Download Keeping the immune system in check: a role for mitophagy

Immunology and Cell Biology (2015) 93, 3–10
& 2015 Australasian Society for Immunology Inc. All rights reserved 0818-9641/15
www.nature.com/icb
REVIEW
Keeping the immune system in check: a role
for mitophagy
Michael Lazarou
Mitochondria play a central role in many facets of cellular function including energy production, control of cell death and
immune signaling. Breakdown of any of these pathways because of mitochondrial deficits or excessive reactive oxygen species
production has detrimental consequences for immune system function and cell viability. Maintaining the functional integrity of
mitochondria is therefore a critical challenge for the cell. Surveillance systems that monitor mitochondrial status enable the cell
to identify and either repair or eliminate dysfunctional mitochondria. Mitophagy is a selective form of autophagy that eliminates
dysfunctional mitochondria from the population to maintain overall mitochondrial health. This review covers the major players
involved in mitophagy and explores the role mitophagy plays to support the immune system.
Immunology and Cell Biology (2015) 93, 3–10; doi:10.1038/icb.2014.75; published online 30 September 2014
Mitochondria are essential cellular organelles that have long been
known for their role in energy production. However, they also serve
many additional roles in maintaining cellular homeostasis by governing cellular signaling pathways such as programmed cell death, cellular
differentiation and aging. Mitochondria harbor their own DNA
encoding 13 polypeptides that synchronously assemble with nuclear
gene encoded proteins to form the respiratory chain (consisting of
complexes I–IV). These molecular machines drive ATP generation via
oxidative phosphorylation (OXPHOS). One of the byproducts of
OXPHOS is the generation of reactive oxygen species (ROS). Excess
ROS from dysfunctional mitochondria can contribute to human
disease by damaging proteins, lipids and DNA. Recent studies have
highlighted important roles for mitochondria in cells of the immune
system, including mediating degradation of pathogens,1 and signaling
in innate immunity.2 In both cases, ROS have been identified to play
an important role. Maintaining mitochondrial fidelity is therefore
critically important to elicit the correct signaling response as well as
immune cell survival.
Two major pathways of mitochondrial quality control serve to
monitor mitochondrial function and elicit a repair response when
damage is identified: (i) the unfolded protein response and
(ii) mitophagy. In the first, unfolded mitochondrial proteins induce
the coordinated activity of molecular chaperones and proteases
(reviewed in Rugarli and Langer3). If the damage is not sufficiently
repaired or continues to accumulate, it can ultimately lead to
breakdown of the respiratory chain and loss of membrane potential.
Under these conditions, mitochondria are recognized by the cell and
removed from the population by the second pathway of mitochondrial
quality control, a selective form of autophagy termed mitophagy.
Removal of damaged mitochondria prevents them from re-fusing with
the healthy network and halts the accumulation of toxic mitochondrial
products. Failure to remove dysfunctional mitochondria can cause an
accumulation of ROS and release of mitochondrial factors that trigger
cell death. In the immune system, this toxic accumulation can also
lead to hyperactivation of inflammatory pathways. This review will
discuss the mechanisms of mitophagy and the major players that are
involved. How the immune system utilizes mitophagy to function
correctly and drive the elimination of pathogens will be subsequently
explored.
MITOPHAGY IS A SELECTIVE FORM OF AUTOPHAGY
The beginning of non-selective autophagy involves the formation of a
phagophore, a curved membranous structure that eventually expands
to engulf cytoplasmic components (reviewed in Mizushima et al.4).
Following expansion and engulfment, the phagophore closes to form
the autophagosome that fuses with a lysosome where the cargo is
degraded. The coordinated action of autophagy-related (Atg) proteins
controls the biogenesis of autophagosomes. Upon stimulation of
autophagy, the transmembrane protein Atg9 and the ULK complex
independently recruit to the site of autophagosome formation. The
VPS34 lipid kinase complex is recruited next and generates phosphatidylinositol 3-phosphate (PI(3)P), a lipid that is essential for complete
autophagosome formation. During the biogenesis of the phagophore
membrane, PI(3)P binding proteins such as WIPI1/2 and DFCP1 help
to generate and remodel the membrane.5,6 Elongation and closure of
the phagophore is controlled by two linked processes: (i) conjugation
of the ubiquitin-like protein Atg12 to Atg5 which, along with Atg16L1,
forms an E3-like ligase complex and (ii) conjugation of Atg8 to the
lipid phosphatidylethanolamine by the Atg16L1 ligase complex. Yeasts
have a single Atg8-encoding gene whereas mammalian cells have six
homologs that belong to the LC3 and GABARAP subfamilies.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia
Correspondence: Dr M Lazarou, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.
E-mail: Michael.Lazarou@Monash.edu
Received 6 August 2014; revised 18 August 2014; accepted 18 August 2014; published online 30 September 2014
Role of mitophagy in immune system
M Lazarou
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The autophagy machinery is also employed for the removal of
damaged mitochondria by the selective process of mitophagy.
Selectivity is achieved by receptors or adaptors that bring together
molecular signals on mitochondria and LC3/GABARAP on phagophores, thereby allowing the autophagic machinery to come in to
proximity with the mitochondria.7 Both autophagy adaptors and
receptors bind to LC3/GABARAP via tetrapeptide consensus
sequences termed Atg8 family-interacting motifs or LC3-interacting
regions (LIR).7–9 In mammalian cells, two main mechanisms of
mitophagy signaling have been identified: ubiquitin- and receptormediated mitophagy. For these, ubiquitin or LIR motif containing
mitochondrial proteins, respectively, act to recruit LC3/GABARAP
family members to mitochondria and thus induce mitophagy. The
mechanisms and factors involved in these mitophagy pathways are
discussed below.
MECHANISMS OF MITOPHAGY
To date, several effectors of mitophagy have been identified in
mammalian cells including the outer mitochondrial membrane
(OMM) receptors NIX1,10 BNIP3,11 and FUNDC1,12 the Parkinson’s
disease proteins PINK1 and Parkin,13–15 and the ubiquitin ligases
Gp78,16 Smurf1,17 and Mul1.18 Furthermore, cardiolipin externalization on damaged mitochondria has also been reported to signal
selective degradation of mitochondria.19
The mitophagy receptors NIX1, BNIP3 and FUNDC1 contain LIR
consensus sequences required for LC3/GABARAP binding and mitophagy signaling. NIX was originally shown to be the key mediator of
mitophagy in reticulocytes where its expression is induced during their
maturation to drive mitochondrial clearance (Figure 1).20,21 In
addition to binding to LC3/GABARAP, NIX controls mitophagy
through a short sequence in its cytoplasmic domain termed the
minimal essential region.22 The factors that bind to the minimal
essential region remain to be identified, but these data point toward
new mechanisms of mitophagy regulation by NIX in addition
to canonical LC3/GABARAP binding. The receptor BNIP3
mediates hypoxia-induced mitophagy to prevent detrimental ROS
accumulation.11 BNIP3 is upregulated during hypoxia by the transcription factor HIF1α and phosphorylation of serine residues near the
BNIP3 LIR promote LC3 binding.23 FUNDC1 has also been reported
to promote mitophagy during hypoxia although it is constitutively
expressed under normoxic conditions.12 Both phosphorylation and
dephosphorylation of the FUNDC1 LIR have been reported to
differentially regulate its binding to LC3 and ultimately mitophagy.24
Although not a receptor in a classical sense, the lipid cardiolipin when
present on the OMM can act as a so called ‘eat me’ signal on damaged
mitochondria.19 Cardiolipin normally resides in the inner membrane
but is externalized on mitochondria of neurons and neuronal-like cells
treated with mitophagic stimuli. Once exposed on the OMM,
cardiolipin binds LC3 to promote engulfment by the autophagosome.
Ubiquitin-mediated mitophagy relies on ubiquitin chains generated
by E3 ligases to act as the ‘eat me’ signal. The most well-characterized
ubiquitin-mediated mitophagy pathway is controlled by PINK1 and
Parkin. Mutations in PINK1 and Parkin are a common cause of
autosomal recessive forms of Parkinson’s disease,25,26 where mitochondrial dysfunction is a prominent disease feature (reviewed in
Narendra et al.27). PINK1 and Parkin coordinately identify damaged
mitochondria and drive their elimination by mitophagy. Typically,
PINK1/Parkin-mediated mitophagy has been studied using immortalized cells overexpressing Parkin. Given this, concerns have been raised
regarding its role in neurons and in vivo; however, recent studies
demonstrate that endogenous PINK1 and Parkin control mitophagy in
Immunology and Cell Biology
flies in vivo,28 and control basal mitophagy in primary mouse
neurons.29 Although additional functions outside of mitophagy have
been ascribed to both PINK1 and Parkin, for the purposes of this
review, their role in mitophagy will be the focus.
PINK1/Parkin mitophagy
PINK1 is a serine/threonine kinase that accumulates on the outer
membrane of damaged mitochondria.30,31 Once on the surface of
mitochondria, PINK1’s kinase activity both activates Parkin’s E3 ligase
activity and recruits Parkin from the cytosol.14,30–32 Parkin then
conjugates ubiquitin onto various OMM proteins to induce mitochondrial engulfment by an autophagosome, and subsequent
fusion with a lysosome to degrade the damaged mitochondrion
(Figure 1).13,33,34
How does PINK1 sense mitochondrial health?
Healthy mitochondria are spared from Parkin mediated mitophagy
through the constitutive proteolysis of PINK1. Mitochondrial membrane potential drives PINK1 import into the inner membrane where
it is cleaved by the rhomboid protease PARL and then degraded by the
N-end rule pathway.35–39 When a mitochondrion sustains damage and
loses its membrane potential, PINK1 import to the inner membrane is
prevented, sequestering PINK1 on the outer membrane away from
PARL.35,40 On the OMM, PINK1 is bound to the translocase of the
outer membrane (TOM) complex as a dimer (Figure 1, panel i).41,42
A genome-wide siRNA screen identified Tom7 (a subunit of the TOM
complex) as an essential factor for PINK1 import and stabilization on
the OMM.43 If a damaged mitochondrion regains function, PINK1’s
association with the TOM complex allows for rapid re-import and
degradation of PINK1 to downregulate the pathway.41 In addition to
loss of membrane potential, PINK1 also accumulates on the OMM in
response to unfolded protein stress in the matrix.44 Misfolded proteins
that aggregate in the matrix may cause PINK1 accumulation by either
shutting down protein import or PINK1 proteolysis. Thus PINK1 is a
molecular sensor of mitochondrial health, flagging only those
mitochondria within the population that become dysfunctional for
Parkin-mediated degradation.
Mechanisms of PINK1/Parkin mitophagy—what is known to date
Although the mechanisms behind PINK1 regulation are well understood, how PINK1 recruits and activates Parkin was until recently,
largely unclear. Biochemical and structural analyses revealed that
under basal conditions, Parkin adopts an auto-inhibited conformation
in which its ubiquitin ligase activity remains latent.32,45–48 Upon
activation, Parkin self-associates and catalyzes ubiquitination through
a conserved cysteine using a HECT-like mechanism.46,49–51 PINK1
phosphorylation of Parkin at serine 65 (S65), located within Parkin’s
ubiquitin-like domain, was shown to activate Parkin’s ligase activity.52
However, phosphorylation at S65 was not essential for Parkin
activation and translocation to mitochondria.33,53 This suggested that
there was an additional unidentified PINK1 substrate. Very recently,
three studies discovered that the identity of the PINK1 substrate is
ubiquitin.53–55 Ubiquitin is phosphorylated by PINK1 at S65,
mirroring PINK1’s phosphorylation of Parkin’s ubiquitin-like domain.
Phosphorylated ubiquitin is sufficient to activate Parkin both in vitro
and in cells.53–55 Despite many new questions arising from the
discovery, a model where PINK1 phosphorylation of both Parkin
and ubiquitin is required for efficient Parkin activation has been
proposed (Figure 1, panel ii).54,55
Once recruited to depolarized mitochondria, Parkin mediates the
ubiquitination of numerous outer membrane substrates and of these,
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i
Ubiquitin mediated mitophagy
Damaged
Healthy
TOM
OMM
i Mitochondrial
damage
TIM
PARL
IMM
PINK1
ii
Inactive
Active
PO4
RBR
PO4
PINK1
ii Parkin
Ubl
PO4
Ubiquitin
Phagophore
Autophagosome
Lysosome
LC3
Ubiquitin
PO4
Autophagy
adaptor
Receptor mediated mitophagy
Phagophore
NIX
Autophagosome
LC3
NIX - Reticulocyte maturation
Figure 1 Ubiquitin-mediated mitophagy: (i) PINK1 is constitutively degraded in healthy mitochondria where it is imported to the inner membrane via the
TOM and TIM complexes and cleaved by the rhomboid protease PARL followed by degradation by the proteasome. When a mitochondrion sustains damage
that leads to a loss of membrane potential or shutdown in protein import, PINK1 import to the inner membrane is blocked and instead PINK1 accumulates
on the outer membrane as a dimer bound to the TOM complex. (ii) Once stabilized on the outer membrane, PINK1 recruits and activates Parkin by
phosphorylating S65 in Ubiquitin and Parkin’s ubiquitin-like domain. Parkin adopts an active conformation as a dimer or multimer. Mitochondrial substrates
on the OMM are then ubiquitinated by Parkin to recruit LC3 conjugated to phagophores via autophagy adaptors such as p62 and NBR1. The phagophore
expands and engulfs the mitochondrion to form an autophagosome that fuses with a lysosome for degradation. Receptor mediated mitophagy: the mitophagy
receptor NIX is highly induced during reticulocyte maturation. This leads to elevated levels of NIX on the OMM where it can bind to LC3 conjugated to a
phagophore via its LIR. The mitochondrion is degraded by lysosomal hydrolases following engulfment by the autophagosome and fusion with a lysosome.
RBR, RING-between-RING domain; TIM, translocase of the inner membrane.
the fusion proteins Mfn1/Mfn2 and the Fe-transport protein
MitoNEET appear to be among the most susceptible.33,34 Lysine (K)
48-linked ubiquitination of substrates by Parkin promotes their
degradation by the proteasome and this plays a permissive role in
mitophagy.34,56,57 Loss of mitochondrial transport has been proposed
to play a role in mitophagy through the degradation of Miro58 and
recruitment of HDAC6.59 Moreover, deubiquinating enzymes such as
USP30 and USP15 regulate mitophagy by modifying ubiquitin chains
on OMM proteins.29,60 Downstream, Rab GTPase-activating proteins
interact with the OMM protein Fis1 and LC3/GABARAP to control
autophagosome formation around the mitochondria.61
The mechanism behind how Parkin signals the recruitment of
autophagic machinery is less understood. A prominent hypothesis is
that non-degradative ubiquitin chain linkages (e.g., K63) bind
autophagy adaptors to mediate selective clearance of mitochondria
via LC3 and/or GABARAP recruitment. The autophagy adaptor p62
translocates to mitochondria upon activation of the PINK1/Parkin
pathway and promotes mitochondrial clustering, but is not essential
for mitophagy.14,62 The autophagy adaptors NBR1, TAX1BP1 and
NDP52 are ubiquitinated by Parkin,33 and the levels of NBR1 increase
on mitochondria following Parkin translocation.34 This indicates
that multiple autophagy adaptors may contribute to PINK1/Parkin
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mitophagy. It is noteworthy that core autophagy effectors including
Ulk1, Atg14, DFCP1, WIPI1 and Atg16L1 all recruit to damaged
mitochondria independently of LC3,63 adding a further layer of
complexity. It is anticipated that specific ubiquitin chains could be
the molecular signal driving LC3/GABARAP-independent recruitment
of the core autophagy proteins. Whether there are also specialized
adaptors for this recruitment remains to be investigated.
MITOPHAGY IN IMMUNE SIGNALING
Innate immunity serves as a front-line defense mechanism against
microbial invasion. Host cells of the innate immune system,
including macrophages, sense invading microorganisms through
pattern-recognition receptors (reviewed in Medzhitov et al.64).
Pattern-recognition receptors can bind to both conserved pathogenassociated molecular patterns such as microbial nucleic acids, proteins
and structural components, as well as damage-associated molecular
patterns, including proteins released from damaged cells. Once
activated, pattern-recognition receptors induce inflammatory
responses mediated by various cytokines and chemokines to eliminate
pathogens. Pattern-recognition receptors include nucleotide
oligomerization domain-like receptors (NLRs), retinoic acidinducible gene I (RIG-I)-like receptors (RLRs), toll-like receptors
and C-type lectin receptors. Mitochondria play important roles to
facilitate innate immune responses by forming platforms required for
signaling, generating activation signals through the production of ROS,
and by providing damage-associated molecular patterns. There is
mounting evidence that mitophagy regulates innate immune responses
primarily by maintaining a functional cohort of mitochondria within
the cell to prevent damaging levels of activation.
Mitophagy versus xenophagy
Given the bacterial ancestry of mitochondria, it is worthwhile to
consider the parallel mechanisms utilized by a cell to control both
bacterial autophagy (xenophagy) and mitophagy. Similarly to mitophagy, bacterial danger signals act to recruit autophagy machinery
through ubiquitin- or receptor-mediated pathways. Bacterial danger
signals come in multiple forms. Foreign bacterial DNA can be
recognized by STING (stimulator of interferon genes), an essential
adaptor protein that functions in type I interferon production and
xenophagy.65 Galectin-8 is a danger receptor that binds exposed host
glycans from damaged vacuoles containing bacteria.66 These detection
processes ultimately tag bacteria with ‘eat me’ signals that recruit the
autophagic machinery.67 Xenophagy effectors include the autophagy
adaptors p62, NBR1 and optineurin that bind ubiquitinated bacteria
and recruit LC3 attached to phagophores.7,68,69 NDP52 functions by
the same mechanism but can also bind to galectin-8 in addition to
ubiquitin chains.66 As discussed above, apart from optineurin, these
adaptors have also been implicated in PINK1/Parkin mitophagy.
Efforts to identify ubiquitin ligases essential for xenophagy have
revealed that LRSAM1 ubiquitinates Salmonella typhimurium,70 leading to the recruitment of p62, NBR1 and optineurin.68,70 Failure to
recruit these adaptors prevents the clearance of S. typhimurium from
cells, demonstrating a clear role for ubiquitination in xenophagy. In a
striking resemblance to mitophagy, Parkin was also recently found
to mediate bacterial clearance.71 Loss of Parkin in cells inhibits
ubiquitination of Mycobacterium tuberculosis ultimately blocking its
clearance by xenophagy. Moreover, loss of Parkin leaves mice and flies
highly vulnerable to various intracellular infections.71 This discovery
may explain the correlation between genetic polymorphisms in the
regulatory region of the Parkin gene and increased susceptibility to
M. lepare and S. enterica serovar Typhi in humans.72,73 Thus, Parkin
Immunology and Cell Biology
has the ability to function in mitophagy as well as in the innate defense
pathway through xenophagy. How Parkin might be activated during
xenophagy is an interesting question. PINK1 is the kinase responsible
for Parkin activation during mitophagy; however, the kinase for Parkin
activation during xenophagy is unknown. A putative candidate is
TANK binding kinase 1 (TBK1). Like Parkin, this serine/threonine
kinase is required to efficiently target M. tuberculosis to autophagosomes,65 and therefore could mirror PINK1 during xenophagy.
NLRP3 inflammasome activation and a role for mitophagy
The NLRP3 (NLR family, pyrin domain-containing 3) inflammasome
is an important mediator of the innate immune response through its
signaling to induce the production of mature interleukin (IL)-1β.
Multiple lines of evidence point toward ROS signaling as a key element
required for NLRP3 inflammasome activation. Cytosolic NADPH
oxidases were initially thought to be the primary source of ROS during
NLRP3 activation.74 However, it was subsequently shown that the
ROS are likely provided by a different source,75 and recently two
independent studies demonstrated that mitochondria are a critical
source of this ROS.76,77 Ablation or reduction of the autophagy
proteins LC3B, Beclin1 or Atg5 increased NLRP3 activation and IL-1β
secretion in response to various stimuli.76,77 This corroborated an
earlier study where Atg16L1 deletion resulted in IL-1β secretion in
response to lipopolysaccharide alone.78 Interestingly, defective clearance of damaged mitochondria through mitophagy was shown to be
the key factor behind the increased levels of ROS and elevated
inflammasome activation.76,77 Mitophagy mediated by RIPK2 also
regulates NLRP3 activation during influenza A virus infection.79
Resting bone-marrow-derived dendritic cells lacking RIPK2 have a
greater mitochondrial content than wild-type cells. Upon IVA infection, RIPK2− / − bone-marrow-derived dendritic cells show a further
accumulation of mitochondria that displayed increased levels of
dysfunction.79 Concurrently, proliferation of damaged mitochondria
resulted in greater ROS production and NLRP3 activation. RIPK2
mediates mitophagy of dysfunctional mitochondria through its kinase
activity by promoting phosphorylation of autophagy effector ULK1.79
Therefore, during influenza A virus infection, RIPK2 functions to
prevent pathologic NLRP3 activation by inhibiting proliferation
of ROS-producing mitochondria. These studies demonstrate that
mitophagy is not only important for basal mitochondrial quality
control, but also during infection to prevent hyperactivation of NLRP3
inflammasomes (Figure 2).
Toxins that inhibit mitochondrial OXPHOS and increase ROS
trigger NLRP3 activity,76 suggesting that mitochondrial dysfunction
alone is sufficient to elicit an inflammatory response. Recent reports
point toward mitochondrial dysfunction and ROS playing a critical
role in inflammatory diseases. Production of proinflammatory
cytokines caused by mitochondrial ROS have been observed in
TNF-receptor-associated periodic syndrome patient monocytes80 and
elevated NLRP3 signaling has been reported in fibromyalgia patient
bone-marrow-derived dendritic cells because of defective OXPHOS
and elevated ROS.81 Furthermore, ROS from damaged mitochondria
was suggested to be involved in deregulated IL-1β secretion from
monocytes in periodic fever disorder.82 In another study, monocytes
from patients with inflammatory bowel disease displayed elevated
levels of mitochondrial ROS as well as increased expression of proteins
involved in OXPHOS.83 Using a mouse model of colitis, Dashdorj
et al.83 were able to ameliorate symptoms using the mitochondrial
antioxidant MitoQ. In addition to antioxidants, it is possible that
stimulating mitophagy pathways to clear damaged mitochondria may
alleviate inflammatory diseases caused by excessive ROS.
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IL1-β
pro IL1-β
Caspase 1
6
Pro caspase 1
HBV & HCV
ASC
NLRP3
NLRP3 inflammasome
Apoptosis
Virus number
Type I IFNs
ROS
RIG-I
MAVS
Gp78
4 Mul1
Smurf1
Phagophore
mtDNA
Ubiquitin
1
Δψm
2
3
LC3
Damaged mitochondrion
Mitophagy
Parkin - Xenophagy
7
Autophagy
adaptor
5
T-cell & M2 macrophage
homeostasis
Bacterium
MHC-1 mitochondrial
antigen presentation
Figure 2 Mechanisms linking mitochondrial dysfunction and mitophagy to immune pathways. (1) Excess mitochondrial ROS from damaged mitochondria
leads to hyperactivation of the NLRP3 inflammasome and increased levels of IL-1β. Mitochondrial DNA released from damaged mitochondria can also
facilitate NLRP3 activation. ROS has additionally been shown to cause elevated RLR signaling resulting in increased type I IFN production during virus
infection or poly (I:C) transfection. (2) RLR signaling through MAVS is lowered when the mitochondrial membrane potential is low. (3) Mitophagy helps to
balance these signaling pathways by removing damaged mitochondria thereby lowering ROS production and mtDNA release, and maintaining a population of
mitochondria with a healthy membrane potential. (4) The ubiquitin ligases Mul1, Smurf1 and Gp78 have all been implicated in mitophagy as well as negative
regulation of MAVS/RIG-I signaling. (5) Mitophagy promotes both T-cell and M2 macrophage homeostasis and has also been reported to promote MHC-1
mitochondrial antigen presentation. (6) During HBV and HCV infection, mitophagy mediated by PINK1/Parkin has been proposed to prevent apoptosis and
thus promote viral replication. (7) Mitophagy and xenophagy share molecular components including Parkin and autophagy adaptors such as p62 and NBR1.
mtDNA, mitochondrial DNA; Δψm, transmembrane potential.
Apart from supplying damage-associated molecular patterns in
the form of ROS, mitochondria can also facilitate inflammasome
activation by releasing mitochondrial DNA (mtDNA).77,82,84,85
Released mtDNA that evades autophagy can elicit damaging levels of
TLR9 inflammatory responses in cardiomyocytes.84 Accumulation
of impaired mitochondria in autophagy-depleted macrophages
(in response to lipopolysaccharide and ATP), leads to NLRP3mediated mtDNA release that facilitates caspase 1 activation and IL-1β
secretion.77 In contrast, it has been reported that NLRP3 stimuli
induce mitochondrial apoptosis to release oxidized mtDNA upstream
of NLRP3 activation.85 Furthermore, the released mtDNA was shown
to be essential for NLRP3 activation as opposed to playing only a
supporting role. Thus, two mutually exclusive mechanisms of mtDNA
release have been proposed; one that requires NLRP3 activation,77 and
the other in which mitochondrial apoptosis liberates mtDNA to
activate NLRP3.85 Using a genetic approach, a recent study by Allam
et al.86 showed that mitochondrial apoptosis was dispensable for
NLRP3 activation. Nevertheless, there is evidence to support that
dysfunctional mitochondria promote pathways that result in mtDNA
release. Mitophagy has been shown to dampen these responses by
maintaining mitochondrial fidelity.77
Links between antiviral immunity and mitophagy
The mitochondrial antiviral signaling (MAVS) protein is an RLR
adaptor molecule located on the OMM.2 MAVS interacts with RIG-I
and MDA5 to activate downstream NF-κB and IRF signaling pathways
for proinflammatory cytokine and type-I interferon production.2
Interestingly, three ubiquitin ligases, Smurf1, Mul1 and Gp78 that
have been implicated in regulation of mitophagy, are also linked to
negative regulation of MAVS signaling. The ubiquitin ligase Smurf1
mediates the degradation of MAVS,87 and Mul1 modulates MAVS
signaling by inhibitory posttranslational modification of RIG-I.88
Furthermore, Gp78 was shown to regulate MAVS using both
ubiquitin-dependent and -independent mechanisms.89 It remains to
be seen whether the dual functions of Smurf1, Mul1 and Gp78 in
mitophagy and MAVS regulation overlap.
Hyperstimulation of RLR signaling has been observed in autophagydeficient ATG5− / − macrophages and mouse embryonic fibroblasts
following vesicular stomatitis virus infection or poly (I:C)
transfection.90 Accumulation of dysfunctional mitochondria in
ATG5− / − cells caused elevated RLR signaling through mitochondrial
ROS and increased levels of MAVS. Wild-type cells maintained
homeostatic regulation of antiviral defense by clearing damaged
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Role of mitophagy in immune system
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mitochondria and modulating MAVS levels through mitophagy.90
Although dysfunctional mitochondria can lead to elevated RLR
signaling through MAVS, a study by Koshiba et al.91 showed that
healthy mitochondria are required to promote MAVS activity. Loss of
mitochondrial membrane potential by chemical uncoupling, overexpression of uncoupling protein-2 or loss of mitochondrial fusion
corresponded with reduced antiviral response through MAVS.91 It is
clear from these studies that controlling and maintaining mitochondrial function is important for antiviral immunity, both in the form of
ROS and the transmembrane potential.
In an interesting twist, hepatitis B and C viruses (HBV and HCV)
utilize mitophagy mediated by PINK1 and Parkin to their benefit.
HCV induces Parkin translocation to damaged mitochondria where it
drives mitophagy.92 Knockdown of PINK1, Parkin or Atg5 to block
mitophagy inhibits HCV replication.92 A follow-up study by the same
group revealed a potential mechanism behind how PINK1/Parkin
mitophagy would help HCV replication. HBV was found to promote
its replication in the cell by circumventing mitochondrial apoptosis
which was achieved, in part, by promoting PINK1/Parkin mitophagy
to prevent pro-apoptotic stimuli from spilling out into the cytosol.93
By manipulating mitophagy and preventing cell death pathways, HBV
promotes the survival and maintenance of persistently infected
hepatocytes.
MITOCHONDRIAL HEALTH AND MITOPHAGY IN IMMUNE CELL
HOMEOSTASIS
Naïve T-cells are quiescent, but after becoming activated or primed,
they undergo a growth phase where they increase in size, proliferate
and differentiate to different T-cell subsets. One of the subsets,
immune-supressive regulatory T-cell, rely heavily on fatty acid
oxidation in mitochondria for energy.94 M2-like macrophages promote tissue repair, and like regulatory T cells, rely predominantly on
fatty acid oxidation and OXPHOS for ATP.95 Moreover, pretreatment
of macrophage precursors with inhibitors of respiration or fatty acid
oxidation blocks M2 activation, highlighting a role for mitochondrial
metabolism during differentiation.95
Given the central role of mitophagy in maintaining mitochondrial
function, it is reasonable to expect that immune cell viability and
differentiation as described above may be disrupted in its absence. This
is supported by the discovery that T-cells depend on autophagy to
maintain homeostasis.96,97 T-cell-specific depletion of Vps3496 or
Atg797 impairs autophagy and leads to an accumulation of damaged
mitochondria and ROS. Although VPS34 is dispensable for T-cell
development, it is important for T-cell survival by removing damaged
mitochondria.96 Atg7 promotes T-cell homeostasis in a similar
manner but was also observed to have an effect on the level of
mitochondrial apoptotic factors.97 In macrophages, presentation of
mitochondrial antigens by MHC-class I molecules is promoted by
mitophagy following TNF-α treatment.98 Treatment of cells
with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone, a compound used to synthetically activate
PINK1/Parkin mitophagy, had a milder but similar effect.95 This
raises the possibility that PINK1 and Parkin partially contribute to
MHC antigen presentation although further studies are required.
CONCLUDING REMARKS
The importance of mitochondria in immune signaling has become
increasingly apparent in recent years. As well as controlling cell fate
through apoptosis, mitochondria provide signaling platforms generated by MAVS and provide damage-associated molecular patterns in
the form of ROS and mtDNA. Many lines of evidence support the idea
Immunology and Cell Biology
that maintenance of mitochondrial fidelity by mitophagy is important
during immune signaling (Figure 2). The cell balances oxidation and
reduction reactions to fine-tune the level of NLRP3 activation.99 The
balance tilts in favor of oxidation and hyperactivation in cells where
dysfunctional mitochondria accumulate. The protective role of mitophagy lies in its ability to degrade dysfunctional, ROS-producing
mitochondria. There is potential to treat inflammatory diseases where
excess ROS and mitochondrial dysfunction are involved by stimulating
mitophagy. This is supported by the observation that mitochondrial
antioxidants help ameliorate symptoms.81,83 In addition, because
defective mitophagy has been implicated in Parkinson’s disease
through PINK1/Parkin, therapeutics targeting this pathway are being
sought.
Much of the research on mitophagy in the immune system to date
has focused on the core components of the autophagy machinery
(Atg5, Atg7, Beclin, VPS34 and LC3). Given that ubiquitinated
inflammasomes can be degraded by autophagy,100 it is important to
delineate the role of specific mitophagy pathways in innate immunity.
To achieve this, future studies will need to focus on the role of
selective autophagy pathways that converge on the mitochondria
during NLRP3 and RIG-I activation. So far, mitophagy during NLRP3
signaling was found to be Parkin-independent.77,86 Conversely,
PINK1/Parkin mitophagy has a specific role to play during HCV
and HBV infection.92,93 Further analysis of the other known mitophagy factors in immune cells is warranted. Indeed, as Nix appears to
be quite specific for mitochondrial clearance in reticulocytes,10 it is
conceivable that the immune system has developed its own, as yet,
uncharacterized pathway of mitophagy.
ACKNOWLEDGEMENTS
Apologies to all colleagues whose work could not be cited because of space
limitations. Work in Michael Lazarou’s laboratory is supported by National
Health and Medical Research Council and Monash University. Thank you to
Lesley Kane and Danielle Sliter for reading the manuscript and invaluable
discussions.
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