Download Mitochondria: an Unexpected Force in Innate Immunity

SHOWCASE ON RESEARCH
Mitochondria: an Unexpected
Force in Innate Immunity
Jing Khoo1, Phillip Nagley2 and Ashley Mansell1*
Centre for Innate Immunity and Infectious Diseases,
Monash Institute of Medical Research, Clayton, VIC 3168
2
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in
Structural and Functional Microbial Genomics, Monash University, Clayton, VIC 3800
*Corresponding author: [email protected]
1
We all know from first year biology that mitochondria
are the cellular powerhouses, generating energy for
physiological processes as well as signalling for apoptotic
cell death. But a role in innate immunity? In the words of
Darryl Kerrigan: ‘tell him he’s dreamin’ (1).
Recent studies have shown us, however, that not only
do mitochondria provide a platform for innate antiviral
signalling but they also take an active role in orchestrating
the innate immune response to disruption of homeostasis.
Furthermore, dysfunctional mitochondria can also act as
activators of innate immunity, thus placing mitochondria
squarely at the interface between cellular function and
immune inflammation.
Pattern Recognition Receptors and Innate Immunity
While only higher vertebrates enjoy the luxury of adaptive
immunity, nearly all organisms rely on innate immunity
to provide protection from pathogens and maintain
homeostasis. Charles Janeway first wrote in 1989 that
“...primitive effector cells bear receptors that allow recognition
of certain pathogen-associated molecular patterns that are
not found in the host. I term these receptors pattern recognition
receptors” (2). Janeway and Ruslan Medzhitov subsequently
published the identification of the first mammalian Toll
homolog hToll in 1997 (3) which was consequently renamed
Toll-like receptor (TLR)-4. TLR4 was next conclusively
identified as the long sought for receptor for the bacterial
product lipopolysaccharide (LPS) (4) which can cause septic
shock. The innate immunity sensors or pattern recognition
receptors (PRRs) had finally been discovered and they
revolutionised our understanding of immunology.
Toll-like Receptors (TLRs): TLRs are evolutionarily
conserved leucine-rich repeat (LRR) transmembrane
receptors that are widely expressed on both immune and
non-immune cells (5). TLRs such as TLR1, TLR2, TLR4,
TLR5 and TLR6 are predominately expressed at the cell
membrane, matching their ability to recognise constituents
of bacterial membranes. In contrast, TLR3, TLR7, TLR8
and TLR9 are found in intracellular compartments such
as endosomes, reflecting their requirement of endosomal
internalisation of their respective ligands, mainly
bacterial and viral nucleic acids. Upon ligand-induced
receptor dimerisation, a series of cytosolic signalling
mediators are recruited to the receptor complex that
initiates a signal transduction pathway culminating in
their nuclear localisation and subsequent transcription of
the prototypic inflammatory transcription factor NF-kB
(Fig. 1). Activation of NF-kB initiates the pro-inflammatory
response characterised by expression of cytokines,
chemokines, leukotrienes, adhesion factors and a host of
genes associated with cell survival. In the case of TLR4 and
TLR3, there is the additive activation of the transcription
factor, interferon regulatory factor (IRF)-3, which leads to
expression of type I interferons (IFNs).
RIG-I-like Receptors (RLRs): RLRs, which include
RIG-I, MDA5 and LGP2 (6), are cytosolically localised
Fig. 1. Toll-like receptors (TLRs) act as the
sentinels to pathogen infection and initiate
the innate immune pro-inflammatory
response.
TLRs (with various numbers) recognise
pathogen products either in the extracellular
or endosomal environment and initiate
activation and nuclear translocation of the
prototypic inflammatory transcription factor
NF-kB to drive the inflammatory response.
Activation of IRF3 by TLR3 and TLR4 also
expresses the antiviral cytokine interferon.
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SHOWCASE ON
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Mitochondria: an Unexpected
Force in Innate Immunity
Fig. 2. Schematic representation of recognition
and response of the RIG-I-like receptor (RLR)
to viral infection.
Cytosolic receptors such as RIG-I and MDA5
recognise RNA produced by replicating viruses,
translocating to the mitochondria to interact
with MAVS and initiate downstream signalling.
Activation of transcription factors such as NFkB and IRF3 induces the pro-inflammatory
responses responsible for clearing the infection.
OMM, outer mitochondrial membrane; IMM,
inner mitochondrial membrane.
RNA helicases that recognise viral single stranded (ss)
RNA species released into the cytoplasm during viral
replication in a variety of cell types. They coordinate
antiviral gene programs via NF-kB and IRF3 induction of
antiviral IFNa/b (Fig. 2). Following detection of ssRNA,
RIG-I and MDA5 undergo post-translational modification
via the addition of ubiquitin chains, inducing association
and subsequent activation of mitochondrial antiviral
signalling (MAVS) protein. As the name suggests, MAVS
(also called IPS1, Cardif or Visa) is found in the outer
mitochondrial membrane and, following activation by
RIG-I or MDA5, interacts with downstream signalling
mediators to induce nuclear translocation of IRF3 and
NF-kB. This leads to induction of antiviral IFNa/b.
This coordinated antiviral response is critical to clearing
pathogens as quickly as possible to prevent exacerbated
viral infection that could be followed by chronic,
persistent infection and inflammation.
Mitochondrial Proteins in the Regulation of RIG-I-like
Receptor Signalling
The first evidence linking mitochondria to innate immune
signalling came with the discovery of MAVS. However,
it was soon found that mitochondria not only provide a
platform to organise signalling, but they also play an active
role in regulating signal transduction. NLRX1 (NODlike receptor X1) is a mitochondrially-localised protein
originally thought to negatively regulate signalling by
interacting with MAVS, thus preventing its interaction with
RIG-I on the outer mitochondrial membrane. However,
a recent analysis of the mitochondrial topology and
targeting sequence of NLRX1 revealed that it is targeted
to the mitochondrial matrix (7), thus making association
with MAVS improbable. NLRX1 may nonetheless behave
like another mitochondrial protein that modulates RLR
signalling, namely the receptor for the globular heads of
C1q (gC1qR). Though found in the mitochondrial matrix or
cytosol, gC1qR is processed upon viral infection and then
it translocates to the outer mitochondrial membrane where
it suppresses MAVS-mediated signalling. Interestingly,
ectopic expression of NLRX1 induces production of reactive
oxygen species (ROS) in response to TNFa stimulation or
Shigella bacterial infection (8). NLRX1 also interacts with
the mitochondrial protein, UQCRC2 (7), a matrix-facing
protein of the respiratory chain complex III involved in
ROS release. Inhibition or ablation of NADPH oxidase,
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an enzyme essential for ROS production, reduces the RLR
antiviral response. NLRX1 may play a modulating role in
RLR signalling; such interference with ROS production
is increasingly recongised as a ‘fine-tuning’ process for
PRR immune responses (9). Recently, our studies have
identified a novel mitochondrial protein, MUL1, which
appears to modulate RLR signalling (10). MUL1 is localised
to mitochondria where it interacts with MAVS and
catalyses RIG-I post-translational modifications that inhibit
RIG-I-dependent cell signalling. Moreover, depletion
of MUL1 boosts the antiviral response and increases
proinflammatory cytokines following challenge with the
RLR ligand poly(I:C) and Sendai virus. This would appear
to identify MUL1 as a novel regulator of RLR signalling,
using mitochondria as a proximal locale to identify and
modulate RIG-I function.
RIG-I-like Receptor Antiviral Responses are Modulated
by Mitochondrial Dynamics
Mitochondria are usually observed as a tubular network
surrounding the nucleus and radiating from the nucleus to
the fringe of the cell. Mitochondria are highly dynamic such
that these organelles can fuse with each other or become
fragmented into individual mitochondria (11). Mitochondrial
fusion is important for maintaining mitochondrial function
in cells. In humans, the mitochondrial fusion machinery
involves two sets of key GTPase proteins: the outer membrane
mitofusins (MFN1 and MFN2) as well as the inner membrane
optic atrophy 1 (OPA1). Conversely, mitochondrial fission
is essential for the division of mitochondria during cell
proliferation. In mammalian cells, mitochondrial fission is
dependent on a key GTPase protein known as dynaminrelated protein 1 (Drp1).
Recent studies have implicated mitochondrial dynamics
in the regulation of RLR antiviral signalling by the
mitochondrial fusion factors MFN1 and MFN2 (Fig. 3).
Arnoult and colleagues initially observed that RIG-I and
MDA5 signalling, triggered by Sendai virus infection and
poly(I:C), respectively were associated with the elongation
of mitochondrial tubules. Depletion by shRNA of MFN1,
OPA1, Drp1 and FIS1, to manipulate mitochondrial
elongation and fragmentation, further demonstrated that
mitochondrial elongation is required for RLR antiviral
and proinflammatory response (12). Alternatively,
encouraging mitochondrial fusion by ablating Drp1 and
FIS1 expression resulted in activation of the signalling
AUSTRALIAN BIOCHEMIST
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SHOWCASE ON
RESEARCH
Mitochondria: an Unexpected
Force in Innate Immunity
Fig. 3. The mitochondria play an integral
role in mediating antiviral signalling.
Following viral recognition by RLRs
and translocation to the mitochondria to
interact with MAVS, the mitochondrial
dynamics, localisation and interaction
with other organelles regulate the spatial
and temporal response to pathogen
challenge and the subsequent immune
response. IFN, interferon; ISG, interferonstimulated gene.
pathway. Critically, mitochondrial elongation enhanced
immune responses to virus or poly(I:C) stimulation.
MAVS was also shown to co-immunoprecipitate MFN1.
Thus, it was proposed that MAVS degradation releases
MFN1 to induce further mitochondrial fusion for the
amplification of downstream responses.
Conversely, a more recent study reporting on the role
of mitochondrial fusion in MAVS signalling presented
slightly different results. Onoguchi and colleagues (13)
demonstrated that, in response to Sendai virus infection,
ectopic expression of MAVS at low levels and endogenous
expression of MAVS led to the formation of clusters on the
mitochondria. MAVS was shown to interact with MFN1
in co-immunoprecipitation studies. Furthermore, MFN1depletion by siRNA was shown to prevent the MAVS
clustering. However, in this study, the clustering of MAVS
appeared to be independent of mitochondrial fusion or
fission, since mitochondrial elongation was not observed
during viral infection. This discrepancy, however, may be
due to viral strain differences.
A further study reported that RLR-mediated antiviral
responses were reduced in mouse embryonic fibroblasts
(MEFs) deficient in both MFN1 and MFN2 (14). Such MEFs
still showed substantial antiviral response, suggesting
that RLR signalling is impaired only when mitochondrial
fusion is completely prevented. Moreover, this study also
showed that the mitochondrial inner membrane potential
is essential for MAVS-mediated antiviral response,
since MFN1/2 double knockout MEFS have defective
membrane potential.
Further muddying the waters, MFN2 was reported as
a negative regulator of MAVS signalling (15). MAVS
was observed to interact with MFN2; further, MFN2
depletion led to increased RLR-mediated antiviral and
proinflammatory responses. To date, the mechanism of
MFN2 regulation of MAVS signalling is also still unclear.
The effect of MFN2 on mitochondrial dynamics during
viral infection has not been studied, so it is unknown if the
effect of antiviral signalling is dependent on mitochondrial
dynamics. However, more recent studies have associated
MFN2 regulation of MAVS signalling via the promotion
by MFN2 of relocalisation of MAVS between mitochondria
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and the endoplasmic reticulum into mitochondrial
associated membranes (see below).
Collectively, these studies indicate that healthy
mitochondria with intact fusion/fission process and
membrane integrity are a prerequisite for RLR- and
MAVS-mediated antiviral responses, although the precise
mechanism and relationship between proteins involved in
mitochondrial dynamics requires further investigation.
Mitochondria Coordinate Antiviral Signalling with
Other Intracellular Compartments
Although RLR signalling converges at the level of MAVS
and mitochondria, other intracellular structures are also
involved in transmitting antiviral signalling. STING
(stimulator of interferon genes) was presented as the
first protein bridging mitochondria and the endoplasmic
reticulum for innate antiviral signalling (16) (Fig. 3).
Although targeted to the endoplasmic reticulum (ER),
STING interacts with MAVS, while STING-deficient
MEFs display impaired antiviral responses to Sendai virus
infection. Importantly, MAVS–STING interaction is more
prominent in the presence of mitochondrial elongation,
while MFN1 was also observed to interact with the MAVS–
STING complex (12). Therefore, MAVS may transmit
downstream IFN responses from the mitochondria–ER
interface in the presence of STING.
MAVS is also targeted to the peroxisomes, where it
can induce antiviral responses in a biphasic manner.
Peroxisomal MAVS was found to trigger a rapid induction
of a subset of IFN-stimulated genes (ISGs), whereas
mitochondrial MAVS induced a sustained expression of
IFN and ISGs (Fig. 3). Expanding on this mitochondrial,
ER and peroxisomal signalling nexus, MAVS was observed
to localise to mitochondrial-associated membrane
(MAM) structures that connect the ER and mitochondria.
Interestingly, MAVS was also found to co-localise with
peroxisomal markers, which were observed proximal to the
MAMs and mitochondrial junction (17). Further enhancing
the role of mitochondrial dynamics in orchestrating
signalling, MFN2 is required for the association of MAVS
to MAMs. MFN2 depletion resulted in reduced MAVS
association with the mitochondria (and thus MAMs),
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SHOWCASE ON
RESEARCH
Mitochondria: an Unexpected
Force in Innate Immunity
with a concomitant increase in the association with the
peroxisomes, suggesting that MFN2 may play a role in
‘sorting’ MAVS between organelles.
Together these studies demonstrate that mitochondrial
signalling may be coordinated by these three different
organelles with the mitochondria a central conduit, possibly
at points of organelle contact.
Good Cop, Bad Cop
Finally, to emphasise the possible coevolutionary paths
of symbiotic incorporation of mitochondria into eukaryotic
cells and the development of an immune system, recent
examples have demonstrated that disrupted mitochondria
themselves can act as inducers of innate inflammation.
Several recent studies have noted that mitochondria harbour
an array of danger-associated molecular patterns which act
as ligands for PRRs (18,19). Mitochondrial DNA and formylpeptides were detected by TLR9 following trauma such as
crush injuries or burns, which induce a clinically dangerous
inflammatory state termed systemic inflammatory response
syndrome (SIRS) (20). SIRS can be caused by both infectious
and non-infectious factors. Mitochondria-derived activators
of PRRs can be released from dysfunctional or necrotic
mitochondria. Products escaping from mitochondrial
autophagy or mitochondrial ROS have now all been
identified as innate immune ‘danger’ signals, causing a
disruption of homeostasis and consequently stimulating
a robust innate immune response via PRR detection and
induction of pro-inflammatory cytokines and chemokines.
Thus, mitochondria are a ‘double-edged’ sword in innate
immunity. Healthy and functional mitochondria are
required to facilitate antiviral immune signalling; conversely
dysfunctional or dysregulated mitochondria may provide
danger-associated ligands that are detected by PRRs as a
disruption to homeostasis. As such, mitochondria play a
critical role in the initiation of innate immune inflammation,
either through dysfunctional mitochondria, or beneficially
via PRR signal transduction through healthy and dynamic
mitochondria.
Concluding Remarks
Despite a billion years of coevolution, the concept of
mitochondria interacting with, and participating in, our
most ancient means of maintaining cellular homeostasis has
not been considered companionable. Recent developments
however have clearly placed two of our most fundamental
processes at the intersection of a robust and vigilant immune
response. Clearly there is a close relationship between
innate immune recognition and signal transduction, on the
one hand, and mitochondrial function and its machinations,
on the other. Research to date has revealed but the tip of
the iceberg. Further studies are required to delineate the role
in innate immunity of the global structure and individual
components of mitochondria, as well as the health and
dysfunction of these organelles. Importantly, given the
expansion and appreciation of the role of PRRs in a plethora
of diseases, further consideration is warranted into the role,
and possible therapeutic targeting, of mitochondria in such
clinical contexts.
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Initially, like Darryl Kerrigan, we may have been dreaming,
speculating that innate immunity and mitochondria are
linked . But stranger combinations such as cola and ice cream,
Shane Warne and Liz Hurley, or vegemite and anything,
appear to work. Contrasting combinations therefore can be
compatible, providing the basis for an efficient and effective
process for the maintenance and regulation of homeostasis.
And, like Shane Warne, we should be thankful that it does.
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