The inflammasome as a target for pain therapy

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1 British Journal of Anaesthesia, 117 (6): (2016) doi: /bja/aew376 Review Article REVIEW ARTICLE The inflammasome as a target for pain therapy H. Zhang 1,,F.Li 1,, W.-W. Li 2, C. Stary 2, J. D. Clark 2,S.Xu 1, * and X. Xiong 3, * 1 Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, PR China, 2 Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, Stanford, CA, USA and 3 Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, Hubei, PR China Co-first author. *Corresponding author. shiyuanxu355@163.com, xiaoxingxiong@whu.edu.cn Abstract The interleukin-1 family of cytokines are potent inducers of inflammation and pain. Proteolytic activation of this family of cytokines is under the control of several innate immune receptors that coordinate to form large multiprotein signalling platforms, termed inflammasomes. Recent evidence suggests that a wide range of inflammatory diseases, cancers, and metabolic and autoimmune disorders, in which pain is a common complaint, may be coordinated by inflammasomes. Activation of inflammasomes results in cleavage of caspase-1, which subsequently induces downstream initiation of several potent pro-inflammatory cascades. Therefore, it has been proposed that targeting inflammasome activity may be a novel and effective therapeutic strategy for these pain-related diseases. The purpose of this narrative review article is to provide the reader with an overview of the activation and regulation of inflammasomes and to investigate the potential therapeutic role of inflammasome inhibition in the treatment of diseases characterized by pain, including the following: complex regional pain syndrome, gout, rheumatoid arthritis, inflammatory pain, neuropathic pain, chronic prostatitis, chronic pelvic pain syndrome, and fibromyalgia. We conclude that the role of the inflammasome in pain-associated diseases is likely to be inflammasome subtype and disease specific. The currently available evidence suggests that disease-specific targeting of the assembly and activity of the inflammasome complex may be a novel therapeutic opportunity for the treatment of refractory pain in many settings. Key words: caspase 1; inflammasomes; inflammation; interleukins; NOD-like receptor; pain The interleukin-1 (IL-1) family of cytokines is composed of 11 isoforms, all of which share the characteristic of their ability to induce inflammation. The principal species, IL-1b, possesses strong pro-inflammatory effects on a variety of cells, and its aberrant production contributes to acute and chronic inflammation and pain. 1 2 Notably, increased IL-1 activity is associated with autoimmune disorders, such as rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis (MS), and Alzheimer s disease (AD), 1 4 which are all conditions in which pain is a common complaint. The pain-inducing mechanisms of IL-1b include the direct activation of neuronal activity in the peripheral and central nervous system, and IL-1b acts as a mediator that stimulates the production of additional algogenic substances Blocking the IL-1b signalling pathways has proved to be highly effective in reducing inflammatory pain sensitization in animal models and patients. However, the mechanisms underlying the aberrant upregulation of IL-1b were largely unknown before the recent discovery of the inflammasome by Martinon and colleagues. 13 Inflammasomes are multiprotein complexes that coordinate to activate caspase-1, an IL-1-converting enzyme Cleavage of the inactive IL-1b and IL-18 precursors (pro-il-1b and pro-il- 18) with caspase-1 is a prerequisite for activation. Like IL-1b, caspase-1 is synthesized as an inactive 45 kda enzyme, VC The Author Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please journals.permissions@oup.com 693

2 694 Zhang et al. pro-caspase-1, which also requires inflammasome activity for cleavage. Given that IL-1b plays a well-established role in inflammation and pain and that inflammasomes are able to sense a wide range of danger signals, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to control IL-1 production, 17 inflammasomes may play a central role in supporting pain in a diverse range of conditions. Predictably, this discovery has promoted research investigating the potential targeting of inflammasomes both to control inflammation and to reduce the associated pain. This type of approach may be particularly important in diseases where inflammasome dysfunction has been shown to play a central role In this review, we present a narrative overview of the current understanding of inflammasome activation and regulation. We then address the question of whether inflammasomes may be a potential target for pain therapy in pain-associated disease by reviewing current literature on the role of inflammasomes in various pain-related disease states. This was accomplished by an electronic database search of PubMed and Google Scholar with several key terms. These terms included the following: inflammasome, inflammation, NLRP, NALP, cytokines, IL-1, IL-18, immune, purinergic receptors, immunoglobulin, pain, hyperalgesia, inflammatory pain, neuropathic pain, complex regional pain syndrome, gout, migraine, chronic prostatitis, chronic pelvic pain syndrome, fibromyalgia, autoimmune disorder, arthritis, multiple sclerosis, Alzheimer s disease, cancer, pathogen associated molecular patterns, damage associated molecular patterns, and toll like receptors. In addition, articles relevant to our discussion were retrieved from the reference list of other online articles on each subtopic. Articles in a language other than English and conference abstracts were excluded from further consideration. In the end, 150 articles closely related to the aims set forth for this review were selected and used. The following questions were addressed in constructing the review. What is our current understanding of inflammasome activation and regulation? What roles does the inflammasome play in pain-associated diseases? Are inflammasomes potential targets for pain-associated diseases? Inflammasome composition and subtypes A summary of inflammasome structure and activation is presented in Fig. 1 and Table 1.//// The multiprotein inflammasome complex contains a nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), an adaptor protein apoptosisassociated speck-like protein (ASC, which contains a caspaseactivating recruitment domain), and pro-caspase-1, pro-caspase 5, or both. NLRs contain the following three domains: a nucleotide-binding domain (NBD), which mediates adenosine triphosphate (ATP)-dependent self-oligomerization; 38 a NALP1/NLRP1 Inflammasome NALP3/NLRP3 Inflammasome NLRC4/IPAF Inflammasome AIM2 Inflammasome P10 P20 CARD CARD PYD PYD NACHT CARD FIIND LRRs P10 P20 CARD P10 P20 CARD CARD PYD PYD NACHT CARD LRRs P10 P20 CARD CARD NACHT LRRs P10 P20 CARD CARD PYD PYD HIN200 ASC PYD CARD CARDINAL FIIND CARD Pro-Caspase CARD P20 P10 Fig 1 Schematic diagram illustrating the composition of inflammasomes. The activation and formation of inflammasome complexes is mediated through several protein domains. NLRs are characterized by the combined presence of a NACHT domain and LRRs. Most NLRs also contain either a CARD or a PYD motif in their amino terminus. AIM2 is composed of an amino-terminal PYD and a carboxy-terminal DNA-binding HIN200 domain. Murine NALP1 lacks the amino-terminal PYD motif found in human NALP1 and is autocatalytically cleaved in its central FII domain. CARD, caspase activation and recruitment domain (orange rectangles); Caspase domain, p20 and p10 as the large and small subunits, respectively (pink and red rounded rectangles); FIIND, function-to-find domain (blue rectangles), which is involved in NALP1 inflammasome activation through auto-proteolysis; HIN: haematopoietic, interferon-inducible, nuclear localization domain (light blue rounded rectangles); LRRs, leucine-rich repeats (white elongated ovals); NACHT, nucleotide-binding and oligomerization domain (light blue and grey rounded rectangles); NLR, NOD-like receptor; PYD, pyrin domain (green rectangles). NLRP1/NALP1, leucine-rich repeat and pyrin domain containing protein 1; NLRP3/ NALP3, leucine-rich repeat and pyrin domain containing protein 3; IPAF/NLRC4, NLR family CARD domain-containing protein 4; ATP, adenosine triphosphate; AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein; CARDINAL, the card containing protein; dsdna, double-stranded DNA.

3 The inflammasome and pain 695 Table 1 Known types of inflammasomes and their activators. NLRP1/NALP1, leucine-rich repeat and pyrin domain containing protein 1; NLRP3/NALP3, leucine-rich repeat and pyrin domain containing protein 3; IPAF/NLRC4, NLR family CARD domain-containing protein 4; ATP, adenosine triphosphate; AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein; CARDINAL, the card containing protein; dsdna, double-stranded DNA Inflammasome Components Activators References NLRP1/NALP1 NLRP1, ASC, caspase-1, caspase-5 Lethal toxin, muramyl dipeptide NLRP3/NALP3 NLRP3, ASC, CARDINAL, caspase-1 Muramyl dipeptide, ATP, b-amyloid, uric acid crystal, alum, low K þ etc. IPAF/NLRC4 NLRC4, caspase-1 Flagellin, T3SS, T4SS AIM2 AIM2, ASC, caspase-1 dsdna C-terminal leucine-rich repeat (LRR) serving as a recognition domain for other receptors (such as Toll-like receptors, TLRs) or microbial ligands; and a variable N-terminal interaction domain responsible for homotypic protein protein interactions. This N- terminal domain can consist of a caspase recruitment domain (CARD), a pyrin domain (PYD), acidic transactivating domains (ADs), or baculovirus inhibitor repeats (BIRs) There seems to be very limited information on how the structure of an inflammasome is related to its activity. The molecular mechanism of inflammasome assembly was only recently elucidated and specifically requires PYD oligomerization. 40 We now know that inflammasome oligomerization requires the following two signals: a priming signal that results in the transcription of pro- IL-1b and pro-il-18; and a second signal that promotes indirect activation of the inflammasome, such as ion or membrane perturbations, reactive oxygen species, or extracellular ATP. 41 Inflammasome oligomerization leads to the cleavage and activation of caspase-1, which subsequently promotes the processing and secretion of IL-1b and IL-18. To date, at least three major inflammasomes have been described, and they are defined by the NLR protein they contain: the NLRP1/NALP1 (leucine-rich repeat and pyrin domain-containing protein 1) inflammasome; the NLRP3/NALP3 inflammasome (leucine-rich repeat and pyrin domain-containing protein 3); and the IPAF/NLRC4 inflammasome (NLR family CARD domain-containing protein 4). These three NLR inflammasomes have two common features: an NBD for self-oligomerization; 38 and a C-terminus LRR for ligand recognition of other receptors (e.g. TLR) or microbial ligands. Additional inflammasome subtypes continue to be identified, including most recently the AIM2 (absent in melanoma 2)-containing subtype and an NALP subtype. The NALP1 inflammasome, the first molecular platform identified, is relatively widely expressed in cells and tissues of the immune system and in non-haematopoeitic tissues. 44 NALP1 directly binds to ASC via its PYD and to caspase-1/ caspase-5 via its CARD domain. A two-step process of activation was proposed, involving mediation by microbial ligands/bacterial toxins, followed by binding of a ribonucleoside triphosphate (rntp) to the nucleotide-binding domain of NALP1. The NALP3 inflammasome, the most studied inflammasome, contains NALP3, the CARD-containing protein CARDINAL (part of the NLRP3 inflammasome complex in humans), ASC, and caspase The NALP3 inflammasome can be activated by diverse ligands and stimuli. This can include exogenous microbial components (RNA and muramyl dipeptide) or endogenous activating signals, such as cholesterol, monosodium urate (MSU) crystals, 28 ATP, 29 low intracellular K þ, 30 aluminum hydroxide (alum), 31 deposition of amyloid polypeptide, 32 and high extracellular glucose. 33 The NALP3 inflammasome isoform shows a restricted tissue distribution, with expression mainly in non-keratinizing epithelia in the oropharynx, oesophagus, ectocervix, in the urothelial layer in the bladder, 44 in myeloid and endothelial cells, and in B cells. 45 The NLRC4 inflammasome, the only member of the NLRC family, contains an amino-terminal CARD, a central NBD domain, and C-terminal LRRs NLRC4 associates with procaspase-1 with its CARD domain, without the need for an adaptor protein. However, interaction with ASC is required for IL-1b secretion. Oligomerization of NLRC4 is triggered by cytosolic flagellin from a variety of bacteria NLRC4 is expressed predominantly in haematopoietic tissues AIM2 is perhaps the best known non-nlr capable of forming an inflammasome In contrast to the NLRs, this pyrin and HIN domain (PYHIN)-family protein binds directly to its ligands, cytosolic double-stranded DNA (dsdna), and associates with ASC through their PYD to form a caspase-1-activating inflammasome, leading to IL-1b secretion AIM2 has been found to be expressed in the cytosol in skin and neural tissues. 46 Inflammasomes and pain-associated diseases Inflammatory pain states Inflammatory hyperalgesia is a complex process that depends on the sensitization of nociceptive neurons, triggered by proinflammatory mediators such as IL-1b, resulting in a heightened sensitivity to pain Using a carrageenan-induced acute inflammatory pain model, Cunha and colleagues 48 demonstrated that the peripheral activation of caspase-1 was implicated in the induction of acute inflammatory pain by promoting the maturation of IL-1b. This finding was confirmed and extended by Lopes and colleagues. 49 In their experiments, wildtype, NALP3 double knockout (NALP3 / ), NLRC4 /, and ASC / mice were subjected to plantar injection of carrageenan or vehicle. Mechanical and thermal nociceptive changes were measured before and 1, 3, and 5 h after injection, and ratios of pre-caspase-1/caspase-1 and pre-il-1b/il-1b in the plantar skin were determined 5 h after the injection. The results indicated that mice deficient in NLRC4 or ASC, but not in NALP3, presented reduced mechanical and thermal acute inflammatory hyperalgesia induced by carrageenan. 49 The reduced hyperalgesia was accompanied by significant reductions in the relative ratios of mature to pro-il-1b (p17) and pro-caspase-1 (p20) at the inflammatory site compared with wild-type mice. The authors concluded that the assembly of these components was responsible for the carrageenan-induced acute inflammatory pain state. 49 However, it remains unknown how carrageenan

4 696 Zhang et al. Table 2 Summary of studies investigating inflammasomes in pain Pain condition studied Animal model/patient Inflammasome targeted Activator Inhibitor Evidence/proposed function of inflammasome in pain Inflammatory pain state Inflammatory pain state Carrageenan-induced inflammatory pain model. Wild-type, NLRC4 /, ASC /, and NALP3 / mice Acute intracranial inflammatory pain model (rat) Neuropathic pain Spared nerve injury neuropathic pain model. Wild-type, NALP3 / mice Neuropathic pain Sciatic nerve chronic constriction injury-induced neuropathic pain model (rat) Neuropathic pain Spinal nerve ligation model (rat) Complex regional pain syndrome Distal tibia fracture with cast immobilization (rat) NLRC4 excluded NALP3 Carrageenan NLRC4- or ASC- but not NALP3-deficient mice exhibited reduced mechanical and thermal hyperalgesia induced by carrageenan NALP3 Low K þ Inflammatory soup induced nuclear cytoplasmic shuttling of NALP3, activated caspase-1, and mature IL-1b in small- to medium-sized C-type neurons in the trigeminal ganglion, and pain behaviour; treatment with the caspase-1 inhibitor Ac-YVAD-CMK diminished these effects Excluded NALP3 SNI-elicited pain behaviours do not require NALP3 expression; intrathecal lipopolysaccharide injection increases apoptosis-associated speck-like protein, caspase-1, and IL-1b NALP1 Aspirin-triggered-15-epilipoxin-A4 (ATL) Only know caspase-1 expression in both wild-type and NALP3 / mice Chronic constriction injury activated NALP1 inflammasome in spinal astrocytes and neurons, leading to an increased production of IL- 1b and neuropathic pain-like behaviour. Repeated intrathecal injection with ATL markedly attenuated the chronic constriction injury-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1b maturation Pannexin-1 (panx1) Nerve injury increased panx1 expression in dorsal root ganglia, and panx1 knockdown reduces caspase-1 release and hyperalgesia NALP1 Substance P Substance P released from peripheral nerve terminal innervated in the fractured hindlimb, which in turn, via NK1 receptors in keratinocytes, stimulated the production of NALP1 inflammasome components, generation of IL- 1b and IL-18, and nociceptive sensitization; LY303870, an NK1 receptor antagonist, blocked the upregulation of activated NALP1 inflammasome components, the production of cytokines, and nociception sensitization in the rat complex regional pain syndrome model References Lopes and colleagues 49 Chen and colleagues 50 Curto-Reyes and colleagues 65 Li and colleagues 61 Zhang and colleagues 87 Li and colleagues 99 (continued)

5 The inflammasome and pain 697 Table 2. Continued Pain condition studied Animal model/patient Inflammasome targeted Activator Inhibitor Evidence/proposed function of inflammasome in pain Complex regional pain syndrome Distal tibia fracture with cast immobilization (mouse) Gout Crystal-induced murine peritoneal model of acute gout Wild-type, caspase-1 /, MyD88 /, ASC / NALP3 / mice Human primary monocyte and THP1 cells Gout Monosodium urate-induced murine peritoneal model of acute gout Gout Subcutaneous air-pouch model of gout (mice) NALP3 / mice and murine bone marrow macrophages Rheumatoid arthritis Spontaneous arthritis model (A20 myel-ko mice) NALP3 / mice Rheumatoid arthritis Rheumatoid arthritis patients Adjuvant Arthritis Adjuvant arthritis model (rats) NALP1 Substance P and calcitonin generelated peptide Both calcitonin gene-related peptide and substance P upregulate NALP1 inflammasome and IL-1b production in a time- and dosedependent manner in vitro and in vivo NALP3 Monosodium urate Monosodium urate engages NALP3 inflammasome activation, resulting in the production of active IL-1b and IL-18. Macrophages from mice deficient in various components of the inflammasome, such as casepase-1 NALP3 Monosodium urate crystals, 5-lipoxygenase-derived leukotriene B 4 NALP3 Monosodium urate crystals NALP3 ATP, migraine, crystalline (silica) NALP3 Polyinosinic-polycytidylic acid, lipopolysaccharide NALP1 Adjuvant Carboxy amidotrazole ASC and NALP3 are defective in crystal-induced IL-1b activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasomedeficient mice or mice deficient in the IL-1b receptor (IL-1R). Provides solid evidence for MSU-mediated inflammation in an inflammasome-dependent manner in gout Monosodium urate induces neutrophil influx in NALP3/ASC/caspase-1/IL-1b/IL-1R1-dependent manners. Inhibition of leukotriene B 4 prevented the neutrophil influx, IL-1b production, and hyperalgesia Monosodium urate crystal-induced inflammatory responses were comparably attenuated in LRR domain-deleted or NALP3 knockout mice and bone marrow-derived macrophages A20 A20 as a negative regulator of NALP3 (but not NLRC4 and AIM2) inflammasome activation and IL-1b production. Arthritis pathology relies crucially on the NALP3 inflammasome IL-1b axis. The P2X7 purinergic receptor, an ATPgated ion channel, is an important cell surface inducer of key Patients with active rheumatoid arthritis have increased expression of NALP3 and NALP3- mediated IL-1b secretion in whole blood cells upon stimulation via TLR3 and TLR4 NALP1 inflammasome was activated in arthritic synovia, which might be a novel therapeutic target for rheumatoid arthritis. Carboxy amidotrazole may have therapeutic value (decreased the arthritis index, improved radiological and histological changes) in rheumatoid arthritis by inhibiting the activation of NALP1 inflammasome and nuclear factor-jb and by decreasing pro-inflammatory cytokines References Shi and colleagues 100 Martinon and colleagues 35 Armaral and colleagues 114 Hoffman and colleagues 116 Vande Walle and colleagues 125 Choulaki and colleagues 126 Zhu and colleagues 128 (continued)

6 698 Zhang et al. Table 2. Continued Pain condition studied Animal model/patient Inflammasome targeted Activator Inhibitor Evidence/proposed function of inflammasome in pain Chronic polyarthritis DNase II-deficiency model of polyarthritis (mice) Chronic prostatitis and chronic pelvic pain syndrome Carrageenan-induced chronic prostatitis and chronic pelvic pain model (rats) Fibromyalgia Fibromyalgia patients and coenzyme Q10 deficiency mouse model Fibromyalgia Fibromyalgia patients and NALP3 / transgenic mice AIM2 Self-DNA Arthritis-prone (Dnase2 / ) mice lacking AIM2 displayed strongly decreased signs of joint inflammation and associated histopathological findings. This was paralleled by a reduction of caspase-1 activation and pro-inflammatory cytokine production in diseased joints NALP1 Carrageenan Increased concentrations of NALP1 (not NALP3), caspase-1, and IL-1b in the cytoplasm of prostate glandular epithelium; treatment with chlorogenic acid altered the expression pattern of NALP1 and reduced caspase-1 and IL-1b expression and pain behaviour NALP3 Coenzyme Q 10 Activation of NALP3 inflammasome and mitochondrial dysfunction were observed in FM patients and the corresponding animal model; NALP3 Adenosine monophosphate-activated protein kinase (AMPK) modulation coenzyme Q 10 deficiency resulted in NALP3 inflammasome activation and marked hyperalgesia in mice. In a placebo-controlled trial, coenzyme Q10 in fibromyalgia patients reduced NALP3 inflammasome activation Impairment in AMPK activation provoked hyperalgesia associated with marked NALP3 inflammasome activation and IL-1b and IL-18 production, which can be rescued by increasing AMPK phosphoralation via metformin treatment, caloric restriction diet, or NALP3 knockout. Deficient AMPK activation and overactivation of NALP3 inflammasome were observed in blood cells from patients with fibromyalgia References Jakobs and colleagues 129 Chen and colleagues 135 Qi and colleagues 137 Cordero and colleagues 141 Bullon and colleagues 142

7 The inflammasome and pain 699 initiates oligomerization of the NOD-like receptor in the NLRC4 inflammasome. Although the NALP3 inflammasome does not appear to contribute to carrageenan-induced inflammatory pain, it may play a role in other inflammatory pain states, as reported by Chen and colleagues. 50 The NALP3 inflammasome has been implicated in acute intracranial inflammatory pain caused by dural inflammation, 50 as occurs during infection or injury to the central nervous system (CNS). Dural inflammation, induced by infusion of a mixture of inflammatory mediators (histamine, serotonin, bradykinin, and prostaglandin E 2, ph 5.5) caused a dose- and time-dependent induction of nuclear cytoplasmic shuttling of NALP3, activated caspase-1, and mature IL-1b proteins in small- to medium-sized C-type neurons in the trigeminal ganglion, withnociceptive sensitization. 50 Application of a selective caspase-1 inhibitor, Ac-YVAD-CMK, diminished these effects. A low intracellular K þ concentration was proposed as a mechanism responsible for the NALP3 inflammasome activation. Collectively, these studies suggest that specific targeting of inflammasome subtypes may be an effective strategy for decreasing inflammatory pain states. Neuropathic pain Approximately 3 4.5% of the global population is afflicted with neuropathic pain, 51 a pain state involving damage or disease affecting the somatosensory nervous system, which is severely disruptive to quality of life Although neuropathic pain is a complicated condition, IL-1b activation has been widely regarded as an important contributory mechanism Enhanced IL-1b expression was observed in damaged nerves, dorsal root ganglia, and spinal nerves in animals and patients with neuropathic pain Ineterleukin-1b was shown to be an active participant in the pathogenesis of neuropathic pain, stimulating a secondary injury cascade However, the mechanisms underlying IL-1b production in neuropathic pain have not been clearly described, particularly in the CNS. Previous research has shown that the NALP1 inflammasome participates in CNS disorders and is mainly present in neurons and astrocytes. 60 Based on this information, Li and colleagues 61 recently examined the role of the NALP1 inflammasome in neuropathic pain using the widely accepted sciatic nerve chronic constriction injury (CCI) model. After CCI surgery, inflammasome platforms consisting of NALP1, caspase-1, and ASC were detected in spinal astrocytes and neurons, particularly in the superficial laminae of the spinal dorsal horn, resulting in increased production of mature IL-1b in the ipsilateral spinal cord and neuropathic pain-like behaviour. 61 Another group also demonstrated the anti-nalp1 effect on the inhibition of IL-1b maturation in a spinal nerve ligation induced neuropathic pain model. 62 Treatment with intrathecal caspase-1 inhibitor Ac-YVAD-CMK significantly reduced the CCI-induced IL-1b formation and the pain behaviour, suggesting that formation of the NALP1 inflammasome platform is central to CCI-induced neuropathic pain. This finding was supported by experiments with aspirintriggered-15-epi-lipoxin A4 (ATL). ATL is an endogenous antiinflammatory lipid capable of reducing IL-1b concentrations, thereby relieving neuropathic pain behaviours The investigators evaluated whether the NALP1 inflammasome plays a role in ATL-induced analgesia in the CCI model by observing the effects of repeated intrathecal injection of ATL on CCI-induced thermal hyperalgesia. In addition to attenuating CCI-induced thermal hyperalgesia, repeated intrathecal injection of ATL inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1b maturation. 61 The authors concluded that the NALP1 inflammasome was central to the development of neuropathic pain, and the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation. A recent study indicated that the NALP3 inflammasome does not regulate neuropathic pain in the mice spared nerve injury model. 65 Several key questions remain unanswered in the literature. For example, how does the NALP1 inflammasome become activated after CCI in specific cell types, such as astrocytes or neurons? What signals are responsible for NALP1 activation? Finally, the observation that IL-1b expression is altered in the brainstem, hippocampus, thalamus, and prefrontal cortex after sciatic nerve injury 66 or spinal nerve ligation 67 suggests that future studies should be directed at understanding inflammasome activation in higher CNS regions. Central neuropathic pain is encountered in chronic neurodegenerative diseases, such as AD and MS, and in post-trauma states, such as spinal cord injury and traumatic brain injury. 1 Consistent with a role for inflammasomes in neuroinflammation, elevated production of IL-1b has been identified in injured CNS tissues after spinal cord injury or traumatic brain injury, 68 and in the brain tissues in patients with MS 69 and AD Notably, NALP1 inflammasome activation was observed in both microglia and astrocytes after spinal cord injury 51 or traumatic brain injury. 56 NALP3 inflammasome activation in microglial cells exacerbated AD 72 and experimental autoimmune encephalomyelitis, an animal model of MS. 73 Further experiments are required to address how the central activation of inflammasomes leads to pain, for instance by supporting the activity of pro-nociceptive circuits or reducing the activity of inhibitory neurons. It is well established that several danger signals can induce NALP inflammasome activation, among them ATP and the P2X receptors (P2XRs), such as P2X4R and P2X7R, key players in IL-1 processing and release. 74 P2X7-induced IL-1b release through activation of the NALP1 or NALP3 inflammasome has been demonstrated extensively in immune cells and spinal neurons. 77 More recently, P2X4 and P2X7 receptors were shown to be expressed on dorsal root ganglion neurons, spinal microglia, and neurons Activation of P2X7R controls the release of pro-inflammatory cytokines (such as IL-1b, IL-6, and tumour necrosis factor-a) involved in nerve-injury-induced pain behaviours and hyperexcitability of dorsal horn neurons and glial cells Suppression of the function or expression of these purinergic receptors, for example by using small interfering RNA or blocking P2X7R activity, strongly suppresses neuropathic pain Finally, inflammasome activation may be associated with increased expression of pannexin 1 (Panx1), a gap junction protein. Nerve injury increased expression of Panx1 in dorsal root ganglia, and Panx1 knockdown reduced caspase-1 release and hyperalgesia. 87 Panx1 has recently been proposed to be the cell membrane pore induced by P2X7R activation, and this in turn may activate the NALP1 inflammasome in astrocytes and neurons. Thus, targeting purinergic signalling may reduce pain in part by reducing inflammasome activation. Complex regional pain syndrome Complex regional pain syndrome (CRPS) is a chronic postinjury pain condition commonly affecting a single limb. 90 Pain is a hallmark feature of this syndrome, and pain-related disability in the setting of CRPS is very common. Unfortunately, the mechanisms supporting these changes remain highly enigmatic, and available clinical treatments are limited in efficacy A tibial fracture model was described that exhibits the principal

8 700 Zhang et al. stigmata of CRPS type I (without associated nerve injury), including chronic oedema, allodynia, epidermal thickening, and periarticular osteoporosis In this model, the investigators observed that activation of the innate system of immunity in skin initiates a cascade of changes supporting a sustained pain response. 96 In relation to IL-1b, Li and colleagues 7 95 demonstrated that tibial fracture induces keratinocyte activation, proliferation, and overexpression of IL-1b and other inflammatory cytokines, which in turn contribute to hindlimb nociceptive and vascular CRPS-like changes. Importantly, continuous infusion of the IL-1 receptor antagonist IL-1ra (anakinra) reduced fractureinduced nociceptive sensitization in the rat fracture model. 7 In situ hybridization and immunostaining demonstrated that epidermal keratinocytes were the main source of IL-1b. 795 Clinical studies have demonstrated elevated IL-1b concentrations in the skin of CRPS patients. 97 Thus, peripheral IL-1b production controlled by the NALP1 inflammasome may support pain in the CRPS model. During neurogenic inflammation, peripheral afferent and efferent neurons induce a rapid release of neural mediators independent of those produced by immune cells. 98 Indeed, Li and colleagues 99 demonstrated tibial fracture-induced substance P (SP) release from peripheral nerve terminals that in turn stimulated the production of NALP1 via the NK1 receptor in keratinocytes, activation of caspase-1, enhanced IL-1b production, and nociceptive sensitization. The neurocutaneous immune regulation of the NALP1 inflammasome in keratinocytes was recently confirmed by Shi and colleagues, 100 who observed that both calcitonin gene-related peptide and SP upregulated NALP1 inflammasome and IL-1 production in a timeand dose-dependent manner in vitro and in vivo. Collectively, these studies demonstrate that the nervous system and innate immune system interact, through inflammasome activation, to form an integrated early host response to injury. It is noteworthy that the inflammasome is not only an innate responder to pathogenic and danger signals, but can also affect adaptive autoimmunity Growing evidence highlights the role of inflammasome-dependent cytokines in shaping the adaptive immune response, as exemplified by the capacity of IL-1b to support T-helper 17 responses or by the finding that IL-18 evokes antigen-independent interferon-c secretion by memory CD8 þ T cells. 102 I.V. immunoglobulin treatment protected neurons in experimental stroke models by a mechanism involving suppression of NALP1 and NALP3 inflammasome activity. 104 Several reports have linked various polymorphisms in different components of inflammasomeforming NLRs, including NALP1 and NALP3, to autoimmunity. 105 Interestingly, autoimmunity has been suggested as one of the pathophysiological mechanisms that might underlie CRPS, because of the compelling evidence that CRPS is associated with an autoantibody-mediated autoimmune process in some patients 106 and in the CRPS animal model. 107 Moreover, anti- CD20 (B cell) treatment reduces IL-1 production and nociceptive responses in a laboratory model of CRPS. 107 Examination and characterization of the interactive pathways between the inflammasome and adaptive immunity leading to CRPS may provide insight into the pathophysiology and optimal treatment of CRPS. Gout Gout is a relatively common inflammatory arthritis, characterized by intense pain in the affected joints, and can greatly impact patient quality of life. It is typically known to occur in middle-aged men, yet is increasing in incidence in the elderly population. 108 Although the initiation and development of gout is not fully understood, the precipitation of MSU crystals in joints appears to be a key factor in induction of gout-associated inflammation. 109 Accumulating evidence indicates that orchestration of the MSU-induced inflammatory response is dependent on IL-1b. In an animal model of gout, wild-type mice pretreated with the IL-1 inhibitor rilonacept demonstrated attenuated inflammation after MSU injection into the ankle joint Correspondingly, mice deficient in IL-1 receptor failed to develop gout-like symptoms fully after MSU injection The recent finding of NALP3 inflammasome in sensing MSU deposition and subsequent activation of the downstream innate immune response further demonstrates a role of IL-1b in gout pathogenesis and hyperalgesia. 35 Monocytes, macrophages, and neutrophils are key cells implicated in the pathogenesis of gout. For instance, macrophages initiate the inflammatory response to MSU crystals and produce inflammatory cytokines and chemokines that induce migration of blood monocytes to amplify the inflammatory response further. 110 Neutrophil influx into the synovium and joint fluid is the pathological hallmark of acute gout It is clear that NALP3 is expressed in all these cell types, with the capability to activate an inflammasome-mediated response. In an MSU-induced murine peritoneal model of acute gout, Martinon and colleagues 35 observed that macrophages from mice deficient in various key components of the NALP3 inflammasome, including caspase-1, ASC, and NALP3, failed to produce active IL-1b in response to MSU. Moreover, animals deficient in NALP3 inflammasome components exhibited impaired neutrophil influx after i.p. MSU injection. These findings have been confirmed by other laboratories However, how MSU crystal deposition induces neutrophil influx was not addressed. Recently, Amaral and colleagues 114 investigated the sequence of events leading to neutrophil influx in a murine model of gout induced by joint injection of MSU crystals. Injection of MSU into joints can also induce leucocyte recruitment in a NALP3/ASC/caspase-1/IL-1b/IL-1R1-dependent manner. Neutrophil influx, dependent on CXCR2 chemokine and CXCR2 ligand production, was driven by NALP3 inflammasomederived IL-1b production. Inhibition of 5-lipoxygenase-derived leukotriene B 4 prevented neutrophil influx, IL-1b production, and hyperalgesia, but this could not be attributed to a direct neutrophil-recruiting ability of leukotriene B 4. Neutrophil influx in gouty inflammation might also be driven by mast cells. 117 Mast cells are able to release IL-1b by activation of the NALP3 inflammasome. 118 Taken together, these results suggest that the NALP3 inflammasome controls CXCR2 ligand production, neutrophil influx, and the subsequent pathological hallmark of an acute gout attack and hyperalgesia. Notably, block of 5-lipoxygenase or leukotriene B 4 was shown to prevent IL-1b production, neutrophil migration, and functional joint impairment. 117 Current strategies for targeting IL-1b have proved successful for alleviating the symptoms of gout in clinical studies, suggesting that targeting this and other components of the pathway may have an important role in gout treatment Interleukin-1 inhibitors currently in clinical trials include anakinra (IL-1 receptor antagonist), rilonacept (IL-1 Trap, a soluble decoy receptor), and canakinumab (anti-il-1b monoclonal antibody). Anakinra has been shown to be efficacious for combating acute gout pain and inflammation, 119 whereas rilonacept has been revealed to be efficacious in reducing the risk of recurrent attacks Canakinumab was found to be efficacious both in inhibiting pain and inflammation in acute attacks, and for

9 The inflammasome and pain 701 reducing the risk of recurrent attacks All three IL-1 inhibitors are generally well tolerated. Rheumatoid arthritis Rheumatoid arthritis (RA) is a chronic autoinflammatory disease that affects 1 2% of the world s population and is characterized by chronic pain, warmth, and swelling. 125 Inflammasomes have been linked to RA. For example, enhanced NALP3 inflammasome activity appears to contribute to the pathology of RA Patients with active RA have increased expression of NALP3 and associated IL-1b secretion in whole blood cells upon stimulation via TLR3 and TLR4 (CFA mice, a model of RA, had analgesic responses to a TLR4 antagonist). 127 In these patients, IL-1b secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NALP3 or downstream caspases may be of benefit in suppressing IL-1b production in RA. 126 Other investigators demonstrated that A20, an RA susceptibility gene, puts a break in NALP3 and reduces inflammasome activation. 125 Likewise, excessive NALP3 inflammasome activation drives arthritis in A20 knockout mice, suggesting that A20 mutant mice might be a suitable preclinical model for validating the effectiveness of experimental therapies for RA-targeting inflammasomes, IL-1 signalling, or both. 125 Interestingly, in adjuvant arthritic rats, a model of RA, Zhu and colleagues 128 demonstrated NALP1 but not NALP3 inflammasome activation in the synovium. The discrepancies may be because arthritis is a complex disease that may be caused by different combinations of genetic and environmental triggers. An additional target may be the AIM2 inflammasome. Arthritisprone mice lacking AIM2 displayed strongly decreased signs of joint inflammation and associated histopathological findings. This was paralleled by a reduction of caspase-1 activation and pro-inflammatory cytokine production in diseased joints. 129 In contrast, systemic signs of inflammation were not dependent on AIM2. These data suggest a tissue-specific role for the AIM2 inflammasome as a sensor for tissue damage. Ultimately, these studies suggest that targeting inflammasome activity may prove beneficial in the clinical treatment of RA. Blocking of IL-1 has been evaluated in the treatment of RA in clinical trials. The efficacy of anakinra in patients with RA, alone or in association with methotrexate, was evaluated in several controlled studies, all of which demonstrated a significant reduction in disease severity and pain, an improvement in the quality of life, and reduction of joint damage and bone erosions The treatment is well tolerated; opportunistic infections are rare compared with those seen with anti-tumour necrosis factor-a agents. Like anakinra, the anti-il-1b monoclonal antibody canakinumab reduces disease severity in RA patients, including those unresponsive to anti-tumour necrosis factor-a therapies. 132 Unlike anakinra, the long-term preservation of joint function with canakinumab remains unstudied. 133 These findings seem to be promising and support the development of a new IL-1b-targeted therapy for more common inflammatory diseases. Chronic prostatitis and chronic pelvic pain syndrome The aetiology, pathogenesis, and optimal treatment of nonbacterial chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) have long been investigated but remain largely unknown. Chronic prostatitis and chronic pelvic pain syndrome is a highly prevalent condition, with detrimental effects on the quality of life. 134 The finding of NALP1 in prostate tissue and NALP3 in bladder urothelium provides a new platform to study innate immunity in CP/CPPS. 44 Recently, Chen and colleagues 135 evaluated NALP1 and NALP3 inflammasome pathways in the development of CP/CPPS. The rat CP/CPPS model that mimics National Insitutes of Health category IIIA prostatitis (i.e. the most common type of prostatitis) was created by injection of carrageenan into each prostatic lobe. Tactile hyperalgesia, local prostate inflammation status, and inflammasome concentrations were determined, and it was observed that rats with CP/CPPS exhibited scrotal static tactile allodynia, increased concentrations of NALP1 (but not NALP3), and downstream inflammasome proteins such as caspase-1 in the cytoplasm of glandular epithelium. Application of chlorogenic acid altered the distribution pattern of NALP1 from glandular epithelium into the interstitial region and induced an increase in total NALP1 in the prostate, while reducing caspase-1, IL-1b, and hyperalgesia. Although elevated concentrations of NALP1 in the interstitial region may be have been the result of clearance of carrageenan by migrating cells (i.e. macrophages or lymphocytes), IL-1b has previously been shown to be a reliable indicator of CP/CPPS in clinical studies. 136 To investigate inflammasome mediation of CP/CPPS further, Qi and colleagues 137 recently established a new animal model using prostate extract with alum. As several reports demonstrate induction of inflammasome activation by alum in a NALP3-dependent manner, 21 this model may help to elucidate both the complex aetiology of CP/ CPPS and the role that inflammasomes are likely to play in the modulation of pain in CP/CPPS. Fibromyalgia Fibromyalgia (FM) represents a prevalent chronic pain syndrome characterized by generalized hyperalgesia associated with a wide spectrum of symptoms, such as fatigue and joint stiffness. 138 Fibromyalgia affects 2 4% of the population, mostly women. Diagnosis and treatment of FM can be difficult, as both the aetiology and the pathogenesis of FM remain largely unclear. Oxidative stress, mitochondrial dysfunction, bioenergetic alterations, and inflammation have all been proposed as contributing to the symptomatology of FM Cordero and colleagues 141 recently investigated the role of coenzyme Q 10 deficiency and mitochondrial dysfunction in inflammasome activation in blood cells from patients with FM, and coenzyme Q 10 deficiency animal models. They observed that mitochondrial dysfunction was accompanied by the activation of NALP3 and caspase-1 and by increased concentrations of IL-1b and IL- 18 in cells from patients with FM and from an animal model of FM. Coenzyme Q 10 deficiency induced by p-aminobenzoate treatment resulted in NALP3 inflammasome activation in blood mononuclear cells that occurred with marked hyperalgesia in mice. A placebo-controlled trial of coenzyme Q 10 in FM patients resulted in reduced NALP3 inflammasome activation and decreased IL-1b and IL-18 serum concentrations, suggesting that NALP3 inflammasome-mediated pathogenesis of FM is likely to be regulated by coenzyme Q 10 activation of the NALP3 inflammasome. The contribution of the NALP3 inflammasome to pain in FM is supported by a recent observation from Bullon and colleagues, 142 who demonstrated that adenosine monophosphate-activated protein kinase (AMPK) phosphoration modulated pain by activation of the NALP3 inflammasome. Deficient AMPK activation and overactivation of the NALP3 inflammasome axis were also observed in blood cells from patients with FM. 129 In addition, metformin treatment, which

10 702 Zhang et al. increased AMPK activation, restored all biochemical alterations in blood cells and significantly improved clinical symptoms, such as pain, fatigue, depression, disturbed sleep, and tender points, in FM patients. 142 A recent report regarding mutation in cytochrome b gene of mitochondrial DNA in FM patients with NALP3 inflammasome activation also supports this hypothesis. 143 Alternatively, elevated substance P concentrations in patients with FM have been the focus of therapeutic trials. It has been shown that substance P enhances the activation of AMPK in vitro, 146 and substance P controls NALP1 inflammasome activation after fracture. 99 Finally, an inflammasome microglial activation mechanism was also proposed in FM. 147 Collectively, these studies suggest that inhibition of NALP3 inflammasome activity by coenzyme Q 10 and AMPK activators may be a potentially effective therapy for the specific and nonspecific pain states associated with FM. Conclusions and future perspectives The discovery of inflammasomes has provided a new avenue for investigating the molecular mechanisms underlying innate immune system activation in infectious, autoinflammatory, metabolic, and neurodegenerative diseases. As these diseases are commonly associated with pain, modulators of innate immunity may provide effective pain relief. Although our understanding of the role of inflammasomes in pain is in the early stages, a review of the currently available literature suggests that collectively, dysregulation in inflammasome activity plays a central role in inflammatory pain, neuropathic pain, CRPS, gout, rheumatoid arthritis, fibromyalgia, and CP/CPPS. Therefore, we conclude from this review of the literature that targeting inflammasomes in these specific pain-associated disease states may indeed prove to be a novel approach to the control of pain. It is also clear from this review that different inflammasome subtypes are recruited and modulate pain in different diseases (Fig. 2). In general IL-1b can act directly or indirectly as an intermediate inflammatory mediator serving to upregulate downstream cytokines and directly activate nociceptive neurons Targeting inflammasome subtypes in specific diseases may more efficiently modulate activation of IL-1 and IL-18 and therefore more effectively inhibit downstream cytokine cascades compared with traditional therapies aimed at countering the effect of individual cytokines. Furthermore, the IL-1 inhibitors anakinra, rilonacept, and canacinumab targeting IL-1 are not specific to NALP inflammasome activity and cause general immune suppression. 148 A therapeutic approach involving the targeting of inflammasome subtypes in specific diseases may be superior to the currently used antibody-based therapies, by avoiding universal suppression of IL-1 activation by other inflammasome-forming NLRs. Unfortunately, no specific inhibitor is available clinically; although both endogenous and synthetic inflammasome inhibitors have been described, this would be a big obstacle for taming the inflammasome via targeted manipulation of the inflammasome complex using a specific inhibitor. However, two potent and specific inhibitors of NALP3 were described recently, thus constituting promising candidates for clinical testing. 149 Other questions also remain. For example, although many NALPs have been demonstrated to modulate caspase-1 activity in animal pain models and in vitro, the precise physiological mechanisms of inflammasome activation in patients with pain remain obscure. Delineating these mechanisms would therefore permit more specific antiinflammasome therapies for specific pain states. Moreover, although four inflammasome subtypes had been identified to play a role in pain (i.e. NLRP1, NLRP3, NLRC4, and AIM2), 150 up to eight inflammasome subtypes have so far been described. 43 Future investigations delineating the specific role of these other Gout, RA, FM, Acute intracranial inflammation, CP/CPPS Neuropathic pain, CRPS Carrageenan induced acute inflammatory pain Chronic polyarthritis MSU Alum LPS Low intracellular [K + ] AMPK CoQ10 deficiency ATP and P2XRs Pannexin SP, CGRP Carrageenan Self- DNA NALP3 inflammasome NALP1 inflammasome NLRC4 inflammasome AIM2 inflammasome? PAIN Fig 2 Schematic overview of proposed inflammasome activation mechanisms in pain. The different inflammasomes are recruited and implicated in the pain in response to a variety of triggers. DAMPs, MSU, alum, low intracellular K þ concentration, AMPK, and coenzyme Q10 (CoQ10) deficiency are thought to activate the NALP3 inflammasome, contributing to pain in gout, FM, acute intracranial inflammation, and CP/CPPS, respectively; Purinergic signalling and P2XRs, pannexin, SP, and calcitonin gene-related peptide are thought to activate the NALP1 inflammasome, contributing to neuropathic pain, CRPS, and adjuvant arthritic pain, respectively. The NLRC4 inflammasome activation contributes to pain in a model of (carrageenan-induced) acute inflammation. The AIM2 inflammasome activation triggered by endogenous DNA drives chronic polyarthritis.