Mechanism and regulation of NLRP3 inflammasome activation.pdf

Mechanism and regulation of NLRP3 inflammasome activationYuan He1,Hideki Hara1,and Gabriel Nez1,*1Department of Pathology and Comprehensive Cancer Center,University of Michigan Medical School,Ann Arbor,MI 48109,USAAbstractMembers of the nucleotide-binding domain and leucine-rich repeat containing(NLR)family and the pyrin and HIN-domain(PYHIN)family can form multiprotein complexes termed“inflammasomes”.The biochemical function of inflammasomes is to activate caspase-1,which leads to the maturation of interleukin 1(IL-1)and IL-18 and induction of pyroptosis,a form of cell death.Unlike other inflammasomes,the NLRP3 inflammasome can be activated by diverse stimuli.The importance of the NLRP3 inflammasome in immunity and human diseases has been well documented,but the mechanism and regulation of NLRP3 inflammasome activation remains unclear.In this review we summarize current understanding of the mechanism and regulation of NLRP3 inflammasome activation,as well as recent advances in the non-canonical and alternative inflammasome pathways.KeywordsNLRP3 inflammasome;K+efflux;Non-canonical inflammasome;Alternative inflammasome;Nek7The NLRP3 inflammasome:A critical component of innate immunity and pathological contributor to human diseasesInflammasomes are a group of cytosolic protein complexes that are formed to mediate host immune responses to microbial infection and cellular damage 1.Assembly of an inflammasome triggers proteolytic cleavage of dormant procaspase-1 into active caspase-1,which converts the cytokine precursors pro-IL-1 and pro-IL-18 into mature and biologically active IL-1 and IL-18,respectively 2,3.Mature IL-1 is a potent proinflammatory mediator in many immune reactions,including the recruitment of innate immune cells to the site of infection and modulation of adaptive immune cells,whereas mature IL-18 is important for the production of interferon-and potentiation of cytolytic activity of natural killer cells and T cells 4.Active caspase-1 also induces a*Correspondence:gabriel.nunezumich.edu(Nez G.),Department of Pathology,University of Michigan Medical School,4215 CC,1500 E.Medical Center Drive,Ann Arbor,Michigan 48109,USA.Publishers Disclaimer:This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting,typesetting,and review of the resulting proof before it is published in its final citable form.Please note that during the production process errors may be discovered which could affect the content,and all legal disclaimers that apply to the journal pertain.HHS Public AccessAuthor manuscriptTrends Biochem Sci.Author manuscript;available in PMC 2017 December 01.Published in final edited form as:Trends Biochem Sci.2016 December;41(12):10121021.doi:10.1016/j.tibs.2016.09.002.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptproinflammatory form of cell death,known as pyroptosis 5.Inflammasome formation requires a pattern recognition receptor(PRR)as the sensor,in most cases the adaptor ASC(apoptosis-associated spec-like protein containing a CARD),and the cysteine protease caspase-1.To date,five PRRs(NLRP1,NLRP3,NLRC4,Pyrin and AIM2)have been shown to form inflammasomes 6,7.Emerging evidence indicates that several other members of the NLR family and the PYHIN family,including NLRP6,NLRP7,NLRP12 and IFI16,can also form inflammasomes,but their composition remains obscure 7.Among these inflammasomes,the NLRP3 inflammasome has been under intensive investigation given its possible involvement in several human diseases.NLRP3 belongs to the NLR protein family,which includes 22 members in human and at least 34 members in mouse 8.Most NLRs have a tripartite structure defined as the following:an amino-terminal caspase-recruitment domain(CARD),pyrin domain,acidic transactivating domain or baculovirus inhibitor repeat that mediates downstream protein-protein interaction;a centrally located nucleotide-binding-and-oligomerization domain that mediates self-oligomerization;and carboxy-terminal leucine-rich repeats(LRRs)that are thought to be involved in recognition of stimuli 9.During microbial infections,the NLRP3 inflammasome promotes host immune defense against infectious microbes,such as Influenza A virus,Staphylococcus aureus and Candida albicans 1.However,dysregulated activation of the NLRP3 inflammasome has been implicated in the pathogenesis of inherited and acquired inflammatory diseases 10,11.The NLRP3 inflammasome was initially shown to be activated by ATP and certain bacterial toxins 12.Subsequently,a wide array of stimuli were identified to activate the NLRP3 inflammasome including multiple microbial products,endogenous molecules or particulate matter 1315.In subsequent studies,however,it became clear that most microbial stimuli,such as Toll-like receptor(TLR)ligands and muramyl dipeptide,do not directly activate the NLRP3 inflammasome,but they instead prime the NLRP3 inflammasome for activation 16.Furthermore,NLRP3 activation induced by ATP,pore-forming toxins and particulate matter requires pretreatment of macrophages with microbial stimuli or cytokines 1618.Therefore,a two-signal model has been proposed for NLRP3 inflammasome activation in macrophages.In this model,the first signal(priming)is provided by microbial or endogenous molecules that induce NLRP3 and pro-IL-1 expression through activation of NF-B;the second signal(activation)is triggered by ATP,pore-forming toxins,viral RNA and particulate matter(Figure 1).Priming the NLRP3 inflammasome:Beyond the induction of NLRP3 and pro-IL-1 Macrophages,such as mouse bone marrow-derived macrophages,show no or minimal activation of the NLRP3 inflammasome when stimulated with NLRP3 activators,whereas pretreatment with microbial ligands strongly enhances NLRP3 inflammasome activation 16.This pretreatment is defined as the priming step,which provides the first signal for NLRP3 inflammasome activation.Unlike ASC and caspase-1,the protein amounts of NLRP3 in resting macrophages are thought to be insufficient for NLRP3 activation.He et al.Page 2Trends Biochem Sci.Author manuscript;available in PMC 2017 December 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptExpression of NLRP3 is induced by priming with microbial components such as TLR ligands or endogenous molecules,such as tumor necrosis factor and IL-,through the activation of NF-B 16,18.Recent studies have shown that FADD and caspase-8 also regulate the induction of NLRP3 expression during priming 1921.Consistent with this transcriptional role in the priming step,macrophages constitutively expressing high levels of NLRP3 via a retroviral vector show inflammasome activation after stimulation with NLRP3 activators in the absence of priming 16,22.Therefore,priming positively regulates the NLRP3 inflammasome through the induction of NLRP3 expression(Figure 1).In addition to this transcriptional role,emerging evidence indicates that priming also regulates NLRP3 activation at the posttranscriptional level.This regulation was first revealed by the finding that priming macrophages for a short time(10 mins),which is not sufficient for the induction of NLRP3 expression,significantly enhances inflammasome activation 22,23.Deeper understanding of the signaling mechanism of priming was provided by experiments using mouse macrophages deficient in signaling molecules of the NF-B pathway.The adaptor MyD88 and the downstream kinases IRAK1 and IRAK4 have been implicated in transcriptional regulation of NLRP3 activation by priming,whereas the adaptor TRIF and IRAK1 play a role in the posttranscriptional regulation of the priming step 16,24,25.Furthermore,NLRP3 is deubiquitinated during priming 23,26,27.BRCC3,a JAMM domain-containing Zn2+metalloprotease,promotes NLRP3 deubiquitination during priming and regulates NLRP3 activation 26.Therefore,the role of priming in NLRP3 activation is more complex than originally appreciated(Figure 1).Transcription-independent signaling of priming may further fine-tune NLRP3 inflammasome activation.Activating the NLRP3 inflammasome:Integration of multiple cellular signaling eventsGiven the chemical and structural diversity of NLRP3-activating stimuli,it is unlikely that NLRP3 physically interacts with its activators.Instead,NLRP3 is likely to sense a common cellular signal induced in response to NLRP3 activators.The identity of this signal is currently under intensive debate.Several molecular and cellular events have been proposed as the trigger(s)for NLRP3 inflammasome activation,including K+efflux,Ca2+signaling,reactive oxygen species(ROS),mitochondrial dysfunction,and lysosomal rupture(Figure 1).K+efflux:a common step for NLRP3 inflammasome activationK+efflux has emerged as a common denominator in the activation of the NLRP3 inflammasome.This was first implicated from an earlier report that potassium ionophores,such as nigericin,trigger IL-1 maturation in LPS-stimulated murine macrophages 28.The importance of K+efflux in NLRP3 inflammasome activation has been further revealed by additional studies 29,30.These studies have shown that a high concentration of extracellular K+blocks NLRP3 inflammasome activation,but not AIM2 or NLRC4 inflammasome activation 31,32.K+efflux is induced in response to most or all NLRP3 stimuli,including ATP,nigericin and particulate matter,and lowering cytosolic K+concentration is sufficient to activate the NLRP3 inflammasome 30.Such a low He et al.Page 3Trends Biochem Sci.Author manuscript;available in PMC 2017 December 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptconcentration of K+can also trigger NLRP3 inflammasome assembly in a cell-free system 31.Therefore,the drop of intracellular K+concentration has been proposed to be a common trigger for NLRP3 inflammasome activation 30.In addition,K+efflux is also required for NLRP3 activation in the non-canonical inflammasome pathway(see next section)33,34.However,the mechanistic link between decreased levels of intracellular K+and NLRP3 activation remains poorly understood.K+efflux is thought to act at or upstream of NLRP3 activation.In support of this notion,K+efflux precedes inflammasome activation 30.Furthermore,activation of the NLRP3 inflammasome in macrophages harboring a NLRP3 activating mutation(NLRP3R258W)occurs in the absence of K+efflux and is not affected by a high concentration of extracellular K+30,35.This suggests that the drop in intracellular K+concentration might induce NLRP3 to undergo conformational changes that are already induced by the presence of NLRP3 activating mutations.Ca2+signaling and NLRP3 inflammasome activationThe involvement of Ca2+signaling in NLRP3 inflammasome activation was suggested by studies showing that the Ca2+chelator BAPTA-AM inhibits IL-1 secretion 3638.Several studies have supported a general requirement for Ca2+signaling in NLRP3 activation 3941.NLRP3 stimuli including ATP,nigericin,and particulate matter induce Ca2+mobilization during the process of NLRP3 inflammasome activation 40.Importantly,inhibition of Ca2+signaling blocks NLRP3 inflammasome activation,but has no effect on the activation of the AIM2 and NLRC4 inflammasomes 39,40.As a major intracellular Ca2+reservoir,the endoplasmic reticulum(ER),appears to be critical for this process,because pharmacological inhibition or small hairpin RNA(shRNA)knockdown of inositol 1,4,5-triphosphate receptor(IP3R),a Ca2+release channel on the ER,attenuates Ca2+mobilization and NLRP3 activation 39.Activation of IP3R is triggered by IP3,a product of phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate(PIP2)cleavage.Consistently,PLC inhibition blocks NLRP3 activation induced by multiple stimuli,whereas direct activation of PLC induces IL-1 secretion in the absence of any other exogenous stimuli 39.How PLC is activated by NLRP3 stimuli remains unclear.Lee et al.reported that the Calcium-Sensing Receptor(CASR),a G protein-coupled receptor(GPCR),acts upstream of PLC to trigger Ca2+mobilization and NLRP3 activation 39.However,another study showed that CASR and a related family member GPRC6A are only required for extracellular Ca2+-induced NLRP3 activation,but not for ATP-induced NLRP3 activation 41.Notably,the addition of Ca2+to extracellular RPMI medium leads to the formation of particulate matter and K+efflux,complicating the interpretation of these experiments 30.The store-operated Ca2+entry(SOCE)channel on the plasma membrane,which is often coupled to ER Ca2+release,has been implicated in Ca2+mobilization and NLRP3 activation 40.How does Ca2+signaling regulate NLRP3 inflammasome activation?Cytosolic Ca2+may promote NLRP3 complex formation 39.Alternatively,Ca2+mobilization and mitochondrial Ca2+overload may cause mitochondrial dysfunction,which may promote NLRP3 activation(see next subsection 40.However,a recent study indicated that NLRP3 inflammasome activation is independent of Ca2+signaling 42.In this study,the authors compared the contribution of decreased cytosolic K+concentrations versus increased cytosolic Ca2+in NLRP3 activation and found that Ca2+is largely dispensable for NLRP3 activation 42.Surprisingly,this study also revealed that BAPTA blocks NLRP3 activation He et al.Page 4Trends Biochem Sci.Author manuscript;available in PMC 2017 December 01.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptindependently of its expected inhibitory effects on Ca2+signaling 42.Thus,the role of Ca2+signaling in NLRP3 activation remains controversial.Mitochondrial dysfunction and ROSThe role of ROS and the mitochondria in NLRP3 inflammasome activation is a topic of long-standing debate 43,44.Cytosolic ROS produced by NAPDH oxidase were initially proposed to be the common signal responsible for NLRP3 inflammasome activation based on studies with chemical inhibitors 45.However,human peripheral blood mononuclear cells and mouse macrophages lacking NAPDH oxidase activity show normal activation of the NLRP3 inflammasome 46,47.The implication of mitochondrial dysfunction in NLRP3 activation was originally based on chemical inhibitors in which the perturbation of mitochondria were found to promote activation of the NLRP3 inflammasome 48.However,chemical inhibitors are prone to causing artifacts,especially at high concentrations.Indeed,at least one study showed that ROS inhibitors also affect the induction of NLRP3 at the priming stage when used at high concentrations 49.Furthermore,although induced by most NLRP3 stimuli,mitochondrial perturbation and associated ROS production appear dispensable for NLRP3 activation 30.In addition,oxidized mitochondrial DNA that can be released into the cytosol has been suggested to interact with and activate NLRP3 50.However,the release of mitochondrial DNA can occur downstream of NLRP3 activation 51.Furthermore,cardiolipin,a mitochondria-specific phospholipid which is nor