Sita Ramyamali Dewamitta,1 Shunsuke Sakai,1 Kohsuke Tsuchiya,1Takamasa Nomura,1 and Masao Mitsuyama1
Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan,1 andDepartment of Microbiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan2
Received 6 January 2009/Returned for modification 25 March 2009/Accepted 2 July 2009
A genomic locus called “region of difference 1” (RD1) in Mycobacterium tuberculosis has been shown tocontribute to the generation of host protective immunity as well as to the virulence of the bacterium. To gaininsight into the molecular mechanism, we investigated the difference in the cytokine-inducing ability betweenH37Rv and a mutant strain deficient for RD1 (�RD1). We found that RD1 is implicated in the production ofcaspase-1-dependent cytokines, interleukin-18 (IL-18) and IL-1�, from infected macrophages. The expressionof these cytokines was similarly induced after infection with H37Rv and �RD1. However, the activation ofcaspase-1 was observed only in H37Rv-infected macrophages. The cytokine production and caspase-1 activa-tion were induced independently of type I interferon receptor signaling events. We also found that theactivation of caspase-1 was markedly inhibited with increasing concentrations of extracellular KCl. Further-more, the production of IL-18 and IL-1� and caspase-1 activation were induced independently of a P2X7purinergic receptor, and the inability of �RD1 in caspase-1 activation was compensated for by nigericin, anagent inducing the potassium ion efflux. Based on these results, we concluded that RD1 participates incaspase-1-dependent cytokine production via induction of the potassium ion efflux in infected macrophages.
Mycobacterium tuberculosis, an etiologic agent of human tu-berculosis, is one of the leading threats to humans. It has beenreported that M. tuberculosis still causes 9.2 million new casesof tuberculosis worldwide and 1.7 million deaths annually (49).The recent emergence of multidrug-resistant and extensivelydrug-resistant M. tuberculosis strains highlights the urgent needfor extensive research unraveling the complex mechanism en-abling the bacterium to be successfully parasitic in humans.
The protective immunity against M. tuberculosis is mediatedmainly by Th1-type CD4� T cells and CD8� T cells. These Tcells produce a large amount of cytokines, including gammainterferon (IFN-�) and tumor necrosis factor alpha (TNF-�),resulting in the enhancement of macrophage bactericidal ac-tivity and the development of granulomas in which M. tuber-culosis is killed and prevented from disseminating to the blood-stream and other tissues (12, 47). It has been demonstratedthat T cells differentiate into Th1 cells in cooperation withseveral proinflammatory cytokines, such as interleukin-12 (IL-12), IL-18, and IFN-�, that are produced by infiltrating den-dritic cells, macrophages, and NK cells. A number of in vitrostudies have shown that these cytokines are produced via rec-ognition of pathogen-associated molecular patterns of M. tu-berculosis by Toll-like receptors (TLRs) (43). However, therole of TLR-mediated signaling pathways in the protectiveimmunity against M. tuberculosis is controversial. Abel et al.
have shown that TLR4-deficient mice display reduced bacterialclearance during a long-term infection and develop chronicpneumonia (2). Drennan et al. have also reported that TLR2-deficient mice initially control an aerosol infection with M.tuberculosis, but develop increased bacterial burden and suc-cumb to chronic pneumonia (8). On the other hand, recentstudies have shown that TLRs are dispensable in the develop-ment of T-cell-mediated adaptive immunity, while myeloid dif-ferentiation protein 88 (MyD88) is required for restriction ofthe intracellular growth of M. tuberculosis (44). In addition,Fremond et al. (16) and Holscher et al. (24) have shown thatmice deficient for IL-1 receptor (IL-1R) succumbed to acuteM. tuberculosis infection in a manner similar to mice deficientfor MyD88, whereas mice deficient for TLR2, TLR4, TLR9, orToll-IL-1R domain-containing adaptor protein could controlacute M. tuberculosis infection to the same extent as wild-typemice. These findings suggest that MyD88 plays a much moreprominent role in adaptive immunity than functioning as anadaptor molecule of TLRs, and the role of the MyD88-depen-dent IL-1R signaling pathway is necessary for induction ofefficient protection against M. tuberculosis.
A genomic locus of M. tuberculosis called “region of differ-ence 1” (RD1) was first discovered as a locus that is absent ina genome of Mycobacterium bovis BCG (30). RD1 is 9.5 kb inlength and comprises nine genes, including the genes thatencode the secretory proteins ESAT-6 (6-kDa early secretedantigen target) and CFP-10 (10-kDa culture filtrate protein).The other genes encode components of a secretion system thatis called ESX-1 (ESAT-6 system 1). It is supposed that morethan 14 proteins contribute to this secretion system (1). Al-
* Corresponding author. Mailing address: Department of Microbi-ology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4447. Fax:81-75-753-4446. E-mail: [email protected].
though all components that are involved in ESX-1 have not yetbeen fully characterized, a number of proteins with knownfunctional domains are identified. They include a putativechaperone with an AAA� ATPase (Rv3868), a subtilisin-likeserine protease (Rv3883c), and an FtsK/SpoIIIE-like ATPase(Rv3870, Rv3871). Rv3877 is predicted to be a membrane-spanning protein that could be part of the translocation pore inthe cytoplasmic membrane. It has also been shown that RD1 isa critical region for bacterial virulence (6, 25, 31, 40). Further-more, intensive studies have demonstrated the role of RD1 inthe generation of protective immunity (5, 41) as well as necro-sis induction (25, 27) and granuloma formation (48). To de-termine how RD1 contributes to the development of hostimmune response, we compared the cytokine-inducing abilitybetween wild-type M. tuberculosis H37Rv and the mutantstrain deficient for RD1. We found that RD1 was essential foractivation of caspase-1 and subsequent secretion of IL-18 andIL-1� from macrophages infected with M. tuberculosis. Theactivation of caspase-1 was induced via a potassium ion effluxthat is highly dependent on RD1 but independent of the P2X7receptor. Moreover, we found that the type I interferon is notrequired for the activation of caspase-1 and cytokine produc-tion.
MATERIALS AND METHODS
Mice. Female C57BL/6 and BALB/c mice were purchased from JapanSLC (Shizuoka, Japan). INF-�, INF-�, and INF-� receptor 1 knockout mice(IFNAR1�/� mice, on a C57BL/6 background) were kindly provided byShigekazu Nagata (Kyoto University Graduate School of Medicine, Kyoto,Japan). P2X7 receptor knockout mice (P2X7R�/� mice, on a C57BL/6 back-ground) were obtained from Tatsuro Ishibashi (Kyushu University, Fukuoka,Japan). Mice were maintained under specific pathogen-free conditions and usedat 7 to 9 weeks of age. All the experimental procedures were approved by theAnimal Ethics and Research Committee of Kyoto University Graduate School ofMedicine, Kyoto, Japan.
Bacterial strains. M. tuberculosis H37Rv, an H37Rv mutant deficient for RD1(�RD1) and an RD1-complemented strain (�RD1::RD1) (pYUB412::Rv3860-Rv3885c) were kindly provided by William R. Jacobs (Albert Einstein Institute,Bronx, NY) (25). These M. tuberculosis strains were grown at 37°C to the mid-logphase in Middlebrook 7H9 broth supplemented with 0.5% albumin, 0.2% dex-trose, 3 �g ml�1 catalase, and 0.2% glycerol. Bacteria were harvested, stirredvigorously with glass beads (3 mm in diameter), and centrifuged at 300 g for3 min to remove the bacterial clumps. The suspension was stored at �80°C inaliquots. After thawing, the viable bacteria were enumerated by counting thecolonies after plating the diluted suspension on Middlebrook 7H10 agar platescontaining 50 �g ml�1 oleic acid, 0.5% albumin, 0.2% dextrose, 4 �g ml�1
catalase, and 0.85 mg ml�1 sodium chloride. In each experiment, bacteria wereadded to the macrophage culture based on the concentration after the absenceof bacterial clumps was confirmed by Kinyoun staining.
Cells. Peritoneal exudate cells of C57BL/6 and BALB/c mice were obtained bya peritoneal lavage 4 days after an intraperitoneal injection with 3 ml of thio-glycolate medium (EIKEN Chemical, Osaka, Japan). Peritoneal exudate cellswere washed and plated at 5.0 105 cells well�1 in 48-well plates and incubatedfor 3 h at 37°C in RPMI 1640 medium supplemented with 10% fetal calf serum.Nonadherent cells were removed by washes with warmed RPMI 1640 medium,and adherent cells were used as macrophages in the following experiments. Bonemarrow cells were collected from tibiae of C57BL/6 mice and cultured with 100ng ml�1 mouse M-CSF (R&D Systems, Minneapolis, MN) for 5 days. Afterwashes, adherent bone marrow-derived macrophages (BMDM) were collectedand seeded at 5.0 105 cells well�1 in 48-well plates (21).
Quantitative real-time RT-PCR. Total cellular RNA was extracted from peri-toneal macrophages 9 h after infection with M. tuberculosis strains by usingNucleospin RNA II (Macherey-Nagel, Duren, Germany). RNA (0.2 �g) wastreated with RNase-free DNase (Promega, Tokyo, Japan) to eliminate contam-inating DNA and then subjected to reverse transcription (RT) using the Super-Script III first-strand synthesis system for RT-PCR (Invitrogen, Tokyo, Japan).Quantitative real-time RT-PCR was performed on ABI PRISM 7000 (Applied
Biosystems, Tokyo, Japan) using Platinum Sybr green quantitative PCR Super-Mix-uracil DNA glycosylase (Invitrogen) according to the manufacturer’s in-structions. The level of each cytokine mRNA expression was normalized on thebasis of �-actin mRNA expression, and results were analyzed with ABI PRISM7000 sodium dodecyl sulfate (SDS) software. The following DNA sequenceswere designed and used as PCR primers: Il-1� (5-AAGCTCTCCACCTCAATGGACAG-3, forward; and 5-CTCAAACTCCACTTTGCTCTTGA-3, re-verse), Il-18 (5-ACTGTACAACCGCAGTAATACGG-3, forward; and 5-AGTGAACATTACAGATTTATCCC-3, reverse), and �-actin (5-TGGAATCCTGTGGCATCCATGAAAC-3, forward; and 5-TAAAACGCAGCTCAGTAACAGTCCG-3, reverse).
Cytokine production and detection of caspase-1. Peritoneal macrophages wereinfected with M. tuberculosis strains at a multiplicity of infection (MOI) of 5 for3 h. Cells were washed to remove extracellular bacteria and then incubated for1, 9, and 21 h in the presence or absence of 500 to 2,000 NU/ml anti-IFN-�immunoglobulin G (IgG) (PBL Biomedical Laboratory, Piscataway, NJ), or 10 to40 mM potassium chloride. Alternatively, macrophages were infected withH37Rv�RD1 at an MOI of 5 for 3 h. Cells were washed to remove extracellularbacteria and incubated for 21 h, and then nigericin (5 �M) and/or KCl (30 mM)was added and incubated for another 3 h. The culture supernatant was collected,and concentrations of cytokines were determined by enzyme-linked immunosor-bent assay (ELISA) as reported previously (17, 19, 20). TNF-�, IL-6, and IL-1�were detected by using ELISA kits (eBioscience, San Diego, CA). IL-18 wasdetected using a pair of biotin-labeled and unlabeled monoclonal antibodiesspecific to IL-18 (MBL, Aichi, Japan).
In order to detect the activated form of caspase-1, 6 ml of the culture super-natants was incubated with 7 �g of rabbit anti-caspase-1 p10 IgG (Santa CruzBiotechnology, Santa Cruz, CA) and protein G Sepharose (GE HealthcareBio-Sciences AB, Uppsala, Sweden) to pull down caspase-1. Concurrently, in-fected macrophages were washed and lysed in phosphate-buffered saline con-taining 1% Nonidet P-40, 1 �g ml�1 leupeptin, 1 �g ml�1 pepstatin A, 1.5 �gml�1 aprotinin, and 2 mM dithiothreitol. The lysate was used for detection ofprocaspase-1. The samples were subjected to SDS-polyacrylamide gel electro-phoresis and transferred to polyvinylidene fluoride membranes by electro-blotting. The membrane was sequentially treated with rabbit anti-caspase-1 p10IgG, anti-rabbit IgG conjugated with peroxidase, and ECL Plus (GE Health-care). The bands representative of procaspase-1 and caspase-1 were detected byLAS-4000 Mini (Fujifilm, Tokyo, Japan). In addition, mature and proform typesof IL-1� were detected by Western blotting using anti-IL-1� antibodies (R&DSystems) in the culture supernatant and the cell lysate, respectively. �-Actin wasdetected using anti-mouse �-actin monoclonal antibody (Sigma-Aldrich, Tokyo,Japan).
Statistical analysis. Student’s t test was used to determine the statisticalsignificance of the values obtained, and a P value of �0.05 was considered to bestatistically significant.
RESULTS
RD1 participates in the production of IL-18 and IL-1�, butnot IL-6 or TNF-�, in macrophages infected with M. tubercu-losis. To investigate whether RD1 in the M. tuberculosis ge-nome contributes to cytokine production, we analyzed the pro-duction of IL-1�, �L-18, IL-6, and TNF-� after infection withM. tuberculosis strains H37Rv, �RD1, and �RD1::RD. Highlevels of cytokine production were detected in peritoneal exu-date macrophages of C57BL/6 mice in response to H37Rvinfection at an MOI of 5 (Fig.1A to D). The production ofthese cytokines was increased in a time-dependent manner,and the significant production was detected later than 12 hafter infection. On the other hand, �RD1 did not induce suchhigh levels of IL-1� and IL-18 production, whereas the pro-duction of TNF-� and IL-6 was comparable with that inducedby H37Rv. In contrast with �RD1, the strain �RD1::RD1, anRD1-complemented strain, was capable of inducing the pro-duction of IL-1� and IL-18 as well as IL-6 and TNF-�, and thelevels were almost similar to those induced by H37Rv. Theresults described above clearly indicate that RD1 is dispens-
able for TNF-� and IL-6 production but that it contributes tothe production of IL-1� and IL-18 from infected macrophages.
It has been shown that virulent M. tuberculosis induces adistinct response in macrophages if cells were infected witheither a high or low dose (29). To rule out the possibility thatthe difference in cytokine production between H37Rv- and
�RD1-infected macrophages is due to a high load of bacteria,we infected macrophages with M. tuberculosis strains at anMOI of 1 and measured the IL-1� production. Similar to theresponse at the high-dose infection, the response from H37Rvand �RD1::RD1 infection showed that cytokine productionwas induced as early as 12 h after infection (Fig. 1E), while
FIG. 1. RD1 participates in the production of IL-1� and IL-18, but not IL-6 or TNF-�, in M. tuberculosis-infected macrophages. Thioglycolate-induced peritoneal exudate macrophages of C57BL/6 mice were infected with H37Rv, �RD1, and �RD1::RD1 at an MOI of 5 for the indicatedperiods of time. The culture supernatant was collected, and the amounts of IL-1� ( ), IL-18 (B), TNF-� (C), and IL-6 (D) were measured byELISA. Peritoneal exudate macrophages were infected with M. tuberculosis strains at an MOI of 1 for the indicated periods of time, and IL-1�production was measured (E). BMDM of C57BL/6 mice (F) and peritoneal exudate macrophages of BALB/c mice (G) were infected with M.tuberculosis strains at an MOI of 5 for 24 h. The culture supernatant was collected, and the amount of IL-1� was measured by ELISA. Datarepresent the mean � standard deviations of triplicate assays and are representative of three independent experiments. *, a P value of �0.05 for�RD1-infected cells compared to either H37Rv-infected cells or �RD1::RD1-infected cells.
�RD1 did not induce production, suggesting that the observedpattern of cytokine production had not resulted from an un-reasonable load of bacteria. In addition, thioglycolate-inducedmacrophages are known to be in an activated state. We thusemployed BMDM to determine whether RD1-dependentIL-1� production is observed in resting macrophages. As ex-pected, infection with H37Rv and �RD1::RD1 induced a sig-nificant level of IL-1� production, but �RD1 infection did not(Fig. 1F). Furthermore, the requirement of RD1 in IL-1�production was observed for peritoneal macrophages ofBALB/c mice, though the magnitude of the cytokine responsewas weaker than that of C57BL/6 macrophages (Fig. 1G).
RD1 contributes to the activation of caspase-1 in macro-phages infected with M. tuberculosis. We next measured theexpression of IL-18 and IL-1� mRNAs by real-time RT-PCRafter infection with M. tuberculosis strains. IL-18 mRNA wasdetected in unstimulated macrophages (Fig. 2A). The level wasalmost similar to that observed after infection with H37Rv and�RD1::RD1. Despite the absence of IL-18 secretion in mac-rophages infected with �RD1 (Fig. 1A), there was no differ-ence in the level of IL-18 mRNA expression between H37Rvand this mutant. Compared to the control response (no infec-tion), IL-1� mRNA expression was dramatically increased af-ter infection with all three strains. This profile showed a sig-
nificant contrast to the profile of secreted IL-1� (Fig. 1). Theseresults clearly showed that though the proforms of IL-18 andIL-1� were generated after �RD1 infection, the mutant failedto induce the secretion of the mature forms of these cytokines.
Both IL-1� and IL-18 are members of the IL-1 family ofcytokines and are produced as immature proteins. It has beenshown that IL-1� and IL-18 are secreted after conversion intomature forms by activated caspase-1 (4, 32). Based on the levelof these cytokine transcripts, it appeared that pro-IL-1� andpro-IL-18 were similarly induced after infection with M. tuber-culosis strains. To determine the level of transcripts, we carriedout Western blotting for IL-1�. As shown in Fig. 2C, the35-kDa band corresponding to pro-IL-1� was similarly de-tected in the lysates of cells infected with three M. tuberculosisstrains (Fig. 2C). We next determined whether the activationof caspase-1 was induced after infection with M. tuberculosisstrains by analyzing the amount of procaspase-1 (p45) and afragment of the activated form of caspase-1 (p10) (Fig. 2D). Ithas been shown that activated caspase-1 is secreted from cellsalong with mature IL-1� and IL-18 (36, 42). Concordantly, wedetected caspase-1 in the culture supernatant but not in thecell lysate, suggesting that caspase-1 is mostly secreted afterconversion from procaspase-1 to activated caspase-1 in thisexperimental system. Therefore, we measured the amount ofprocaspase-1 in the cell lysate and evaluated the activation ofcaspase-1 by measuring the amount of p10 in the culture su-pernatant. A large amount of procaspase-1 (p45) was detectedin the lysate of uninfected macrophages. A similar amount ofp45 was detected in macrophages infected with three M. tuber-culosis strains. On the other hand, p10 was detected only in theculture supernatant of macrophages infected with H37Rv or�RD1::RD1 and was hardly detected in the culture superna-tant of �RD1-infected macrophages. In proportion to thecaspase-1 activation, the mature IL-1� was detected in theculture supernatant of cells infected with H37Rv and �RD1::RD1 (Fig. 2C). The results clearly showed that RD1 contrib-utes to the activation of caspase-1, leading to the secretion ofIL-1� and IL-18 from H37Rv-infected macrophages.
Endogenous IFN-� does not participate in the activation ofcaspase-1 in macrophages infected with M. tuberculosis. Henryet al. have shown that IFN-� is necessary for the activation ofcaspase-1 in macrophages infected with Francisella tularensisand Listeria monocytogenes, whereas Salmonella enterica sero-var Typhimurium, another intracellular bacterium, induces ac-tivation of caspase-1 independently of IFN-� (22). We exam-ined whether IFN-� contributes to the activation of caspase-1in macrophages infected with M. tuberculosis. We first infectedmacrophages with H37Rv in the presence of anti-IFN-� IgGand measured the production of IL-1� and IL-18. As shown inFig. 3A and B, neutralization of IFN-� did not affect theproduction of these cytokines. The antibody employed in thisstudy could block the secretion of IL-1� and IL-18 from mac-rophages infected with Listeria monocytogenes at the concen-trations used in this experiment (data not shown). We alsodetermined the effect of anti-IFN-� IgG on the activation ofcaspase-1. The Western blot clearly showed that the antibodydid not affect the amount of p10 fragment of caspase-1 re-leased after infection with H37Rv (Fig. 3C). The absence of anIFN-� contribution, as suggested by the findings describedabove, could be further confirmed by using type I IFN receptor
FIG. 2. RD1 contributes to secretion of IL-1� and IL-18 throughthe activation of caspase-1. Peritoneal macrophages were infected withM. tuberculosis strains at an MOI of 5 for 9 h. Total RNA was extractedand subjected to quantitative real-time RT-PCR to compare the ex-pression levels of IL-18 ( ) and IL-1� (B). Peritoneal macrophageswere infected with M. tuberculosis strains at an MOI of 5 for 24 h. Thecell lysate was prepared, and the amounts of proIL-1� (C) and pro-caspase-1 (D) were determined by Western blotting. (C) To detectmature IL-1�, the culture supernatant was collected and Westernblotting was done. As the direct detection of the activated form ofcaspase-1 in the supernatant was difficult, the culture supernatant wastreated with anti-caspase-1 p10 antibodies plus protein G Sepharosebeads to enrich caspase-1. (D) The sample was then subjected toSDS-polyacrylamide gel electrophoresis, and the relative amount ofmature caspase-1 (p10) was determined by Western blotting. �-Actinwas used as a loading control of the cell lysate.
knockout (IFNAR1�/�) macrophages. As shown in Fig. 3Dand E, the level of these cytokines produced from IFNAR1�/�
macrophages was comparable to that of wild-type macro-phages. We also analyzed the activation of caspase-1 afterinfection with H37Rv. There was no difference in the amountof the fragment of activated caspase-1 (p10) between wild-typeand IFNAR1�/� macrophages (Fig. 3F). Therefore, we con-cluded that IFN-� was not necessary for caspase-1 activation inM. tuberculosis infection.
Activation of caspase-1 in M. tuberculosis infection is depen-dent on potassium ion efflux. It has been shown that nigericin,anthrax lethal toxin, monosodium urate, and ATP efficientlyinduce the activation of caspase-1. These reagents cause a
temporal decrease in intracellular potassium concentrationand then promote the activation of caspase-1 (38, 39). To knowwhether potassium efflux is involved in the activation ofcaspase-1, we examined the effect of increasing concentrationsof extracellular KCl on the production of cytokines. The sig-nificant levels of IL-1� and IL-18 secretion induced after in-fection with H37Rv were clearly decreased with increasingconcentrations of KCl (Fig. 4A and B). On the other hand, theproduction of TNF-� and IL-6 was not affected by even thehighest concentrations of KCl (Fig. 4C and D). Using Westernblot analysis, we determined the effect of extracellular KCl onthe activation of caspase-1 in H37Rv-infected macrophages.As expected, the amount of p10 fragment was decreased with
FIG. 3. IFN-� does not contribute to the activation of caspase-1 in M. tuberculosis-infected macrophages. Peritoneal macrophages were infectedwith H37Rv at an MOI of 5 for 24 h in the presence or absence of anti-IFN-� IgG or control IgG. The amount of IL-1� (A) and IL-18 (B) in theculture supernatant was measured by ELISA. Data represent the mean � standard deviations of triplicate assays and are representative of threeindependent experiments. Peritoneal macrophages were infected with H37Rv at an MOI of 5 for 24 h in the presence or absence of anti-IFN-�IgG (2 103 NU/ml) or control IgG. Procaspase-1 (p45) in the culture supernatant and the activated form of caspase-1 (p10) in the cell lysatewere detected by Western blotting (C). Peritoneal exudate macrophages from C57BL/6 and IFNAR1�/� mice were infected with H37Rv at an MOIof 5 for 24 h. The production of IL-1� (D) and IL-18 (E) in the culture supernatant was measured by ELISA. In addition, the amounts ofprocaspase-1 (p45) in the cell lysate and the fragment (p10) of mature caspase-1, which was immunoprecipitated from the culture supernatant,were detected by Western blotting (F). �-Actin was used as a loading control of the cell lysate. WT, wild type; Ab, antibody.
increasing concentrations of KCl and was diminished to thelevel of the noninfected control when cells were cultured with40 mM KCl (Fig. 4E). These results suggested that the potas-sium ion efflux is essential for the secretion of IL-1� and IL-18and that the inability of �RD1 to induce the production ofthese cytokines may be due to the inability of the potassium ionefflux to be induced.
P2X7 receptor does not participate in the activation ofcaspase-1 in macrophages infected with M. tuberculosis. TheP2X7 receptor was identified as an important component forcaspase-1 activation through promotion of potassium efflux(10, 26). Recently, it has been shown that in response to TLRagonists or infection with Staphylococcus aureus or Escherichiacoli, caspase-1 activation is triggered by the addition of ATP, a
signal that promotes caspase-1 activation through depletion ofintracellular potassium caused by stimulation of the P2X7 re-ceptor. On the other hand, caspase-1 activation induced bySalmonella or Listeria was not affected in macrophages defi-cient in the P2X7 receptor (15). In view of this reported find-ing, we analyzed whether the P2X7 receptor contributes toM. tuberculosis-induced caspase-1 activation by measuring thecytokine response in P2X7 receptor knockout (P2X7R�/�)macrophages after H37Rv infection. As shown in Fig. 5, therewas no difference in the secretion of IL-1� and IL-18 (Fig. 5Aand B), and the activation of caspase-1 (Fig. 5C), betweenwild-type and P2X7R�/� macrophages. The result indicatedthat the P2X7 receptor does not play any role in caspase-1activation induced by M. tuberculosis.
FIG. 4. Potassium ion efflux is essential for the secretion of IL-1� and IL-18 and activation of caspase-1 in M. tuberculosis-infected macro-phages. Peritoneal macrophages were infected with H37Rv at an MOI of 5 for 24 h in the presence or absence of KCl at 0 mM (black columns),10 mM (dark gray columns), 20 mM (light gray columns), and 40 mM (white columns). The amounts of IL-1� ( ), IL-18 (B), TNF-� (C), and IL-6(D) were measured by ELISA. Data represent the mean � standard deviations of triplicate assays. *, a P value of �0.05 for H37Rv-infected cellsin the presence of 40 mM KCl compared to H37Rv-infected cells in the absence of KCl. Peritoneal macrophages were infected with H37Rv at anMOI of 5 for 24 h in the presence of increasing concentrations of KCl. The amounts of procaspase-1 (p45) in the cell lysate and the fragment (p10)of mature caspase-1, which was immunoprecipitated from the culture supernatant, were detected by Western blotting (E). �-Actin was used as aloading control of the cell lysate.
Inability of �RD1 to induce production of IL-18 and IL-1�is compensated for by nigericin. The findings described aboveimplied that M. tuberculosis-induced caspase-1 activation isthrough the induction of the potassium ion efflux that is de-pendent on RD1 but not on the P2X7 receptor. If this is thecase, the inability of �RD1 to induce cytokine maturation maybe compensated for by the induction of the potassium ionefflux by some means. To test this possibility, macrophageswere infected with �RD1 and then stimulated with nigericin, apotassium ionophore. The culture supernatant was collected,and the production of cytokines and the activation of caspase-1were measured. The production of IL-1� and IL-18 was notinduced by infection with only �RD1 or treatment with nigeri-cin alone (Fig. 6A and B). However, a strong cytokine responsewas observed when �RD1-infected cells were stimulated withnigericin. Furthermore, the enhanced cytokine response wasdiminished mostly by the addition of 30 mM KCl. In propor-tion to the cytokine production, the amount of p10 was alsoincreased by treatment with nigericin and was reduced by theaddition of 30 mM KCl (Fig. 6C). Based on these data, weconcluded that RD1 participates in the caspase-1-dependentcytokine production via induction of the potassium ion efflux ininfected macrophages.
DISCUSSION
In the present study, the RD1 locus in the M. tuberculosisgenome is implicated in the activation of caspase-1 via induc-tion of the potassium ion efflux in infected macrophages. Kooet al. have shown recently that M. tuberculosis stimulates the
secretion of IL-1� and IL-18, and the activity is closely relatedto the RD1 locus (28). Our results are consistent with theirfindings. In addition, we newly demonstrated in this study thatM. tuberculosis-induced caspase-1 activation is not dependenton IFN-�. Furthermore, M. tuberculosis caused a potassium ionefflux independently of the P2X7 receptor. We also found thatH37Rv has a higher ability to induce cell death of infectedmacrophages than �RD1 (data not shown). ESX-1-dependentcytolysis may be involved in the cytokine and lysosome secre-tion, as reported previously (28). However, the exact relation-ship between the intracellular molecular events and the cyto-kine secretion still remains to be elucidated.
It has been shown that caspase-1 is activated after infectionwith several bacteria, including Salmonella enterica serovar Ty-phimurium, Pseudomonas aeruginosa, Listeria monocytogenes,Shigella flexneri, and Francisella tularensis, and that variouscomponents or cascades are involved in the activation ofcaspase-1 (32, 50) Recently, Henry et al. have shown thatIFN-� participates in the activation of caspase-1 in macro-phages infected with Francisella tularensis and Listeria mono-cytogenes, whereas it is dispensable for the activation ofcaspase-1 in infections with Salmonella enterica serovar Typhi-murium (22). In the case of M. tuberculosis infection, it hasbeen shown that IFN-� is produced from macrophages in-
FIG. 5. P2X7 receptor does not contribute to the activation ofcaspase-1 in M. tuberculosis-infected macrophages. Peritoneal exudatemacrophages from C57BL/6 and P2X7R�/� mice were infected withH37Rv at an MOI of 5 for 24. The levels of IL-1� (A) and IL-18 (B) inculture supernatants were determined by ELISA. (C) The amounts ofprocaspase-1 (p45) in the cell lysate and the fragment (p10) of maturecaspase-1, which was immunoprecipitated from the culture superna-tant, were detected by Western blotting. �-Actin was used as a loadingcontrol of the cell lysate. WT, wild type.
FIG. 6. The inability of H37Rv�RD1 to induce production ofIL-18 and IL-1� is compensated by the addition of potassium iono-phore (nigericin). Peritoneal macrophages were infected with �RD1 atan MOI of 5 for 21 h. Nigericin (5 �M) and/or KCl (30 mM) wasadded, and the culture was continued for another 3 h. The culturesupernatant was collected, and the production of IL-1� (A) and IL-18(B) was measured by ELISA. Data represent the mean � standarddeviations of triplicate assays. *, a P value of �0.05 for �RD1-infectedcells in the presence of nigericin compared to either �RD1-infectedcells in the absence of nigericin or �RD1-infected cells in the presenceof both nigericin and KCl. (C) The amounts of procaspase-1 (p45) inthe cell lysate and the fragment (p10) of mature caspase-1, which wasimmunoprecipitated from the culture supernatant, were detected byWestern blotting. �-Actin was used as a loading control of the celllysate.
fected with M. tuberculosis and that the production is depen-dent on ESX-1 (46). Our preliminary study also showed thatH37Rv induced higher IFN-� production than �RD1 (data notshown). However, the present study clearly showed that IFN-�was not required for the activation of caspase-1 in M. tubercu-losis infection. It has been shown that M. tuberculosis mayinduce the formation of the NALP3/ASC inflammasome (28).As reported previously, L. monocytogenes induces caspase-1activation via the formation of the NALP3/ASC inflamma-some. However, there was a difference in the requirement ofIFN-� in the formation of inflammasome after infection withM. tuberculosis and L. monocytogenes. Although the nature ofIFN-�-dependent signaling events is not known, it is probablethat the inflammasome is completed via the formation of amultiprotein complex. There may be differences in the com-position of inflammasome induced by infection with differentbacterial species.
Franchi et al. have shown that the requirement for the P2X7receptor and intracellular potassium in caspase-1 activation isdifferent between infection with intracellular and extracellularparasitic bacterial species (15). According to their report, re-garding infection with Staphylococcus aureus and Escherichiacoli, caspase-1 activation was triggered by P2X7 receptor-me-diated intracellular potassium depletion, which is induced bythe addition of ATP. In contrast, infection with Salmonellaenterica serovar Typhimurium and Listeria monocytogenes in-duced both caspase-1 activation and the cytokine secretionindependently of the P2X7 receptor and potassium ion efflux.In M. tuberculosis infection, as shown here, the P2X7 receptorwas not required for caspase-1 activation. However, a potas-sium ion efflux was necessary for caspase-1 activation, and RD1was implicated in triggering the intracellular event. Severalstudies revealed that bacteria or bacterial components secretedin the cytosol induced caspase-1 activation (3, 19, 35) In thiscontext, it has been shown that flagellin secreted by a type IIIsecretion system of S. enterica serovar Typhimurium and list-eriolysin O produced by L. monocytogenes are identified aseffector molecules for inducing caspase-1 activation (14, 21).Recent evidence suggested that the ESX-1 secretory system ofM. tuberculosis is capable of delivering several effector proteinsto the host cytosol (1). Therefore, it is likely that some bacterialcomponents induce caspase-1 activation during M. tuberculosisinfection by intracellular potassium ion efflux.
It has been found that NALP3 and ASC are involved in theactivation of caspase-1 in M. tuberculosis-infected macrophages(28). Petrilli et al. have shown that the formation of the NALPinflammasome (NALP1 or NALP3) is induced under a lowintracellular potassium concentration (39). Hentze et al. fur-ther demonstrated that cathepsin B contributes to the forma-tion of the NALP3 inflammasome that is induced by potassiumion efflux (23). Taken together, it is probable that an ESX-1-dependent potassium ion efflux might cause the formation ofthe NALP3/ASC inflammasome through a release of cathepsinB from the lysosomal compartment. On the other hand, Fer-nandes-Alnemri et al. have shown that potassium depletioninduces the formation of a pyroptosome, distinct from an in-flammasome, which is largely composed of oligomerized ASCand can activate caspase-1 and release IL-1� (9). Although theformation of an inflammasome or pyroptosome may be in-volved in the activation of caspase-1 in infected macrophages,
there was no information about the M. tuberculosis factorsresponsible for the potassium ion efflux followed by the acti-vation of caspase-1. In this study, we demonstrated the closerelationship between RD1 and the potassium ion efflux. M.tuberculosis and Mycobacterium marinum have been shown toinduce permeation of the cell membrane (13, 18). Smith et al.have shown that the permeation is caused by pore formation(45). It has been suggested that ESAT-6 of M. tuberculosis hasa membrane-lysing activity and that ESAT-6 of M. marinumcould play a direct role in causing pore formation (7, 45). Inaddition, there are other M. tuberculosis components that aresecreted through the ESX-1 secretion system, although theirfunctions have not yet been identified (13, 34). Therefore, it isprobable that one or more of the effector proteins secreted bythe ESX-1 secretory system cause changes in the membraneintegrity, leading to a decrease in the intracellular potassiumlevel. We are attempting to identify the M. tuberculosis factorswhich may lead to such intracellular potassium perturbationsin our future study.
The roles of IL-18 and IL-1� in the pathogenesis of tuber-culosis still remain controversial. There is one recent reportdemonstrating that M. tuberculosis and Mycobacterium bovisBCG actively prevent inflammasome activation by use of aputative Zn2� metalloprotease (33), while another report (28)and ours demonstrate that RD1-sufficient strains of mycobac-teria can induce inflammasome activation. From our point ofview, however, it can be suggested that IL-1� and IL-18 in-duced by M. tuberculosis are important for the protection (16,44) and formation (37) of tuberculous granuloma. These cyto-kines, in concert with other cytokines or chemokines, may exertboth beneficial and detrimental effects to the host, resulting ina complex pathology. Considering the fact that a RD1-defi-cient strain of M. tuberculosis cannot induce a strong activationof caspase-1 and secretion of IL-18 and IL-1�, it is tempting toassume that the limited efficacy of the BCG vaccine againstadult pulmonary tuberculosis (11) is due, at least in part, to theabsence of RD-1-dependent induction of mature IL-1� andIL-18. In fact, it has been shown that BCG or Mycobacteriummicroti strains that were transformed with the RD1 regionshow enhanced efficacy of vaccination in animal models (5, 41),although it is still unknown to what extent IL-1� and IL-18 playroles in this vaccination. Further studies are needed to obtaina comprehensive idea about the roles played by IL-1� andIL-18 in the pathogenesis of tuberculosis and to develop effec-tive vaccines against tuberculosis.
ACKNOWLEDGMENTS
We thank William R. Jacobs (Albert Einstein Institute) for providingthe M. tuberculosis strains (H37Rv, H37Rv�RD1, and H37Rv�RD1::RD1), Shigekazu Nagata (Kyoto University Graduate School of Med-icine) for providing the IFNAR1 knockout mice, and Tatsuro Ishibashi(Kyushu University) for providing the P2X7 receptor knockout mice.
This study was supported by a Grant-in-Aid for Scientific Researchon Priority Areas from the Ministry of Education, Science, Culture,and Sports of Japan; Grants-in-Aid for Scientific Research (B) and (C); aGrant-in-Aid for Research on Emerging and Re-emerging InfectiousDiseases from the Ministry of Health, Labor, and Welfare of Japan; andThe Waksman Foundation of Japan.
REFERENCES
1. Abdallah, A., N. Gey van Pittius, P. Champion, J. Cox, J. Luirink, C.Vandenbroucke-Grauls, B. Appelmelk, and W. Bitter. 2007. Type VII secre-tion—mycobacteria show the way. Nat. Rev. Microbiol. 5:883–891.
2. Abel, B., N. Thieblemont, V. J. F. Quesniaux, N. Brown, J. Mpagi, K. Miyake,F. Bihl, and B. Ryffel. 2002. Toll-like receptor 4 expression is required tocontrol chronic Mycobacterium tuberculosis infection in mice. J. Immunol.169:3155–3162.
3. Amer, A., L. Franchi, T. D. Kanneganti, M. Body-Malapel, N. Ozoren, G.Brady, S. Meshinchi, R. Jagirdar, A. Gewirtz, S. Akira, and G. Nunez. 2006.Regulation of Legionella phagosome maturation and infection throughflagellin and host Ipaf. J. Biol. Chem. 281:35217–35223.
4. Barksby, H., S. Lea, P. Preshaw, and J. Taylor. 2007. The expanding familyof interleukin-1 cytokines and their role in destructive inflammatory disor-ders. Clin. Exp. Immunol. 149:217–225.
5. Brodin, P., L. Majlessi, R. Brosch, D. Smith, G. Bancroft, S. Clark, A.Williams, C. Leclerc, and S. Cole. 2004. Enhanced protection against tuber-culosis by vaccination with recombinant Mycobacterium microti vaccine thatinduces T cell immunity against region of difference 1 antigens. J. Infect. Dis.190:115–122.
6. Brodin, P., L. Majlessi, L. Marsollier, M. de Jonge, D. Bottai, C. Demangel,J. Hinds, O. Neyrolles, P. Butcher, C. Leclerc, S. Cole, and R. Brosch. 2006.Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact onimmunogenicity and virulence. Infect. Immun. 74:88–98.
7. de Jonge, M., G. Pehau-Arnaudet, M. Fretz, F. Romain, D. Bottai, P. Brodin,N. Honore, G. Marchal, W. Jiskoot, P. England, S. Cole, and R. Brosch.2007. ESAT-6 from Mycobacterium tuberculosis dissociates from its putativechaperone CFP-10 under acidic conditions and exhibits membrane-lysingactivity. J. Bacteriol. 189:6028–6034.
8. Drennan, M. B., D. Nicolle, V. J. F. Quesniaux, M. Jaacobs, N. Allie, J.Mpagi, C. Fremond, H. Wagner, C. Kirschning, and B. Ryffel. 2004. Toll-likereceptor 2-deficient mice succumb to Mycobacterium tuberculosis infection.Am. J. Pathol. 164:49–57.
9. Fernandes-Alnemri, T., J. Wu, J. Yu, P. Datta, B. Miller, W. Jankowski, S.Rosenberg, J. Zhang, and E. Alnemri. 2007. The pyroptosome: a supramo-lecular assembly of ASC dimers mediating inflammatory cell death viacaspase-1 activation. Cell Death Differ. 14:1590–1604.
10. Ferrari, D., C. Pizzirani, E. Adinolfi, R. M. Lemoli, A. Curti, M. Idzko, E.Panther, and F. Di Virgilio. 2006. The P2X7 receptor: a key player in IL-1processing and release. J. Immunol. 176:3877–3883.
11. Fine, P. 1988. BCG vaccination against tuberculosis and leprosy. Br. Med.Bull. 44:691–703.
12. Flynn, J. 2006. Lessons from experimental Mycobacterium tuberculosis infec-tions. Microbes Infect. 8:1179–1188.
13. Fortune, S., A. Jaeger, D. Sarracino, M. Chase, C. Sassetti, D. Sherman, B.Bloom, and E. Rubin. 2005. Mutually dependent secretion of proteins re-quired for mycobacterial virulence. Proc. Natl. Acad. Sci. USA 102:10676–10681.
14. Franchi, L., A. Amer, M. Body-Malapel, T. D. Kanneganti, N. Ozoren, R.Jagirdar, N. Inohara, P. Vandenabeele, A. Coyle, E. P. Grant, and G. Nunez.2006. Cytosolic flagellin requires Ipaf for activation of caspase-1 and inter-leukin 1� in Salmonella-infected macrophages. Nat. Immunol. 7:576–582.
15. Franchi, L., T. Kanneganti, G. R. Dubyak, and G. Nunez. 2007. Differentialrequirement of P2X7 receptor and intracellular K� for caspase-1 activationinduced by intracellular and extracellular bacteria. J. Biol. Chem. 282:18810–18818.
16. Fremond, C., D. Togbe, E. Doz, S. Rose, V. Vasseur, I. Maillet, M. Jacobs, B.Ryffel, and V. Quesniaux. 2007. IL-1 receptor-mediated signal is an essentialcomponent of MyD88-dependent innate response to Mycobacterium tuber-culosis infection. J. Immunol. 179:1178–1189.
17. Fukasawa, Y., I. Kawamura, R. Uchiyama, K. Yamamoto, T. Kaku, T. To-minaga, T. Nomura, S. Ichiyama, T. Ezaki, and M. Mitsuyama. 2005. Strep-tomycin-dependent exhibition of cytokine-inducing activity in streptomycin-dependent Mycobacterium tuberculosis strain 18b. Infect. Immun. 73:7051–7055.
18. Gao, L.-Y., S. Guo, B. McLaughlin, H. Morisaki, J. N. Engel, and E. J.Brown. 2004. A mycobacterial virulence gene cluster extending RD1 is re-quired for cytolysis, bacterial spreading and ESAT-6 secretion. Mol. Micro-biol. 53:1677–1693.
19. Gavrilin, M. A., I. J. Bouakl, N. L. Knatz, M. D. Duncan, M. W. Hall, J. S.Gunn, and M. D. Wewers. 2006. Internalization and phagosome escaperequired for Francisella to induce human monocyte IL-1� processing andrelease. Proc. Natl. Acad. Sci. USA 103:141–146.
20. Hara, H., I. Kawamura, T. Nomura, T. Tominaga, K. Tsuchiya, and M.Mitsuyama. 2007. Cytolysin-dependent escape of the bacterium from thephagosome is required but not sufficient for induction of the Th1 immuneresponse against Listeria monocytogenes infection: distinct role of listeriolysinO determined by cytolysin gene replacement. Infect. Immun. 75:3791–3801.
21. Hara, H., K. Tsuchiya, T. Nomura, I. Kawamura, S. Shoma, and M. Mit-suyama. 2008. Dependency of caspase-1 activation induced in macrophagesby Listeria monocytogenes on cytolysin, listeriolysin O, after evasion fromphagosome into the cytoplasm. J. Immunol. 180:7859–7868.
22. Henry, T., A. Brotcke, D. Weiss, L. Thompson, and D. Monack. 2007. TypeI interferon signaling is required for activation of the inflammasome duringFrancisella infection. J. Exp. Med. 204:987–994.
23. Hentze, H., X. Lin, M. Choi, and A. Porter. 2003. Critical role for cathepsin
B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin. Cell DeathDiffer. 10:956–968.
24. Holscher, C., N. Reiling, U. Schaible, A. Holscher, C. Bathmann, D. Korbel,I. Lenz, T. Sonntag, S. Kroger, S. Akira, H. Mossmann, C. Kirschning, H.Wagner, M. Freudenberg, and S. Ehlers. 2008. Containment of aerogenicMycobacterium tuberculosis infection in mice does not require MyD88 adap-tor function for TLR2, -4 and -9. Eur. J. Immunol. 38:680–694.
25. Hsu, T., S. Hingley-Wilson, B. Chen, M. Chen, A. Dai, P. Morin, C. Marks,J. Padiyar, C. Goulding, M. Gingery, D. Eisenberg, R. Russell, S. Derrick, F.Collins, S. Morris, C. King, and W. J. Jacobs. 2003. The primary mechanismof attenuation of bacillus Calmette-Guerin is a loss of secreted lytic functionrequired for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA100:12420–12425.
26. Kahlenberg, J. M., and G. R. Dubyak. 2004. Mechanism of caspase-1 acti-vation by P2X7 receptor-mediated K� release. Am. J. Physiol. Cell Physiol.286:C1100–C1108.
27. Kaku, T., I. Kawamura, R. Uchiyama, T. Kurenuma, and M. Mitsuyama.2007. RD1 region in mycobacterial genome is involved in the induction ofnecrosis in infected RAW264 cells via mitochondrial membrane damage andATP depletion. FEMS Microbiol. Lett. 274:189–195.
28. Koo, I., C. Wang, S. Raghavan, J. Morisaki, J. Cox, and E. Brown. 2008.ESX-1-dependent cytolysis in lysosome secretion and inflammasome activa-tion during mycobacterial infection. Cell. Microbiol. 10:1866–1878.
29. Lee, J., H. G. Remold, M. H. Ieong, and H. Kornfeld. 2006. Macrophagesapoptosis in response to high intracellular burden of Mycobacterium tuber-culosis is mediated by a novel caspase-independent pathway. J. Immunol.176:4267–4274.
30. Mahairas, G., P. Sabo, M. Hickey, D. Singh, and C. Stover. 1996. Molecularanalysis of genetic differences between Mycobacterium bovis BCG and viru-lent M. bovis. J. Bacteriol. 178:1274–1282.
31. Majlessi, L., P. Brodin, R. Brosch, M. Rojas, H. Khun, M. Huerre, S. Cole,and C. Leclerc. 2005. Influence of ESAT-6 secretion system 1 (RD1) ofMycobacterium tuberculosis on the interaction between mycobacteria and thehost immune system. J. Immunol. 174:3570–3579.
32. Martinon, F., and J. Tschopp. 2007. Inflammatory caspases and inflamma-somes: master switches of inflammation. Cell Death Differ. 14:10–22.
33. Master, S., S. Rampini, A. Davis, C. Keller, S. Ehlers, B. Springer, G.Timmins, P. Sander, and V. Deretic. 2008. Mycobacterium tuberculosis pre-vents inflammasome activation. Cell Host Microbe 3:224–232.
34. McLaughlin, B., J. Chon, J. MacGurn, F. Carlsson, T. Cheng, J. Cox, and E.Brown. 2007. A mycobacterium ESX-1-secreted virulence factor with uniquerequirements for export. PLoS Pathog. 3:e105.
35. Miao, E. A., C. M. Alpuche-Aranda, M. Dors, A. E. Clark, M. W. Bader, S. L.Miller, and A. Aderem. 2006. Cytoplasmic flagellin activates caspase-1 andsecretion of interleukin 1� via Ipaf. Nat. Immunol. 7:569–575.
36. Pan, Q., J. Mathison, C. Fearns, V. Kravchenko, J. Da Silva Correia, H.Hoffman, K. Kobayashi, J. Bertin, E. Grant, A. Coyle, F. Sutterwala, Y.Ogura, R. Flavell, and R. Ulevitch. 2007. MDP-induced interleukin-1betaprocessing requires Nod2 and CIAS1/NALP3. J. Leukoc. Biol. 82:177–183.
37. Pechkovsky, D., T. Goldmann, E. Vollmer, J. Muller-Quernheim, and G.Zissel. 2006. Interleukin-18 expression by alveolar epithelial cells type II intuberculosis and sarcoidosis. FEMS Immunol. Med. Microbiol. 46:30–38.
38. Petrilli, V., C. Dostert, D. Muruve, and J. Tschopp. 2007. The inflamma-some: a danger sensing complex triggering innate immunity. Curr. Opin.Immunol. 19:615–622.
39. Petrilli, V., S. Papin, C. Dostert, A. Mayor, F. Martinon, and J. Tschopp.2007. Activation of the NALP3 inflammasome is triggered by low intracel-lular potassium concentration. Cell Death Differ. 14:1583–1589.
40. Pym, A., P. Brodin, R. Brosch, M. Huerre, and S. Cole. 2002. Loss of RD1contributed to the attenuation of the live tuberculosis vaccines Mycobacte-rium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46:709–717.
41. Pym, A., P. Brodin, L. Majlessi, R. Brosch, C. Demangel, A. Williams, K.Griffiths, G. Marchal, C. Leclerc, and S. Cole. 2003. Recombinant BCGexporting ESAT-6 confers enhanced protection against tuberculosis. Nat.Med. 9:533–539.
42. Qu, Y., L. Franchi, G. Nunez, and G. Dubyak. 2007. Nonclassical IL-1 betasecretion stimulated by P2X7 receptors is dependent on inflammasome ac-tivation and correlated with exosome release in murine macrophages. J. Im-munol. 179:1913–1925.
43. Quesniaux, V., C. Fremond, M. Jacobs, S. Parida, D. Nicolle, V. Yeremeev,F. Bihl, F. Erard, T. Botha, M. Drennan, M. Soler, M. Le Bert, B. Schnyder,and B. Ryffel. 2004. Toll-like receptor pathways in the immune responses tomycobacteria. Microbes Infect. 6:946–959.
44. Reiling, N., S. Ehlers, and C. Holscher. 2008. MyDths and un-TOLLedtruths: sensor, instructive and effector immunity to tuberculosis. Immunol.Lett. 116:15–23.
45. Smith, J., J. Manoranjan, M. Pan, A. Bohsali, J. Xu, J. Liu, K. L. McDonald,A. Szyk, N. LaRonde-LeBlanc, and L.-Y. Gao. 2008. Evidence for poreformation in host cell membranes by Esx-1 secreted ESAT-6 and its role inMycobacterium marinum escape from the vacuole. Infect. Immun. 76:5478–5487.
46. Stanley, S., J. Johndrow, P. Manzanillo, and J. Cox. 2007. The type I IFNresponse to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178:3143–3152.
47. Ulrichs, T., and S. Kaufmann. 2006. New insights into the function ofgranulomas in human tuberculosis. J. Pathol. 208:261–269.
48. Volkman, H., H. Clay, D. Beery, J. Chang, D. Sherman, and L. Ramakrish-
nan. 2004. Tuberculous granuloma formation is enhanced by a mycobacte-rium virulence determinant. PLoS Biol. 2:e367.
49. World Health Organization. 2008. Global tuberculosis control: surveillance,planning, financing. WHO report 2008. World Health Organization, Geneva,Switzerland.
50. Yu, H., and B. Finlay. 2008. The caspase-1 inflammasome: a pilot of innateimmune responses. Cell Host Microbe 4:198–208.
