REACTIVE OXYGEN SPECIES IN INFLAMMASOME ACTIVATION SHORT REVIEW OF REACTIVE OXYGEN SPECIES IN NOD-LIKE RECEPTOR P3 SIGNALING

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1 REACTIVE OXYGEN SPECIES IN INFLAMMASOME ACTIVATION SHORT REVIEW OF REACTIVE OXYGEN SPECIES IN NOD-LIKE RECEPTOR P3 SIGNALING 2009, MSc. I&I. Thesis. J.J.W.Kuiper, Under supervision of Dr. J.Frenkel, Associated Professor at General Pediatrics and Pediatric Immunology, University Medical Centre, Utrecht 1

2 INDEX Reactive Oxygen Species in Inflammasome Regulation...3 IL IL IL Pattern Recognition Receptors...6 Toll-Like Receptors...6 NOD-Like Receptors...6 NLRC4 inflammasome...7 NLRP1 inflammasome...7 NLRP1 in Ischemia...7 NLRP3 inflammasome...7 NLRP3 in human pathology...8 NLRP3 activation...8 Reactive Oxygen Species (ROS)...9 UA, ROS and NLRP NLRX1 and ROS ERK1/ APANAREI-Axis Remark

3 REACTIVE OXYGEN SPECIES IN INFLAMMASOME ACTIVATION APANAREI-axis: ATP-mediated P2X 7-receptor Activation of NAPH- Oxidase induced ROS production that activates ERK1/2, which activates caspase-1 by the NLRP3- Inflammasome; ASC Apoptosisassociated speck-like protein containing a ; ATP Adenosine Triphosphate; Caspase Recruiting Domain; CAPS Cryopyrinassociated periodic syndromes; CAT Catalase; CREB camp response element-binding protein; ERK1/2 Extracellular signal-regulated protein kinases 1 and 2; FCAS Familial cold-induced autoinflammatory syndrome; GPX Glutathione peroxidase; IPAF ICEprotease activating factor; LPS Lipopolysaccharide; Leucinerich repeats; MAPK mitogenactivated protein kinases; MDP Muramyl dipeptide; MWS Muckle- Wells syndrome; NAC N- acetylcysteine; NAIP neuronal apoptosis inhibitor protein; NF-kB Nuclear Factor kb; NLR NOD-like receptor; NLRX1 NOD-like receptor X1; NOD Nucleotide-binding and oligomerization domain; NOMID neonatal onset multisystem inflammatory disorder; PAMP Pathogen-associated molecular pattern; PAPA Pyogenic arthritis, pyoderma gangrenosum, and acne; PMN Polymorphonuclear leukocytes/ neutrophils; PRR Pattern Recognition Receptor; PYD Pyrin-domain; ROS Reactive oxygen species; SOD Superoxide dismutase; TLR Toll-like Receptors; UA Uric Acid; WT Wild-type; XO Oxidoreductase. IL-1 The fundamental role of the innate immune system is to form the first barrier in bacterial and viral defence. It is based upon the recognition of pathogenic-associated molecular patterns (PAMPs) by germ-encoded pattern recognition receptors (PRRs)(1). Recognition of PAMPs by leukocytes, monocytes or dendritic cells induce the production of pro-inflammatory cytokines that initiate biological effects associated with inflammation. The most prominent pro-inflammatory cytokines belong to the interleukin-1-like family, which includes interleukin-1α, interleuking- 1β, interleukin-18, and more recently identified interleukin-33 (2;3). Binding of IL-1 to the IL-1-receptor (IL-1R) activates its accessory protein (IL-1RAcP), inducing a wide variety of responses. These responses act locally as well as systemic and range from fever to leukocyte and lymphocyte activation and migration(4). IL-1 is a pro-inflammatory and anti-apoptotic agent that induces cytokine production by activating NF-κB and MAP-Kinase signaling(3;5). These signal-transduction pathways are known to influence; cytoskeleton reorganization, inflammatory protein and mrna synthesis, and Ca 2+ or K + -fluxes(6;7). This results in the secretion of numerous leukocyte and monocyte chemokines, vascular adhesion molecules, phospholipases and prostaglandins(8;9). Both IL-1α and IL-1β interact with the same IL-1R(10;11). However, there is strict discrimination in the distribution and post-translational processing. IL-1α is far less potent than IL-1β and constitutively secreted by epithelial skin cells and endothelium(10-12). IL-1β is primarily expressed by innate immune cells such as monocytes and dendritic cells. IL-1β mrna is present in resting Polymorph nuclear neutrophils (PMN)(13), but also tissue specific immune cells like Astrocytes(14). Pro-IL-1β is cleaved in its 17-kDa active form by caspase-1(15-17). Due to lack of secretory signal peptides(18), IL-1β is secreted via an unconventional, unidentified pathway(19). IL-1β secretion is associated with the P2X 7 -receptor and multi-vesicular bodies(20). 3

4 The significant role of IL-1β in innate defence is typically demonstrated in IL-1β -/- mice, which have severely impaired bacterial clearance(21). In contrast, high levels of IL-1β cause numerous clinical manifestations in patients suffering from rare autoinflammatory syndromes. IL-1β disorders include Familial Mediterranean Fever (FMF), Pyogenic arthritis pyoderma gangrenosum and acne(papa), and Cryopyrinopathies; like cold-induced auto-inflammatory syndrome(fcas)(22), Muckle-Wells Syndrome(MWS) and Neonatal onset multisystem inflammatory disease(nomid). Patients suffering from these disorders have circulating blood monocytes which secrete high levels of IL-1β(23). IL-1β is also associated with inflammatory diseases like gout(24) Schnitzler s syndrome (25), Adult-onset Still s disease and juvenile idiopathic arthritis (JIA)(26). Further, environmental acquired inflammatory syndromes like silicosis or asbestosis(27) and damage following ischemic insults (28-30) are associated with elevated IL-1β expression. In addition, IL-1β is linked to diabetes(31), neuroinflammatory diseases (32) and auto-immunity (e.g. Vitiligo)(33). Recently, Aksentijevich et al.(34) described that mutations in the IL-1RN-gene lead to absence of functional endogenous IL-1R antagonist and consequently hypersensitivity to IL-1β stimulation. Deficiency of Interleukin-1 Receptor Antagonist (DIRA)-patient s clinical presentation resembles that of severe cryopyrinopathy. The involvement of IL-1β in general inflammation in numerous disorders makes the cytokine a valid target for therapeutic intervention(28). Indeed, a recombinant IL-1 receptor antagonist, Anakinra, does exist and books remarkable results in therapy for auto-inflammatory disorders, stroke and arthritis(29;30). IL-18 Interleukin-18 shows structural resemblance with IL-1β(3). Macrophages, monocytes and epithelial cells produce IL-18(35). Like pro-il-1β, pro-il-18 is proteolytically activated by caspase-1(36). Pro-IL-18 can also be cleaved by metalloproteinase meprin-β subunit (MEP1B), which is present in intestinal and renal tissues(37). IL-1β can also be activated by MEP1B in vitro(38). The significance of this enzyme for IL-1β activation in vivo, however, has not yet been demonstrated. Cathelicidin and β-defensin, and several bacterial proteins induce IL-18 secretion independently of caspase-1(39;40). IL-18 secretion pathway(s) are unknown, but uncleaved IL-18 is also secreted and biologically functional(41-45). - independent IL-18 secretion was suggested to be mediated by the FAS-pathway(46). IL-18 stimulates IFN-γ, Th1 and Th2 cytokines, including IL-2, -4,-5,-10,-12,-13 and IL- 15. IL-18 is therefore linked to T-helper cell(th)1 dominant diseases like psoriasis and auto-immune disease(35;41), and Th2 dominant diseases like atopic dermatitis and general allergy(43;47). 4

5 IL-33 The latest member of the IL-1 family is Interleukin-33. IL-33 is thought to be involved in Arthritis, Alzheimer disease and allergy, and is expressed primarily by epithelial cells(48-51). Although relatively new, thus far, the cytokine has been associated with Th2-mediated immune reactions by stimulating Eosinophils, Basophils and Mastcells(52-55). CASPASE-1 Maturation of IL-1β is accomplished by caspase-1, a member of the Cysteinyl aspartate-specific proteases family (caspases). Caspases are proteases that activate or inactivate their substrate via cleavage and initiate key events in inflammation and apoptosis(56). In humans, 11 caspases have been identified up to date. The inflammatory caspases are caspase-1,4 and 5 in humans, corresponding to caspase- 1,11 and-12, in mice(57). Other caspases belong to the apoptotic caspases and are classified as initiator(caspase-2,8 and-9) or executive(3,6 and-7) caspases(31). Caspases are synthesized as in-active zymogens, or pro-caspases, which are constitutively expressed in the cytosol(58). Maturation is achieved in sophisticated multi-protein complexes, which activate the zymogens by conformational processing. These complexes are named after their associated pathways, of which the apoptosis-inducing apoptosome is the most commonly known(59). The complex that leads to the activation of caspase-1 is called the inflammasome(17;60;61). The substrates of caspase-4 and -5 are still unknown. can cleave the proforms of IL-1β, IL-18 and IL-33(27;36;62). As described, IL-18 is only partly activated by caspase-1(39), while IL-1β is primarily activated by this caspase. Inhibition of caspase-1 decreases IL-1β secretion by 53% - 81%(63). Furthermore, all known microbial, environmental and endogenous inducers of IL-1β depend on caspase-1 activation. This was demonstrated by caspase-1-, and inflammasome-knock-out models(64). In contrast to caspase-1, caspase-3 can deactivate IL-1β and IL-18(37). Interestingly, has also recently been found to activate caspase-7 after exposure to microbial PAMPs. Caspase-7 regulates apoptosis independently of IL-1β activation(65). Acute cell-death during inflammation is called Pyroptosis. Caspases consist of a large and a small catalytic subunit, plus an interacting Caspase Recruiting-Domain(). -domains are also present on other proteins that regulate caspases. For example, Receptor Interacting Protein (RIP)-2 contains a -domain(66) and regulates caspase-1 activity. Fever is a well known clinical and systemic effect of IL-1β. In vitro heat shocked (41 45 C) macrophages no longer secrete IL-1β upon pathogen exposure(67). It was therefore suggested that caspase- 1 is regulated by a negative feedback loop from fever to IL-1β. 5

6 PATTERN RECOGNITION RECEPTORS (PRRS) The PRRs are implicated in recognition of PAMPs. These innate receptors are subdivided into distinct families of which the majority belongs to the Toll-like receptors(tlrs) and Nod-like receptors(nlrs)(68;69). TOLL-LIKE RECEPTORS TLRs are membrane-associated receptors activated by a great variety of PAMPs derived from bacteria, viruses, fungi and parasites. The extracellular binding domain consists of leucine-rich repeats(). Upon activation, the cytosolic receptor domain activates its Toll/IL-1 receptor interaction (TIR)-domain, which forms a complex with either myeloid differentiation primary response protein 88(MyD88)- or TIR domaincontaining adaptor inducing IFN-β (TRIF)-adaptor domain. Interaction with MyD88 activates NF-κB pathways, while TRIF pathways initiate anti-viral pathways. Lipopolysaccharide (LPS), the major component of the Gram-negative bacteria outer membrane, specifically binds and stimulates the TLR-4(70). Stimulation of macrophages with LPS induces production of pro-inflammatory cytokines like TNF-α and IL-1β(23). Other studies claim that LPS is not capable of inducing IL-1β secretion on its own(23). Nonetheless, IL-1β secretion is more efficient in LPS-primed macrophages(71;72). The end derivate of purine metabolism, uric acid (UA), stimulates pro-il-1β production(73). Increase in pro-il-1β is also seen after exposure to vaccine adjuvants(74). Like pro-il-1β, pro-il-18 is also induced by TLRs(75). The secretion of IL-1β by macrophages in MyD88 -/- mice is not different from WT mice after pathogen or UA stimulation (17;64). The knock-out model of this adaptor protein separates IL-1β production from IL-1β secretion. Indeed, the activation of pro-il1β by caspase-1 is TLR-independent(76). Summarized, TLRs are necessary in pro-il-1β production, but are not involved in IL- 1β secretion. NOD-LIKE RECEPTORS Intracellular PAMP recognition is accomplished by cytosolic nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs).The structural organization is phylogenetically conserved, considering the resemblance with plant diseaseresistance genes(77). NLRs consists out of an N-terminal recruitment domain for downstream signaling, a central self-activating -domain and a C-terminal PAMP-recognition-domain. The N-terminal contains a -domain, Baculovirus inhibition repeat (BIR)-domain, or Pyrin (PYD)-domain. The C-terminus consists of s which function in ligand recognition(78). NLRs are associated with caspase activation and play key roles in host defence, apoptosis, but also developmental and reproductive biology(31). The NLRs family contains 23 members in humans and is subdivided into 2 NODs, 14 NLRPs, NLRC4 (or ICE-protease-activating factor, IPAF), Neural Apoptosis inhibitor protein(naip), and recently discovered NLRX1(61;77;79-81). NOD1 specifically recognizes peptidoglycans, whereas NOD2 recognizes muramyl dipeptide(mdp), both bacterial compounds. NOD2 can activate caspase-1 via NLRP1(82-84). NLRP1, NLRP3, NLRC4 and NAIPs are involved in inflammatory caspase maturation by forming inflammasomes(17;31). They recruit different proteins to the multi-protein 6

7 platform for conformational activation of pro-caspase-1. The inflammasome is TLRindependently activated(76). NLRP-inflammasomes differ in composition from NLRC4- and NAIPs- inflammasomes in their dependency on the adaptor protein apoptosis-associated speck-like protein containing a (ASC). This adaptor protein forms the backbone of the NLRP1- and NLRP3-inflammasome. ASC -/- mice resemble the IL-1 deficient phenotype in mice(21). In addition, in vitro IL-1β production by macrophages is ASC dependent (10;11;71). NLRC4 INFLAMMASOME NLRC4 (IPAF) directly recruits pro-caspase-1 to the NLRC4-inflammasome (figure 1). Although ASC is was not suggested to form the NLRC4-inflammasome, ASC is involved. NAIPs were suggested to associate with NLRC4 in the inflammasome(85). NLRC4 activation is involved in specific and acute forms of bacterial induced Pyroptosis. Furthermore, NLRC4 is specifically activated by several bacteria that use type III/IV secretion systems and forms the cytoplasmatic counterpart of TLR5 in flagellin recognition from Salmonella typhimurium and Legionella pneumophila (83;86-88). NLRP1 INFLAMMASOME NLRP1 was the first NLRP discovered by genetic analysis of chromosome 11 after observed differences in susceptibility to systemic anthrax infections in mice(89). The gram-positive bacillus anthracis produces Lethal Toxin (LT), which induces caspase-1 activation via NLRP1b (murine homolog of human NLRP1)(90). When NLRP1 binds ligand it recruits ASC by its PYD-domain. ASC contains a domain and recruits pro-caspase-1(figure 1). The C-terminus of NLRP1 contains an additional -domain by which is was thought to recruit caspase-5, although this domain may also recruit caspase-1 directly and independently of ASC.(31;78;91). Murine NLRP1b does not contain a PYD-domain(92), therefore, the exact formation of the NLRP1b- and NLRP1-inflammasomes is still under intense investigation. NLRP1 IN ISCHEMIA When blood circulation is blocked, tissue will get damaged by an acute lack of oxygen, accumulation of CO 2 and nutritional starvation, collectively called Ischemia(9;28). During an ischemic insult, K +, Ca 2+ and ATP concentrations alter which activates the NLRP1 inflammasome(28;30). After ischemic stroke, NLRP1 inflammasome components become present in neuronal tissues, Astrocytes and Microglia. Recently, Anakinra is being used in first phase clinical trials as therapy for ischemic or hemorrhagic stroke, which has improved clinical prognosis(30;93). NLRP3 INFLAMMASOME The most studied NLRP is NLRP3, which is alternatively called NALP3, CIAS-1 or Cryopyrin(94). NLRP3 recruits ASC and an additional adaptor protein called INAL, which resemble the C-terminus of NLRP1(95). This forms the NLRP3- inflammasome(figure1). NLPR3 -/- mice show impaired leukocyte and lymphocyte migration and decreased IL-1β production(21;27;64). 7

8 PAMPs DAMPs IL-1β INAL IL-1β pro- A ASC NLRP3 PYD PYD PYD INAL NLRP3 PYD PYD pro-il-1β INAL IL-1β B PYD NLRP1 PYD PYD NLRP1 PYD PYD pro-iil-1β NLRC4 pro-il-1β IL-1β C NLRC4 Figure 1. Cytosolic NOD-Like receptors form unique multi-protein complexes or inflammasomes. When PAMPs or DAMPs bind a membrane receptor, are endocytosed, or directly penetrate the membrane and enter the cytosol, the NLRs will be activated upon recogniztion. A) NLRP3 contains a Leucinrich repeat ()-domain for recognition of PAMPs and DAMPs, a self-oligmerization and regulating ()-domain, and a Pyrin-domain(PYD), which functions in recruitment of accessory proteins and regulation of the inflammasome formation. Upon activation, INAL is recruited to the NLPR. I- NAL contains a -domain. ASC is recruited by its PYD domain and also contains a -domain. The formed NLRP3-inflammasome recruits pro-caspase-1 with its free -domains. Activated caspase-1 cleaves pro-il-1β into its active form. Matured IL-1β is secreted extracellularly and functions proinflammatory. B) NLRP1, like NLRP3, consists out of a PYD-, - and -domain. Additional to NLRP3, it contains a -domain, by which it can directly recruit pro-caspase-1. The PYD-domain also recruits ASC, by which another pro-caspase-1 is recruited. The NLRP1-inflammasome activates caspase-1 and the following IL-1β cleavage. C) NLRC4, like NLRP1, contains -domains and forms the NLRC4-Iinflammasome by oligormerization with multiple NLRC4s (only two shown), and activate resulting in IL-1β maturation.

9 PYRIN Another protein associated with the NLRP3 inflammasome is Pyrin. Patients suffering from Familial Mediterranean Fever (FMF) have mutations in the MEFV (from MEditerranean FeVer) gene, that encodes for Pyrin(96). FMF is an autoinflammatory syndrome, causing episodic fever and systemic inflammation(97). Pyrin has a PYD-domain by which it binds to ASC and regulates the NLRP3- inflammasome. This precise role of this protein is unknown, since it was reported to both enhance(98) and inhibit the inflammasome(99). NLRP3 IN HUMAN PATHOLOGY NLRP3 associated disorders are collectively called Cryopyrinopathies or Cryopyrin- Associated Periodic Syndromes (CAPS). Gain-of-function mutations in NLRP3 lead to constitutive activation of the inflammasome(100) and high IL-1β production by blood monocytes(23). These rare auto-inflammatory disorders are characterized by recurrent periodic systemic inflammation with fever, urticarial rash, arthropathy, severe neurological dysfunctions and inflammatory deafness(101). Autoinflammatory syndromes differ from auto-immune disease in their lack of autoreactive T-cells or auto-antibodies. FCAS, MWS, NOMID (also called Chronic Infantile onset Neurological Cutaneous Articular, CINCA) are all characterized by NLRP3 mutations. These syndromes have overlapping clinical presentations, with an increase in severity from FCAS, MWS to NOMID, respectively. Furthermore, NLRP3 is linked to Crohn s disease(102), Alzheimer s disease(103), (Pseudo)gout(72), Asbestosis and Silicosis (104). NLRP3 ACTIVATION The activation of the NLP3 inflammasome is still subject of intense debate. Beside Silica, Asbestos, bacterial nigericin, maitotoxin and MDP, the NLRP3-inflammasome recognizes endogenous danger signals (DAMPs) like uric acid, K + and ATP(71;105). ATP AND LPS Extracellular ATP is an inflammatory mediator and can bind the purinergic receptor, P2X 7 (106). P2X 7 -receptor is present on immune cells like macrophages and epithelial cells(106;107). Binding of ATP to P2X 7 -receptor activates signaling pathways leading to ion-fluxes and activation of the NLPR3-inflammasome( ). Priming of macrophages with LPS increases ATP-mediated inflammasome (caspase- 1) activation(112). LPS binds TLR4 and induces pro-il-1β production, while ATP mediates secretion of matured IL-1β. This states that two distinct signals initiate a IL- 1β response by immune cells. In contrast to macrophages, monocytes produce endogenous ATP(113). Netea et al.(23) demonstrated that monocytes only need TLR stimulation for IL-1β secretion, because they have constitutively activated NLRP3. Macrophages need extracellular ATP as a second signal for IL-1β secretion. The difference in regulation of IL-1β in these immune cells, could reflect the differential function of these cells in their natural micro-environment(23). 8

10 POTASSIUM(K + ) K + -fluxes are altered after activation of the P2X 7 -receptor(114), which is associated with NLRP3 activation(figure2)(114;115). In vitro, cytosolic K + -depletion leads to activation of caspase-1, IL-1β and IL-18. This could be an indication that the effect of ATP is basically an induction of K + -efflux, instead of direct binding of ligand to NLRP3(64). Indeed, most NLRP3 activators including monosodium urate crystals (MSU), asbestos fibres, and vaccine adjuvants co-initiate a K + -efflux (74; ). Interestingly, there have been reports on differential activation of chemically related substances. MSU and UA are DAMPs(119) that activate NLRP3. MSU induces K + - efflux, while UA crystals do not and actually have to be endocytosed to activate NLRP3(71;115). This could explain contradicting reports about TLR-signaling in NLRP3 activation via UA(73;120). Recall that NLRs are cytosolic PRRs. Bacterial and viral compounds like DNA and RNA that activate NLRP3 are recognized in the cytosol. Thereby, bypassing the P2X 7 - receptor (and therefore membrane)-mediated K + -efflux. Still, the commonly shared intracellular depletion of K + is an indication that it takes part upstream the cascade of NLRP3 activation. Alternatively, redundant pathways could co-exist for some specific PAMPs or DAMPs. Other membrane associated receptors like CXCR16 (63) and CD36 (13) have been linked to IL-1β regulation. Inhalation of pro-fibrotic agents like asbestos or silica damage lung and pleura and can develop chronic respiratory pathologies(121;122). Exposure to asbestos or silica activates the NLRP3-inflammasome(72), however the mechanism of NLR activation differ. Activation by asbestos fibres does not require endocytosis and can be blocked by inhibition of the K + -efflux(64). In contrast, silica needs to be endocytosed for activation of the NLRP3-inflammasome(27). Also vaccine adjuvant micro-particles are dependent on endocytosis for NLRP3 activation. Vaccine adjuvants may induce lysosomal damage, where after the inflammasome is activated(74). REACTIVE OXYGEN SPECIES (ROS) Interestingly, macrophages that have been exposed to extracellular ATP produce high levels of ROS, which play a role in inflammatory signaling pathways(58) Oxygen is one of the essential elements in life. Due to its low threshold for biochemical reactions (e.g. oxidation of iron) it is involved in many biological processes like energy metabolism, cellular defence, but also immune-pathology and degradation (aging) of tissue. Highly reactive forms of oxygen (i.e. its derivatives) are called reactive oxygen species (ROS) and include hydrogen peroxide (H 2 O 2 ), superoxide anion (O * 2 ) and hydroxyl radical (OH * )(123). The main source of ROS production is the electron transport chain within the mitochondria. In addition, some enzymes primarily produce ROS (NADPH-Oxidase(124;125), Xanthine oxidoreductase(xo), Cycloxygenases and Lipoxygenases)(123;126). ROS are abundantly produced by PMNs and Monocytes(127) when exposed to PAMPs(128) and pro-inflammatory cytokines(129). 9

11 Tight regulation of ROS by anti-oxidant systems is accomplished by several enzymes including N-acetyl cysteine(nac)(126), Superoxide dismutases(sods), Glutathione peroxidase(gpx) and Catalase(CAT). The latter three form a enzymatic reaction chain which catalyses the transition of H 2 O 2 into neutral H 2 O and O 2. Activation of caspase-1 and IL-1β in stressed mice is ROS dependent(130;131) In return, IL-1β stimulates intracellular ROS accumulation(132) by uncoupling SOD, GPX and CAT. This inhibits the dismutasis of the H 2 O 2 (133). ROS AND CASPASE-1 Inhibition of caspase-1 decreases ROS production(134), while IL-1β stimulates ROS production. This may suggest a positive loop from IL-1β to caspase-1 via ROS. However, IL-1β initiates fever and this results in cellular heat shock. Heat shock changes the redox-state of the cell and thereby inhibits caspase-1 and inflammasome activities(67). ROS have been linked to caspase-1 regulation in apoptosis, before the discovery of the inflammasome(135). The origin of this ROS was assumed to be mitochondrial, since no NADPH-Oxidase was involved. This could link ROS to caspase-1 (via caspase-7) induced Pyroptosis. NLRP3-inflammasome activators like silica, asbestos and ATP all require ROS production for their activity(27;58;136). Dostert et al.(64) demonstrated that the iron chelation (by deferoxamine) of asbestos fibres results in a loss of IL-1β maturation. This indicates that NLRP3 activation by asbestos is primarily dependent on ROS. ROS production, upon silica exposure, has been demonstrated in WT macrophages(137), as well as in NLRP3 -/- macrophages. Therefore, ROS are upstream of NLRP3. UA, ROS AND NLRP3 Most mammals, but men, still have functional Uricase that degrades UA. Therefore, humans are sensitive to an increase in serum levels of UA(138). UA triggers NF-κB and MAP-Kinase activation(139). In Gout, UA is massively deposited in joints, causing inflammation through NLRP3(72). Processing of UA from Hypoxanthine by XO is accompanied by ROS production(126;140;140). The inflammatory capacity of UA is inhibited by NAC-antioxidant(126). This confirms that ROS production is prerequisite for NLRP3 inflammasome(27). NLRX1 AND ROS NLRX1, the newest member of the NLR family, seems to primarily function in ROS production. NLRX1 differs structurally from the other NLRs, but shows the most homology with the NOD-subfamily(80). While the other NLRs are localized within the cytosol, NLRX1 is specifically associated with the mitochondrial membrane. NLRX1 is necessary for ROS production upon inflammatory signals like TNF-α. ASC has been associated with the mitochondrial membrane(141), but the existence of NLRX1- inflammasomes has not yet been reported. Future research must determine the importance of NLRX1 in caspase-1 activation and IL-1β signaling. 10

12 ERK1/2 Oxidative stress can activate each of the three super families of membrane receptors; Receptor tyrosine kinases, G-protein-coupled receptors, or epidermal growth factor receptors. These receptors activate downstream effectors in intracellular signaling, most importantly the mitogen-activated protein kinases (MAPK)(142). Raf MAPK Kinase Kinase activates MEK1/2 MAPK kinase, which in turn activates Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). There are numerous excellent reviews about ERK1/2 in multiple signaling pathways and associated diseases, however little is known about ERK1/2 in IL-1β signaling and ROS activation(143;144). Few studies show that exposure to ROS activates ERK1/2 and associated MAPK p38(133;145). Interestingly, ERK1/2 induces IL-1β secretion, while IL-1β binding to IL-1R activates ERK1/2. The biological effects of IL-1β on cell proliferation are therefore similar to ERK1/2 (14). ERK1/2 is involved in directing the cell towards a pro- or anti-apoptotic state during inflammation by regulating expression of pro-inflammatory cytokines like TNF-α, IL- 1β and IL-18 (13;14;39;146). Ikeda et al(13). demonstrated that when ROS production decreases, ERK1/2 activation, as well as IL-1β production decreases. ROS-ERK1/2 -dependant activation of IL-1β is also reported in bacteria(128), LPS (147), silica(148) and Dextran Sulfate Sodium (causes Crohn s disease in mice)(63). Considering its redox-sensitivity(149), caspase-1 may also be directly activated by ROS instead of via ERK1/2. Interestingly, ERK1/2 can also activate Caspase-3, which inactivates IL-1β(150). Other studies claim that the supposedly ERK1/2 activation by ROS is actually mediated by p53, as a consequence of the damaging effect of ROS on DNA(151). The exact interactions between ERK1/2 and have not yet been determined (152). APANAREI-AXIS I suggest a model for NLRP3 activation in macrophages which includes the P2X 7 - receptor, NADPH-oxidase, ROS, ERK1/2, NLRP3 and caspase-1 (figure 2). In this model I hypothesize, ATP binds P2X 7, which up regulates NADPH-oxidase-, PI3Kinase-activity, Ca 2+ fluxes, and MAP-Kinases including ERK1/2. Consequently, caspase-1 is activated in the inflammasome and IL-1β is cleaved(58; ). We will call this IL-1β secretion axis the APANAREI-axis. APANAREI stands for; ATPmediated P2X 7 -receptor Activation of NAPH-Oxidase induced ROS production, which activates ERK1/2 and thereby caspase-1 in the NLRP3-Inflammasome. When the APANAREI-axis (from Apañarei, future tense of the Spanish verb apañar, translated as to boost ) is activated, it boosts the innate inflammatory response. 11

13 A ATP P2X7 -receptor H NADPH-oxidase B + K C + K + + K K O2 O2* G NLRP3 Inflammasome IL-1β ERK1/2? ASC Cardinal D NLRP3 F E Figure 2. ATP-P2x7-receptor Activation mediated NADPH-oxidase induced ROS production, which activates ERK1/2 and consequently the NLRP3-Inflammasome (APANAREI)-axis. (A) Micro-environmental stress is accompanied by increase of extracellular ATP that binds the purenergic P2x7-receptor. Activation of the receptor leads to conformational changes in the receptor-coupled cation-channel and opens a pore. (B) Cellular ion-fluxes are altered upon P2x7-receptor activation. The consequently depletion of intracellular pottasium is linked to NLRP3 activation, although the subsequent mechanism remains unknown. (C) Initiation of several signaling pathways i.e. target the membrane associated NADPH-Oxidase. Several phox-subunits cluster and function in mutase of oxygen, leading to production of reactive oxygen species. (D) Intracellular reactive oxygen species function in NLRP3 signaling by activating MAP-kinases including the extracellular signal regulated kinases(erk1/2). E) Inactive cytosolic NLRP3 is somehow activated by ERK1/2, following recruitment of adaptor proteins (F). Activated NLRP3 oligomerzises to form the NLRP3-inflammasome(G). H) Cleaved IL-1β is secreted into the extracellular environment to initiate inflammation.

14 REMARK The suggestion of NLRP3 activation by ROS via ERK1/2 in the APANAREI-axis is still based on observational studies, claiming co-expression of NLRP3 inflammasome, components of the APANAREI-axis, and ROS. However, the exact mechanisms of signaling downstream of ERK1/2 to caspase-1 are still unknown. Therefore, the exact role of ROS signaling in inflammasome activation needs to be explored in future research. ~ 12

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