Mitochondrial control of the NLRP3 inflammasome - Nature

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have found that ROS derived from complex I and complex III (but not those derived from complex II) stimulate activation of the NLRP3 inflammasome and release ...

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Mitochondrial control of the NLRP3 inflammasome Oliver Kepp, Lorenzo Galluzzi & Guido Kroemer During the past few decades, the vital and lethal functions of mitochondria have been investigated extensively. Data now demonstrate that these organelles also regulate the innate immune response by modulating inflammasomemediated generation of proinflammatory cytokines.

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any metabolic circuitries and signaling pathways involve mitochondria. Just a few examples of these include the following: oxidative phosphorylation generates most intracellular ATP; mitochondria participate in the biosynthesis of many molecules (such as heme, steroids and iron-sulfur clusters) as well as in many catabolic pathways (such as the β-oxidation of fatty acids); mitochondria regulate calcium homeostasis; and mitochondria produce most of the cell’s reactive oxygen species (ROS). Mitochondrial membrane permeabilization represents the point of no return of programmed cell death pathways that culminate in apoptosis or regulated necrosis1,2 (Fig. 1a). New data from Nakahira et al. in this issue of Nature Immunology3 and Zhou et al. in Nature4 now indicate that mitochondria also orchestrate the innate immune response. A plethora of distinct stimuli can ­activate innate immunity, including pathogen­associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs such as bacterial lipopolysaccharide (LPS) or viral double-stranded RNA are of microbial origin, whereas DAMPs such as uric acid, extracellular ATP and heatshock proteins are host-intrinsic signals that accumulate with stress5. Both PAMPs and DAMPs bind to a series of specific patternrecognition receptors (PRRs), which are either expressed on the cell surface, such as Toll-like receptors, or are present in the cytoplasm, such as Nod-like receptors (NLRs) and the RNA helicase DDX58 (better known as RIG-I)6. The engagement of Toll-like receptors at the cell surface and the activation of RIG-I can stimulate the activation of ­proinflammatory Oliver Kepp and Lorenzo Galluzzi are with Institut National de la Santé et de la Recherche Médicale, U848, Villejuif, France, Institut Gustave Roussy, Villejuif, France, and Université Paris-Sud, Villejuif, France. Guido Kroemer is with Institut National de la Santé et de la Recherche Médicale, U84s8, Villejuif, France, Metabolomics Platform, Institut Gustave Roussy, Villejuif, France, Centre de Recherche des Cordeliers, Paris, France, Pôle de Biologie, Hôpital Européen Georges Pompidou, Paris, France, and Université Paris Descartes, Paris, France. e-mail: [email protected]

genes via the transcription factor NF-κB6. Conversely, some NLRs, including NLRP3 (also known as NALP3), respond to PAMPs and/or DAMPs by orchestrating the assembly of a supramolecular platform (the socalled ‘inflammasome’) for the activation of caspase-1, which is required for the proteolytic maturation and release of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18 (ref. 5). In 2005, a mitochondrial polypeptide required for RIG-I-mediated activation of NF-κB and secretion of interferon-γ in response to viral infection was described for the first time and was dubbed ‘mitochondrial antiviral signaling protein’7 (Fig. 1a). That work already suggested that mitochondria can modulate innate immunity, a hypothesis that has now been confirmed and extended by Nakahira et al.3 and Zhou et al.4. Published

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work has indicated that ROS are the common integrator across several stimuli that activate the NLRP3 inflammasome8; however, the source of NLRP3-activating ROS and the underlying molecular mechanisms have remained obscure. To address those questions, Zhou and colleagues have now studied the response of THP-1 human macrophages to inhibitors specific for complex I, II or III of the mitochondrial respiratory chain and have found that ROS derived from complex I and complex III (but not those derived from complex II) stimulate activation of the NLRP3 inflammasome and release of IL-1β. As ROSgenerating mitochondria are normally eliminated by mitophagy, a specialized branch of autophagy (mitochondrial autophagy), the authors hypothesize that inhibition of autophagy would also affect IL-1β production. Indeed, they find that ­pharmacological

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Figure 1 Mitochondrial death and danger signaling. (a) Intracellular stress signals can induce mitochondrial membrane permeabilization (MMP), which leads to the cytosolic release of toxic proteins that are normally confined in mitochondria, followed by the activation of several classes of proteases and execution of the cell death program. During viral infection, single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) rigger RIG-I and allow its recruitment to the outer mitochondrial membrane by mitochondrial antiviral signaling protein (MAVS); binding of RIG-I to mitochondrial antiviral signaling protein leads to the activation of interferon-regulatory factors (IRFs) and NF-κB, which drives the production of type I interferon (IFN) and proinflammatory cytokines, respectively. (b) Danger signals, including several DAMPs, can stimulate the overproduction of ROS via a VDACregulated mechanism. ROS promote MPT, favoring mitochondrial uncoupling and further ROS generation in a feed-forward circuitry. After MPT, the PRR NLRP3 facilitates the cytosolic release of mtDNA, which stimulates activation of the NLRP3 inflammasome and the production of IL-1β and IL-18. Mitophagy inhibits this process by removing ROS-generating mitochondria. (c) Environmental stress, mitochondrial diseases (mitochondriopathies) and cellular aging have all been associated with inhibition of mitophagy and the accumulation of damaged mitochondria. In this context, excess generation of ROS can trigger the activation of PRRs and the production of proinflammatory cytokines, thereby sustaining the onset of inflammation.

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ne w s and v ie w s or genetic inhibition of the autophagic ma­chinery results in a small amount of inflam­masome activation and IL-1β release. Moreover, suppression of autophagy strongly potentiates the proinflammatory response to the NLRP3 activators monosodium urate and nigericin4. These data are compatible with the published observation that macrophages from Atg16l1–/– mice (which are characterized by impaired autophagy) have exaggerated release of IL-1β in response to LPS9. Zhou et al. further substantiate the involvement of mitochondria in regulating the NLRP3 inflammasome at two levels. First, they find that in response to monosodium urate and nigericin, both NLRP3 and PYCARD (another component of the inflammasome, also known as ASC) relocalize to the mitochondria and ­mitochondria-associated endoplasmic reticulum membranes. Second, they observe that activation of the NLRP3 inflammasome by several stimuli, including monosodium urate, silica, alum and nigericin, is impaired after depletion of the voltage-dependent anion channel (VDAC) by RNA-mediated interference or after over­expression of the VDACclosing protein Bcl-2, two interventions that nearly completely abolish the mitochondrial generation of ROS4. Intrigued by the observation that autophagy-deficient mice are hypersensitive to LPS and other PAMPs9, Nakahira and colleagues tackle essentially the same problem as Zhou et al. do in Nature but from a different perspective. First, they demonstrate that bone marrow–derived macrophages (BMDMs) isolated from mice lacking the ­autophagosomal component LC3B or haploinsufficient for the essential regulator of autophagy Beclin 1 (Becn1+/–) release more IL-1β and IL-18 in response to LPS and ATP (which act on the PRRs TLR4 (Toll-like receptor 4) and P2X7 (purinergic receptor), respectively) than do wild-type BMDMs3. As autophagy is critical for the removal of damaged organelles, the authors study the ultrastructural phenotype of Lc3b–/– and Bcn1+/– macrophages responding to LPS and ATP and find that these cells show signs of defective mitochondrial homeostasis, including swollen and severely disrupted mitochondria and greater mitochondrial generation of ROS3. These results prompt investigation of the relationship between various aspects of mitochondrial metabolism and activation of the NLRP3 inflammasome and lead to several important discoveries. First, similar to Zhou et al.4, Nakahira et al. find that the

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­ itochondria-targeting drug rotenone exacerm bates the secretion of proinflammatory cytokines by BMDMs challenged with LPS and ATP. Second, these authors find that this response is abolished in macrophages rendered respiration deficient by prolonged culture in ethidium bromide (which blocks the replication of mitochondrial DNA (mtDNA), yielding so-called ‘ρ0 cells’), as well as in BMDMs treated with the mitochondria-targeted antioxidant Mito-TEMPO. Third, Mito-TEMPO and cyclosporine A, a potent inhibitor of the so-called mitochondrial permeability transition (MPT; a phenomenon that mediates some intrinsic apoptosis)1, block the release of IL-1β driven by LPS and ATP in both autophagy-sufficient and autophagy-deficient BMDMs4. Together with the results from Zhou et al., who also identify a role for MPTmediating proteins, in particular VDAC, these data indicate that NLRP3 activation requires mitochondrial generation of ROS and MPT, at least in some cases (Fig. 1b). Nakahira and colleagues go one step further and examine the potential involvement of mtDNA, which has been shown to act as a DAMP, at least under certain conditions10, in the activation of the NLRP3 inflammasome. Indeed, they find that mtDNA is released into the cytosol of BMDMs responding to LPS and ATP and that such cytosolic mtDNA underpins the optimal release of IL-1β and IL-18. Intriguingly, they also find that NLRP3 is required for MPT and the subsequent translocation of mtDNA from the mitochondrial matrix to the cytosol3, which identifies an unprecedented circuitry in which NLRP3 might act upstream and also downstream of mitochondria. Next Nakahira and colleagues examine the role of autophagic proteins in caspase-1mediated inflammation in endotoxemic mice (a commonly used model of septic shock) and in a model using cecal ligation and puncture (which is a clinically relevant model of polymicrobial sepsis). In line with the in vitro data, Lc3b–/– and Bcn1+/– mice have higher serum concentrations of IL-1β and IL-18 during the septic response than their wild-type counterparts do, and Lc3b–/– mice are more susceptible to LPS-induced mortality3. Collectively these results demonstrate that the autophagic machinery operates in vivo to regulate inflammation and in particular the circulating concentrations of IL-18 and IL-1β3. A pathologically relevant link between autophagy and innate immunity has been identified by the demonstration that the

intracellular PRRs Nod1 and Nod2 recruit the autophagy protein ATG16L1 to the plasma membrane at the site of bacterial invasion11,12. Together, the work by Zhou et al. and Nakahira et al. underscores the intimate link between autophagy and innate immunity. More importantly, both papers unequivocally place mitochondria at the crossroads of bioenergetic metabolism, cell death signaling and the innate immune response. Although the articles by Zhou et al. and by Nakahira et al. have identified a common role for the generation of ROS by the respiratory chain as well as for MPT in activation of the NLRP3 inflammasome, several enigmatic issues remain to be resolved. For example, it is unknown whether mtDNA directly activates the inflammasome by binding to one (or more) of its known (or unknown) components or whether mtDNA acts on an as-yet-unidentified cytoplasmic receptor that relays to the inflammasome by indirect mechanisms. Moreover, it remains to be determined whether pro- and antiapoptotic proteins of the Bcl-2 family modulate activation of the inflammasome directly or at the level of MPT. Regardless of which proves true, it turns out that MPT induced by cellular stress can either elicit the cell’s demise1 or stimulate an innate immune response3,4. Whether cell type–specific factors and/or quantitative thresholds determine which consequence ensures MPT remains an open issue. Further comprehension of these possibilities and choices may yield important insights into the question of how mitochondrial ­damage that arises during normal or pathological aging leads to cell loss or to smoldering or overt inflammation (Fig. 1c). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Kroemer, G., Galluzzi, L. & Brenner, C. Physiol. Rev. 87, 99–163 (2007). 2. Vandenabeele, P., Galluzzi, L., Vanden Berghe, T. & Kroemer, G. Nat. Rev. Mol. Cell Biol. 11, 700–714 (2010). 3. Nakahira, K. et al. Nat. Immunol. 12, 222–230 (2011). 4. Zhou, R., Yazdi, A.S., Menu, P. & Tschopp, J. Nature 469, 221–225 (2011). 5. Zitvogel, L., Kepp, O. & Kroemer, G. Cell 140, 798–804 (2010). 6. Kawai, T. & Akira, S. Int. Immunol. 21, 317–337 (2009). 7. Seth, R.B., Sun, L., Ea, C.K. & Chen, Z.J. Cell 122, 669–682 (2005). 8. Dostert, C. et al. Science 320, 674–677 (2008). 9. Saitoh, T. et al. Nature 456, 264–268 (2008). 10. Zhang, Q. et al. Nature 464, 104–107 (2010). 11. Galluzzi, L., Kepp, O., Zitvogel, L. & Kroemer, G. Curr. Biol. 20, R106–R108 (2010). 12. Travassos, L.H. et al. Nat. Immunol. 11, 55–62 (2010).

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