Inflammasomes in Mycobacterium tuberculosis-Driven Immunity

The development of effective innate and subsequent adaptive host immune responses is highly dependent on the production of proinflammatory cytokines that increase the activity of immune cells. The key role in this process is played by inflammasomes, multimeric protein complexes serving as a platform for caspase-1, an enzyme responsible for proteolytic cleavage of IL-1β and IL-18 precursors. Inflammasome activation, which triggers the multifaceted activity of these two proinflammatory cytokines, is a prerequisite for developing an efficient inflammatory response against pathogenic Mycobacterium tuberculosis (M.tb). This review focuses on the role of NLRP3 and AIM2 inflammasomes in M.tb-driven immunity.


Introduction
Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is a facultative intracellular bacterium that can survive and replicate within host macrophages [1,2]. By avoiding critical components of macrophage-killing repertoire such as phagosome-lysosome fusion, phagosome acidi cation, activity of lysosomal enzymes or reactive oxygen, and nitrogen intermediates, M.tb evades killing and eradication [3]. In addition to phagocytic activity and ability to present antigens to T-cells, macrophages are key cells that regulate the antimycobacterial immune response via secreted cytokines. e functional capacity of macrophages in ghting infection depends on the degree of their activation. Inactive macrophages have limited ability to inhibit the growth of ingested mycobacteria, thereby serving as a safe life niche. After activation by interferon-gamma (IFN-γ) that is secreted by T-cells, macrophages acquire enhanced bactericidal strength enabling them to kill mycobacteria growing intracellularly [4]. e IFN-γ-driven antimicrobial properties of phagocytes are augmented by IL-18 and IL-1β, two proin ammatory cytokines processed by caspase-1 that are recruited to the in ammasomes, multiprotein platforms composed inter alia of intracellular sensors for pathogen-or host-derived molecules. IL-18, belonging to the IL-1 family, is produced by a wide range of immune and nonimmune cells [5][6][7]. e IL-18 precursor (pro-IL-18) is converted by caspase-1 into an active molecule, which forms a signaling complex with IL-18R [8,9]. e receptor is composed of two chains: alpha (IL-18Ra) and beta (IL-18Rb). IL-18Rb is a signal transduction chain, essential for the formation of a high a nity complex and cell activation. e primary role of IL-18 is to induce IFN-γ production in cooperation with IL-12 or IL-15, although immunological e ects exerted by IL-18 are dependent on the cytokine microenvironment. IL-18 is able to polarize T lymphocyte response towards 1, induce T-cell proliferation, activate NK cells, enhance CD8(+) T cytolytic activity, and augment, apart from IFN-γ, the production of varied cytokines including tumor necrosis factor-α (TNF-α), interleukin-(IL-) 4, IL-5, IL-13, IL-17, and granulocyte-macrophage colony stimulating factor (GM-CSF) [8,10,11].
us, the multifaceted activity of IL-18 seems to play a prominent role in host defense against both extracellular and intracellular pathogens, including M.tb. However, an excessive IL-18 response might contribute to the induction of pathomechanisms leading to the damage of cells and tissues [12,13]. erefore, the proin ammatory activity of IL-18 is balanced by a constitutively secreted IL-18 binding protein (IL-18BP), whose binding to IL-18 decreases the production of IFN-γ and other cytokines, thereby reducing the risk of immunopathology [14]. e other in ammasome-dependent cytokine, IL-1β, which is mainly produced by monocytes and macrophages, plays an important role in in ammation and host immune response by a ecting the function of various cells, either alone or in combination with other cytokines [15][16][17].
e activity of IL-1β is tightly regulated at the levels of its transcription and release. e production of IL-1β is regulated by several proteins including pyrin, PI-9 (the caspase-1 inhibitor proteinase inhibitor 9), and some CARD-containing proteins, which interfere with the recruitment of caspase-1 or directly neutralize its activity [18]. e e ects of IL-1β are exerted via binding speci c cell surface receptors-IL-1RI and IL-1RII [19]. As in the mature IL-18 form, active IL-1β is created after the proteolytic cleavage of its precursor by in ammasomedependent caspase-1. Mature IL-1β plays important homeostatic functions in organisms and is implicated in the initiation of antimicrobial immunity via the induction of TNF-α and IL-6 release and polarization of 17 response, which improve protective mucosal host defense by the secretion of IL-17 and IL-22 [20,21]. e proin ammatory role of IL-1β in the resistance against M.tb has been con rmed by the observation that IL-1β or IL-1R knockout mice were found to be more susceptible to TB showing high mortality and increased bacterial burden in the lungs [22]. Additionally, double-de cient IL-1α/β mice had signicantly larger granulomas, and their alveolar macrophages produced less nitric oxide than the cells from wild-type animals [23].

Inflammasomes-Mediators of Inflammation
In ammation is an evolutionarily conserved protective response to noxious stimuli mounted by the innate immune system of the host. Immune de ciencies leading to insu cient development of in ammation processes may result in severe and recurrent infections, although overly intense activation of the in ammation cascade may be a cause of chronic systemic in ammatory disorders [24,25]. e development of innate immunity starts from the recognition of conservative antigenic structures called DAMPs (dangerassociated molecular patterns) and PAMPs (pathogenassociated molecular patterns) by pattern recognition receptors (PRRs) presented on the surface of rst-line defense immune cells-macrophages and neutrophils. Activation of these receptors triggers a cascade of signals that results in the induction of multiple proin ammatory cytokines. e nal step of the activation is the production of oxygen and nitrogen radicals, essential elements of the intracellular killing system. e secretion of these radicals is under strict control of a variety of monocyte/macrophage-derived cytokines such as IL-1β and IL-18. e key role in this process is played by structures called in ammasomes, multimeric protein complexes that control many aspects of innate and adaptive immunity. rough their cooperation with PRRs, in ammasomes activate host defense pathways resulting in clearance of various viral and bacterial infections, including those caused by mycobacteria. ey function as an activating sca old for in ammatory caspases that play an essential role in the maturation and secretion of proinammatory cytokines as well as in pyroptosis, an inammatory death of infected cells [26,27]. Caspases are produced as inactive proenzymes that dimerize and undergo cleavage to form active molecules. Assembly into dimers, facilitated by various adaptor proteins binding to speci c regions of their precursor forms-procaspases, is achieved through in ammasome formation [28]. Activated in ammatory caspases, typically caspase-1, lead to the generation of active IL-1β, IL-18, and IL-33 from their proprotein precursors. e mature cytokines are engaged in the recruitment of immune cells to the sites of infection and enhancement of the host's defensive responses against invading pathogens [26]. e in ammasomes are activated by multiple recognition receptors, which determine their structure and function. e canonical in ammasome sensors are nucleotide-binding domain-like (NLR) proteins and absent in melanoma 2-like (ALR) proteins and PYRIN. All of them have the ability to assemble in ammasomes and activate the in ammatory caspase-1.
e NLR family contains the NLRPs (or NALPs) and the IPAF (ICE-protease-activating factor) subfamilies [29,30]. Each NLR molecule (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, or NAIP/NLRC4) recognizes speci c ligands that activate the assembly of the in ammasome. NLR proteins consist of the conserved nucleotide-binding and oligomerization domain (NACHT or NOD), an N-terminal caspase recruitment domain (CARD) or pyrin domain (PYD) or baculovirus inhibitor repeat-(BIR-) like domain, and C-terminal leucine rich repeats (LRRs) [26,[31][32][33][34][35]. LRRs are responsible for the recognition of PAMPs, while the NACHT domain activates proin ammatory cytokine pathways via ATP-dependent oligomerization [26,29]. e NLRP1 in ammasome has a CARD that activates caspase-1 [36,37], and therefore the recruitment of ASC is not required to interact directly with procaspase-1. However, it has been shown that the participation of ASC in the process enhanced the activation of the enzyme. In contrast, NLRP3 contains no typical CARD domain that contributes to the activation of caspase-1 through the interaction of the PYD domain of NLRP3 with ASC [25]. Compared with NLRP1 and NLRP3, the IPAF protein does not contain a PYD but instead has a CARD that interacts directly with procaspase-1 without the need for ASC [38]. e members of the ALR group (known as the PYHIN family) are characterized by the presence of the pyrin domain (PYD) and one or two hematopoietic IFN-inducible nuclear antigens with 200 amino acid repeat (HIN-200) domains [26]. e PYD recruits proteins for the formation of in ammasomes, while the HIN domain recognizes and binds to DNA that can be found in the cytosol [26]. e best-known ALRs, absent in melanoma 2 (AIM2) and IFN-γ inducible protein 16 (IFI16), function as intracellular immune sensors that detect microbial DNA. e PYHIN proteins di er in their localization in the cell compartments; AIM2 can be found in the cytosol, whereas IFI16 is usually localized in the nucleus [39].
PYRIN, another canonical in ammasome-activating protein, is composed of an N-terminal PYD followed by two central B-box zinc nger and coiled-coil domains and in humans, a C-terminal B30.2/rfp/SPRY domain [40]. PYRIN associates through a PYD-PYD interaction with ASC protein, leading to its oligomerization that results in caspase-1 activation and interleukin-1β processing [40]. e activation of the PYRIN in ammasome is induced by the inactivation of RhoA GTPase by bacterial toxins [26,41]. e process of activation has been detected in both mice and humans, suggesting that the B30.2/rfp/SPRY domain is not necessary for its initiation.
e NLRP3 in ammasome-activated responses result in the release of signi cant amounts of caspase-1, which leads to maturation and secretion of IL-1β and IL-18 and activation of pyroptosis [26]. e process of NLRP3 activation is triggered by at least two signals: (1) a priming signal eliciting the expression of NLRP3, pro-IL-1β, and pro-IL-18 genes after TLR stimulation and (2) an activation signal leading to the autocatalytic activation of procaspase-1 and proteolytic cleavage of pro-IL-1β and pro-IL-18. In most cell types, NLRP3 priming is a prerequisite for deubiquitination and assembly of the NLRP3 in ammasome. Relocalization of NLRP3 to the mitochondria is followed by the secretion of mitochondrial factors into the cytosol, potassium e ux through membrane ion channels, and release of cathepsin resulting in destabilization of lysosomal membranes. Apoptosis-associated speck-like protein (ASC) plays an important role in the formation of an e ective in ammasome. ASC recruits procaspase-1 through its C-terminal caspase recruitment domain (CARD) and interacts with NLRP3 via its pyrin domain (PYD), serving as a bridge between these two molecules. e autocatalysis of procaspase-1 results in its cleavage and transformation into active caspase-1, which in turn cleaves the precursors of two proin ammatory cytokines, IL-1β and IL-18, leading to their secretion into the cytoplasm or induction [24,25,48,54,55]. However, the mechanism of triggering the NLRP3 in ammasome complex activation cascade is still a subject of debate, and at least three models for the process have been proposed. e rst suggestion is that the activation mechanism is associated with an e ux of potassium ions out of the cell and a reduction in their intracellular concentration. Such a model of activation occurs in monocytes/macrophages after stimulation with numerous stimuli including ATP, nigericin, bacterial cells, or their components [56,57]. Recently, NEK7 protein, a member of the family of NIMA-related kinases (NEK proteins), has been identi ed as an NLRP3-binding protein that acts downstream of potassium e ux to regulate NLRP3 assembly and activation [58]. He et al. demonstrated that in the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3 [58]. According to the second suggested mechanism, in ammasome activation is a result of lysosomal membrane damage and release of the phagosome content into cytosol [22,59]. e third and most accepted model assumes that the induction of the NLRP3 in ammasome complex is caused by mitochondrial reactive oxygen species (ROS) [60][61][62][63]. e common nal step in all of these models is the release of cathepsins into the cytosol leading to the lysosomal destabilization and conversion of procaspase-1 into a biologically active caspase-1 form. It should also be mentioned that formation of the NLRP3 in ammasome and cytokine release occur independently of transcriptional upregulation [64]. Juliana et al. showed that TLR4 signaling through MyD88 nontranscriptionally primed the NLRP3 in ammasome by its deubiquitination. e mechanism was dependent on mitochondria-derived reactive oxygen species and was involved in the secretion of cytokines, such as IL-18, and other in ammatory mediators such as high-mobility group protein 1 (HMGB1) [64,65]. e AIM2 (absent in melanoma 2) receptor, possessing a C-terminal HIN-200 domain and an N-terminal pyrin domain (PYD), triggers AIM2 in ammasome activation, in ammatory cell death (pyroptosis), and release of IL-1β and IL-18 in response to cytosolic double-stranded (ds) DNA [66,67]. Studies of gene-targeted AIM2-de cient mice have shown that AIM2 in ammasomes play a role in host defense against viruses and intracellular bacterial pathogens such as listeriae and mycobacteria [68][69][70]. AIM2 in ammasomes can be activated by DNA sequences having at least 80 base pairs in length in a sequence-independent manner [71,72]. e HIN-200 and PYD domains take part in forming a complex, which is maintained in an inactive state during homeostasis [71,73]. Binding of dsDNA to HIN-200 facilitates oligomerization of AIM2, and the resulting conformational change exposes the N-terminal PYD to allow the recruitment of the adaptor protein ASC. e CARD of ASC binds the CARD of procaspase-1, that forms an active AIM2 platform. Upon autoactivation, caspase-1 directs maturation and secretion of proin ammatory cytokines [48,55,66,68,74]. e latest data suggest that NLRP3-or ASC-de cient animals are characterized by impaired in ammasome formation and increased susceptibility to TB [20,54,68,75,76]. However, NLRP3 −/− and ASC −/− mice produced IL-18 and IL-1β levels comparable to those of wild-type mice, which  suggests the involvement of in ammasome-independent pathways in the secretion of these cytokines [21,42,47]. Many reports have demonstrated that a wide range of microorganisms are able to inhibit in ammasome activation and function. Viruses and many bacterial pathogens develop several mechanisms of repression of in ammasome folding; however, not all mechanisms are clearly understood. Yersinia enterocolitica produce YopE and YopT proteins that supress caspase-1 maturation, whereas YopK protein of Y. pseudotuberculosis binds to the type III secretion system, thereby preventing the recognition of the pathogen by host cell in ammasome. Pseudomonas aeruginosa mediates suppression of NLRC4-in ammasome by secreting ExoU and ExoS e ectors, whose mechanism of action still needs elucidation. Virulent M.tb can inhibit the formation of AIM2 and NLRP3 in ammasomes both directly and indirectly, but the factors responsible for the inhibition have not been recognized thus far. One of the likely mechanisms is the activity of Zn-metalloprotease called ZMP1, which inhibits the activation of NLRP3 in ammasome and, as a consequence, leads to the reduction of caspase-1 activity [77][78][79]. Master et al. showed that infection of mice macrophages with zmp1-deleted M.tb induces activation of the in ammasome, resulting in enhanced maturation of phagosomes, increased IL-1β secretion, and better M.tb clearance in lungs [79]. It is probable that M.tb is able to restrain the activation of other in ammasome types, but evidence is needed to con rm this hypothesis. In addition to the induction of in ammasome activation via PRRs, M.tb antigens can modulate other innate immunity-associated functions. One recently identi ed protein, tyrosine phosphatase (Ptp) A, enters the nucleus of the host cells and regulates the transcription of many host genes involved in the mechanisms of innate immunity, cell proliferation, and migration [80]. e enzyme is also able to dephosphorylate certain host proteins (p-JNK, p-p38, and p-VPS33B), leading to inhibition of phagosome-lysosome fusion and blocking the acidi cation of phagosomes. Both activities are crucial for M.tb virulence in vivo through the promotion of M.tb's intracellular survival in macrophages [80]. M.tb often escapes from the phagosome within a few days of the invasion of the host organism and creates di culties in assessing the potential role of in ammasomes during the initial stages of mycobacterial infection. Moreover, the evaluation of IL-1β and IL-18 produced as a result of in ammasome activation is inadequate in revealing the signi cance of formed multiprotein platforms in the course of developing infection. e initiation of phagocytosis causes a decrease in the levels of potassium ions in macrophages, which have been found to be one of the crucial in ammasome activators during infections with M.tb and nontuberculous mycobacteria [81].
Other regulators such as thioredoxin-interacting proteins, activated by the increase in reactive oxygen species in cytosol, are thought to have minor e ect on the formation of in ammasomes in M.tb infection [47]. e signaling cascade can also be activated by the mycobacterial type VII secretion system (ESX-1), which is responsible for translocation of extracellular DNA (eDNA) in cytosol and the production of IFN-β. Many studies have demonstrated that, at the molecular level, IFN-β regulates the AIM2 in ammasome activity [82,83]. Some ESX-1-de cient M. smegmatis mutants have been shown to possess limited capacity for AIM2 in ammasome activation. However, in contrast to nontuberculous mycobacteria (NTM), M.tb mutants lacking ESX-1 system failed to inhibit AIM2 formation, while the wild-type strain inhibited the in ammasome activation [47,84]. e suggested mechanism of inhibition involves the IFN-β-mediated induction of IL-10, which in turn suppresses IL-1β production [85,86]. However, further investigation is needed to elucidate the molecular mechanism of M.tb-driven AIM2 inhibition and its consequences for bacterial virulence. M. bovis BCG vaccine strain, which does not possess the ESX-1 system, poorly activates multiple NLR and in ammasome complex components including caspase-1 [87]. e bacilli repress the expression of thioredoxin-interacting protein (TXNIP), an antioxidant inhibitor recruiting caspase-1 to the NLRP3 in ammasome. e inhibition of TXNIP by BCG limits NLRP3 activation and restrains pyroptosis following mycobacterial infection. Proin ammatory responses to BCG bacilli was found to be driven primarily through Toll-like receptors (TLRs), since BCG does not activate expression of genes downstream of TLR/MyD88-and NOD-2-driven NF-κβ and AP-1 pathways. However, BCG is still able to induce moderate IL-1β secretion as measured by transcription of in ammasome network genes [87,88]. Understanding BCG-induced pathways of in ammasome activation can be helpful in improving the existing vaccine or developing new antituberculous vaccines. e recombinant BCG ΔureC::hly vaccine candidate (VPM1002) has been shown to induce improved protection against TB over the parental BCG strain [4]. Saiga et al. demonstrated that VPM1002 activated the AIM2 in ammasome and caspase-1 through the ability of listeriolysin to perforate phagosome membranes, which is encoded by the hly gene integrated into BCG genome [4]. e perforation facilitates the release of mycobacterial DNA into the cytosol, in a way that is similar to the ESX-1 system of M.tb. Mice vaccinated with VPM1002 showed increased production of IL-1β and IL-18 as well as induction of the stimulator of IFN genes (STING)dependent autophagy, which promotes delivery of BCG antigens to MHC molecules and improves their presentation to T-cells [4].
Apart from direct induction of proin ammatory cytokine secretion, the activated caspase-1 triggers the pyroptotic death of infected cells. e cytosolic protein Gasdermin D (GSDMS) is a key mediator of this process. e cleavage of GSDMD by activated caspase-1 results in the release of its N-terminal fragment (GSDMD-NT), which forms pores in the plasma membrane of the infected cell leading to the elimination of the pathogen [26,[89][90][91]. e pores disrupt cell membrane integrity allowing water in ux, cell swelling, and osmotic lysis together with an e ux of small molecules, including proin ammatory cytokines. GSDMD-NT is able to kill both cell-free and intracellular microorganisms and can be thought as a new antibacterial agent. However, it is still not known whether GSDMD-NT is able to permeabilize the membrane of the phagosomes and kill the bacteria

Therapeutics Targeting Inflammasome Pathways
Biologic agents interfering with in ammasome activation may provide new means of therapeutical interventions for many diseases. ese agents may target either upstream processes of in ammasome regulation or downstream IL-1 signaling [41]. Inappropriate activity of in ammasomes has been found to be involved in the pathogenesis of certain autoin ammatory skin disorders such as cryopyrinassociated periodic syndrome (CAPS) or familial Mediterranean fever (FMF) as well as a number of chronic in ammatory diseases such as multiple sclerosis, gouty arthritis (gout), atherosclerosis, type 2 diabetes, and obesity [29,93,94]. Moreover, mechanisms controlling the NLRP3 in ammasome arrangement have also been implicated in the development of lung, kidney, and liver diseases [95][96][97]. Colchicine, a drug used for treatment of gout, has been shown to inhibit macrophage NLRP3 in ammasome assembly and activation in vitro and in vivo [98]. Colchicine blocks monosodium urate crystal-induced NLRP3 in ammasome-driven caspase-1 activation and IL-1β processing and release, suppresses the expression of genes involved in cell regulation, and inhibits IL-1-induced L-selectin expression on neutrophils [99]. Other therapeutics that target in ammasome-driven end products include VX-765 (inhibitor of caspase-1 activation), Anakinra (recombinant form of IL-1 receptor antagonist), Canakinumab (monoclonal antibody against IL-1β), Rilonacept (IL-1 inhibitor), IL-18 binding protein, and anti-IL-18 receptors antibodies [8,41,100]. A number of new molecules have been identi ed as inhibitors of IL-1β processing (glyburide, parthenolide, CRID3, aurano n, isoliquiritigenin, β-hydroxybutyrate, and MCC950); however, con rming their clinical utility will require additional time and research [24].

Conclusion
In ammasomes have been implicated as specialized signaling platforms critical for the regulation of both innate immunity and in ammation. M.tb has been shown to modulate the host innate immune response by delaying cell death systems of the host, thereby facilitating its own proliferation. Understanding the molecular mechanisms of in ammasome activation during intracellular pathogen infections such as with M.tb, and the evasive mechanisms employed by this evading pathogen, may lead to development of more potent therapies to combat the proliferation of M.tb.

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this article.