The role of IL-1β throughout infection of the host with Mtb is complex with some evidence in support of a host protective role and other data supporting a role in increasing host susceptibility (3). First, we will discuss the data demonstrating that IL-1β is of importance for host resistance against infections with Mtb and the possible mechanisms (3, 102–106). Mouse studies demonstrate the hyper susceptibility of mice deficient in the expression of either IL-1α/-β or the IL-1β receptor (107–112). The mechanism of protection conferring host resistance by IL-1β has been proposed to involve cell intrinsic mechanisms via the increase in host cell apoptosis (113) or autophagy signaling (114). The inflammasome activation has been linked to increasing maturation of Mtb-containing phagosomes and thus limiting bacterial growth (115, 116). Nevertheless, another study demonstrates that cell intrinsic mechanisms are not how IL-1β confers host resistance but instead it is mediated via trans-protection of infected cells (117). Although the precise mechanism is unclear, in vivo, a major function of IL-1β seems to be to suppress necrosis of lung cells (110, 118). This seems counterintuitive since inflammasome-mediated IL-1β production is associated with cell death (pyroptosis) but it was shown that, at least in the mouse model, IL-1β production is independent of the inflammasome during Mtb infections (111).

It is well-established that, at least partially, the protective effect of IL-1β in vivo is linked to its capacity to suppress IFN-β expression since increased IFN-β production increases host susceptibility (for review (3, 102, 104, 106, 119, 120). Importantly, IL-1β can suppress IFN-β expression and vice versa (121). This interdependence can be exploited for HDT approaches to boost IL-1β production, reduce IFN-β expression and reduce the associated morbidities and mortalities (118). Interestingly, IFN-β-mediated signaling is associated with increased necrotic cell death during ex vivo Mtb infections which could provide a mechanism for the in vivo observed immunopathologies associated with high IFN-β expression (122). Mtb clinical isolates associated with severe TB evade NLRP3 inflammasome activation suggesting a host protective role of IL-1β (123). Macrophages isolated from patients with inflammatory disease carrying gain-of-function genetic variants in inflammasome genes (NLRP3 and/or CARD8) subsequently infected with Mtb displayed increased growth restriction of Mtb in human macrophages (116). In the zebrafish/Mmar infection model, treatment with the drug clemastine modulates the host innate immunity via potentiation of P2RX7 that enhances calcium transients within infected macrophages in vivo. P2RX7 potentiation augments inflammasome activation, resulting in constraint of mycobacterial growth in zebrafish larvae (124).

In contrast, other data from mouse and human studies point towards a role of IL-1β in increasing host susceptibility (3). Some of the strongest evidence for a positive correlation between increased IL-1β and severity of disease in humans comes from several studies analyzing genetic variability and clinical outcomes. The analysis of single nucleotide polymorphisms (SNP) in the human IL1B gene identified 3 SNPs in the genes promoter region that results in increased IL-1β expression and was associated with more severe tuberculosis possibly due to the increased infiltration of neutrophils (125). A polymorphism in the IL-1 receptor agonist (IL1RA) gene resulted in population with decreased IL1RA and increased IL1B gene expression that was more commonly found in patients with tuberculoid pleurisy (126). Furthermore, several studies using the mouse model suggest a detrimental role of IL-1β to host defense. For example, it was demonstrated that the primary protective mechanism of nitric oxide (NO) during Mtb infection is not antibacterial activity but instead the suppression of inflammasome activation (127, 128). MCC950 inhibits the NLRP3 inflammasome activation in Mtb-infected BMDMs and results in decreased survival of Mtb in addition to reduced processing of IL-1β (129).

In conclusion, it is very likely that, similar to the situation with TNF and IFN-β (130, 131), also for the production of the IL-1β the Goldilocks principle applies with just the right amount of cytokine being produced in the right context at the right time during infection in order to produce a host protective outcome.

Different mycobacterial species express different proteins and lipids which may affect their recognition by NLR/ALR proteins (132) ( Table 1 ). Secretion of proinflammatory mature IL-1β or IL-18 during Mtb infection requires activation of NLRP3 inflammasome (9–11, 17, 19, 22, 133). The activation of the NLRP3 inflammasome after Mtb infection is conserved across various cell types: mouse bone marrow derived macrophages (BMDMs) (4, 5, 8–10, 20), peritoneal exudate macrophages (4, 8), THP-1 human macrophages (5, 7, 8, 10, 11, 19), mouse retinal pigment epithelium (RPE) cells (6), primary microglia (15), Ana-1 mouse macrophage (16), mouse bone marrow derived dendritic cells (BMDCs) (10, 22), J774A.1 mouse macrophages (5), PBMCs (7) and human monocyte derived macrophages (hMDMs) (11) (see also Table 1 ). The adaptor ASC, NLRP3 and caspase-1/11 are required for the secretion of IL-1β in Mtb-infected BMDCs (22). Mtb infection induces increased K⁺ and Cl^- efflux but does not affect Ca²⁺ flux (134). The phagosomal and mitochondrial ROS are not involved in the activation of the NLRP3 inflammasome upon Mtb infection but instead it is cytosolic ROS, generated by the xanthine oxidase (XO) (134). Interestingly, plasma membrane damage mediated by ESX-1 system triggers increase in K⁺ efflux, consequently activating the NLRP3 inflammasome and pyroptosis, which permits the spreading of Mtb to neighboring cells (135).

The activation of the NLRP3 inflammasome has also been demonstrated for non-tuberculous mycobacteria (NTM) species including Mycobacterium marinum (Mmar) (12, 17), Mycobacterium abscessus (Mab) (13, 21) and Mycobacterium kansasii (Mkan) (14) and Mycobacterium ulcerans (26) ( Table 1 ). More specifically, Mab leads to caspase-1 activation and release of IL-1β in human macrophages. Dectin-1/Syk-dependent signaling, increased expression of the cytoplasmic scaffold protein p62/SQSTM1 and potassium efflux are implicated in Mab mediated NLRP3 inflammasome activation (13). Consistently, it has been demonstrated that Mab results in increased production of IL-1β in murine macrophages (21). Mab induces mitochondrial ROS and thereby leads to enhanced NLRP3 inflammasome activation (21). A recent study has demonstrated that infection of microglia with the attenuated Mtb H37Ra strain triggers NLRP3 mediated secretion of IL-1β and IL-18 (15). Moreover, they also found that by inhibiting NF-κB (signal 1) and P2X7R (signal 2) they can alter the secretion of IL-1β and IL-18 and thereby regulate the NLRP3 inflammasome pathway in microglia during Mtb infection (15) ( Table 1 ). Interestingly, irrespective of the overexpression of NLRP3 and inflammatory caspases-4/5 detected in the lepromatous pole, low expression of caspase-1, IL-1β, and IL-18 were observed in leprosy and therefore these results indicate that NLRP3 inflammasome does not actively contribute to the innate immune response in leprosy, suggesting immune evasion of M. leprae (136).

In addition to NLRP3 inflammasome, different reports have implicated the critical role for the AIM2 inflammasome following mycobacterial infection. Activation of AIM2 inflammasome has been reported in several cell types during infection with various mycobacterial species, including Mycobacterium smegmatis (Msme) (23), Mycobacterium fortuitum (Mfor) and Mkan in BMDCs (23) ( Table 1 ). In BMDCs about 40–50% of the production of IL-1β following Msme, Mfor, and Mkan infection was dependent on the AIM2 inflammasome and moreover there was an inverse correlation between virulence of the mycobacterial species and the amount of IL-1β release, with the least virulent species inducing the highest levels of IL-1β (23). Mycobacterium bovis (Mbov) infection activated the AIM2 inflammasome in BMDMs and J774A.1 mouse macrophage (25) ( Table 1 ). Mbov infection augments the mRNA expression of AIM2 and ASC in both BMDMs and J774A.1 mouse macrophage (25). Potassium efflux and mycobacterial escape into the cytosol are the two essential triggers in the activation of AIM2 inflammasome during Mbov infection. Additionally, infection of J774A.1 mouse macrophage with Mbov results in activation of caspase-1 as early as 6h post-infection (25). The transfection of Mtb genomic DNA into LPS-primed peritoneal macrophages results in AIM2-dependent caspase-1 activation and subsequent secretion of mature IL-1β and IL-18 (24). This is not surprising since AIM2 recognizes any type of dsDNA but interesting because Mtb does not activate the AIM2 inflammasome upon infection of BMDCs or BMDMs (23).

The Proline-Proline-Glutamate (PPE) family protein, PPE13, participates in the assembly of NLRP3 inflammasome complex via its C-terminal repetitive major polymorphic tandem repeat (MPTR) domain by directly interacting with the NACHT and Leucine-rich repeat (LRR) domains of NLRP3 (5) ( Figure 2 and Table 1 ). A recombinant Msme strain expressing the Mbov PPE13 induces increased cell death and increased secretion of IL-1β in J774A.1, BMDMs, and THP-1 macrophages but to different levels depending on the cell type (5). The release of IL-1β was dependent on activation of NLRP3 inflammasome as confirmed by caspase-1 and NLRP3 inflammasome inhibitor studies (5). The role of PPE13 in inflammasome signaling needs further validation by creating a gene specific knockout in Mtb.

Recent studies show that Rv1579c, located within the Mtb H37Rv region of difference 3 (RD3), encodes for a protein (EST12) which acts as a pyroptosis-inducing protein (8) ( Figure 2 and Table 1 ). Indeed, EST12 interacts with the host protein receptor for activated C kinase 1 (RACK1) and forms a EST12-RACK1 complex in macrophages. The EST12-RACK1 dimer recruits the deubiquitinase UCHL5 to stimulate the K48-linked deubiquitination of NLRP3 and consequently triggers the NLRP3 inflammasome mediated pyroptosis and IL-1β secretion (8) ( Figure 2 ). Mice infected with an Mtb strain lacking EST12 showed significant increase in the bacterial growth in the lungs and lower levels of serum IL-1β compared to wild-type Mtb-infected mice. Consistently, mice infected with BCG or Msme strains overexpressing EST12 showed lower bacterial burden in lungs or spleen and increased levels of IL-1β compared to mice infected with control bacteria. Consequently, Mtb EST12 increases mycobacterial clearance in mice and is responsible to activate the host’s immunity (8). It will be interesting to see in future studies which Mtb secretion system is responsive for the secretion of EST12.

The Mtb lipoprotein, LpqH, activates the NLRP3 inflammasome via a mechanism that involves activation of the TLR-2 receptor (16) ( Figure 2 and Table 1 ). The treatment of LPS-primed Ana-1 mouse macrophages with purified LpqH protein results in increased expression of NLRP3, ASC and caspase-1 proteins in a dose-dependent manner (16). Moreover, potassium efflux acts as an important trigger for LpqH-mediated activation of the NLRP3 inflammasome (16). However, the underlying mechanism of how LpqH influences potassium efflux has not been explored in this study. The work was performed in mouse macrophages and thus further validation in human macrophages would be valuable. It will also be crucial to test a lpqH deletion mutant of Mtb for changes in NLRP3 inflammasome activation to confirm that the observed activity of purified proteins is conserved within the context of a whole bacterium.

The Mtb RD1 locus which encodes for the ESX-1 secretion system is required for activation of NLRP3 inflammasome and subsequent release of IL-1β in human PBMCs, THP-1 cells and mouse BMDMs and retinal pigment epithelium (RPE) cells (4, 6, 7, 9, 11, 14, 19, 20) ( Figure 2 and Table 1 ). The ESX-5a is a duplicated region of 4 genes out of the ESX-5 secretion system which is important for the secretion of subset of ESX-5-secreted proteins and its deletion results in reduced inflammasome activation (10) ( Table 1 ).

EsxA is one of the main substrates secreted by the ESX-1 system and consequently many studies have been performed by adding purified EsxA to cells during ex vivo experimentations. EsxA interacts with TLR-2 and TLR-4 receptors to induce host cell signaling (141–144). One report shows that treatment of mouse RPE cells with different doses of EsxA results in caspase-1 activation in a dose dependent manner and that this activation is dependent on TLR/MyD88 signaling and the NLRP3 inflammasome (6). Other studies reveal that stimulation of either PBMCs (7) or THP-1 macrophages (7, 19) with EsxA results in increased release of proinflammatory cytokine IL-1β. Furthermore, stimulation of PBMCs and THP-1 derived macrophages with either Mtb protein EsxA or heat inactivated Mtb lysates induces increase in expression of MFN2 and results in increased release of IL-1β. Therefore, these findings suggest that MFN2 is required for the assembly and activation of NLRP3 inflammasome during Mtb infection (7). Transcriptional profiling in human PBMCs from active tuberculosis patients and healthy controls determined that a mitochondrial outer membrane protein, MFN2 expression was significantly upregulated in TB patients compared to healthy controls (7). Intriguingly, another study reported the importance of EsxA and RD1 locus in secretion of IL-1β, since BMDMs when infected with Mtb strains lacking esxA or RD1 showed a significant reduction in the secretion of IL-1β compared to the BMDMs infected with Mtb (20). EsxA was partially responsible for the release of mature cathepsin B by the lysosomes during Mtb infection and further leads to NLRP3 inflammasome activation in BMDMs (20). In addition to lysosomal permeabilization, EsxA also triggers phagosome damage and Syk activation in human macrophages that result in NLRP3 mediated necrotic cell death (11). Additionally, EsxA facilitates the translocation of other immunostimulatory Mtb components such as Ag85 into the macrophage cytosol, resulting in increased activation of caspase-1 and subsequent secretion of IL-1β (19).

Critical role for the ESX-1/EsxA in NLRP3 activation has also been reported during the infection with non-tuberculous mycobacteria species including Mmar (12, 17, 18), and Mkan (14) ( Table 1 ). In addition to ESX-1, ESX-5 secreted substrates also play a role in activation of NLRP3 inflammasome in response to infection with Mmar (12) ( Table 1 ). Additionally, transfection of mouse RPE cells with mycobacterial dsRNA induces NLRP3 inflammasome-dependent caspase-1 activation via an uncharacterized mechanism (6).

The role of mycobacterial cell wall lipids in NLRP3 inflammasome activation has also been studied in addition to mycobacterial secreted effector proteins (145). Trehalose-6,6’-dibehenate (TDB), a synthetic analogue of Trehalose-6,6’-dimycolate (TDM) also known as mycobacterial cord factor has been developed as an effective adjuvant for tuberculosis subunit vaccine and both act as a potent proinflammatory pathogen-associated molecular pattern (PAMP) which is recognized by macrophage inducible C-type lectin (Mincle) receptor on innate immune cells and triggers host innate immune response. TDB induces the NLRP3/ASC/Caspase-1 mediated increase in production of IL-1β in BMDCs. Activation of NLRP3 inflammasome by TDB involves numerous triggers such as lysosomal permeabilization, increased ROS generation and increased potassium efflux (145).

It is important to remember that the NLRP3 inflammasome is acting most likely as the cells stress or danger sensor and thus any bacterial induction of the NRLP3 inflammasome might not be mediated by a direct interaction with the inflammasome complex but through interaction with other cellular components/pathways that then trigger the stress/danger signal.

As discussed before many studies have shown that Mtb causes NLRP3 inflammasome activation and even that the deletion of certain Mtb genes led to an increased activation of the NLRP3 inflammasome ( Figure 2 and Table 1 ). Nevertheless, not until recently was it shown that Mtb infection can inhibit activation of the NLRP3 inflammasome via either LPS/Nigericin or LPS/ATP stimuli (134). Mtb inhibits the NLRP3 inflammasome activation via a mechanism that is independent of the ESX-1 secretion system which is opposed to the capacity of Mtb to inhibit the AIM2 inflammasome in an ESX-1 dependent mechanism (23, 134). Mtb infection inhibits the LPS/ATP-induced K⁺ efflux and increase in xanthine oxidase (XO) activity leading to decreased cytosolic ROS levels (134). The Mtb serine threonine kinase, PknF, mediates inhibition of NLRP3 inflammasome dependent production of IL-1β and pyroptosis in both mouse and human derived primary macrophages (134). Moreover, K⁺ efflux, Cl²⁺ efflux and ROS generation are implicated in the PknF-mediated NLRP3 inflammasome inhibition (134) ( Figure 2 ). Additionally, the Mtb pknF mutant induces an increase in the XO activity compared to Mtb-infected cells and thus increasing XO-mediated ROS production (134). Altogether it seems that PknF is inhibiting the exact NLRP3 inflammasome activation pathway that is being only slightly activated upon Mtb infection.

The BCG gene zmp1 encodes a zinc metalloprotease that has been shown to inhibit the NLRP3 inflammasome dependent processing of IL-1β (115). However, these findings could not be independently confirmed since generation of the zmp1 Mtb deletion mutant strain did not show any effect on pyroptosis, on the caspase-1 activation nor the release of IL-1β (11). The inconsistency between these two publications might be due to a difference in the background of the zmp1 mutant strain because most of the studies performed by Master et al. were done using the BCG strain that, unlike Mtb, lacks a functional ESX-1 secretion system. Another possibility may be the difference in cell types used to study the strains lacking zmp1.

The Mtb serine hydrolase, Hip1, inhibits the NLRP3 inflammasome activation by dampening the TLR2-dependent cell signaling in BMDMs (148) ( Figure 2 ). Hip1 not only affects the secretion of inflammasome dependent cytokines but also result in decrease of other proinflammatory cytokines thus suggesting Hip1 modulates the proinflammatory responses in macrophages by preventing the activation of TLR2 dependent cell signaling (148). The mechanism involves Hip1-mediated proteolytical cleavage of GroEL2 from multimer to monomer and the cleaved monomeric GroEL2 subsequently contributes to dampening of proinflammatory responses in macrophages mediated by TLR2 signaling (149). Therefore, it is not possible to attribute the in vivo attenuation of the hip1 Mtb deletion mutant to its increase in inflammasome activation since also other important proinflammatory cytokines such as TNF are upregulated (148).The Mtb protein, Rv3364c, binds to and inhibits the membrane associated host serine protease cathepsin G which leads to suppression of caspase-1 activity and pyroptosis in macrophages (150) ( Figure 2 ).

Host cell derived NO acts as a negative regulator of NLRP3 inflammasome activation and inhibits processing of IL-1β (127, 151) ( Figure 2 ). Stimulation of Mtb-infected macrophages with IFN-γ showed iNOS-dependent thiol nitrosylation of NLRP3 which leads to inhibition of NLRP3 inflammasome dependent maturation of IL-1β and minimize inflammatory tissue damage during chronic Mtb infection (127). Indeed, the NO-mediated nitrosylation inhibits the assembly of NLRP3 inflammasome complex (127, 151).

During mycobacterial infection, a host serine/threonine protein kinase, the oxidative stress responsive kinase 1 (OXSR1), inhibits K⁺ channels responsible for K⁺ efflux (152). Indeed, the mycobacterial infection leads to the upregulation of host OXSR1 and this host cell manipulation is dependent on the mycobacterial ESX-1 secretion system. The immunomodulatory role of OXSR1 is conserved in both zebrafish and humans (152). Furthermore, inhibition or depletion of OXSR1 results in diminished levels of intracellular potassium and hence limits the growth of mycobacteria. Therefore, targeting of OXSR1 might be a valuable approach for host-directed therapy. Micheliolide (MCL), a sesquiterpene lactone, act as an anti-inflammatory molecule by inhibiting PI3K/Akt/NF-κB and NLRP3 inflammasome signaling during Mtb infection (153). However, previous report showed that RAW264.7 cells do not release mature IL-1β since they do not express ASC as determined by immunoblot analysis with an ASC-specific antibody (154). The study by Zhang et al. did not address this problem since all experiments were performed in RAW264.7 macrophages and hence there remains some questions on the validity of their results.

Overall, there has been notable progress but still the molecular mechanisms by which Mtb evades NLRP3 inflammasome activation and the importance of this manipulation for virulence of Mtb remain poorly understood.