SHOWCASE ON RESEARCH Mitochondria: an Unexpected Force in Innate Immunity Jing Khoo1, Phillip Nagley2 and Ashley Mansell1* 1Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Clayton, VIC 3168 2Department 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] We all know from first year biology that mitochondria receptors (PRRs) had finally been discovered and they are the cellular powerhouses, generating energy for revolutionised our understanding of immunology. physiological processes as well as signalling for apoptotic cell death. But a role in innate immunity? In the words of Toll-like Receptors (TLRs): TLRs are evolutionarily Darryl Kerrigan: ‘tell him he’s dreamin’ (1). conserved leucine-rich repeat (LRR) transmembrane Recent studies have shown us, however, that not only receptors that are widely expressed on both immune and do mitochondria provide a platform for innate antiviral non-immune cells (5). TLRs such as TLR1, TLR2, TLR4, signalling but they also take an active role in orchestrating TLR5 and TLR6 are predominately expressed at the cell the innate immune response to disruption of homeostasis. membrane, matching their ability to recognise constituents Furthermore, dysfunctional mitochondria can also act as of bacterial membranes. In contrast, TLR3, TLR7, TLR8 activators of innate immunity, thus placing mitochondria and TLR9 are found in intracellular compartments such squarely at the interface between cellular function and as endosomes, reflecting their requirement of endosomal immune inflammation. internalisation of their respective ligands, mainly bacterial and viral nucleic acids. Upon ligand-induced Pattern Recognition Receptors and Innate Immunity receptor dimerisation, a series of cytosolic signalling While only higher vertebrates enjoy the luxury of adaptive mediators are recruited to the receptor complex that immunity, nearly all organisms rely on innate immunity initiates a signal transduction pathway culminating in to provide protection from pathogens and maintain their nuclear localisation and subsequent transcription of homeostasis. Charles Janeway first wrote in 1989 that the prototypic inflammatory transcription factor NF-kB “...primitive effector cells bear receptors that allow recognition (Fig. 1). Activation of NF-kB initiates the pro-inflammatory of certain pathogen-associated molecular patterns that are response characterised by expression of cytokines, not found in the host. I term these receptors pattern recognition chemokines, leukotrienes, adhesion factors and a host of receptors” (2). Janeway and Ruslan Medzhitov subsequently genes associated with cell survival. In the case of TLR4 and published the identification of the first mammalian Toll TLR3, there is the additive activation of the transcription homolog hToll in 1997 (3) which was consequently renamed factor, interferon regulatory factor (IRF)-3, which leads to Toll-like receptor (TLR)-4. TLR4 was next conclusively expression of type I interferons (IFNs). identified as the long sought for receptor for the bacterial product lipopolysaccharide (LPS) (4) which can cause septic RIG-I-like Receptors (RLRs): RLRs, which include shock. The innate immunity sensors or pattern recognition 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. Vol 44 No 1 April 2013 AUSTRALIAN BIOCHEMIST Page 17 Mitochondria: an Unexpected SHOWCASE ON Force in Innate Immunity RESEARCH 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 NF- kB 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) an enzyme essential for ROS production, reduces the RLR RNA species released into the cytoplasm during viral antiviral response. NLRX1 may play a modulating role in replication in a variety of cell types. They coordinate RLR signalling; such interference with ROS production antiviral gene programs via NF-kB and IRF3 induction of is increasingly recongised as a ‘fine-tuning’ process for antiviral IFNa/b (Fig. 2). Following detection of ssRNA, PRR immune responses (9). Recently, our studies have RIG-I and MDA5 undergo post-translational modification identified a novel mitochondrial protein, MUL1, which via the addition of ubiquitin chains, inducing association appears to modulate RLR signalling (10). MUL1 is localised and subsequent activation of mitochondrial antiviral to mitochondria where it interacts with MAVS and signalling (MAVS) protein. As the name suggests, MAVS catalyses RIG-I post-translational modifications that inhibit (also called IPS1, Cardif or Visa) is found in the outer RIG-I-dependent cell signalling. Moreover, depletion mitochondrial membrane and, following activation by of MUL1 boosts the antiviral response and increases RIG-I or MDA5, interacts with downstream signalling proinflammatory cytokines following challenge with the mediators to induce nuclear translocation of IRF3 and RLR ligand poly(I:C) and Sendai virus. This would appear NF-kB. This leads to induction of antiviral IFNa/b. to identify MUL1 as a novel regulator of RLR signalling, This coordinated antiviral response is critical to clearing using mitochondria as a proximal locale to identify and pathogens as quickly as possible to prevent exacerbated modulate RIG-I function. viral infection that could be followed by chronic, persistent infection and inflammation. RIG-I-like Receptor Antiviral Responses are Modulated by Mitochondrial Dynamics Mitochondrial Proteins in the Regulation of RIG-I-like Mitochondria are usually observed as a tubular network Receptor Signalling surrounding the nucleus and radiating from the nucleus to The first evidence linking mitochondria to innate immune the fringe of the cell. Mitochondria are highly dynamic such signalling came with the discovery of MAVS. However, that these organelles can fuse with each other or become it was soon found that mitochondria not only provide a fragmented into individual mitochondria (11). Mitochondrial platform to organise signalling, but they also play an active fusion is important for maintaining mitochondrial function role in regulating signal transduction. NLRX1 (NOD- in cells. In humans, the mitochondrial fusion machinery like receptor X1) is a mitochondrially-localised protein involves two sets of key GTPase proteins: the outer membrane originally thought to negatively regulate signalling by mitofusins (MFN1 and MFN2) as well as the inner membrane interacting with MAVS, thus preventing its interaction with optic atrophy 1 (OPA1). Conversely, mitochondrial fission RIG-I on the outer mitochondrial membrane. However, is essential for the division of mitochondria during cell a recent analysis of the mitochondrial topology and proliferation. In mammalian cells, mitochondrial fission is targeting sequence of NLRX1 revealed that it is targeted dependent on a key GTPase protein known as dynamin- to the mitochondrial matrix (7), thus making association related protein 1 (Drp1). with MAVS improbable. NLRX1 may nonetheless behave Recent studies have implicated mitochondrial dynamics like another mitochondrial protein that modulates RLR in the regulation of RLR antiviral signalling by the signalling, namely the receptor for the globular heads of mitochondrial fusion factors MFN1 and MFN2 (Fig. 3). C1q (gC1qR). Though found in the mitochondrial matrix or Arnoult and colleagues initially observed that RIG-I and cytosol, gC1qR is processed upon viral infection and then MDA5 signalling, triggered by Sendai virus infection and it translocates to the outer mitochondrial membrane where poly(I:C), respectively were associated with the elongation it suppresses MAVS-mediated signalling. Interestingly, of mitochondrial tubules. Depletion by shRNA of MFN1, ectopic expression of NLRX1 induces production of reactive OPA1, Drp1 and FIS1, to manipulate mitochondrial oxygen species (ROS) in response to TNFa stimulation or elongation and fragmentation, further demonstrated that Shigella bacterial infection (8). NLRX1 also interacts with mitochondrial elongation is required for RLR antiviral the mitochondrial protein, UQCRC2 (7), a matrix-facing and proinflammatory response (12). Alternatively, protein of the respiratory chain complex III involved in encouraging mitochondrial fusion by ablating Drp1 and ROS release. Inhibition or ablation of NADPH oxidase, FIS1 expression resulted in activation of the signalling Page 18 AUSTRALIAN BIOCHEMIST Vol 44 No 1 April 2013 SHOWCASE ON Mitochondria: an Unexpected RESEARCH Force in Innate Immunity Fig. 3. The mitochondria