The RD1 locus in the Mycobacterium tuberculosis genome contributes to the maturation and secretion of IL-1α from infected macrophages through the elevation of cytoplasmic calcium levels and calpain activation
Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing, China
Department of Microbiology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan
This is a well-written and detailed report proceeds logically with a series of in vitro experiments that show that release of mature IL-1alpha by macrophages following a Mycobacterium tuberculosis infection is dependent on RD1. The requirements for the cysteine protease calpain and for RD1-dependent increase of intracellular Ca2+ levels for cleavage of pro-IL-1alpha are demonstrated.
Region of difference 1 (RD1) is a genomic locus in the Mycobacterium tuberculosis genome that has been shown to participate in the virulence of the bacterium, induction of cell death, and cytokine secretion in infected macrophages. In this study, we investigated the role of RD1 in interleukin-1α (IL-1α) secretion. M. tuberculosis H37Rv strain, but not a mutant strain deficient for RD1 (∆RD1), significantly induced IL-1α secretion from infected macrophages. Although IL-1α secretion was only observed in H37Rv-infected macrophages, there was no difference in the level of IL-1α transcription and pro-IL1α synthesis after infection with H37Rv and ∆RD1. Interestingly, ∆RD1 infection did not increase intracellular Ca2+ levels, and Ca2+ chelators markedly inhibited IL-1α secretion in response to H37Rv infection. Moreover, the inability of ∆RD1 to induce IL-1α secretion was restored by treatment with the calcium ionophore A23187. A significant increase in calpain activity was detected in macrophages infected with H37Rv, but not with ∆RD1, and calpain inhibitors abrogated IL-1α secretion. Taken together, these results suggest that in M. tuberculosis-infected macrophages, RD1 contributed to maturation and secretion of IL-1α by enhancing the influx of Ca2+ followed by calpain activation.
Mycobacterium tuberculosis, an etiologic agent of tuberculosis, is one of the leading threats to humans. Based on estimates by the World Health Organization (WHO), M. tuberculosis caused 8.7 million new cases of tuberculosis and 1.4 million deaths worldwide in 2011 (WHO, 2012). The recent emergence of extensively drug-resistant M. tuberculosis and its co-infection with HIV highlights the urgent need for an improved understanding of the pathogenesis of M. tuberculosis, which may identify novel approaches to treat and prevent tuberculosis (O'Donnell et al., 2013).
A genomic locus of M. tuberculosis called ‘region of difference 1′ (RD1) is approximately 9.5 kb in length, and it was first discovered as a region that is absent in a genome of M. bovis BCG (Mahairas et al., 1996). RD1 is critical for bacterial virulence (Pym et al., 2002; Stanley et al., 2003, 2007; Gao et al., 2004; Guinn et al., 2004; Majlessi et al., 2005; Brodin et al., 2006), necrosis induction (Hsu et al., 2003; Junqueira-Kipnis et al., 2006; Chen et al., 2007; Kaku et al., 2007), granuloma formation (Volkman et al., 2004), and the generation of protective immunity (Pym et al., 2003; Brodin et al.,2004a, 2004b). RD1 is located in the ESX-1 locus (Feltcher et al., 2011), which is one of the specialized export systems of M. tuberculosis. The ESX-1 system is named for the secretory component, the 6-kDa early secreted antigenic target (ESAT-6) of M. tuberculosis and has been referred to as a type VII secretion system (Abdallah et al., 2007). The ESX-1 locus contains genes encoding secretory proteins and a suite of genes encoding the secretion machinery (Feltcher et al., 2011). Although all the components have not yet been fully characterized, proteins with known functional domains were identified. They include putative chaperones with an AAA+ ATPase (Rv3868; EccA1, Rv3871; EccCb1), a subtilisin-like serine protease (Rv3883c; MycP1), and FtsK/SpoIIIE-like ATPase (Rv3870; EccCa1). EccB1 and EccE1 are shown to serve as core components of ESX-1. rv3877 (eccD1) is located in the RD1 region and predicted to be a membrane-spanning protein that could be part of the translocation pore in the cytoplasmic membrane. The RD1 region also contains genes coding for two secretory components, ESAT-6 (EsxA) and culture filtrate protein of 10 kDa (CFP-10; EsxB). Therefore, deletion of the RD1 locus completely abrogates the ESX-1 secretory system.
Upon infection with M. tuberculosis, macrophages secrete a variety of cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, IL-12, and IL-18 (Patel et al., 2007; Kurenuma et al., 2009). Mycobacterium tuberculosis infection is accompanied by an intense local inflammation, which is likely due to these proinflammatory cytokines. In fact, TNF-α, IL-1, and IL-6 mediate the tissue injury in animal models of tuberculosis (Grover et al., 2008). IL-1 is a prototypic multifunctional cytokine and was initially characterized as a factor that causes fever and augments lymphocyte responses (Dinarello, 1996). In addition, several studies have shown that IL-1α is recognized as a danger signal that is released from necrotic cells and functions as a key mediator of the inflammatory response (Chen et al., 2007; Sims & Smith, 2010). Furthermore, IL-1α secretion could be of significance in terms of spreading proinflammatory signals to healthy neighboring cells in vivo (Schaub et al., 2003), suggesting that IL-1α plays a key role in the pathological outcome. On the other hand, recent reports have shown that IL-1 receptor knockout mice are highly susceptible to M. tuberculosis infection (Fremond et al., 2007), and both IL-1α and IL-1β serve as mediators of protective immune responses to M. tuberculosis (Mayer-Barber et al., 2001; Guler et al., 2011). These findings suggest that IL-1α, together with IL-1β, is critical to controlling acute M. tuberculosis infection and that coordinated regulation of IL-1 activity may be necessary to govern host immune response to M. tuberculosis.
Mycobacterium tuberculosis infection induces the production of the proinflammatory cytokines IL-1α, IL-1β, and IL-18, which belong to the IL-1 superfamily (Sims & Smith, 2010). IL-1β and IL-18 are produced as proforms that are cleaved by caspase-1 and secreted as an active form. Previous studies demonstrated the activation of caspase-1 by the induction of potassium ion efflux in M. tuberculosis-infected macrophages, and the RD1 locus of M. tuberculosis was required for the induction this ion efflux (Kurenuma et al., 2009). In addition to IL-1β, a large amount of IL-1α is produced by infected macrophages, and calpain, a Ca2+-dependent cysteine protease, is predicted to contribute to the processing and secretion of IL-1α (Carruth et al., 1991; Goll et al., 2003). However, the precise mechanism underlying IL-1α secretion remains undefined. In this study, we determined that M. tuberculosis infection induced IL-1α mRNA expression and pro-IL1α synthesis independently of RD1. Furthermore, the RD1 gene locus was indispensable for the induction of Ca2+ influx and the activation of calpain, which were required for the processing and secretion of IL-1α.
Materials and methods
EGTA was purchased from Nacalai Tesque Inc. (Kyoto, Japan). BAPTA-AM and A23187 were obtained from Sigma-Aldrich (St. Louis, MO). Calpain inhibitors, MDL28170 (carbobenzoxy-valyl-phenylalanial), EST ((2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester), and calpain inhibitor IV (Z-LLY-FMK), were obtained from Merck Chemicals (Tokyo, Japan).
Female C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). Mice were maintained under specific pathogen-free conditions and used at 7–9 weeks of age. All animal experiments were approved by the Animal Ethics and Research Committee of Kyoto University Graduate School of Medicine.
The following M. tuberculosis strains were kindly provided by Prof. William R. Jacobs (Albert Einstein Institute, Bronx, NY) (Hsu et al., 2003): H37Rv strain, an H37Rv mutant strain deficient for the RD1 locus (∆RD1), and an RD1-complemented strain (∆RD1::RD1). These M. tuberculosis strains were grown at 37 °C to mid-log phase in Middlebrook 7H9 broth supplemented with 0.5% albumin, 0.2% dextrose, 3 μg mL−1 catalase, and 0.2% glycerol. Bacteria were harvested, stirred vigorously with glass beads (3 mm in diameter), and centrifuged at 300 × g for 3 min to remove the bacterial clumps. The suspension was stored at −80 °C in aliquots. After thawing, the number of viable bacteria was enumerated by counting the colonies after plating the diluted suspension on Middlebrook 7H10 agar plates containing enrichments (50 μg mL−1 oleic acid, 0.5% albumin, 0.2% dextrose, 4 μg mL−1 catalase, and 0.85 mg mL−1 sodium chloride).
Peritoneal exudate cells were obtained by a peritoneal lavage 4 days after an intraperitoneal injection with 3 mL of thioglycollate medium (EIKEN Chemical, Osaka, Japan) and plated at 5.0 × 105 cells well−1 in 48-well tissue culture plates. After incubation for 3 h, nonadherent cells were removed and adherent cells were used as macrophages. Based on Giemsa staining and a phagocytosis assay using latex beads, more than 98% of adherent cells were identified as macrophages.
Intracellular growth of bacteria
Macrophages were infected with H37Rv, ∆RD1, and ∆RD1::RD1 at a multiplicity of infection (MOI) of 5 for 3 h. Cells were washed to remove extracellular bacteria and cultured for 21 h at 37 °C. Cells were then lysed in phosphate-buffered saline (PBS) containing 0.05% Triton X-100, and the number of viable bacteria was enumerated by inoculating the cell lysate on Middlebrook 7H10 agar plates containing enrichments and counting colonies 3 weeks later.
Macrophages were infected with H37Rv, ∆RD1, and ∆RD1::RD1 at a MOI of 5 for 3 h, washed, and cultured for 3 h. Total RNA was extracted using the Nucleospin RNA II kit (Macherey-Nagel, Duren, Germany). Total RNA (0.2 μg) was treated with an RNase-free DNase (Promega, Tokyo, Japan) and subjected to reverse transcription (RT) using a Super-Script III first-strand synthesis system for RT-PCR (Invitrogen, Tokyo, Japan). Quantitative real-time RT-PCR was performed on an ABI Prism 7000 (Applied Biosystems, Foster City, CA) using a Platinum SYBR green qPCR SuperMix-UDG (Invitrogen) according to the manufacturer's instructions. The level of IL-1α mRNA was normalized on the basis of β-actin mRNA expression and analyzed with ABI Prism 7000 sds software. DNA sequences of the PCR primers are as follows: IL-1α, 5′-CTCTAGAGCACCATGCTACAGAC-3′ (forward) and 5′-TGGAATCCAGGGGAAACACTG-3′ (reverse); and β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (forward) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse).
Macrophages were infected with H37Rv, ∆RD1, and ∆RD1::RD1 at a MOI of 5 for 3 h. Cells were washed to remove extracellular bacteria and cultured for 21 h. The culture supernatant was collected, and the levels of IL-1α and IL-12/23p40 were determined by the OptEIA mouse IL-1α ELISA set and the OptEIA mouse IL-12/23p40 ELISA set (BD Bioscience Pharmingen, San Diego, CA), respectively. In one experiment, macrophages were infected with each of three M. tuberculosis strains for 3 h, washed, and cultured for 3, 6, 9, 12, and 21 h. The culture supernatant was collected, and the level of IL-1α was measured. Concomitantly, cells were lysed in PBS containing 1% Triton X-100 at each time point. The lysate was centrifuged to prepare the cleared cell lysate, and the level of IL-1α in the cell lysate was also measured. Alternatively, macrophages were infected with ∆RD1 at a MOI of 5 for 3 h. Cells were washed and incubated for 21 h in the presence or absence of 10 μM A23187. The culture supernatant was collected, and IL-1α concentration was determined. In addition, macrophages were infected with M. tuberculosis strains and cultured as described above. The culture supernatant was collected, and cells were lysed in 10 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, and a protease/phosphatase inhibitor cocktail (Nacalai Tesque Inc., Kyoto, Japan). The culture supernatant and the cell lysate were subjected to SDS-PAGE, and separated proteins were transferred to a polyvinylidene difluoride membrane by electroblotting. To detect pro-IL1α (35 kDa) and mature IL-1α (17 kDa), the membrane was treated sequentially with biotinylated goat anti-IL-1α polyclonal antibody (Genzyme, Cambridge, MA) and streptavidin–horseradish peroxidase (HRP) conjugate (Invitrogen). After a brief incubation of the membrane in ECL Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), the bands representative of pro-IL-1α and mature IL-1α were detected in a LAS-4000 mini imager (Fuji Film, Tokyo, Japan).
Macrophages were infected with H37Rv for 3 h. Cells were washed to remove extracellular bacteria, cultured for 3 h, and then treated for 21 h with or without 1 mM EGTA, 20 μM BAPTA-AM, 10–40 μM MDL28170, 100 μM calpain inhibitor IV, and 100 μM EST. The culture supernatant was collected, and the level of IL-1α was measured by ELISA or Western blotting. Preliminary studies showed that the concentration ranges of the Ca2+ chelators and calpain inhibitors exhibited no direct cytotoxicity to macrophages (data not shown). In one experiment, macrophages were infected with three M. tuberculosis strains for 3 h, washed, and cultured for 3, 6, 9, 12, and 24 h. The culture supernatants were collected, and the cell lysates were prepared for each culture period. To evaluate calpain activity, the culture supernatants and the cell lysates were subjected to SDS-PAGE and Western blotting using anti-α-fodrin monoclonal antibody (Chemicon International, Temecula, CA) and HRP-conjugated anti-mouse IgG1 antibody (Invitrogen). We monitored the amount of the proteolytic fragment of α-fodrin, an internal substrate of calpain, in the culture supernatant and that of full-length α-fodrin in the cell lysate.
Lactate dehydorogenase (LDH) release
Macrophages were infected with H37Rv, ∆RD1, and ∆RD1::RD1 for 3 h, washed, and then cultured for 21 h. The LDH activity in the culture supernatant was monitored at 3-h intervals after infection using a LDH cytotoxicity detection kit (TaKaRa BIO). In one experiment, cells were infected with H37Rv for 3 h, washed, and subsequently cultured with or without 1 mM EGTA, 40 μM MDL28170, and 100 μM calpain inhibitor IV for 21 h. The culture supernatant was collected, and LDH activity was measured. The maximum LDH release referred to the LDH value obtained from the supernatant of cells treated with 1% Triton X-100.
Intracellular Ca2+ level
Macrophages were plated at 1.25 × 105 cells well−1 in 96-well black microplates (Invitrogen) and infected with M. tuberculosis H37Rv, ∆RD1, and ∆RD1::RD1 at a MOI of 5 for 3 h. The intracellular Ca2+ concentration was monitored at 3-h intervals for 21 h using a Fluo-4 NW Ca2+ assay kit (Invitrogen) according to the manufacturer's instructions.
Statistical analysis was performed using Student's t-test. A value of < 0.05 was considered to be statistically significant.
The RD1 locus is indispensable for IL-1α secretion from M. tuberculosis-infected macrophages
Based on our investigation into the mechanism underlying the bacterial virulence factors that contribute to cytokine production by infected macrophages, we recently identified that the RD1 locus in the M. tuberculosis genome was critically involved in IL-1β and IL-18 secretions through the activation of caspase-1 (Kurenuma et al., 2009). Furthermore, we also found that IL-1α secretion was highly dependent upon the RD1 locus. In this current study, therefore, we analyzed the mechanism underlying IL-1α secretion from M. tuberculosis-infected macrophages, specifically the role of the RD1 locus. IL-1α secretion by macrophages infected with the H37Rv strain, an RD1-deficient mutant (∆RD1) strain, and an RD1-complemented (∆RD1::RD1) strain was measured. We found that H37Rv, but not △RD1, induced a high level of IL-1α secretion from infected macrophages (Fig. 1a). Furthermore, complementation with the RD1 locus mostly restored the inability of ∆RD1 to induce IL-1α secretion (Fig. 1b). Interestingly, H37Rv and △RD1 induced comparable levels of IL-12/23p40 production. These results clearly demonstrated that RD1 is required for the secretion of IL-1α from infected macrophages, whereas it is dispensable for IL-12/23p40 production. To rule out the possibility that the difference between the induction of IL-1α in response to H37Rv and ∆RD1 infection was due to the efficacy of infection and replication of these bacteria strains, we measured their cfu numbers at 3 h and 24 h after infection. There was no significant difference in the number of bacteria recovered for these M. tuberculosis strains (Fig. 1d). Taken together, these results indicated that RD1 is indispensable for the induction of IL-1α secretion from H37Rv-infected macrophages.
RD1 is involved in the maturation and secretion of IL-1α, but does not participate in the induction of IL-1α transcription in H37Rv-infected macrophages
To analyze the mechanism by which RD1 facilitates IL-1α secretion from H37Rv-infected macrophages, we first determined the level of IL-1α mRNA expression after infection with H37Rv and ∆RD1 by real-time RT-PCR. A significant increase in IL-1α mRNA expression was detected in macrophages infected with H37Rv, and the levels were almost 20-fold higher than that observed in noninfected macrophages (Fig. 2a). Similarly, significantly high levels of IL-1α mRNA expression were also detected in macrophages infected with △RD1. We next compared the level of IL-1α synthesis and its secretion from macrophages infected with H37Rv and ∆RD1. IL-1α is synthesized as a 35-kDa precursor protein (pro-IL1α) that is subsequently converted into the mature IL-1α (17 kDa) and secreted into the extracellular space. To analyze the kinetics of IL-1α production in response to H37Rv and ∆RD1 infection, we quantified the total amount of IL-1α by determining the titer of mature IL-1α in the culture supernatant plus the titer of pro-IL-1α in the cell lysate. Consistent with the pattern of mRNA expression, IL-1α production was similarly observed as early as 3 h after infection with H37Rv and ∆RD1, and the levels gradually increased up to 21 h. There was no difference in the kinetics of IL-1α production after infection with H37Rv and ∆RD1 (Fig. 2b). On the other hand, IL-1α was detected only in the culture supernatant of H37Rv-infected macrophages, and ∆RD1 did not exert the ability to induce IL-1α secretion (Fig. 2c). Western blot analysis revealed similar levels of pro-IL1α synthesis in macrophages after infection with H37Rv and ∆RD1; however, significant IL-1α secretion was only observed in macrophages infected with H37Rv (Fig. 2d). These results indicated that while IL-1α transcription and pro-IL1α synthesis were induced independently of RD1 in H37Rv-infected macrophages, the gene locus plays a role in the maturation and secretion of IL-1α.
Calpain is required for the secretion of IL-1α from H37Rv-infected macrophages
It has been shown that calpain, a Ca2+-dependent cysteine protease, is activated by various stimuli and contributes to the maturation and secretion of IL-1α (Kobayashi et al., 1990). To determine whether calpain is also involved in IL-1α secretion from H37Rv-infected macrophages, we investigated the effect of several calpain inhibitors on IL-1α secretion. As shown in Fig. 3, MDL28170 dramatically inhibited IL-1α secretion from H37Rv-infected macrophages in a dose-dependent manner. Furthermore, two other calpain inhibitors, calpain inhibitor IV and EST, also impaired IL-1α secretion. In addition, the Ca2+ chelators EGTA and BAPTA-AM markedly inhibited cytokine secretion. On the other hand, these reagents did not affect IL-12/23p40 secretion from H37Rv-infected macrophages. Taken together, these results suggested that calpain is involved in the secretion of IL-1α from H37Rv-infected macrophages. To further clarify the contribution of calpain to H37Rv-induced IL-1α secretion, we measured the activity of calpain in infected macrophages. It has been shown that α-fodrin, a 240-kDa cytoskeletal protein, is an endogenous substrate of calpain, and the amount of the degraded fragment (145 kDa) released in the culture supernatant is indicative of calpain activity (Wang, 2000; Goll et al., 2003). Thus, we analyzed the amount of fragmented α-fodrin in the culture supernatant after infection with H37Rv, ∆RD1, and ∆RD1::RD1. As shown in Fig. 4a, the amount of fragmented α-fodrin was detected in the culture supernatant as early as 6 h after infection with H37Rv and gradually increased until 24 h. However, ∆RD1 hardly induced the breakdown of α-fodrin. The kinetics of degraded product largely paralleled time-dependent IL-1α secretion. Moreover, H37Rv markedly decreased the amount of full-length α-fodrin in the cell lysate and increased the small fragment of α-fodrin in the culture supernatant (Fig. 4b). However, ∆RD1 infection induced a marginal level of α-fodrin fragmentation. On the other hand, significant levels of fragmented α-fodrin were observed in macrophages infected with ∆RD1::RD1. These results suggested that calpain was involved in IL-1α secretion from H37Rv-infected macrophages with RD1 playing a critical role in calpain activation.
RD1 is implicated in calpain activation and IL-1α secretion through the elevation of intracellular Ca2+ levels in H37Rv-infected macrophages
Calpain is a Ca2+-dependent cysteine protease, and an increase in the cytosolic Ca2+ level plays a major role in calpain activation (Goll et al., 2003). Therefore, we next analyzed the kinetics of cytosolic Ca2+ levels in macrophages infected with H37Rv, ∆RD1, and ∆RD1::RD1 using the Fluo-4 NW Ca2+ assay kit. H37Rv-infection markedly increased cytosolic Ca2+ levels as indicated by the high fluorescence intensity of Fluo-4. The fluorescence intensity was markedly enhanced 6 h after infection and maintained until 21 h (Fig. 5a). In contrast, ∆RD1 induced a weak fluorescence intensity compared with H37Rv, and it was sustained, albeit at a lower level, during the culture period. On the other hand, ∆RD1::RD1 induced a high level of fluorescence intensity with similar kinetics as that of H37Rv, Therefore suggesting that RD1 was involved in the induction of Ca2+ influx in H37Rv-infected macrophages. To further elucidate the contribution of RD1, we investigated whether A23187, a calcium ionophore, restored the inability of ∆RD1 to induce IL-1α secretion. As shown in Fig. 5b, ∆RD1 induced a low level of IL-1α secretion; however, treatment with A23187 significantly enhanced IL-1α secretion. These data suggested that RD1 contributes to the increase in cytosolic Ca2+ levels in infected macrophages, and the RD1-dependent Ca2+ influx leads to calpain activation and IL-1α secretion from infected macrophages.
In this study, we examined the role of RD1 in IL-1α secretion from H37Rv-infected macrophages. Our results clearly showed that H37Rv infection induced a high level of IL-1α secretion, and RD1 was indispensable for the maturation and secretion of IL-1α; however, RD1 did not contribute to the induction of IL-1α transcription and pro-IL1α synthesis. Finally, RD1 was implicated in calpain activation through the increase of cytosolic Ca2+ levels.
Recent studies have shown that M. tuberculosis has TLR ligands, and the cytokine production by TLR2- and TLR4-deficient macrophages in response to M. tuberculosis is markedly decreased compared with that of WT macrophages (Means et al., 2001). Therefore, it is highly likely that IL-1α production is mainly induced by a direct interaction of macrophages with M. tuberculosis. Furthermore, previous studies have shown that RD1 contributes to bacterial virulence, the generation of protective immunity (Pym et al., 2003; Brodin et al., 2006), granuloma formation (Volkman et al., 2004), and necrosis induction (Hsu et al., 2003; Junqueira-Kipnis et al., 2006; Chen et al., 2007; Kaku et al., 2007). In addition, we have found that RD1 played a pivotal role in caspase-1 activation through facilitating K+ efflux from the cytoplasm of infected macrophages (Kurenuma et al., 2009). In this study, we demonstrated the active participation of RD1 in Ca2+ influx in infected macrophages. Based on these findings, we speculated that RD1 may provoke a perturbation of cytoplasmic membrane integrity, thus causing the transmembrane fluxes of calcium and potassium ions.
RD1 is located in the ESX-1 locus of the M. tuberculosis genome and includes genes encoding ESAT-6 and CFP-10 (Feltcher et al., 2011). Both ESAT-6 and CFP-10, which belong to the WXG-100 superfamily, are secreted through the ESX-1 secretion system and form a protein complex (Brodin et al., 2004a, 2004b). Recent studies have shown that ESAT-6 exerts membrane lytic activity (Hsu et al., 2003; de Jonge et al., 2007) and influences a variety of signaling pathways (Yu & Xie, 2012). Therefore, it is probable that ESAT-6 may provoke the morphological alteration not only in the phagosomal membrane but also in the cytoplasmic membrane, resulting in the influx of intracellular Ca2+ level. Alternatively, recent reports have shown that the ESX-1 system may facilitate the diffusion of some bacterial products into the cytoplasm. Indeed, Mishra et al. have shown that ESAT-6 contributes to caspase-1 activation by facilitating the spread of immunostimulatory components into the cytoplasm (Mishra et al., 2010). Wang et al. reported that ESAT-6 induced IL-1β secretion from human dendritic cells (Wang et al., 2012). ESX-1 is required for the disruption of the phagosomal membrane and translocation of mycobacteria. Bacteria that have escaped from the phagosome subsequently secrete components through the ESX-5 system, which induce inflammasome activation, IL-1β secretion, and caspase-independent cell death (Abdallah et al., 2011). Therefore, it is possible that RD1 may function in IL-1α production by inducing phagosomal membrane damage and the diffusion of some mycobacterial components that are required for Ca2+ influx, as well as calpain activation in the cytoplasm of infected macrophages. Alternatively, it has been shown that nucleotides including ATP and ADP induce calcium signaling in immune cells (Marteau et al., 2004; Bendz et al., 2008). Because wild-type M. tuberculosis causes necrosis of infected macrophages with a high frequency, therefore, it is probable that nucleotides released from necrotic cells may partly contribute to the intracellular Ca2+ mobilization. To clarify the precise mechanism of RD1 in IL-1α secretion, further studies are required to identify mycobacterial components that are directly responsible for the induction of Ca2+ influx.
RD1 is involved in the induction of necrotic cell death of infected macrophages by causing mitochondrial membrane damage and ATP depletion (Kaku et al., 2007). Thus, we determined whether calpain participates in cell death of infected macrophages by measuring the release of LDH from cells infected with H37Rv in the presence of the calpain inhibitors MDL28170, inhibitor IV, and EGTA. The calpain inhibitors weakly, but significantly, prevented LDH release and largely abrogated IL-1α secretion (Fig. 2 and Supporting Information, Fig. S1). In this regard, previous studies reported that ESAT-6 was a potent activator of the NLRP3 inflammasome, and consequently, necrotic cell death of infected macrophages was triggered by ESAT-6-dependent NLRP3 inflammasome activation (Mishra et al., 2010; Wong & Jacobs, 2011). Therefore, calpain was necessary to catalyze pro-IL-1α, but it likely only contributes to necrotic cell death of infected macrophages. On the other hand, EGTA also inhibited LDH release from infected macrophages, and its inhibitory activity was much stronger than those of the calpain inhibitors, suggesting that Ca2+ signaling was required for the cell death.
Pro-IL-1α, unlike pro-IL-1β, is thought to activate IL-1 receptor-dependent signal pathway in a similar manner as mature IL-1α. Therefore, the passive release of pro-IL-1α upon cell death serves as a danger signal and is capable of causing inflammation. Indeed, a sterile inflammatory response can be triggered by IL-1α released from dying cells (Chen et al., 2007). In H37Rv-infected macrophages, a large portion of IL-1α secreted into the culture supernatant was converted to the mature form, while most IL-1α detected in the cell lysate remained in the proform, indicating an intracellular process that resulted in the preferentially secretion of mature IL-1α. Recent reports indicated that IL-1α secretion was closely related to IL-1β secretion. Gross et al. identified a distinct IL-1α secretion pathway that was dependent on inflammasome (Gross et al., 2012). Fettelschoss et al. also showed that mature IL-1α secretion required activation of the inflammasome, and IL-1β served as a shuttle for IL-1α (Fettelschoss et al., 2012). Furthermore, Zheng et al. recently showed that an intracellular form of IL-1 receptor 2 (IL-1R2) interacted with pro-IL-1α, preventing calpain cleavage. Caspase-1 cleaves IL-1R2, which allows cleavage of pro-IL-1α by calpain (Zheng et al., 2013). Therefore, it appeared that caspase-1 and the inflammasome were not involved in the maturation of IL-1α, but the secretion of mature IL-1α was likely dependent on inflammasome, caspase-1, and IL-1β.
Calpains are intracellular Ca2+-dependent cysteine proteases that are ubiquitously expressed, and more than 15 proteins belong to the calpain family (Croall & DeMartino, 1991). There is mounting evidence that indicates that calpains break down a wide variety of cytoskeletal, membrane-associated, and regulatory proteins and participate in signal transduction, apoptosis, and IL-1α secretion (Czuprynski & Brown, 1987; Carruth et al., 1991; Chua et al., 2000; Croall & Ersfeld, 2007; Dewamitta et al., 2010; Sato et al., 2011). In general, calpain activity is tightly regulated by the level of transcription, Ca2+ availability, autoproteolysis, phosphorylation, intracellular distribution, and endogenous inhibitors (Goll et al., 2003). Our results showed that RD1 contributes to calpain activation through the increase in cytosolic Ca2+ levels in infected macrophages. Furthermore, IL-1α secretion by infected macrophages was significantly inhibited by MDL28170, which exerts broad-spectrum activities, and also by EST and calpain inhibitor IV, which preferentially inhibit calpain I and calpain II, respectively. The results suggested that calpain I and calpain II, two major calpains that are distinguished by the optimal Ca2+ concentration required for maximal activity, are possibly involved in the processing of IL-1α during M. tuberculosis infection. However, we cannot exclude the possibility that other calcium-dependent proteases may also contribute to the processing of IL-1α either alone or in combination with calpain.
Although it has long been believed that both pro- and mature IL-1α exert a similar activity through binding to IL-1 receptor, recent reports demonstrated that pro-IL-1α accumulated in the nucleus where it induced biological responses that were dependent upon the nuclear localization (Stevenson et al., 1997; Cheng et al., 2008; Luheshi et al., 2009). Therefore, IL-1α can be regarded as a dual function cytokine, and it is unknown whether these two forms of IL-1α similarly contribute to the induction of host resistance to M. tuberculosis, as it has been shown that IL-1R-deficient mice are susceptible to M. tuberculosis infection and blocking of IL-1α increases their susceptibility. Therefore, IL-1 signaling is definitely important for host resistance to M. tuberculosis (Fremond et al., 2007; Guler et al., 2011). Thus, it may be interesting to determine the role of pro- and mature IL-1α for the primary host resistance to M. tuberculosis infection.
In conclusion, our study clearly indicated that M. tuberculosis infection induced IL-1α mRNA expression and pro-IL-1α synthesis. These processes were independent of RD1. In contrast, RD1 was implicated in calpain activation, as well as the maturation and secretion of IL-1α through the elevation of intracellular Ca2+ level. These findings provide further insight into the interaction between the RD1 locus and the host cytokine response to M. tuberculosis infection.
This study was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Culture and Sports of Japan, grants-in-aid for scientific research (B and C) and a grant-in-aid for young scientists (B) from the Japan Society for the Promotion of Science, and a grant-in-aid for research on emerging and reemerging infectious diseases from the Ministry of Health, Labor and Welfare of Japan.