The Roles of Inflammasomes in Host Defense against Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) infection is characterized by granulomatous lung lesions and systemic inflammatory responses during active disease. Inflammasome activation is involved in regulation of inflammation. Inflammasomes are multiprotein complexes serving a platform for activation of caspase-1, which cleaves the proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 into their active forms. These cytokines play an essential role in MTB control. MTB infection triggers activation of the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes in vitro, but only AIM2 and apoptosis-associated speck-like protein containing a caspase-activation recruitment domain (ASC), rather than NLRP3 or caspase-1, favor host survival and restriction of mycobacterial replication in vivo. Interferons (IFNs) inhibits MTB-induced inflammasome activation and IL-1 signaling. In this review, we focus on activation and regulation of the NLRP3 and AIM2 inflammasomes after exposure to MTB, as well as the effect of inflammasome activation on host defense against the infection.


Introduction
Despite the development of chemotherapy and vaccine programs, tuberculosis (TB) continues to lead to increasing death tolls and poses a serious threat to global public health [1]. It is one of the top 10 causes of mortality and the leading cause from a single infectious pathogen. WHO estimated that 1.5 million people died from TB in 2018 (https:// www.who.int/news-room/fact-sheets/detail/tuberculosis). Approximately one-third of the world's population is infected with MTB, the main causative agent of TB, and 5-10% of the population develops active TB [2]. MTB can infect the host for decades without causing clinical manifestations, only to reactivate in compromised immunity. Bacterial replication results in a robust granulomatous inflammatory response in immunocompromised patients. Inflammation is indispensable for initial control of infection, and also helps disseminate MTB to susceptible individuals in the community [3] IL-1β and IL-18, members of IL-1 family, are potent proinflammatory cytokines [4][5][6]. They play a critical role in host defense against MTB infection. Mice deficient in IL-1β or IL-1 receptor type I (IL-1R1) have been shown to be highly susceptible to infection with MTB, as reflected by decreased survival time, increased bacterial burden in lungs and bronchoalveolar lavage fluid (BALF) and extensive pulmonary necrosis [7,8]. IL-18 deficiency in mice elicits higher bacterial burden in lung tissues and larger granulomas in the lungs and spleens. Administering exogenous recombinant IL-18 subcutaneously to IL-

A Brief Introduction to Inflammasome
Inflammasomes, major components of the innate immune system, consist of sensor proteins, ASC that is not necessary for all inflammasomes such as the NLRP1 and NLRC4 inflammasomes, and executor caspase-1. The sensors interacts with ASC and caspase-1 following detecting pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), leading to assembly of inflammasomes and activation of caspase-1 [45]. Active caspase-1 mediates maturation and release of proinflammatory cytokines such as IL-1β and IL-18 as well as pyroptosis, a programmed necrotic cell death which is mediated via gasdermin D's membrane pore-forming activity [46,47]. Among inflammasomes, the NLRP3 and AIM2 inflammasomes are extensively described. Upon exposure to chemically-and structurally-unrelated agonists, NLRP3 is activated via its association with mitochondrion-derived molecules, such as cardiolipin [48] and mitochondrial DNA (mtDNA) [49]. Its activation requires two signals: signal I contributes to upregulation of IL-1β and NLRP3 in an NF-κB-dependent manner, which is called priming, and signal II induces activation of the NLRP3 inflammasome characterized by maturation of IL-1β and caspase-1 [50]. Reactive oxygen species (ROS) production controls the priming step in most circumstances [51]. Several organelles, including mitochondria [52], endoplasmic reticulum [53], mitochondria-associated ER membranes (MAMs) [53] and the Golgi apparatus [54,55], participate in NLRP3 inflammasome activation. AIM2 senses non-sequence-specific DNA via electrostatic attraction between the double-stranded DNA (dsDNA) sugar-phosphate backbone and the positively charged hematopoietic expression, interferon-inducible nature and nuclear localization (HIN) domain residues [56,57] oligomerizes at multiple binding sites in dsDNA [58] and recruits ASC and caspase-1 to assemble the AIM2 inflammasome [59].
Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that sense specific invariant elements from pathogens and activate NF-κB signaling [76]. They are necessary for acute activation [76,77] and priming step of the NLRP3 inflammasome [78]. TLRs participate in macrophage activation upon MTB infection [79]. Multiple ligands expressed by MTB bind to TLRs, activating proinflammatory immune responses ( Figure 1). TLRs play a vital role in host defense against MTB infection. TLR2 recognizes diacylated or triacylated lipoproteins by forming heterodimers with TLR6 or TLR1 [80]. TLR2 -/mice are more susceptible following aerosol infection with 2000 CFU MTB per lung, and the serum level of proinflammatory cytokine IL-12p40 is lower than that of WT mice 10 days postinfection [81]. The role of TLR2 in protection against MTB infection was confirmed by Andre and colleagues' study [82]. The innate immune system senses lipopolysaccharide (LPS) via TLR4 [83]. TLR4 knockout leads to decreased level of IL-12p40 in lung homogenate supernatant 4 weeks postinfection, higher mortality and shorter survival time after intranasal inoculation with 10 5 or 5 × 10 5 CFU [84] or aerosol infection with 2000 CFU MTB [85], but the Reiling and colleagues study showed that TLR4 -/mice succumb to aerosol infection with 2000 CFU MTB with similar kinetics as WT mice [81]. TLR9 recognizes unmethylated CpG dinucleotides in microbial DNA sequences [86]. The genome of MTB possesses highly immunostimulatory CpG motifs [87]. DNA from MTB induces production of IL-12p40 and IL-6 in BMDCs and BMDMs. TLR9 -/mice are more susceptible after aerosol infection with 50-100 or 500 CFU MTB, although only infection with the high dose causes higher bacterial burden in lungs. Compared to TLR2 -/or TLR9 -/mice, TLR2/9 -/mice are more susceptible [82]. Deficiency of TLR1 surface expression coupled with specific genotypes is associated with susceptibility to TB [88]. TLR6 -/mice display similar bacterial burden in lungs and spleens, mRNA expression of proinflammatory cytokines and pulmonary histopathology compared with WT mice [89].
Adaptor TIRAP mediates MyD88 transport to plasma membrane to mount an inflammatory response. However, association between Tirap polymorphism and TB risk are controversial, perhaps due to the different ethnicities. The heterozygous genotypes of Tirap C539T (also known as rs8177374 or S180L) in south Indian [132], G286A in China [133] and C558T in Vietnam [134] are associated with high risk for pulmonary tuberculosis (PTB), while the C539T variant in UK, Vietnam, several African countries [135], Colombia [136], Italy, Romania and Ukraine [137] was found to be a protective factor against PTB. Tirap polymorphisms in South Africa [138], Russia, Ghana, Indonesia [139], Colombia [140] and Zhengzhou, China [141] are not involved in TB susceptibility. In addition, Tirap -/mice possess the similar capacity to control acute MTB infection [142]. Liu and colleagues conducted a meta-analysis to evaluate the association between TIRAP C539T polymorphism and TB risk based on the data from 16 studies published from 2006 to 2013. The results indicates that TIRAP C539T is associated with decreased risk for PTB, especially in Europe [143].
In spite of the role of the NLRP3 inflammasome in host defense against MTB which is demonstrated by plenty of in vitro studies, in vivo studies show that only ASC mediates host protection during chronic MTB infection, while NLRP3 and caspase-1 are dispensable [35]. MTB bacterial burden in lungs and spleens, IL-1β and IL-1α concentrations in lung homogenates, the size, morphology and cellular composition of the lung lesions are not affected by NLRP3 absence following infection with virulent MTB via aerosol [63]. Nlrp3 -/mice also have a similar survival profile to WT controls. Compared to WT mice, Caspase-1 -/mice display similar levels of bacteria in the lungs and survival profile as well as even higher levels of IL-1β in lung homogenate extracts. ASC disruption leads to decreased survival time and fewer granulomas, although it has no effect on mycobacterial burden in the lungs [35]. Thus, Nlpr3 -/and caspase-1 -/mice have compensatory mechanisms of processing IL-1β and forming organized granulomas, and ASC is involved in host defense against MTB in NLRP3-and caspase-1-independent manners.

MTB and the AIM2 Inflammasome
If MTB infection activates or inhibits the AIM2 inflammasome is of debate. On the one hand, MTB residing in the phagosomes permeabilizes the phagosomal membrane early after infection via the ESX-1 secretion system, which results in release of phagosomal contents, including MTB and its DNA, into the cytosol [148]. How DNA is liberated from MTB is still unclear. Saiga and colleagues found that released DNA is sensed by and co-localized with AIM2, provoking AIM2 inflammasome activation. Compared to peritoneal macrophages from WT mice, cleavage of caspase-1 and expression of IL-1β and IL-18 at both the mRNA and protein levels are reduced in the cells from Aim2 -/mice following infection with MTB [149]. M. bovis, a member of the MTB complex, is also able to cause TB in human beings. Its genome sequence is more than 99.95% identical to that of MTB [150]. Yang and colleagues found that M. bovis challenge induces upregulation of AIM2 at or after 24 hpi in J774A.1 macrophages and BMDMs. The siRNA-mediated knockdown of AIM2 expression impairs caspase-1 activation and IL-1β secretion, as well as release of lactate dehydrogenase (LDH) at 24 hpi in J774A.1 cells [151]. On the other hand, Shah and colleagues found that IL-1β release is inversely correlated with the virulence in mycobacterial species based on the detection of IL-1β levels in the culture supernatant following infection with Mycobacterium smegmatis, Mycobacterium fortuitum, M. kansasii, MTB H37Ra and MTB H37Rv. Aim2 deletion makes no change to IL-1β secretion in LPS-primed BMDCs at 16 hpi after challenging with MTB H37Rv. LPS-primed cells pretreated with MTB H37Rv, but not ESAT-6 deletion mutant, secrets less IL-1β and IL-18 in response to M. smegmatis or poly(dA:dT), indicating that virulent MTB strains inhibits AIM2-dependent IL-1β release [152]. These two different conclusions may result from the two following reasons: firstly, Shah and colleagues used LPS-primed BMDCs, while Saiga et al. and Yang et al. utilized the cells that have not been pretreated with LPS. MTB infection activates the NLRP3 inflammasome, and LPS is supposed to promote NLRP3-dependent IL-1β secretion for its function in priming, which is required for NLRP3 activation [51]. This may decrease the contribution of AIM2 to MTB-mediated IL-1β release. In addition, the priming step is dispensable for AIM2 inflammasome activation, and poly(dA:dT) is able to activate caspase-1 in an AIM2-dependent manner in the absence of LPS [51]. Whether MTB pretreatment induces reduced IL-1β release in response to only poly(dA:dT) is still unclear. M. smegmatis without LPS induces little IL-1β release in J774A.1 cells and BMDMs [153]. Thus, more evidence is needed to support that the MTB infection inhibits AIM2 inflammasome activation and resultant IL-1β release. Secondly, Saiga and colleagues used BMDCs, while BMDM and J774A.1 macrophages were used in the former two studies. In addition to AIM2, MTB DNA released into the cytosol can also be sensed by cyclic GMP-AMP synthase (cGAS) [154,155] and interferon-γ inducible protein 204 (IFI204) [154,156]. This triggers activation of type I IFNs signaling and autophagy.
AIM2 is indispensable for host defense against MTB infection. Aim2 -/mice succumb within 7 weeks following intratracheal infection with MTB H37Rv, while WT mice are able to survive at least 8 weeks. At 4 weeks postinfection, higher bacterial load in the lungs and livers, more evident granulomatous changes and increased inflammatory cell infiltration in the lungs were found in Aim2 -/mice. At 3 weeks after infection, the levels of IL-1β in BALF and IL-18 in serum from Aim2 -/mice are lower than that from WT mice [149].

Regulation of Inflammasome Activation during MTB Infection
IFNs inhibit MTB-mediated inflammasome activation. Type I IFNs inhibits production of IL-1α and IL-1β in macrophages and DCs in lungs of MTB-infected mice [8]. cGAS generates the second messenger cGAMP after recognizing and binding MTB or M. bovis DNA released into the cytosol. Stimulator of interferon genes (STING) interacts with cGAMP, contributing to production of type I IFNs through activating interferon regulatory factor 3 (IRF3) [154,157]. Type I IFNs are detrimental for the control of MTB [158,159]. They play an inhibitory role in IL-1β production at its mRNA level. Addition of exogenous IFN-β or supplementation of culture medium with neutralizing antibody for IFN-α/βreceptor 2 (IFNABR2) affects the expression of IL-1β mRNA, rather than caspase-1 cleavage. M. bovis BCG does not trigger significant mRNA expression of type I IFNs [160]. Guarda and colleagues proposed that Type I IFNs inhibit inflammasome activation and IL-1β production through two independent mechanisms. On the one hand, Type I IFNs bind IFNAR, inducing secretion of anti-inflammatory cytokine IL-10. IL-10 interacts with its receptor IL-10R, decreasing the expression of pro-IL-1 at the protein level via activation of signal transducers and activators of transcription 3 (STAT3). The inhibitory effect of IFN-α or IFN-β on expression of pro-IL-1α and pro-IL-1β becomes less prominent in BMDMs iso-lated from Stat3 -/or Il-10 -/mice. Compared to control Stat3 flox/− BMDMs, the NLRP3 agonist aluminum slats-mediated caspase-1 cleavage is not altered in the presence of type I IFNs in Stat3 -/cells. On the other hand, STAT1 is phosphorylated at tyrosine 701, which mediates inhibition of NLRP3-dependent caspase-1 activation. IFN-α or IFN-β fails to induce inhibition of activated caspase-1 in Stat1 -/-BMDMs in response to aluminum salts. IFN-β inhibits activation of the NLRP1b and NLRP3 inflammasomes, but not the AIM2 and IPAF inflammasomes. IFN-β inhibits caspase-1 activation following stimulation with NLRP3 inducers, including monosodium urate crystals, asbestos, nigericin, ATP and Candida albicans, and the NLRP1b inducer Bacillus anthracis lethal toxin, rather than the AIM2 agonist poly(dA:dT) or the IPAF agonist Salmonella typhimurium, though amounts of mature form and precursor of IL-1β are diminished in all cases [161]. IL-1 and type I IFNs mutually regulate each other via prostaglandin E2 (PGE2) to control the balance. Ifnar1 knockout results in increased PGE2 and IL-1β in BALF, and addition of exogenous IFN-β to MTB-infected BMDMs or human MDMs reduces PGE2. Knockout of Il1r1 or IL-1α/β enhances IFN-α and IFN-β at both the mRNA and protein levels [162]. CD4 + T cell-derived IFN-γ plays a protective role in MTB control [163]. It inhibits expression of IL-1α and IL-1β only in inflammatory monocytes [8] and does not influence pro-IL-1 expression as well as caspase-1 activation and IL-1β maturation in BMDMs [161]. Meanwhile, IFN-γ facilitates iron export through control of the expression of iron regulatory proteins hepcidin and ferroportin, and prevents MTB-induced intracellular iron sequestration, retarding the bacterial growth by decreasing iron availability [164].

Concluding Remarks
Remarkable advances in MTB-host interaction have been made. Many studies identified the roles of certain cytokines in host defense against MTB infection. IL-1 plays a protective role, while type I IFNs have a detrimental effect. Most reports demonstrated that MTB triggers NLRP3 inflammasome activation and subsequent maturation and release of proinflammatory cytokines via ESX-1 secretion system and its substrate ESAT-6 in vitro, but NLRP3 and caspase-1 are dispensable for control of MTB in vivo. AIM2 facilitates to restrict MTB replication both in vitro and in vivo. Type I IFNs suppress IL-1β activity through interaction with IFNAR. However, the mechanisms by which IL-1β is regulated is still unclear. AIM2 is indispensable for activities of IL-1β and IL-18, but caspase-1 does not contribute to higher levels of IL-1β in vivo, implicating that AIM2 exerts its protective function in a caspase-1-independent manner after sensing MTB DNA released into the cytosol. Exploration of the role of inflammasome in host defense against MTB infection, especially the regulation of IL-1, contributes to a better understanding of MTB-host interaction and provides potential therapeutic targets for treating TB.

Conflicts of Interest:
The authors declare no conflict of interest.