and
beclin 1 (REF. 119).
Autophagy affects conventional MHC class I presen-
tation by competing with the proteasome for the deg-
radation of newly synthesized cytoplasmic proteins120
(FIG.  1c) . Autophagy may also contribute to uncon-
ventional MHC class I presentation pathways in ways
that are yet to be fully understood121,122. In principle,
autophagy could broadly affect MHC class I peptide rep-
ertoires; for example, NDP52‑dependent autophagy tar-
gets DICER and argonaute (AGO)123, which are involved
in miRNA processing and function. This might correlate
with the abundance of MHC class I‑associated peptides
that seem to be derived from transcripts containing
miRNA response elements (FIG. 1c).
Autophagy in T cell homeostasis and TH17 cell polari-
zation. In addition to its role in antigen presentation,
autophagy affects both the homeostasis and the func-
tion of T cells (FIG. 1c). Autophagy and clearance of
mitochondria are needed for normal haematopoietic
stem cell (HSC) maintenance and function, which
are essential for the production of both myeloid and
lymphoid progenitor cells124. After exiting the thymus,
naive T cells depend on autophagy and mitochon-
drial content reduction for their maturation125. T cells
require maintenance of an ER that is capable of calcium
homeostasis126. Calcium ions are sequestered in ER‑like
structures that accumulate in ATG7‑deficient T cells.
This curtails calcium fluxes in response to TCR engage-
ment 126. In naive T cells, autophagy is suppressed by
cellular FLICE-like inhibitory protein (CFLIP; also
known as CFLAR)127,128, but TCR signalling and CD28
co‑stimulation induce autophagy in activated T cells129.
Autophagy is a pro-survival process in activated T cells
and counteracts the pro-apoptotic function of CD95
(also known as FAS) and CD95L (also known as FASL),
which are upregulated by TCR stimulation128.
© 2013 Macmillan Publishers Limited. All rights reserved
Following T cell activation, autophagy can influence
TH cell polarization, partly by controlling the inflamma-
tory function of innate immune cells; for example, the
excessive secretion of IL‑1α and IL‑1β from autophagy-
deficient macrophages, functions together with IL‑6
and transforming growth factor‑β (TGFβ), to promote
TH17 cell responses109 (FIG. 1c). Indeed, the levels of IL‑17
are increased in the lungs of mice with ATG5‑deficient
myeloid cells during M.  tuberculosis infection 109.
CD4+ T cells show increased IL‑17 expression when
lung-infiltrating leukocytes from mice with an ATG5
deletion in the myeloid lineage are restimulated with
mycobacterial antigens ex vivo109. The increased TH17
cell response might also result from the increased dura-
bility of immunological synapses between T cells and
autophagy-defective DCs109,118.
Autophagy in plasma cells and humoral immunity.
Although autophagy seems not to be important for the
survival of the majority of mature B cells130, lymphoid pre-
cursor stages are affected by the absence of autophagy 131.
Autophagy is needed for the survival of B1 cells, which
are a subset of self-renewing B cells that cannot be
replenished from the adult bone marrow 130. Moreover,
autophagy has a role in ER maintenance in plasma cells
under conditions of high secretory demands. Somewhat
paradoxically, absence of autophagy leads to excessive
immunoglobulin secretion132. Autophagy is important
not only for the function of plasma cells but also for their
survival and homeostasis; it is especially important for
the preservation of the bone marrow plasma cell pool
and, thus, is relevant for the maintenance of long-lived
humoral immunity 132 (FIG. 1c).
In summary, autophagy influences adaptive immune
responses through its effects on antigen presentation,
naive T cell repertoire selection, T cell homeostasis and
TH cell polarization. Remaining questions include whether
autophagy affects T cell polarization beyond TH17 cell
responses — for example, whether it affects regulatory
T cells and other T cell subsets — and how autophagy
in one cell type affects other cells and immune networks
in specific anatomical sites. Answers to these questions
should help to explain the complex phenotypes that are
associated with risk mutations in autophagy genes.
Secretion of immune mediators
In addition to its intracellular actions, autophagy influ-
ences the extracellular release of immune mediators133–135
(FIG. 1d), some of which are stored as pre-made secretary
granules. The defective secretion of lysozyme from
Paneth cells in ATG16L1‑hypomorphic mice results in an
intestinal phenotype that corresponds to that of patients
with Crohn’s disease133. Autophagy modulates the secre-
tion of low-molecular-mass immune mediators such as
ATP, which function extracellularly — either directly or
following ectonucleotidase action — on purinergic P2Y
and P2X receptors to promote immune cell chemotaxis
and inflammasome activation135. Autophagic processes
in senescent cells affect the secretion of IL‑6 and IL‑8
through the constitutive ER‑to‑Golgi apparatus secre-
tory pathway 136. Autophagy reduces immunoglobulin
 REVIEWS

secretion from plasma cells and, thus, seems to be a
mechanism that prevents excessive antibody produc-
tion132. The effects of autophagy on secretory func-
tions are not limited to immune effectors: for example,
autophagy factors and LC3 have a role in the exocytosis
of secretory lysosomes, as has been observed for a form
of LAP during osteoclastic bone resorption137.
Autophagy may contribute to the unconventional
secretion of cytosolic proteins that have extracellular
immune functions. The exact mechanism by which this
occurs is not fully understood. IL‑1β and IL‑18 do not
have signal peptides that facilitate entry to the ER and
the conventional secretory pathway (ER to Golgi appa-
ratus to plasma membrane). Instead, they are delivered
following inflammasome activation to the extracellular
environment via unconventional secretion. Although
autophagy suppresses inflammasome activation under
normal nutrient-rich conditions, autophagy can contrib-
ute to the unconventional secretion of IL‑1β under stress
conditions, for example, following nutrient starvation106.
It is possible that autophagy functions as a double-switch
mechanism to repress the inflammasome under basal con-
ditions but to temporarily increase its physiological out-
put in response to DAMPs or PAMPs during infection.
The pro-inflammatory role of autophagy also extends to
the unconventional secretion of IL‑18 and the DAMP
HMGB1 (REF. 106). This role depends on ATG factors
and on a specialized unconventional secretion regulator
Golgi reassembly-stacking protein (GRASP; for example
GRASP of 55 kDa (GRASP55)). GRASP is important not
only in unconventional secretion but also affects canonical
starvation-induced autophagy in mammalian cells106.
Our understanding of the molecular and cellular
mechanisms that are involved in the secretory role of
autophagy is still in its infancy. It nevertheless extends
the idea of the importance of autophagy in the intracel-
lular space to the extracellular milieu, where it affects
tissue organization, remodelling and the delivery of
extracellular mediators of immunity and inflammation.
Conclusions
Autophagy influences many aspects of innate and adap-
tive immunity. Indeed, it is possible that autophagy
evolved as one of the first antimicrobial defences in
eukaryotic cells, and that it was shaped early on in evolu-
tion from what may initially have arisen as a metabolic
and quality control pathway. Autophagy uses a distinct
set of adaptors, the SLRs, to eliminate invading micro­
organisms and, in turn, pathogens have evolved strategies
to evade autophagic capture. It seems that as evolution
has progressed, nearly all components of the innate
immune system, such as conventional PRRs and inflam-
masomes, have become integrated with autophagy. In the
chordate lineage, this has extended to adaptive immunity,
as is best shown in mammalian systems. As a result, in
humans, a failure in parts of the autophagic apparatus can
lead to inflammatory, autoimmune or general immune
disorders.
The current knowledge of immunological autophagy
is still in its infancy and important questions remain. As
IKKs and TBK1 regulate autophagy, why is autophagy
often at cross-purposes with type I IFN-mediated inflam-
mation? Is the ubiquitylation of microbial components the
main process that leads to pathogen recognition by SLRs,
or does it result from a failure to detect microorganisms
by other means? How are the recognition of microbial
entry and the subsequent targeting of microorganisms
for autophagy integrated through the functions of host
membrane damage sensing, E3 ubiquitin ligases, and pro-
tein and lipid kinases? Are mitochondria and mitophagy
the present-day manifestation of an evolutionary battle
between autophagy and the bacterium endosymbiont
precursor to mitochondria? The increased understanding
of the roles of autophagy in secretion during inflamma-
tion and tissue remodelling is an important development.
In this context, the relationship between autophagy and
unconventional secretion, including the secretion of
DAMPs and of inflammasome-dependent substrates
such as IL‑1β, needs further work. Finally, although early
progress has been made in understanding the roles of
autophagy in adaptive immunity, this area warrants
more study. Some of the questions that have recently
been asked in this area refer to the role of autophagy in the
polarization of TH17 cells and possibly other T cell subsets,
along with the role of autophagy in general lymphocyte
homeostasis and differentiation.
In summary, autophagy and immunity are fully
integrated and our continuing study of the interface
between these processes will be a fruitful area of sci-
entific inquiry for many years to come. In translational
terms, autophagy is undoubtedly an attractive target for
developing new treatments in inflammatory disorders
and autoimmunity, and may eventually be harnessed as
an anti-infective mechanism.

1. Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of
atg proteins in autophagosome formation. Annu.
Rev. Cell Dev. Biol. 27, 107–132 (2011).
2. Codogno, P., Mehrpour, M. & Proikas-Cezanne, T.
Canonical and non-canonical autophagy: variations on
a common theme of self-eating? Nature Rev. Mol. Cell
Biol. 13, 7–12 (2012).
3. Virgin, H. W. & Levine, B. Autophagy genes in
immunity. Nature Immunol. 10, 461–470 (2009).
4. Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in
immunity and inflammation. Nature 469, 323–335
(2011).
5. Hamasaki, M. et al. Autophagosomes form at
ER‑mitochondria contact sites. Nature 495, 389–393
(2013).
6. Itakura, E., Kishi-Itakura, C. & Mizushima, N. The
hairpin-type tail-anchored SNARE syntaxin 17 targets
to autophagosomes for fusion with endosomes/
lysosomes. Cell 151, 1256–1269 (2012).
7. Tattoli, I. et al. Amino acid starvation induced by
invasive bacterial pathogens triggers an innate host
defense program. Cell Host Microbe 11, 563–575
(2012).
8. Shelly, S., Lukinova, N., Bambina, S., Berman, A. &
Cherry, S. Autophagy is an essential component of
Drosophila immunity against vesicular stomatitis
virus. Immunity 30, 588–598 (2009).
9. Shi, C. S. & Kehrl, J. H. TRAF6 and A20 regulate lysine
63‑linked ubiquitination of Beclin‑1 to control
TLR4‑induced autophagy. Sci. Signal. 3, ra42 (2010).
10. Nazio, F. et al. mTOR inhibits autophagy by controlling
ULK1 ubiquitylation, self-association and function
through AMBRA1 and TRAF6. Nature Cell Biol. 15,
406–416 (2013).
11. Chaturvedi, A., Dorward, D. & Pierce, S. K. The B cell
receptor governs the subcellular location of Toll-like
receptor 9 leading to hyperresponses to DNA-
containing antigens. Immunity 28, 799–809 (2008).
12. Biswas, D. et al. ATP-induced autophagy is associated
with rapid killing of intracellular mycobacteria within
human monocytes/macrophages. BMC Immunol. 9, 35
(2008).
13. Tang, D. et al. Endogenous HMGB1 regulates
autophagy. J. Cell Biol. 190, 881–892 (2010).
14. Pilli, M. et al. TBK‑1 promotes autophagy-mediated
antimicrobial defense by controlling autophagosome
maturation. Immunity 37, 223–234 (2012).
15. Gutierrez, M. G. et al. Autophagy is a defense
mechanism inhibiting BCG and Mycobacterium
tuberculosis survival in infected macrophages.
Cell 119, 753–766 (2004).

NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 735

© 2013 Macmillan Publishers Limited. All rights reserved

REVIEWS
16. Harris, J. et al. T helper 2 cytokines inhibit autophagic
control of intracellular Mycobacterium tuberculosis.
Immunity 27, 505–517 (2007).
17. Singh, S. B. et al. Human IRGM regulates autophagy and
cell-autonomous immunity functions through
mitochondria. Nature Cell Biol. 12, 1154–1165
(2010).
18. Zalckvar, E. et al. DAP-kinase-mediated phosphorylation
on the BH3 domain of beclin 1 promotes dissociation
of beclin 1 from Bcl‑XL and induction of autophagy.
EMBO Rep. 10, 285–292 (2009).
19. Fabri, M. et al. Vitamin D is required for IFNγ-
mediated antimicrobial activity of human
macrophages. Sci. Transl. Med. 3, 104ra102 (2011).
This is a translationally important study showing
that vitamin D has a key cooperative role with IFNγ
in inducing antimicrobial autophagy in human cells,
whereas vitamin D metabolites are diminished in
tuberculosis and HIV.
20. Mostowy, S. et al. p62 and NDP52 proteins target
intracytosolic Shigella and Listeria to different
autophagy pathways. J. Biol. Chem. 286,
26987–26995 (2011).
21. Van Grol, J. et al. HIV‑1 inhibits autophagy in
bystander macrophage/monocytic cells through Src-
Akt and STAT3. PLoS ONE 5, e11733 (2010).
22. Shen, S. et al. Cytoplasmic STAT3 represses
autophagy by inhibiting PKR activity. Mol. Cell 48,
667–680 (2012).
23. Scherz-Shouval, R. et al. Reactive oxygen species are
essential for autophagy and specifically regulate the
activity of Atg4. EMBO J. 26, 1749–1760 (2007).
24. Huang, J. et al. Activation of antibacterial autophagy
by NADPH oxidases. Proc. Natl Acad. Sci. USA 106,
6226–6231 (2009).
25. Tal, M. C. et al. Absence of autophagy results in
reactive oxygen species-dependent amplification
of RLR signaling. Proc. Natl Acad. Sci. USA 106,
2770–2775 (2009).
26. Yang, C. S. et al. Autophagy protein Rubicon mediates
phagocytic NADPH oxidase activation in response
to microbial infection or TLR stimulation.
Cell Host Microbe 11, 264–276 (2012).
27. Sarkar, S. et al. Complex inhibitory effects of nitric
oxide on autophagy. Mol. Cell 43, 19–32 (2011).
28. Maiuri, M. C. et al. Functional and physical interaction
between Bcl‑XL and a BH3‑like domain in Beclin‑1.
EMBO J. 26, 2527–2539 (2007).
29. Wei, Y., Pattingre, S., Sinha, S., Bassik, M. & Levine, B.
JNK1‑mediated phosphorylation of Bcl‑2 regulates
starvation-induced autophagy. Mol. Cell 30, 678–688
(2008).
30. Egan, D. F. et al. Phosphorylation of ULK1 (hATG1)
by AMP-activated protein kinase connects energy
sensing to mitophagy. Science 331, 456–461
(2011).
31. Sanjuan, M. A. et al. Toll-like receptor signalling in
macrophages links the autophagy pathway to
phagocytosis. Nature 450, 1253–1257 (2007).
32. Henault, J. et al. Noncanonical autophagy is required
for type I interferon secretion in response to DNA-
immune complexes. Immunity 37, 986–997 (2012).
This is an important study that differentiates LAP
from conventional autophagy by defining it at the
molecular level as a process that is independent of
ULK1 but that is dependent on beclin 1.
33. Johansen, T. & Lamark, T. Selective autophagy
mediated by autophagic adapter proteins.
Autophagy 7, 279–296 (2011).
34. Dupont, N. et al. Shigella phagocytic vacuolar
membrane remnants participate in the cellular
response to pathogen invasion and are regulated by
autophagy. Cell Host Microbe 6, 137–149 (2009).
35. Thurston, T. L., Ryzhakov, G., Bloor, S., von
Muhlinen, N. & Randow, F. The TBK1 adaptor and
autophagy receptor NDP52 restricts the proliferation
of ubiquitin-coated bacteria. Nature Immunol. 10,
1215–1221 (2009).
36. Wild, P. et al. Phosphorylation of the autophagy
receptor optineurin restricts Salmonella growth.
Science 333, 228–233 (2011).
37. Shahnazari, S. et al. A diacylglycerol-dependent
signaling pathway contributes to regulation of
antibacterial autophagy. Cell Host Microbe 8,
137–146 (2010).
38. Thurston, T. L., Wandel, M. P., von Muhlinen, N.,
Foeglein, A. & Randow, F. Galectin 8 targets damaged
vesicles for autophagy to defend cells against bacterial
invasion. Nature 482, 414–418 (2012).
39. Deretic, V. & Levine, B. Autophagy, immunity, and
microbial adaptations. Cell Host Microbe 5, 527–549
(2009).
736 | O CTOBER 2013 | VOLUME 13 www.nature.com/reviews/immunol
40. Saitoh, T. & Akira, S. Regulation of innate immune
responses by autophagy-related proteins. J. Cell Biol.
189, 925–935 (2010).
41. Travassos, L. H. et al. Nod1 and Nod2 direct
autophagy by recruiting ATG16L1 to the plasma
membrane at the site of bacterial entry. Nature
Immunol. 11, 55–62 (2010).
42. Cooney, R. et al. NOD2 stimulation induces autophagy
in dendritic cells influencing bacterial handling and
antigen presentation. Nature Med. 16, 90–97
(2010).
43. Birmingham, C. L., Smith, A. C., Bakowski, M. A.,
Yoshimori, T. & Brumell, J. H. Autophagy controls
Salmonella infection in response to damage to the
Salmonella-containing vacuole. J. Biol. Chem. 281,
11374–11383 (2006).
44. Birmingham, C. L. et al. Listeriolysin O allows Listeria
monocytogenes replication in macrophage vacuoles.
Nature 451, 350–354 (2008).
45. Watson, R. O., Manzanillo, P. S. & Cox, J. S.
Extracellular M. tuberculosis DNA targets bacteria
for autophagy by activating the host DNA-sensing
pathway. Cell 150, 803–815 (2012).
This study shows that microbial DNA that is
released into the host cell cytosol can induce
autophagy via STING, which forms an important
intersection with type I IFN signalling. It also shows
the in vivo role of autophagy in protection against
M. tuberculosis.
46. Yoshikawa, Y. et al. Listeria monocytogenes ActA-
mediated escape from autophagic recognition.
Nature Cell Biol. 11, 1233–1240 (2009).
47. Ogawa, M. et al. A Tecpr1‑dependent selective
autophagy pathway targets bacterial pathogens.
Cell Host Microbe 9, 376–389 (2011).
48. dependent on stimulator of IFN genes. J. Immunol.
Talloczy, Z., Virgin, H. W.t. & Levine, B. PKR-dependent
autophagic degradation of herpes simplex virus
type 1. Autophagy 2, 24–29 (2006).
49. Orvedahl, A. et al. HSV‑1 ICP34.5 confers
neurovirulence by targeting the beclin 1 autophagy
protein. Cell Host Microbe 1, 23–35 (2007).
50. Lee, H. K., Lund, J. M., Ramanathan, B.,
Mizushima, N. & Iwasaki, A. Autophagy-dependent
viral recognition by plasmacytoid dendritic cells.
Science 315, 1398–1401 (2007).
51. Kyei, G. B. et al. Autophagy pathway intersects with
HIV‑1 biosynthesis and regulates viral yields in
macrophages. J. Cell Biol. 186, 255–268 (2009).
52. Gannage, M. et al. Matrix protein 2 of influenza A
virus blocks autophagosome fusion with lysosomes.
Cell Host Microbe 6, 367–380 (2009).
53. Lee, H. K. et al. In vivo requirement for Atg5 in
antigen presentation by dendritic cells. Immunity 32,
227–239 (2010).
54. Blanchet, F. P. et al. Human immunodeficiency virus‑1
inhibition of immunoamphisomes in dendritic cells
impairs early innate and adaptive immune responses.
Immunity 32, 654–669 (2010).
55. Orvedahl, A. et al. Autophagy protects against
sindbis virus infection of the central nervous system.
Cell Host Microbe 7, 115–127 (2010).
56. Yordy, B., Iijima, N., Huttner, A., Leib, D. & Iwasaki, A.
A neuron-specific role for autophagy in antiviral
defense against herpes simplex virus. Cell Host
Microbe 12, 334–345 (2012).
57. Shoji-Kawata, S. et al. Identification of a candidate
therapeutic autophagy-inducing peptide. Nature 494,
201–206 (2013).
This study is a key step towards customizing the
induction of autophagy without using mTOR
inhibitors. The peptide that was designed on the
basis of the Nef-binding site on beclin 1 showed
efficacy in various infections and in other in vitro
and in vivo models.
58. Xu, Y. et al. Toll-like receptor 4 is a sensor for
autophagy associated with innate immunity.
Immunity 27, 135–144 (2007).
59. Delgado, M. A., Elmaoued, R. A., Davis, A. S., Kyei, G.
& Deretic, V. Toll-like receptors control autophagy.
EMBO J. 27, 1110–1121 (2008).
60. Alonso, S., Pethe, K., Russell, D. G. & Purdy, G. E.
Lysosomal killing of Mycobacterium mediated by
ubiquitin-derived peptides is enhanced by
autophagy. Proc. Natl Acad. Sci. USA 104,
6031–6036 (2007).
61. Ponpuak, M. et al. Delivery of cytosolic components
by autophagic adaptor protein p62 endows
autophagosomes with unique antimicrobial
properties. Immunity 32, 329–341 (2010).
62. Kim, B. H. et al. A family of IFNγ-inducible 65‑kD
GTPases protects against bacterial infection. Science
332, 717–721 (2011).
© 2013 Macmillan Publishers Limited. All rights reserved
63. Jagannath, C. et al. Autophagy enhances the efficacy
of BCG vaccine by increasing peptide presentation in
mouse dendritic cells. Nature Med. 15, 267–276
(2009).
64. Yano, T. et al. Autophagic control of listeria through
intracellular innate immune recognition in drosophila.
Nature Immunol. 9, 908–916 (2008).
65. Moreau, K., Ravikumar, B., Renna, M., Puri, C. &
Rubinsztein, D. C. Autophagosome precursor
maturation requires homotypic fusion. Cell 146,
303–317 (2011).
66. Wellcome Trust Case Control Consortium. Genome-
wide association study of 14,000 cases of seven
common diseases and 3,000 shared controls. Nature
447, 661–678 (2007).
67. Lei, Y. et al. The mitochondrial proteins NLRX1 and
TUFM form a complex that regulates type I interferon
and autophagy. Immunity 36, 933–946 (2012).
68. Jounai, N. et al. NLRP4 negatively regulates
autophagic processes through an association with
beclin1. J. Immunol. 186, 1646–1655 (2011).
69. Jounai, N. et al. The Atg5 Atg12 conjugate associates
with innate antiviral immune responses. Proc. Natl
Acad. Sci. USA 104, 14050–14055 (2007).
This is a key study that led to the appreciation of
the interference between autophagy and type I IFN
signalling.
70. McFarlane, S. et al. Early induction of autophagy
in human fibroblasts after infection with human
cytomegalovirus or herpes simplex virus 1.
J. Virol. 85, 4212–4221 (2011).
71. Rasmussen, S. B. et al. Activation of autophagy by
α-herpesviruses in myeloid cells is mediated by
cytoplasmic viral DNA through a mechanism
187, 5268–5276 (2011).
72. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J.
Cyclic GMP-AMP synthase is a cytosolic DNA sensor
that activates the type I interferon pathway.
Science 339, 786–791 (2013).
73. Zheng, Y. T. et al. The adaptor protein p62/SQSTM1
targets invading bacteria to the autophagy pathway.
J. Immunol. 183, 5909–5916 (2009).
74. Yang, J. Q., Liu, H., Diaz-Meco, M. T. & Moscat, J.
NBR1 is a new PB1 signalling adapter in Th2
differentiation and allergic airway inflammation
in vivo. EMBO J. 29, 3421–3433 (2010).
75. Bjorkoy, G. et al. p62/SQSTM1 forms protein
aggregates degraded by autophagy and has a
protective effect on huntingtin-induced cell death.
J. Cell Biol. 171, 603–614 (2005).
This is a seminal study that led to the identification
of autophagic receptors and mechanisms of
selective autophagy in mammalian cells.
76. Kirkin, V. et al. A role for NBR1 in autophagosomal
degradation of ubiquitinated substrates. Mol. Cell 33,
505–516 (2009).
77. Newman, A. C. et al. TBK1 kinase addiction in lung
cancer cells is mediated via autophagy of Tax1bp1/
Ndp52 and non-canonical NF‑κB signalling.
PLoS ONE 7, e50672 (2012).
78. Moscat, J. & Diaz-Meco, M. T. p62 at the crossroads
of autophagy, apoptosis, and cancer. Cell 137,
1001–1004 (2009).
79. Li, S. et al. Sterical hindrance promotes selectivity of
the autophagy cargo receptor NDP52 for the danger
receptor galectin‑8 in antibacterial autophagy.
Science signaling 6, ra9 (2013).
80. Husnjak, K. & Dikic, I. Ubiquitin-binding proteins:
decoders of ubiquitin-mediated cellular functions.
Annu. Rev. Biochem. 81, 291–322 (2012).
81. von Muhlinen, N. et al. LC3C, bound selectively by
a noncanonical LIR motif in NDP52, is required for
antibacterial autophagy. Mol. Cell 48, 329–342
(2012).
This was an important step in defining the roles of
different mammalian ATG-8 paralogues in
autophagic antimicrobial defence.
82. Matsumoto, G., Wada, K., Okuno, M., Kurosawa, M.
& Nukina, N. Serine 403 phosphorylation of p62/
SQSTM1 regulates selective autophagic clearance of
ubiquitinated proteins. Mol. Cell 44, 279–289
(2011).
83. Huett, A. et al. The LRR and RING domain protein
LRSAM1 is an E3 ligase crucial for ubiquitin-
dependent autophagy of intracellular Salmonella
Typhimurium. Cell Host Microbe 12, 778–790 (2012).
This paper identifies an E3 ubiquitin ligase that
recognizes cytoplasmic bacteria, that ubiquitylates
autophagic targets and that cooperates with other
receptors to guide autophagic capture and
elimination of the invading microorganisms.

84. Huang, W. et al. Crystal structure and biochemical
analyses reveal Beclin 1 as a novel membrane binding
protein. Cell Res. 22, 473–489 (2012).
85. Craddock, N. et al. Genome-wide association study
of CNVs in 16,000 cases of eight common diseases
and 3,000 shared controls. Nature 464, 713–720
(2010).
86. Brest, P. et al. A synonymous variant in IRGM alters a
binding site for miR‑196 and causes deregulation of
IRGM-dependent xenophagy in Crohn’s disease.
Nature Genet. 43, 242–245 (2011).
87. Orvedahl, A. et al. Image-based genome-wide siRNA
screen identifies selective autophagy factors. Nature
480, 113–117 (2011).
88. Mesquita, F. S. et al. The Salmonella deubiquitinase
SseL inhibits selective autophagy of cytosolic
aggregates. PLoS Pathog. 8, e1002743 (2012).
89. Jostins, L. et al. Host-microbe interactions have
shaped the genetic architecture of inflammatory
bowel disease. Nature 491, 119–124 (2012).
90. Henckaerts, L. et al. Genetic variation in the
autophagy gene ULK1 and risk of Crohn’s disease.
Inflamm. Bowel Dis. 17, 1392–1397 (2011).
91. Ramos, P. S. et al. A comprehensive analysis of
shared loci between systemic lupus erythematosus
(SLE) and sixteen autoimmune diseases reveals
limited genetic overlap. PLoS Genet. 7, e1002406
(2011).
92. Harley, J. B. et al. Genome-wide association scan in
women with systemic lupus erythematosus identifies
susceptibility variants in ITGAM, PXK, KIAA1542 and
other loci. Nature Genet. 40, 204–210 (2008).
93. Han, J. W. et al. Genome-wide association study in a
Chinese Han population identifies nine new
susceptibility loci for systemic lupus erythematosus.
Nature Genet. 41, 1234–1237 (2009).
94. Yang, W. et al. Meta-analysis followed by replication
identifies loci in or near CDKN1B, TET3, CD80,
DRAM1, and ARID5B as associated with systemic
lupus erythematosus in Asians. Am. J. Hum. Genet.
92, 41–51 (2013).
95. Martin, L. J. et al. Functional variant in the autophagy-
related 5 gene promotor is associated with childhood
asthma. PLoS ONE 7, e33454 (2012).
96. Raychaudhuri, S. et al. Genetic variants at CD28,
PRDM1 and CD2/CD58 are associated with
rheumatoid arthritis risk. Nature Genet. 41,
1313–1318 (2009).
97. Zhao, H. et al. Mice deficient in Epg5 exhibit selective
neuronal vulnerability to degeneration. J. Cell Biol.
200, 731–741 (2013).
98. Cullup, T. et al. Recessive mutations in EPG5 cause
Vici syndrome, a multisystem disorder with defective
autophagy. Nature Genet. 45, 83–87 (2013).
99. Yang, C. S. et al. The autophagy regulator Rubicon is
a feedback inhibitor of CARD9‑mediated host innate
immunity. Cell Host Microbe 11, 277–289 (2012).
100. Saitoh, T. et al. Atg9a controls dsDNA-driven
dynamic translocation of STING and the innate
immune response. Proc. Natl Acad. Sci. USA 106,
20842–20846 (2009).
101. Li, S., Wang, L., Berman, M., Kong, Y. Y. & Dorf, M. E.
Mapping a dynamic innate immunity protein
interaction network regulating type I interferon
production. Immunity 35, 426–440 (2011).
102. Saitoh, T. et al. Loss of the autophagy protein
Atg16L1 enhances endotoxin-induced IL‑1β
production. Nature 456, 264–268 (2008).
This is a key study that recognizes the
anti-inflammatory role of autophagy.
103. Rathinam, V. A., Vanaja, S. K. & Fitzgerald, K. A.
Regulation of inflammasome signaling. Nature
Immunol. 13, 333–332 (2012).
104. Zhou, R., Yazdi, A. S., Menu, P. & Tschopp, J. A role
for mitochondria in NLRP3 inflammasome activation.
Nature 469, 221–225 (2011).
This is a conceptually important study that shows
that housekeeping functions, such as the autophagic
clearance of depolarized mitochondria when they are
failing, can lead to endogenous sterile inflammation.
105. Nakahira, K. et al. Autophagy proteins regulate
innate immune responses by inhibiting the release
of mitochondrial DNA mediated by the NALP3
inflammasome. Nature Immunol. 12, 222–230
(2011).
106. Dupont, N. et al. Autophagy-based unconventional
secretory pathway for extracellular delivery of IL‑1β.
EMBO J. 30, 4701–4711 (2011).
107. Shi, C. S. et al. Activation of autophagy by
inflammatory signals limits IL‑1β production by
targeting ubiquitinated inflammasomes for
destruction. Nature Immunol. 13, 255–263 (2012).
NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | O CTOBER 2013 | 737
108. Lupfer, C. et al. Receptor interacting protein kinase
2‑mediated mitophagy regulates inflammasome
activation during virus infection. Nature Immunol. 14,
480–488 (2013).
109. Castillo, E. F. et al. Autophagy protects against
active tuberculosis by suppressing bacterial burden
and inflammation. Proc. Natl Acad. Sci. USA 109,
E3168–E3176 (2012).
This paper is an in vivo demonstration of the role
of autophagy in controlling both inflammation and
bacteria during infection.
110. Paul, S., Kashyap, A. K., Jia, W., He, Y. W. &
Schaefer, B. C. Selective autophagy of the adaptor
protein Bcl10 modulates T cell receptor activation
of NF‑κB. Immunity 36, 947–958 (2012).
111. Shibata, Y. et al. p47 negatively regulates IKK
activation by inducing the lysosomal degradation of
polyubiquitinated NEMO. Nature Commun. 3, 1061
(2012).
112. Fliss, P. M. et al. Viral mediated redirection of NEMO/
IKKγ to autophagosomes curtails the inflammatory
cascade. PLoS Pathog. 8, e1002517 (2012).
113. Wang, C. et al. Atg16L1 deficiency confers protection
from uropathogenic Escherichia coli infection in vivo.
Proc. Natl Acad. Sci. USA 109, 11008–11013
(2012).
114. Paludan, C. et al. Endogenous MHC class II processing
of a viral nuclear antigen after autophagy. Science
307, 593–596 (2005).
115. Nedjic, J., Aichinger, M., Emmerich, J., Mizushima, N.
& Klein, L. Autophagy in thymic epithelium shapes the
T‑cell repertoire and is essential for tolerance. Nature
455, 396–400 (2008).
116. Schmid, D., Pypaert, M. & Munz, C. Antigen-loading
compartments for major histocompatibility complex
class II molecules continuously receive input from
autophagosomes. Immunity 26, 79–92 (2007).
117. Ireland, J. M. & Unanue, E. R. Autophagy in antigen-
presenting cells results in presentation of
citrullinated peptides to CD4 T cells. J. Exp. Med.
208, 2625–2632 (2011).
118. Wildenberg, M. E. et al. Autophagy attenuates
the adaptive immune response by destabilizing the
immunologic synapse. Gastroenterology 142,
1493–1503 e6 (2012).
119. Akalay, I. et al. Epithelial-to‑mesenchymal transition
and autophagy induction in breast carcinoma promote
escape from T‑cell-mediated lysis. Cancer Res. 73,
2418–2427 (2013).
120. Wenger, T. et al. Autophagy inhibition promotes
defective neosynthesized proteins storage in ALIS, and
induces redirection toward proteasome processing
and MHCI-restricted presentation. Autophagy 8,
350–363 (2012).
121. Fiegl, D. et al. Amphisomal route of MHC class I
cross-presentation in bacteria-infected dendritic cells.
J. Immunol. 190, 2791–2806 (2013).
122. Tey, S. K. & Khanna, R. Autophagy mediates
transporter associated with antigen processing-
independent presentation of viral epitopes through
MHC class I pathway. Blood 120, 994–1004
(2012).
123. Gibbings, D. et al. Selective autophagy degrades
DICER and AGO2 and regulates miRNA activity.
Nature Cell Biol. 14, 1314–1321 (2012).
124. Mortensen, M. et al. The autophagy protein Atg7 is
essential for hematopoietic stem cell maintenance.
J. Exp. Med. 208, 455–467 (2011).
125. Pua, H. H., Guo, J., Komatsu, M. & He, Y. W.
Autophagy is essential for mitochondrial clearance in
mature T lymphocytes. J. Immunol. 182, 4046–4055
(2009).
126. Jia, W., Pua, H. H., Li, Q. J. & He, Y. W. Autophagy
regulates endoplasmic reticulum homeostasis and
calcium mobilization in T lymphocytes. J. Immunol.
186, 1564–1574 (2011).
127. Lee, J. S. et al. FLIP-mediated autophagy regulation in
cell death control. Nature Cell Biol. 11, 1355–1362
(2009).
128. He, M. X. & He, Y. W. A role for c‑FLIPL in the regulation
of apoptosis, autophagy, and necroptosis in
T lymphocytes. Cell Death Differ. 20, 188–197
(2013).
129. Hubbard, V. M. et al. Macroautophagy regulates
energy metabolism during effector T cell activation.
J. Immunol. 185, 7349–7357 (2010).
130. Miller, B. C. et al. The autophagy gene ATG5 plays an
essential role in B lymphocyte development.
Autophagy 4, 309–314 (2008).
131. Arsov, I. et al. A role for autophagic protein beclin 1
early in lymphocyte development. J. Immunol. 186,
2201–2209 (2011).
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
132. Pengo, N. et al. Plasma cells require autophagy
for sustainable immunoglobulin production.
Nature Immunol. 14, 298–305 (2013).
133. Cadwell, K. et al. A key role for autophagy and the
autophagy gene Atg16l1 in mouse and human
intestinal Paneth cells. Nature 456, 259–263 (2008).
This is a key study that shows the general secretory
defects in autophagy-deficient cells that are of
consequence for inflammatory bowel diseases such
as Crohn’s disease.
134. Ushio, H. et al. Crucial role for autophagy in
degranulation of mast cells. J. Allergy Clin. Immunol.
127, 1267–1276 e6 (2011).
135. Michaud, M. et al. Autophagy-dependent anticancer
immune responses induced by chemotherapeutic
agents in mice. Science 334, 1573–1577 (2011).
136. Narita, M. et al. Spatial coupling of mTOR and
autophagy augments secretory phenotypes.
Science 332, 966–970 (2011).
137. DeSelm, C. J. et al. Autophagy proteins regulate the
secretory component of osteoclastic bone resorption.
Dev. Cell 21, 966–974 (2011).
138. Chauhan, S. & Boyd, D. ZKSCAN3 is a master
transcriptional repressor of autophagy.
Mol. Cell 50, 16–28 (2013).
139. Settembre, C. et al. TFEB controls cellular lipid
metabolism through a starvation-induced
autoregulatory loop. Nature Cell Biol. 15, 647–658
(2013).
140. Russell, R. C. et al. ULK1 induces autophagy by
phosphorylating Beclin‑1 and activating VPS34 lipid
kinase. Nature Cell Biol. 15, 741–750 (2013).
This is a study that functionally links two key
branches of autophagy regulators, the ULK1
branch and the beclin 1 branch.
141. Takaesu, G., Kobayashi, T. & Yoshimura, A.
TGFβ-activated kinase 1 (TAK1)-binding proteins
(TAB) 2 and 3 negatively regulate autophagy.
J. Biochem. 151, 157–166 (2012).
142. Zalckvar, E., Berissi, H., Eisenstein, M. & Kimchi, A.
Phosphorylation of Beclin 1 by DAP-kinase promotes
autophagy by weakening its interactions with Bcl‑2
and Bcl‑XL. Autophagy 5, 720–722 (2009).
143. Criollo, A. et al. Inhibition of autophagy by TAB2
and TAB3. EMBO J. 30, 4908–4920 (2011).
144. Radtke, A. L., Delbridge, L. M., Balachandran, S.,
Barber, G. N. & O’Riordan, M. X. TBK1 protects
vacuolar integrity during intracellular bacterial
infection. PLoS Pathog. 3, e29 (2007).
145. Ku, B. et al. Structural and biochemical bases for the
inhibition of autophagy and apoptosis by viral BCL‑2
of murine γ-herpesvirus 68. PLoS Pathog. 4, e25
(2008).
146. Choy, A. et al. The Legionella effector RavZ inhibits
host autophagy through irreversible Atg8
deconjugation. Science 338, 1072–1076 (2012).
This is an elegant recent example of how bacteria
defend themselves against autophagy by
preventing LC3 lipidation, which thus stalls the
autophagic response.
147. Dortet, L. et al. Recruitment of the major vault protein
by InlK: a Listeria monocytogenes strategy to avoid
autophagy. PLoS Pathog. 7, e1002168 (2011).
148. Niu, H., Xiong, Q., Yamamoto, A., Hayashi-Nishino, M.
& Rikihisa, Y. Autophagosomes induced by a bacterial
Beclin 1 binding protein facilitate obligatory
intracellular infection. Proc. Natl Acad. Sci. USA 109,
20800–20807 (2012).
149. Ogawa, M. et al. Escape of intracellular Shigella
from autophagy. Science 307, 727–731 (2005).
150. Gregoire, I. P. et al. IRGM is a common target of
RNA viruses that subvert the autophagy network.
PLoS Pathog. 7, e1002422 (2011).
Acknowledgements
We apologize to our colleagues whose work or specific studies
have not been cited. This is not a reflection of lack of interest
on the authors’ part or on the significance for the field but
was dictated by the article scope and reference number limi-
tations. V.D. is supported by US National Institutes of Health
grant AI042999 and by a grant from the Bill and Melinda
Gates Foundation.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
Expressed sequence tags NCBI database:
http://www.ncbi.nlm.nih.gov/nucest
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