Novel insights into the innate immune response to non‐tuberculous Mycobacteria

Inflammasomes are cytosolic protein complexes that regulate the activity of caspase-1, and thereby trigger the caspase-1-dependent maturation and secretion of potent pro-inflammatory mediators.1 Inflammasomes are activated upon cellular infection with specific pathogens, and inflammasome-dependent cytokines such as interleukin (IL)-1β mediate protective host responses to clear infection. Precise mechanisms directing pathogen-dependent NLRP3 inflammasome activation are currently unclear. In this issue of Immunology and Cell Biology, Lee et al.2 demonstrate that the bacterial pathogen Mycobacterium abscessus (Mabc) activates the NLRP3 inflammasome, and intriguingly, reveal a function for the signal scaffold p62, also known as sequestosome (SQSTM1), in this process. Inflammasomes are formed by specific members of the NOD-like receptor (NLR) family, and the HIN200 family member, AIM2.1 The most fully characterised inflammasome scaffold is NLRP3, which contains a C-terminal string of leucine-rich repeats thought to sense ligand, a central oligomerisation (NACHT) domain, and an N-terminal pyrin domain that enables interaction with the pro-form of caspase-1 via a small adaptor, ASC. Upon activation, NLRP3 oligomerises, triggering pro-caspase-1 clustering and proximity-induced auto-cleavage. Active caspase-1 then in turn cleaves cellular targets such as pro-IL-1β. Because of its potent pro-inflammatory activity, IL-1β action is tightly regulated. The availability of the IL-1β pro-form is modulated independently of the inflammasome, by nuclear factor κB (NF-κB)-activating pathways such as those triggered by Toll-like receptors (TLRs; signal 1), whereas IL-1β maturation and secretion is controlled by the inflammasome (signal 2). Although many pathogen components solely trigger inflammasome activation (signal 2) without concurrent induction of pro-IL-1β (signal 1), Syk-coupled C-type lectin (CTL) receptors can engage both pathways.3 Receptor ligation triggers Syk kinase activation, and then signal transduction bifurcates: Syk-dependent activation of the Card9–Bcl10–Malt1 complex triggers NF-κB signalling and pro-IL-1β induction (contributing to signal 1 in collaboration with other pathways, for example, TLR2), and in parallel, Syk-dependent inflammasome activation mediates IL-1β maturation and release (signal 2). The NLRP3 inflammasome is activated by a variety of whole bacteria, fungi and viruses. In some cases, the individual pathogen motifs that drive inflammasome activation are defined (for example, bacterial toxins such as nigericin from Streptomyces hygroscopicus). Synthetic irritants (for example, aluminium hydroxide) and host-derived molecules indicative of cellular injury or stress (for example, ATP released from necrotic cells) can also activate the NLRP3 inflammasome. The precise molecular mechanisms triggering NLRP3 activation remain to be definitively established.1 Pathogens of the bacterial genus Mycobacteria are responsible for serious human infectious diseases, including tuberculosis and leprosy.4 Although the bacteriology and immunology of Mycobacterium tuberculosis (Mtb) infection has been a focus of intensive research,5 the host–pathogen interactions occurring during atypical (non-tuberculous) mycobacterial infection have remained poorly characterised. Such atypical mycobacteria include Mabc, an emerging pathogen that causes a spectrum of human diseases, including infections of the skin, soft tissues and the lung.4 Prior research has identified a complex interplay of innate immune signalling pathways triggered by Mtb infection.5 Mtb activates the NLRP3 inflammasome leading to IL-1β secretion in vitro,6,7 and IL-1β deficiency renders mice susceptible to aerosol Mtb infection.6 However, the relative contribution of inflammasome-dependent versus inflammasome-independent pathways in mediating IL-1β action during in vivo Mtb infection remains controversial.6,7 Although signalling pathways triggered by Mtb are relatively well characterised, information regarding the specific innate immune signalling pathways mediating detection and elimination of Mabc is somewhat limited. An earlier study demonstrated that TLR2 and Dectin-1 collaborate to drive efficient bacterial phagocytosis and inflammatory cytokine production.8 In their current study, Lee et al.2 extend the current knowledge of innate immune receptors activated by Mabc to the inflammasome/IL-1β axis. Infection of human monocyte-derived macrophages with Mabc causes pro-IL-1β induction (signal 1) and, interestingly, also triggers robust activation and secretion of both caspase-1 and IL-1β (signal 2; see Figure 1). The authors used small interfering RNA technology in the human myeloid cell line, THP-1, to demonstrate that both Dectin-1 and TLR2 are required for pro-IL-1β induction (Signal 1),2 consistent with previous studies for Mabc-dependent induction of other cytokines (tumor necrosis factor, IL-6 and IL-12p40).8 The authors also show that Mabc triggers caspase-1 activation; this response requires both Syk kinase activity and Dectin-1-dependent Ca2+ flux. Mabc-dependent caspase-1 cleavage is blocked by knockdown of NLRP3 or ASC, indicating that Mabc is a novel infectious trigger for the NLRP3 inflammasome.2 Furthermore, Mabc-dependent caspase-1 activation is inhibited when cells are cultured in high extracellular K+,2 supporting previous studies proposing K+ efflux as an important event upstream of NLRP3 activation.1 Intriguingly, Lee et al.2 reveal that both Mabc-dependent pro-IL-1β induction (signal 1) and inflammasome activation (signal 2) rely upon the signalling adaptor p62, a ubiquitin-binding protein involved in a number of cell signalling pathways.9 p62 knockdown partially blocks Mabc-dependent upregulation of pro-IL-1β (signal 1),2 consistent with reports suggesting a function for p62 in promoting NF-κB signalling by mediating the aggregation and subsequent activation of TRAF6.9 p62 also modulates other antimicrobial pathways such as that of autophagy pathways.9,10 p62 targets polyubiquitinated protein aggregates and whole intracellular pathogens to the autophagic machinery, and traffics antimicrobial peptides to autophagolysosomes, where they become proteolytically activated to aid in the destruction of Mtb.10 In the current study, Lee et al.2 made the surprising finding that p62 knockdown prevents Mabc-dependent secretion of mature caspase-1 and IL-1β, suggesting that p62 is required for inflammasome activity (signal 2) during Mabc infection. Although clear links between inflammasome activation, autophagy and Mtb bacterial control exist,1,10 it is unlikely that p62 mediates Mabc-dependent inflammasome activation via modulation of autophagic pathways, as autophagy blockade promotes, rather than represses, inflammasome activation.1 Importantly, p62 was dispensable for the NLRP3 agonist activity of ATP and nigericin,2 raising the question of how exactly p62 participates in NLRP3 activation downstream of Mabc but not soluble NLRP3 agonists. In a recent report, p62 was shown to localise to the same subcellular compartment as inflammasome components NLRP3, ASC and caspase-1 in another infectious model, Shigella flexneri.11 This observation, coupled with the characterised function of p62 in promoting protein aggregation, and the known importance of oligomerisation for inflammasome-dependent caspase-1 activation suggest the possibility that p62 may function to promote clustering of inflammasome components downstream of syk-coupled CTL receptors. In earlier studies, NLRP3 agonists that require internalisation (such as particulate agonists) were proposed to trigger inflammasome activation through a mechanism involving phagosome destabilisation;1 it is also possible that p62 participates in this specific arm of the NLRP3 activation scheme. A further possibility is that p62 is required for Mabc phagocytic uptake, or engagement of the CTL signalling pathway. In summary, the report by Lee et al.2 detailing NLRP3 inflammasome activation via Dectin-1/Syk significantly advances our understanding of human innate immune responses to the atypical Mycobacterium, Mabc. The study also implicates the versatile ubiquitin-binding protein, p62, as a new factor in the inflammasome activation pathway for a subset of NLRP3 agonists. Future experiments should examine this aspect of inflammasome biology more carefully, to determine whether p62 is a general requirement for all NLRP3 activators requiring phagocytic internalisation and/or phagosomal destabilisation. Such future studies may illuminate NLRP3 activation mechanisms that are currently poorly defined. Mabc regulates IL-1β action via multiple mechanisms that require the ubiquitin-binding protein, p62. IL-1β bioavailability is controlled by at least two mechanisms. Mabc triggers signalling by innate immune receptors, TLR2 and Dectin-1, thereby inducing pro-IL-1β transcription in an NF-κB-dependent manner (signal 1). Although TLR2 engages the MyD88 adaptor protein and IRAK kinases for signal transduction, Dectin-1 signalling requires ITAM-dependent activation of the Card9–Bcl10–Malt1 complex, via the kinase, Syk. In parallel, Syk-dependent formation of a NLRP3 inflammasome (composed of NLRP3, ASC and pro-caspase-1) leads to caspase-1 activation, which then cleaves pro-IL-1β to its active form, IL-1β, and also triggers its secretion (signal 2). Ionic flux (K+ efflux and Ca2+ influx) is critical to Mabc-dependent inflammasome activation. The ubiquitin-binding protein, p62, promotes both signals 1 and 2.