Seminars in Immunology 21 (2009) 233–241
Contents lists available at ScienceDirect
Seminars in Immunology
journal homepage: www.elsevier.com/locate/ysmim
Review
Autophagy as an emerging dimension to adaptive and innate immunity
Séamus Hussey a,b,c,d,1 , Leonardo H. Travassos c,1 , Nicola L. Jones a,b,d,∗
a
Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, Toronto, Canada
Cell Biology Programme, Research Institute, Hospital for Sick Children, Toronto, Canada
c
Department of Immunology, University of Toronto, Toronto, Canada
d
Departments of Pediatrics and Physiology, University of Toronto, Toronto, Canada
b
a r t i c l e
i n f o
Keywords:
Autophagy
ATG16L1
Innate immunity
Antigen presentation
Pattern recognition molecules
a b s t r a c t
Autophagy is an evolutionary conserved cellular process during which cytoplasmic material is engulfed
in double membrane vacuoles that then fuse with lysosomes, ultimately degrading their cargo. Emerging evidence, however, now suggests that autophagy can form part of our innate and adaptive immune
defense programs. Recent studies have identified pattern recognition molecules as mediators of this process and shown that intracellular pathogens can interact with and even manipulate autophagy. Recent
translational evidence has also implicated autophagy in the pathogenesis of several immune-mediated
diseases, including Crohn disease. In this review, we present autophagy in the context of its role as an
immune system component and effector and speculate on imminent and future research directions in
this field.
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Throughout evolution, facets of our cellular biology have been
conserved and adapted. One process at the forefront of homeostasis
and environmental interaction is autophagy. This complex process in eukaryotic cells involves the trafficking of cellular elements
from the cytosol to the lysosome wherein they are degraded and
processed. Ongoing developments in this field point to an inextricable link between autophagy and the innate and adaptive immune
system. In this review, we summarize the pertinent research findings to date and suggest future research directions in this dynamic
field, especially with respect to pattern recognition receptor
interaction.
Three sub-types of autophagy have been described—chaperonemediated autophagy, microautophagy and macroautophagy (hereafter called autophagy). The term ‘autophagy’ was first suggested
by de Duve over 45 years ago [1]. Lamellar vesicles that encapsulated portions of the cytosol and organelle remnants had been
described in early electron microscopy studies as vacuoles and
lysosomes, and were speculated to arise from focal cytoplasmic
degradation [2–4]. Such vesicles bore the hallmarks of what are
now termed ‘autophagosomes’, the characteristic vacuoles synonymous with autophagy. Metabolic manipulation was shown to affect
∗ Corresponding author at: Hospital for Sick Children, 555 University Avenue,
Toronto, ON, Canada M5G 1X8. Tel.: +1 416 813 7734; fax: +1 416 813 6531.
E-mail address: nicola.jones@sickkids.ca (N.L. Jones).
1
These authors contributed equally to this manuscript.
1044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.smim.2009.05.004
autophagy induction, demonstrating that autophagy was a malleable rather than a static process. The catabolic hormone glucagon
and deprivation of amino acids and nutrients were shown to
induce autophagy while insulin and certain exogenous amino acids
impaired autophagy and proteolysis, defining a role for autophagy
in adaptation to cellular stresses [2,5–11]. The complexity of signaling molecules that influence autophagy is an ongoing focus of
research and will be discussed later.
The stepwise process of autophagosome biogenesis is a cornerstone of autophagy. Over thirty governing autophagy genes
(ATG) and their proteins (Atg) have been identified in elegant studies in yeast species [12–14]. While not all mammalian orthologs
have been identified, some have numerous mammalian paralogs
with striking similarities in structure and/or function to their
yeast counterparts [15,16]. Ultra-structural studies of autophagosome membranes have shown that they harbor relatively few
transmembrane proteins and are thinner than other cellular membranes e.g. the plasma membrane [17]. The earliest identifiable
structure in the sequence of autophagosome formation is the diskshaped, isolation membrane or phagophore (Fig. 1). Once formed,
this membrane progressively elongates, encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol, eventually
sealing to complete the autophagosome. Speculation continues
whether the foundation template for the isolation membrane originates from the endoplasmic reticulum, golgi, mitochondria, a
pre-formed organelle membrane or even de novo [18–25]. The
molecular mechanisms that lead to isolation membrane appearance continue to be elucidated in both yeast and mammalian cell
systems.
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2. Machinery of autophagosome formation
Two ubiquitin-like conjugation systems are pivotal to
autophagosome formation and completion. The first system
modifies a core autophagy protein–microtubule-associated protein 1 light chain 3 (LC3). Multiple paralogs of Atg8 exist in
mammals – LC3A, LC3B, GATE16, GABARAP – hereafter referred to
collectively as LC3 [26,27]. LC3 has a diffuse cytosolic distribution.
It is cleaved at its c-terminus by the cysteine protease Atg4 and
in turn undergoes sequential ubiquitin-like modifications by the
E1-like enzyme, Atg7, and the E2-like enzyme, Atg3, to form LC3-1.
The c-terminal carboxyl group of LC3-I is ultimately conjugated
to the amine of phosphatidylethanolamine, forming LC3-II. This
lipidation of LC3-I to form LC3-II is notable in that LC3-II is
exclusively found on autophagosome membranes. The conjugated
yeast ortholog of LC3, Atg8, is known to have membrane tethering
properties, which may explain one of its roles in autophagosome
formation [28]. Atg4 also deconjugates LC3-II on the autophagosome membrane, releasing LC3, highlighting the plasticity of
this process. The multifunctional protein p62 interacts with both
ubiquitinated proteins and LC3, whereby it is incorporated into
autophagosomes. p62 accumulates during autophagy inhibition
and has been implicated in targeting proteins to autophagosomes,
although it is not itself essential for autophagosome formation
[29].
In the second conjugation system, the ubiquitin-like autophagy
protein Atg12 is covalently conjugated to Atg5 via its c-terminal
glycine, forming the dimeric Atg12–Atg5 complex, following
ubiquitin-like reactions involving Atg7 and Atg10. The autophagy
scaffold protein Atg16L1 is then conjugated to Atg5 via its Nterminus, forming the Atg12–Atg5–Atg16L1 complex. The Atg16L1
complex self-multimerizes, forming large 800 kDa complexes.
These are found in the cytosol and on the evolving isolation membrane, and are likely necessary for the ultimate conjugation of LC3-I.
Fujita et al. showed that the Atg16L1 complex behaves as an E3-like
enzyme and targets LC3-I to its membrane site of lipid conjugation [30]. The Atg16L1 complexes dissociate from autophagosome
membranes as they near completion.
Other essential groups of autophagy proteins participate in isolation membrane formation. The mammalian autophagy proteins
ULK1 (Unc-51-like kinase), FIP200 (focal adhesion kinase family
interacting protein) and Atg13 were recently identified in a complex
which subsequently co-localized at the nascent isolation membrane on autophagy induction, similar to the Atg1–Atg13–Atg17
complex in yeast [31]. The c-terminus of ULK-1 binds to FIP200 and
Atg13, and ULK-1 also interacts with LC3 [32]. Mammalian studies
of the trans-membrane protein Atg9 have similarly underscored
its essential role early in autophagosome formation. It associates
with the trans-golgi network, late endosomes, LC3, the Rab-GTPase
proteins (Rab7 and Rab9) and re-distributes following autophagy
induction, localizing to the nascent autophagosome [33]. Yeast Atg9
was recently shown to self-multimerise via its c-terminus, facilitating its intra-cellular trafficking, independent of other autophagy
proteins. This novel finding suggests a potential further role for
such Atg9 complexes in contributing to early isolation membrane
formation [25].
3. Controlling autophagy
The discovery of the target of rapamycin in yeast (TOR)
and mammalian cells (mTOR) led to significant advances in
understanding autophagy regulation, through the family of phosphatidylinositol kinase-related kinases [34–36]. These signaling
networks are involved in broad cellular functions from metabolic
responses to growth and proliferation.
The key serine/threonine kinase, Akt, links the mTOR and
phosphatidylinositol-3 kinase (PI3K) pathways which are activated by a diverse array of stimuli, including cytokine receptors
and toll-like receptors (TLR) [37]. Following receptor activation,
class-I PI3Ks are recruited by receptor adaptor molecules to phosphorylate phosphatidylinositol-4,5-bisphosphate, which in turn
phosphorylates and activates Akt [38,39]. The mTOR complexes,
down-stream positive effectors of Akt, integrate multiple cellular
signals, including those from growth factors, amino acids and intracellular ATP. mTOR activation increases cellular anabolic activity
and protein translation [40–42]. Autophagy is under negative regulation by activated Akt and mTOR [43]. Recently, mTOR was shown
to phosphorylate and therefore inhibit the ULK kinase-complex
activity, disrupting autophagosome formation [44,45]. Rapamycin
inhibition of mTOR and amino acid deprivation reversed these
effects. mTOR may further affect autophagy through its control of
autophagy gene transcription [40,46].
The class III PI3K enzyme, Vps34 (vacuolar protein sorting 34),
solely phosphorylates phosphatidylinositol and is involved in regulating vesicular trafficking, nutrient sensing and autophagy [47,48].
The pharmacological agent 3-methyadenine (3-MA) inhibits its
function in vitro. Together with Vps15 (another kinase), Beclin1, UVRAG (ultraviolet radiation resistance associated gene) and
Ambra-1, Vps34 forms a multiprotein complex that is necessary
for early stages of autophagosome biogenesis and can up-regulate
autophagy overall [49–51]. However, its seemingly paradoxical role
in signal transduction to the mTOR complex following amino acid
sensing suggests that its signaling function may depend on the
nature of its interacting protein complexes [52,53].
Beclin-1, a tumor suppressor protein, is itself also involved
in modulating autophagy through its interaction with Bcl-2, an
anti-apoptotic protein that inhibits both autophagy and apoptosis. The Beclin-1/Bcl-2 interaction is an evolutionary conserved
phenomenon, the balance of which determines either up- or downregulation of autophagy. Silencing or over-expression of Bcl-2
was shown to enhance or suppress starvation-induced autophagy
respectively [54]. These effects were specifically dependent on
Beclin-1/Bcl-2 interaction, suggesting that nutrient sensing affects
the equilibrium of the Beclin-1/Bcl-2 interaction. Bcl-2 dominant
interactions with Beclin-1 likely disrupt Beclin-1/Vps34 complex
formation, leading to autophagy suppression, although the mechanism has not been fully elucidated. Recently, the toll-like receptor
(TLR) signaling molecules MyD88 and TRIF were shown to modulate the Beclin-1/Bcl-2 interaction, enhancing their interaction with
Beclin-1 to induce autophagy [55].
A myriad of other signal transduction and effector molecules
influence autophagy regulation, directly or indirectly. The Akt
and JNK pathways have been shown to enhance or reduce
expression of LC3 and Beclin-1 in response to tumor necrosis
factor-␣ (TNF-␣) and insulin-like growth factor-1 respectively [56].
The mammalian transcription factor, NFB, is a key regulator
of gene expression, modulating physiological processes including inflammation, apoptosis and also autophagy. TNF␣-induced
NFB activation suppresses autophagy, while NFB suppression
enhances starvation-induced autophagy [57,58]. NFB may signal
through mTOR activation or by affecting enhanced Bcl-2 expression to modulate autophagy. Autophagy itself may in turn influence
NFB activity since it is involved in degradation of IB kinase, the
upstream activator of NFB, through association with the heatshock protein, Hsp90 [59,60]. Reactive oxygen species (ROS) are
highly reactive molecules generated from mitochondrial respiratory activity and the products of oxidase enzymes, including NADPH
oxidase, and are capable of modulating autophagy [61,62]. Atg4
is redox-regulated via a conserved cysteine residue and, furthermore, starvation-induced autophagy depends on H2 O2 signaling
[62]. Starvation lead to local H2 O2 formation, partly dependent on
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class III PI3K activity, and anti-oxidant treatment in vitro attenuated
autophagy induction. Recently, a transgenic mouse model harboring a mutant form of super-oxide dismutase, a key anti-oxidant
enzyme, also showed increased autophagic activity due to ROS
accumulation [63]. Evidence also suggests that autophagic (type
II) cell death may stem from ROS accumulation, as seen following
in vitro treatments with TNF␣ and LPS [57,64].
4. Pathogen recognition of autophagy
Microbial invasion of the cytosol presents a serious challenge to
our innate defenses, including autophagy. While several agents succumb to autophagic destruction (xenophagy), others have evolved
mechanisms of autophagy evasion and manipulation. Various
Gram+ and Gram− bacteria, viruses and protazoa are known
autophagy targets (Table 1). The mechanisms by which microbes
are selectively sequestered in autophagosomes remain elusive and a
combination of host and microbial factors are likely to be necessary.
Microbial molecular motifs themselves may solicit autophagosome
formation. Alternatively, the up-regulation of autophagy through
activating multiple pattern recognition receptors could culminate
in xenophagy or perhaps organelle or compartmental damage
may lead to targeting by autophagic machinery. Microbial factors may be of equal importance for autophagy activation. For
example, Group A streptococcus (GAS) is sequestered in autophagosomes following escape from its early endosomal compartment
into the cytosol [65]. Lysosomal degradation of bacteria-containing
autophagosomes ensues, effects not observed in autophagy deficient cells. Strains of GAS lacking the streptolysin O toxin remain
within endosomes and avoid autophagic destruction indicating a
role for streptolysin O in induction of autophagy.
The Gram− human diarrheal agent Shigella flexneri is a highly
adapted pathogen harboring a type III secretion system (TTSS) for
delivery of its effector proteins to host cells. In epithelial cells,
wild-type (WT) strains secreting the effector IcsB are capable of
evading entrapment in autophagosomes, in comparison to mutant
strains lacking IcsB [66]. Interestingly, IcsB did not appear to confer autophagy protection in a subsequent study in murine marrow
derived macrophages, suggesting a cell-type specific phenomenon
[67]. The intracellular bacterium Burkholderia pseudomallei, also
avoids autophagic destruction in murine macrophages through
secretion of its TTSS-delivered effector protein BopA, which shares
some homology with IcsB [68].
Salmonella enterica serovar Typhimurium resides within
salmonella-containing vacuoles following intracellular invasion. Salmonella employs its TTSS to disrupt these vacuoles,
facilitating cytoplasmic entry. Autophagy promptly contributes to
subsequent restriction of intracellular proliferation by targeting
bacteria from damaged vacuoles—effects that were dependent on a
functioning TTSS and reversed in autophagy deficient cells [69,70].
Listeria monocytogenes, a Gram+ bacillus, replicates within the
host cytoplasm following phagosome escape, evading autophagic
destruction [71,72]. The virulence factors listeriolysin O, ActA
and phospholipase C were recently shown to be of importance
in modulating Listeria-containing phagosomal compartments,
blocking lysosomal degradation and facilitating replication and
survival [73,74]. Mycobacterium tuberculosis has adapted to survive
within host macrophages by interfering with and blocking phagosome fusion with lysosomes [75]. Autophagy up-regulation with
rapamycin or IFN-␥ overcame this evasion, and lead to phagosome
degradation [76,77].
Secreted bacterial toxins are themselves capable of interacting with the autophagy pathway. The non-invasive pathogen,
Vibrio cholerae, causes a potentially fatal secretory diarrhea. Its
secreted exotoxin, VCC, induces vacuole formation consistent with
autophagy induction [78]. Furthermore, cell viability was adversely
affected following autophagy inhibition, suggesting that in this situation, autophagy may defend against cell toxicity. Our group has
recently reported autophagy induction following infection with
Helicobacter pylori, which was dependent on the vacuolating cytotoxin, VacA. Autophagy limited the stability of intracellular VacA,
again suggesting a cytoprotective function of autophagy in response
to secreted toxins [79].
Viruses also interact with autophagy. The herpes virus HSV1 evades autophagy in part through Beclin-1 inhibition by
its neurovirulence protein ICP34.5 [80]. Poliovirus manipulates
autophagic machinery following cellular infection, as evidenced
by a marked reduction in viral release following pharmacological
Table 1
Microbial agents interacting with autophagy.
Microbe
Host autophagy interaction
Biological factors and outcomes
Bacteria
Streptococcus pyogenes
Staphlococcus aureus
Francisella tularensis
Salmonella Typhimurium
Rickettsia conorii
Escherichia coli
Mycobacterium tuberculosis
Vibrio chloerae (exotoxin)
Legionella pneumophilia
Brucella abortus
Coxiella burnetti
Listeria monocytogenes
Shigella flexneri
Burkholderia pseudomallei
Induction
Induction
Induction
Induction
Induction
Induction
Induction
Induction
Manipulation
Manipulation
Manipulation
Evasion
Evasion
Evasion
Bacterial clearance
Bacterial clearance
Bacterial clearance
Bacterial clearance
Bacterial clearance
Bacterial clearance
IFN␥ treatment enhances clearance
Limits cytotoxicity, enhances survival
Autophagosome maturation delayed
Autophagy harnessed for replication
Autophagosome maturation delayed
Dependent on ActA, PLC
Bacterial escape, dependent on IcsB
Bacterial escape. Dependent on BopA
Viruses
Parvovirus B19
Herpes simplex virus
Kaposi sarcoma-associated virus
Rotavirus
Human poliovirus
Hepatitis C virus
Coxsackievirus
Induction
Evasion
Evasion
Manipulation
Manipulation
Manipulation
Manipulation
Cell cycle arrest, virus sequestration
Dependent on neurovirulence factor
Viral Bcl-2 inhibits Beclin-1
Impaired autophagosome maturation
Autophagy harnessed for replication
Autophagosome maturation delayed
Autophagy harnessed for replication
Protozoa
Toxoplasma gondii
Induction
Parasite elimination
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inhibition of autophagy and siRNA silencing of key autophagy proteins [81]. Rotavirus has been suggested to harness the autophagic
apparatus to facilitate replication. Its enterotoxin, NSP4, was
found to co-localize with LC3+ structures on immunofluorescence microscopy, and the study authors speculate that NSP4
may interfere with autophagosome–lysosome fusion, enabling
viral recruitment of autophagosomes as replication niches [82].
The antiviral protein kinase, PKR, participates in viral induced
autophagy, functioning upstream of Beclin-1. It is possible that
other viruses which inhibit PKR function, including Influenza and
Ebstein–Barr virus, may in turn inhibit autophagy to enhance
their own survival [83,84]. The possibility of a cell-type dependent
autophagy response to viral infection was suggested by coronavirus
studies, wherein mouse hepatitis virus replication was impaired in
ATG5−/− stem cells, but not in ATG5−/− embryonic fibroblasts or
marrow derived macrophages [85–87].
These diverse examples of autophagy–microbial interactions
underpin the conserved primary innate role of autophagy as an
anti-microbial, protective mechanism and how certain pathogenic
organisms have evolved to recognize and commandeer this process
for their own advantage.
5. Autophagy and innate immunity
The innate immune system is responsible for the early detection and destruction of pathogens. This first line of defense relies
mostly on a set of receptors called pattern recognition molecules
(PRM) that sense molecular motifs that are common to a wide range
of pathogens, triggering different signaling cascades that culminate
with the elimination of pathogens and the initiation of an adaptive
response [87,88]. The findings that autophagy can specifically target cytosolic pathogens immediately prompted the investigation
of the role of PRM in the autophagic detection and elimination of
intracellular microbes.
The TLRs are transmembrane proteins, mostly located at the cell
surface, with a Toll-IL-1 receptor (TIR) domain facing the cytosol.
This domain is able to recruit four different adapter molecules: the
myeloid differentiation primary response protein 88 (MyD88), the
TIR domain-containing adaptor protein (TIRAP, also called MyD88
adaptor-like—MAL), the TIR domain-containing adaptor-inducing
IFN--Trif, also called TIR-domain-containing adaptor molecule
1—TICAM-1) and the Trif-related adaptor molecule (TRAM or
TICAM2) [88,89]. As we will see in this section, recent data suggest that induction of autophagy after TLR engagement requires
the recruitment of specific adaptors (Fig. 2).
Eissa and colleagues provided the first evidence that TLRs are
able to trigger an autophagic response by showing the formation of
numerous autophagosomes in response to LPS stimulation in the
murine macrophage RAW264.7 cell line [90]. Furthermore, silencing TLR4 using RNA interference resulted in significant reduction
in autophagosomes. The TLR4-induced autophagic response was
dependent on p38, RIP1 and Trif-, but not MyD88. As TLR4 can use
Fig. 1. Autophagosome biogenesis. The earliest identifiable structure in the initiation (nucleation) sequence of autophagosome formation is the crescent-shaped isolation
membrane or phagophore. Key elements include Atg9, the ULK1–FIP200–Atg13 complex, LC3-II, the Atg12–Atg5–Atg16L complex. Once formed, this membrane progressively
elongates (elongation), encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol. The membrane tips fuse and eventually seal, forming the autophagosome
(completion). The autophagosome may fuse with the endosomal compartment, forming an amphisome, prior to its ultimate maturation step, whereby its outer membrane fuses
with the lysosome to form an autolysosome (also termed autophagolysosome). This facilitates degradation, processing and recycling of the contents of the autophagosome.
S. Hussey et al. / Seminars in Immunology 21 (2009) 233–241
both MyD88 and Trif adapter molecules for downstream signaling,
the authors proposed that by recruiting both signaling cascades,
TLR4 could promote both a fast phagocytic response (through
MyD88) and a slower autophagic response (via Trif).
Other TLR family members have also been implicated in the
control of autophagy (Fig. 2). Deretic and colleagues have recently
demonstrated that when RAW264.7 macrophages were stimulated
with a panel of TLR ligands such as Pam3 CSK4 (TLR2), flagellin
(TLR5), CpG DNA (TLR9), poly (I:C) (TLR3), LPS (TLR4) and ssRNA
(TLR7), the latter three were able to up regulate autophagy [91].
In contrast to TLR3 (that recruits only Trif) and TLR4 (that recruits
both MyD88 and Trif), TLR7 recruits only MyD88, suggesting that
MyD88 may trigger autophagy after TLR7 activation. However, TLR9
activation by CpG DNA also activates MyD88 but did not induce
autophagy. Therefore, a simple analysis of which downstream adaptor protein is recruited by TLRs does not fully explain the induction
of autophagy by some pathogen associated molecular patterns
(PAMPs) and not others. The mechanistic explanation is still elusive
and seemingly conflicting evidence remains difficult to reconcile.
For example, TLR7 recruitment of MyD88 also leads to the activation
NFB, which is thought to inhibit autophagy [57]. Trif-dependent
signaling leads to the induction of type I interferon, which was
previously shown not to affect autophagy [76,88]. Two recent studies have proposed a mechanism by which TLRs might regulate
autophagy. Kehrl and Shi demonstrated that not only Trif, but also
MyD88 targets Beclin1 and reduces its binding to Bcl-2, upon stimulation with an array of TLR ligands [54,55]. Alternatively, Wagner
and colleagues observed that TLR activation leads to the activation of mTOR, which in turn interacts with the adaptor proteins
MyD88 and interferon-regulatory factors (IRFs) 5 and 7, thus controlling the transcription of cytokines such as TNF-␣, IL-10, IL12,
type I interferons but, surprisingly, not IL-1 [92]. These lines of evidence suggest a more elaborate TLR control of autophagy whereby
TLR-adapter molecules interact with proteins from the autophagic
237
pathway rather than by simply activating the classic hierarchical
signaling cascades described heretofore.
In contrast with the general notion that TLR ligands up regulate
autophagy, Green and colleagues suggested a model in which some
TLRs, when engaged by their cognate ligands, usurp the autophagic
pathway, recruiting LC3 to the phagosome membrane instead of
forming classic autophagosomes [93]. However, as pointed out
by the authors, it is not possible to exclude the possibility that
the LC3 recruited to phagosomes has its origin in rapidly forming autophagosomes. If confirmed, these data would have a deep
impact on the understanding on the role of autophagy in the
enhancement of antigen presentation for example.
PAMP recognition as an autophagy trigger seems to be an evolutionary conserved feature. In Drosophila, peptidoglycan-recognition
protein (PGRP) family members sense peptidoglycan (PG) from
gram-negative bacteria [94]. One of the PGRP family members,
PGRP-LE was recently implicated in PG sensing and induction of
autophagy upon infection with L. monocytogenes thereby leading to clearance of bacteria [95]. Cytosolic PRMs have also been
implicated in regulation of autophagy. Suzuki and colleagues
demonstrated that Ipaf, a Nod-like protein previously shown to
sense flagellin, down regulates autophagy during infection with
the non-flagellated bacterium S. flexneri [67]. The down regulation
of autophagy did not involve the ASC adapter protein, normally
required for the induction of IL-1 after Ipaf activation. One can
speculate that Nod proteins, which sense PG in mammalian cells,
may play a similar role in the regulation of autophagy. Up-coming
studies addressing this question are eagerly awaited.
In summary, the data above suggest a dynamic interaction
between receptors from the innate immune system and regulation of autophagy. Additional studies with knockout mice are now
needed in order to demonstrate a definitive role for TLR- or NLR
in autophagy during infection with pathogens known to activate
specific PRMs.
Fig. 2. TLR activation triggers autophagy. LPS triggers autophagy after recruitment of Trif (also RIP1 and p38, not shown) and MyD88. The latter seems to interact with Beclin-1,
reducing its binding to the anti-autophagic molecule BCL-2. TLR2 engagement induces the incorporation of LC3 to phagosomes (unkown mechanism). Viruses are able to
induce autophagy through TLR3, RIG-I (dsRNA) or TLR7/8-MyD88 (ssRNA). Conventional DCs sense viral ligads through the RIG-I/MAVS axis to secrete type I interferon, while
the conjugate ATG5/12 seems to be a down regulator of such response. Plasmocytoid DCs deliver TLR7 ligands from the cytosol to the compartments containing TLR7 using
basal autophagy. IPAF inhibits autophagy through an unclear mechanism.
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6. Autophagy and cytokine responses
In the last few years, autophagy induction has been frequently reported as a consequence of innate immune system
activation. However, there is compelling evidence that the relationship between autophagy and the immune system is reciprocal.
Cytokines from the innate and adaptive systems regulate autophagy
by different mechanisms. Two of the prototypical Th1 cytokines,
IFN-␥ and TNF-␣, were shown to up-regulate autophagy. Gutierrez
and colleagues first demonstrated that mouse macrophages harboring Mycobacterium within phagosomes were able to clear bacteria
after stimulation with IFN-␥ in an autophagy-dependent manner
[76]. Follow up studies implicated GTPases in this process. The
mouse genome contains 23 different immunity-related GTPases,
most of which respond to IFN-␥ stimulation and play a role in the
defense against intracellular pathogens via a mechanism which is as
yet unclear [96]. The studies from Deretic’s group showed that both
mouse immunity-related GTPase (Irgm1) and its human ortholog
IRGM are the key molecules driving the induction of autophagy
upon IFN-␥ stimulation, leading to the clearance of Mycobacterium
from infected macrophages [76,97].
The other Th1 cytokine shown to stimulate autophagy is TNF-␣.
Codogno and colleagues observed that cells stimulated with TNF␣ are committed to die when NFB is blocked [57,59,60]. These
findings are of great interest as the activation of autophagy may
represent a way to overcome the resistance of cancer cells to anticancer drugs targeting NFB.
In contrast to the autophagy enhancing effect of some Th1
cytokines, Th2 cytokines such as IL-4 and IL-13, seem to counteract starvation and IFN-␥-induced autophagy by different pathways
[97]. While IL-4 and IL-13 block starvation-induced autophagy
by activating the Akt-mTOR axis, these cytokines inhibit IFN-␥induced autophagy in an Akt-independent but STAT6-dependent
manner.
The regulation of cytokine secretion by autophagy, has also been
reported. Jounai and colleagues demonstrated that in response
to infection with RNA viruses or immunostimulatory RNA, IFN levels were increased in ATG5 knockout embryonic fibroblasts
[98]. The authors demonstrated that the Atg5–Atg12 conjugate
negatively regulates the antiviral immune response by interacting with the RIG-I-like receptor (s protein retinoic acid-inducible
gene I (RIG-I) and IFN- promoter stimulator 1 (IPS-1) thus,
implying autophagy contributes to viral replication. Iwasaki and
colleagues showed that in autophagy-impaired cells the increased
cytokine secretion in response to immune-stimulatory RNA is due
to the accumulation of defective mitochondria and consequent
IPS-1 and ROS accumulation, further strengthening the importance of autophagy in the maintenance of cellular homeostasis
[99].
Autophagy has also been proposed to regulate cytokine secretion in Crohn disease. Crohn disease (CD) is a chronic inflammatory
intestinal disease with a complex and multifactorial etiology. Several recent independent genome wide association studies have
implicated a number of heretofore unappreciated biological pathways in CD pathogenesis, including autophagy [100–103]. Since
the identification of a non-synonymous single nucleotide polymorphism in the ATG16L1 gene as a causal risk variant for CD, several
groups have sought to elucidate its functional impact on development of CD. Akira and colleagues generated mice lacking the
coiled–coil domain of ATG16L1 and observed aberrant IL-1 secretion upon LPS stimulation of fetal derived liver macrophages. In
contrast to previous studies, LPS did not induce autophagy in control macrophages indicating the enhanced IL-1 was not due to
disruption of LPS-mediated autophagy [104]. Chimeric mice with
ATG16L1-deficient hematopoietic cells had an unremarkable baseline intestinal phenotype, but displayed increased susceptibility to
DSS-induced colitis compared with controls. Even though this study
used mice expressing a truncated form of ATG16L1, rather than the
ATG16L1 risk allele, the results point to the importance of functional
autophagy machinery for normal intestinal function. Using an alternative mouse model hypomorphic for ATG16L1 protein expression,
Cadwell and colleagues noted paneth cell-specific abnormalities
including degenerating mitochondria, loss of lysozyme granule
integrity and absence of apical microvilli [105]. Parallel findings
were observed when intestinal ATG5 expression was suppressed.
Transcriptional profiling analysis revealed that, among other differences, transcripts for the adipocytokines leptin and adiponectin
were highly enriched. Similar increased expression profiles were
observed previously in patients with CD [106,107]. The above findings, while not specific to ATG16L1 suppression, underscore the
importance of autophagy pathway integrity to normal paneth cell
function. Interestingly, ATG7 knockout of pancreatic islet cells
resulted in abnormal cellular morphology on EM, including mitochondrial swelling, distension of the endoplasmic reticulum and
a paucity of insulin granules when compared with controls [108].
It remains unclear why paneth cells, above others, are susceptible to autophagy interference and how autophagy is involved in
maintaining integrity of its lysozyme exocytosis pathway. However, the interaction between autophagy and multivesicular body
biogenesis may provide a potential explanation for abnormal granule formation and exocytosis. Once again, the paneth cell is placed
at the convergence of several innate immune pathway aberrations and CD pathogenesis. Translational clinical data are keenly
awaited.
7. Autophagy and antigen presentation
The products of the two main cellular degradation systems – the
proteasome and the lysosome – are not merely unwanted material but are, instead, key molecules utilized to instruct the immune
system. This instruction step is achieved by the presentation of
these products to cells from both innate and adaptive immune systems. CD8+ T cells monitor mainly cytosolic and nuclear antigens
degraded by the proteasome (a large cytosolic enzyme complex)
and loaded into MHC class I. In contrast, CD4+ T cells respond to
extracellular or membrane peptides generated by lysosomal degradation and presented in the context of MHC class II at the cell
surface [109,110]. However, this paradigm has been challenged by
the demonstration that dendritic cells (DCs) are also capable of
presenting extracellular antigens on MHC class I, and not just on
MHC class II as initially thought, through a mechanism called crosspresentation. Cross-presentation allows DCs to instruct also CD8+
T cells, generating a more efficient T-cell response [111].
Functional evidence for the presentation of endogenous antigens on MHC class II was first provided by Long and colleagues,
who demonstrated that measles and influenza antigens could be
presented in the context of MHC class II [112,113]. Indeed, the
affinity purification of MHC class II from Epstein–Barr virus (EBV)transformed B lymphoblastoid cells, murine B cell lymphoma and
myeloid cells showed that more than 20% of natural MHC class II ligands had their origin in intracellular proteins [109]. Together these
studies suggested that an alternative and unknown route could
deliver antigens from the cytosolic compartment for presentation
on MHC class II. Knecht and colleagues were the first to suggest a
role for autophagy in this process by showing that glyceraldehyde3-phosphate dehydrogenase, an important source of human MHC
class II ligands, is degraded via chaperone-mediated autophagy
[114]. Additionally, peptides from two Atg8 homologues, LC3 and
GABARAP, have been isolated from human and mouse MHC class II
molecules, respectively, providing further support to the notion of
autophagy as an alternative route for delivery of cytosolic antigens
for MHC class II.
S. Hussey et al. / Seminars in Immunology 21 (2009) 233–241
More direct evidence came from studies using pharmacological
inhibition of macroautophagy with PI3K inhibitors (such as 3-MA
and wortmannin), which are thought to block the sequestration
step of autophagy. Stockinger and colleagues demonstrated that
over-expressed C5 was processed and loaded onto MHC class II in an
autophagy-dependent manner, as the loading was decreased in the
presence of 3-MA [114]. A similar approach was used to show that
an endogenously expressed bacterial peptide, NeoR (neomycinphosphotransferase II), was sequestered in autophagosomes and
processed in endosomal/lysosomal compartments for loading onto
MHC class II. Brossart and colleagues also used pharmacological
inhibition to demonstrate that DCs electroporated with RNA coding
for the tumor-associated antigen Muc-1 requires not only lysosomal
antigen degradation and processing, but also autophagy in order to
prime CD4+ T cells [115].
Further evidence that autophagy contributes to MHC class II presentation came from Munz and colleagues, in which they analyzed
the endogenous MHC class II processing of the nuclear antigen 1
(EBNA 1) from EBV, the dominant EBV-latent antigen for CD4+ T
cell. Inhibition of autophagy by Atg12siRNA in EBV-transformed
B cells reduced recognition by EBNA1-specific CD4+ T cells [116].
In another study, the same group demonstrated that the fusion
of influenza matrix protein 1 (MP1) with Atg8/LC3 drives this
molecule to autophagosomes in different cell types and enhances
recognition by antigen specific CD4+ T cells [117]. Knockdown of
Atg12 confirmed that the localization of the fusion proteins with
MHC class II molecules was dependent of autophagy. Importantly,
these results represent great potential for vaccine design, since targeting antigens to autophagosomes induces a more robust T cell
response. In support of this contention, Jagannath et al. demonstrated that induction of autophagy enhances BCG vaccine efficacy
in a murine model [118].
In contrast to model or viral antigens, very little is known about
bacterial antigens requiring autophagy for proper presentation on
MHC class II. So far, only the 85B antigen from M. tuberculosis
was shown to be presented more efficiently on MHC class II upon
induction of autophagy [119]. Accordingly, Atg6 silencing dampened this process, while rapamycin treatment enhanced priming of
85B-specific CD4+ T cells, strongly suggesting a role for autophagy
in MHC class II presentation of antigens of bacterial origin. Even
though studies with other bacterial models are lacking, it is possible to speculate a role for autophagy in MHC class II presentation
during infections with bacteria that escape from phagosomes, such
as L. monocytogenes
8. Conclusion
Autophagy is steadily emerging from its historic ‘house-keeping’
role as a new dimension in our host defense program. Taken
together, the data highlighted above suggest that autophagy
impacts on the development of both innate and adaptive immune
responses to diverse pathogens and that, conversely, components
of the immune system themselves also regulate autophagy. This
biological process is now a major target for researchers who want
to enhance understanding of and develop strategies to modulate
immune responses in a variety of inflammatory and infectious conditions.
Acknowledgements
Funding sources: S.H. is supported by a research fellowship award
from CIHR, Canadian Association of Gastroenterology and Crohn’s
and Colitis Foundation of Canada. L.H.T is supported by a research
fellowship award from CIHR. N.L.J. is supported by operating grants
from CIHR and CCFC.
239
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