Nod like receptors (NLRs) are a versatile family of relatively recently discovered intracellular receptors which have a broad range of functions in humans, ranging from the regulation of antigen presentation and modulation of inflammation to embryonic development and sensing cellular metabolic changes (Motta et al 2015). Evolutionarily, NLRs are well conserved and orthologs are found across much of the animal kingdom (Lange et al 2011).
The NLR family in humans is relatively diverse, comprising of 22 members with specific functions dependent on ligand specificity and effector functions, each determined by individual domains the in the basic molecular tripartite structure. Upon activation, the autoinhibited monomeric NLR’s oligomerise to form the characteristic pinwheel shaped active inflammasome structure leading to cell signalling and cytokine secretion events (Lechtenberg et al 2014).
NLRP3 and inflammasome cytokine secretion
As mentioned, a large number of NLRs perform vital roles within the body, however the most well studied, ubiquitously activated and pathologically relevant inflammasome is NLRP3. Whilst NLRs perform many critical cellular roles, dysregulation of NLRP3 can perpetuate chronic inflammation through cytokine maturation and secretion and the induction of pyroptosis.
This dysregulation of NLR signalling has been implicated in both classical cryopryrinopathies such as Muckle-Wells syndrome and neonatal-onset multi-system inflammatory disease (NOMID) as well as more common pathologies including cardiovascular, metabolic and rheumatic conditions (Rosential, Till and Schrieber 2007) (Li et al 2014) (Tong et al 2015).
NLRP3 inflammasome tools
NLRP3 inflammasome structure
NLRs are comprised of a distinct structure, which in the case of NLRP3 is tripartite, however other inflammasomes such as NLRP1 can contain additional domains. The Leucine rich repeat region (LRR), the nucleotide binding and oligomerisation domain (NACHT) and N terminal effector domain (a pyrin or a caspase activation and recruitment domain – PYD and CARD respectively) which co-operatively determine both ligand specificity and the presence or absence of a linker between the inflammasome and associated caspase, which in the case of NLRP3 is caspase-1. Due to NLRP3 being the most widely studied and implicated in disease, this review will predominantly focus upon this particular inflammasome, however similarities with other family members are still retained.
Figure 1 – Schematic of the molecular structure of NLR inflammasomes. The C terminal LRR domain has been demonstrated to be central for ligand recognition and inflammasome specificity. It bares structural comparability with the LRR’s of TLR’s, however the superhelical topology is right handed compared to flat / left handed in TLR’s. The central NACHT/NBD domain is responsible for the ATPase function of the protein family, which has been demonstrated to be central for oligomerisation and conformational change from the autoinhibited resting state. The final conserved domain is the N terminal PYD/CARD domain which are responsible for interacting directly (CARD) or indirectly via a linker (PYD) with the caspase effector in the inflammasome complex which upon binding allows the cleavage of inflammasome substrates (eg. Pro-IL1β) in to their mature forms. Image adapted from Stutz et al 2009.
Canonical inflammasome activation
In order to activate the inflammasome, induce oligomerisation and effectively secrete IL-1b and IL-18 a two-step signalling process is generally required; with an initial priming step induced by TLR, C5a receptor or cytokine receptor signalling. This results in an upregulation of the substrate pro-IL1β / pro-IL18 as well as NLRP3 itself, but not upregulation of the inflammasome components ASC or Caspase-1 (Hornung and Latz 2010).
The canonical priming signal occurs through the Nf-κb signalling pathway was traditionally thought to simply induce the transcription of necessary components for inflammasome activation. However, activation of Nf-κb has also been implicated to have a direct effect upon NLRP3 through the generation or ROS, which in turn activate the BRCC3 deubiquitinase and the resultant deubiquitination of NLRP3 yielding a primed state (Juliana et al 2012). Further studies have also implied that transcriptional upregulation may not be an essential feature for NLRP3 activation, however the classical priming followed by activation mechanism is predominantly still utilised in experiments (Gross et al 2010) (Lin et al 2013).
NLRP3 inflammasome priming by TLR4 activation
The classical cell signalling underlying the priming of the NLRP3 inflammasome is mediated by TLR4 activation by a range of ligands, including PAMPS such as the bacterial cell wall component lipopolysaccharide (LPS) and DAMPS including HMGB1 (Frank et al 2013). TLR4 is a single pass transmembrane receptor comprising of a 608aa extracellular domain, a transmembrane domain and an 187aa intracellular domain (Carpenter S, O’Neill L 2009). The recognition and interaction of TLR4 with LPS is mediated by MD-2 which undergoes conformational change upon interaction with LPS, which then allows phosphate groups on the LPS molecule to induce receptor multimerisation through ionic interactions with a pocket of positive residues on TLR4/MD-2 (Park et al 2009).
The engagement of the TLR then induces the cytoplasmic TIR domain to recruit various signalling adaptors including MYD-88 and or TRIF/TRAM (Lim and Staudt 2013). Dependent on the signalling adaptor engaged, a range of potential effectors can then be activated, including the IRAK kinase family and the ubiquitin ligases TRAF6 and Pellino 1 (Butler et al 2005). The activation of these pathways then culminates in the engagement of NF-κb, JNK and p38 MAPK signalling pathways to regulate gene transcription, including inflammasome related genes.
NF-kb Signalling through TLR4
The induction of NF-κb signalling through TLR4 activation is via the canonical pathway, which results in the phosphorylation of IKKβ. This in turn then phosphorylates IKKα Ser 32 and 36, which induces IKKα proteosomal degradation, freeing NF-κb from the inhibitor complex. This then allows NF-κB P50/P65 heterodimer translocatation to the nucleus to induce the transcription of a host of pro-inflammatory genes (Hoesel and Schmid 2013).
NLRP3 expression & priming
As previously mentioned, the requirement for transcriptional priming in some situations is sometimes bypassed, however in most cell types under most conditions the upregulation of NLRP3 gene expression is required. This has been demonstrated to be around 10 fold via a luciferase reporter assay in PBMC’s, and is generally required for effective inflammasome activation, the bypassing of which may be due to the cell type constitutively expressing higher NLRP3 levels or a stimuli activating NF-κb through the discussed mechanisms (Bauernfiend et al 2009).
Figure 2 – The activation of the canonical NF-κB signalling pathway through TLR4. Ligand binding to the TLR4 induces conformational change in the receptor and allows homodimerization. The TLR4 homodimers then recruit the adaptor protein MYD88. The binding and activation of MYD88 to the TLR4 homodimer activates the cascade, allowing phosphorylation of IRAK4 and subsequent downstream targets. The phosphorylation of IKKβ regulatory subunits of the NF-κB complex allows the active P50/ P65 heterodimer to translocate in to the nucleus to function as transcription factors. The downstream effects of NF-κB activation on the inflammasome are twofold, firstly to induce the expression of pro-IL1β, pro-IL18 and NLRP3 which are generally necessary for effective inflammasome activation, and secondly to potentially induce post translational modification of previously translated NLRP3 (Lim and Staudt 2013).
The NLRP3 inflammasome, once primed in either a transcription dependent or independent manner, can become activated by a plethora of activating stimuli which break down in to 3 main categories – intracellular ionic fluxes, ROS generation and mitochondrial dysfunction and lysosomal rupture and damage (de Zoete et al 2014). Due to the diversity of these activating stimuli, it would appear that a conserved activation mechanism should exist, however as of yet this is still unclear.
The concept that lysosomal destabilisation, notably through inefficient phagocytosis of crystals such as oxidised LDL or aggregated protein such amyloid β has been demonstrated in multiple studies, predominantly ustilising the specific Cathepsin B inhibitor Ca074Me. However, Cathepsin B KO cells fail to fully recapitulate the effects, suggesting off target effects of Ca074Me on other Cathepsins or further afield targets may be responsible (Hornung et al 2008) (Orlowski et al 2015). The exact mechanisms by which Cathepsins modulate NLRP3 activation remain unclear, however it has been shown that Cathepsins are involved in both signal one and signal two of inflammasome activation, and that NLRP3 and Cathepsin B can directly interact and co-precipitate with IP (Orlowski et al 2015) (Bruchard et al 2013).
Potassium efflux inflammasome
The hypothesis that ionic flux is a necessary and sufficient signal to induce NLRP3 inflammasome activation has been one of the most strongly supported amongst the potential activation mechanisms, however the exact nature of the flux, whether it is dependent on potassium efflux, calcium influx, or a combination of both is still largely contested and unclear (Chae et al 2015) (Katsnelson et al 2015) (Yaron et al 2015).
The hypothesis that potassium efflux is solely responsible for the activation of the NLRP3 inflammasome has been well established, with the recent paper by Katsnelson et al systematically demonstrating that there was no role in Ca 2+ influx in the activation of NLRP3. This was done through utilisation of the fluo-4-am calcium detection system to show nigericin induced calcium influx occurred downstream of caspase-1 activation and may well have been due to the potential of caspase-1 to alter membrane permeability. The group went on to demonstrate that alteration and depletion calcium from the stimulation media, depletion of ER Ca2+ stores and Ca 2+ specific ionophores were all incapable of affecting IL-1β secretion. Furthermore, the group sought to demonstrate commonly used Ca2+ chelator BAPTA which has previously been shown to strongly inhibit NLRP3 activation exerted pleitropic inhibitory effects away from canonical calcium chelation by demonstrating BAPTA inhibited NLRP3 activation in response to Nigericin even in Ca2+ depleted media (Katsnelson et al 2015). The exact mechanism by which K+ efflux is postulated to activate the inflammasome was not demonstrated in this paper, however future work by He et al implicating NEK7 as a downstream activator in response to K+ efflux may prescribe the mechanism for these observations.
Despite this, other groups have strongly conflicting data observing a crucial role for Ca2+ with multiple studies showing it is indispensable for activation (Sutterwala et al 2014). An intriguing example of which is the study by Lee et al in 2012 which postulates that the intracellular calcium receptor (CASR) was able and sufficient to activate the NLRP3 inflammasome in instances of elevated cytosolic calcium. Furthermore, a concomitant decrease cAMP which is believed to be a direct inhibitory modulator of NLRP3 by retaining it in an inactive conformation was observed. Interestingly, they reported a paradoxical response to increased extracellular calcium, which they found to inhibit NLRP3 activation by extracellular ATP but not flagellin or dsDNA – and may begin to explain some of the discrepancies in the literature. Furthermore, as Katnelson et al described that the IP3R and ER Ca2+ store release had no effect on the activation of the NLRP3 inflammasome as they dismissed effects of the chosen inhibitor 2-APB as off target due to their chronology of the activation process, Lee et al found that knockdown of the IP3r markedly decreased IL-1β secretion – again in stark contrast between the two papers (Lee et al 2012).
Some groups such as Yaron et al have attempted to unify the contrasting theories on the role of ionic fluxes in NLRP3 inflammasome activation utilising new real time imaging techniques to link the potassium and calcium fluxes and determine that NLRP3 activation is dependent on both ions, in this paper proposing a model where K+ efflux was the regulator for Ca 2+, which was then in turn proposed to induced mitochondrial dysfunction and ROS generation to induce the final activation signal (Yaron et al 2015).
A role for mitochondria in NLRP3 inflammasome activation
The concept that mitochondria may be intrinsically linked with NLRP3 activation has strong support, with the mitochondria acting as a nexus between Calcium, ROS and cell metabolism – all of which have been shown to activate or regulate NLRP3 activation. The concept that ROS, predominantly produced by the mitochondria, is an activator of the NLRP3 inflammasome is controversial, with studies presenting arguments both for and against the case (Rubartelli 2012) (Iyer et al 2013). Whether ROS act as an activator of NLRP3 or are generated as a consequence of the activation remain unclear, with real time ROS production imaging being required to delineate the chronology of events.
However further to mitochondrial ROS, other molecules associated with the organelle including Cardiolipin, MAVS, Mitofusins and mitochondrial DNA have all been shown to act as activators of NLRP3. This, coupled with the interesting developments that sphingolipids and other metabolic intermediaries can activate the inflammasome, whilst the ketone hydroxybutyrate can downregulate NLRP3 activation demonstrates that the NLRP3 inflammasome stands at the crossroads of immunity, metabolism and cell signalling cascades, which aligns it with the mitochondria (Camell et al 2015) (Gurung et al 2015).
Figure 3: Schematic of multiple inflammasome activation pathways of inflammasome activation. A primed cell (with NLRP3 and pro IL-1β/ IL-18) can become activated through a diverse array of stimuli. Ion flux, both potassium and calcium have been hypothesised to drive inflammasome activation, however the only downstream mechanism so far described is activation of NEK7 which independently of its kinase domain was essential for NLRP3 activation in macrophages. ROS production / mitochondrial dysfunction has been hypothesised to activated through TXNIP in response to oxidative stress or through direct interaction of molecules such as cardiolipin. Lysosomal destabilisation is thought to activate inflammasome predominantly through the release of lysosome localised proteases such as cathepsin B are implicated in both signal one and activation of inflammasome responses
Therefore, the processes underlying the activation of the NLRP3 inflammasome are both as diverse as they are obscure. The contrasting evidence for various stimuli makes a singular mechanism difficult to decipher, however in all probability inflammasome activation is a multifaceted process comprising an element of each of the described mechanisms. The recent discovery of NEK7, a kinase with a role in mitosis, as an indispensable component of the NLRP3 inflammasome in macrophages downstream of potassium efflux begins to suggest that the finer details of the general mechanisms are beginning to be elucidated (He et al 2016). Currently, a single specific NLRP3 inhibitor, MCC950, is undergoing clinical trials and showing promise in a range of lab based studies, however with increased understanding of activation processes and tailoring towards the activating stimuli in an individual disease may pave the way for a new wave of therapeutics pertinent to a range of pathologies.
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