Phosphorylation of the adaptor ASC acts as a molecular switch that controls the formation of speck-like aggregates and inflammasome activity.

The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.

Serum Syndecan-4 as a Possible Biomarker in Patients With Acute Pneumonia.

BACKGROUND: Syndecan-4 is a transmembrane heparan sulfate proteoglycan expressed in a variety of cells, and glycosaminoglycan side chains of syndecan-4 bind to several proteins, suggesting several biological functions. However, the role of syndecan-4 in acute bacterial pneumonia has not yet been elucidated. METHODS: Serum syndecan-4 levels were measured in patients with acute pneumonia, and the relationships between serum syndecan-4 levels and clinical parameters were analyzed. Next, we treated wild-type and syndecan-4-deficient mice with Streptococcus pneumoniae intranasally and analyzed the phenotype of syndecan-4-deficient mice. RESULTS: In the patients with acute pneumonia, serum syndecan-4 levels were significantly higher than in the healthy volunteers and correlated negatively with the pneumonia severity score. In addition, in patients who improved with short-term antibiotic therapy, serum syndecan-4 levels were higher on admission and gradually increased during antibiotic therapy. Furthermore, in syndecan-4-deficient mice, the survival rate was significantly worse, and total neutrophil counts in bronchoalveolar lavage fluid, bacterial counts in blood, and plasma levels of inflammatory cytokines were significantly higher than in wild-type mice. CONCLUSIONS: These results suggest that syndecan-4 has an anti-inflammatory function in acute pneumonia and could serve as a useful biomarker in these patients.

The adaptor ASC exacerbates lethal Listeria monocytogenes infection by mediating IL-18 production in an inflammasome-dependent and -independent manner.

Listeria monocytogenes induces the formation of inflammasomes and subsequent caspase-1 activation, and the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is crucial for this response. However, the role of ASC in L. monocytogenes infection in vivo is unclear. In this study, we demonstrate that ASC has a detrimental effect on host defense against L. monocytogenes infection at a lethal dose (10(6) CFU), but not at a sublethal dose (10(3) CFU). During lethal L. monocytogenes infection, serum levels of IL-18 and IL-10 were markedly elevated in WT mice, but not in ASC KO mice. IL-18 KO mice were more resistant to lethal L. monocytogenes infection than WT mice and had lower levels of serum IL-10. Furthermore, blockade of IL-10 receptor resulted in a reduction in bacterial counts, suggesting that ASC and IL-18 might exacerbate L. monocytogenes infection through induction of IL-10. We noticed that maturation of IL-18 during lethal infection was partially independent of caspase-1, but was critically dependent on ASC. ASC was required for the elevation of serum neutrophil serine protease activity, which correlated with caspase-1-independent IL-18 maturation and IL-10 production. Collectively, these results suggest that ASC plays a detrimental role in lethal L. monocytogenes infection through IL-18 production in an inflammasome-dependent and -independent manner.

Type I interferon signaling regulates activation of the absent in melanoma 2 inflammasome during Streptococcus pneumoniae infection.

Streptococcus pneumoniae, a Gram-positive bacterial pathogen, causes pneumonia, meningitis, and septicemia. Innate immune responses are critical for the control and pathology of pneumococcal infections. It has been demonstrated that S. pneumoniae induces the production of type I interferons (IFNs) by host cells and that type I IFNs regulate resistance and chemokine responses to S. pneumoniae infection in an autocrine/paracrine manner. In this study, we examined the effects of type I IFNs on macrophage proinflammatory cytokine production in response to S. pneumoniae. The production of interleukin-18 (IL-18), but not other cytokines tested, was significantly decreased by the absence or blockade of the IFN-α/ß receptor, suggesting that type I IFN signaling is necessary for IL-18 production. Type I IFN signaling was also required for S. pneumoniae-induced activation of caspase-1, a cysteine protease that plays a central role in maturation and secretion of IL-18. Earlier studies proposed that the AIM2 and NLRP3 inflammasomes mediate caspase-1 activation in response to S. pneumoniae. From our results, the AIM2 inflammasome rather than the NLRP3 inflammasome seemed to require type I IFN signaling for its optimal activation. Consistently, AIM2, but not NLRP3, was upregulated in S. pneumoniae-infected macrophages in a manner dependent on the IFN-α/ß receptor. Furthermore, type I IFN signaling was found to contribute to IL-18 production in pneumococcal pneumonia in vivo. Taken together, these results suggest that type I IFNs regulate S. pneumoniae-induced activation of the AIM2 inflammasome by upregulating AIM2 expression. This study revealed a novel role for type I IFNs in innate responses to S. pneumoniae.

Cutting edge: nitric oxide inhibits the NLRP3 inflammasome.

Although the NLRP3 inflammasome plays a pivotal role in host defense, its uncontrolled activation is associated with inflammatory disorders, suggesting that regulation of the inflammasome is important to prevent detrimental effects. Type I IFNs and long-term LPS stimulation were shown to negatively regulate NLRP3 activation. In this study, we found that endogenous NO is involved in the regulation of NLRP3 inflammasome activation by either IFN-ß pretreatment or long-term LPS stimulation. Furthermore, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, markedly inhibited NLRP3 inflammasome activation, whereas the AIM2 and NLRC4 inflammasomes were only partially inhibited by SNAP. An increase in mitochondrial reactive oxygen species induced by ATP was only modestly affected by SNAP treatment. Interestingly, S-nitrosylation of NLRP3 was detected in macrophages treated with SNAP, and this modification may account for the NO-mediated mechanism controlling inflammasome activation. Taken together, these results revealed a novel role for NO in regulating the NLRP3 inflammasome.

Mice deficient in ficolin, a lectin complement pathway recognition molecule, are susceptible to Streptococcus pneumoniae infection.

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)-deficient (Fcna(-/-)) and FcnA/ficolin B double-deficient (Fcna(-/-)b(-/-)) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna(-/-), Fcnb(-/-), and Fcna(-/-)b(-/-)) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna(-/-) but not in Fcna(-/-)b(-/-) mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

Critical roles of ASC inflammasomes in caspase-1 activation and host innate resistance to Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1ß and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3(-/-) macrophages, whereas knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC(-/-) mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1ß and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.

Listeria monocytogenes strain-specific impairment of the TetR regulator underlies the drastic increase in cyclic di-AMP secretion and beta interferon-inducing ability.

Among a number of laboratory strains of Listeria monocytogenes used in experimental infection, strain LO28 is highly capable of inducing robust beta interferon (IFN-ß) production in infected macrophages. In this study, we investigated the molecular mechanism of the IFN-ß-inducing ability of LO28 by comparing it with that of strain EGD, a low-IFN-ß-inducing strain. It was found that LO28 secretes a large amount of IFN-ß-inducing factor, which turned out to be cyclic di-AMP. The secretion of cyclic di-AMP was dependent on MdrT, a multidrug resistance transporter, and LO28 exhibited a very high level of mdrT expression. The introduction of a null mutation into mdrT abolished the ability of LO28 to induce IFN-ß production. Examination of genes responsible for the regulation of mdrT expression revealed a spontaneous 188-bp deletion in tetR of LO28. By constructing recombinant strains of LO28 and EGD in which tetR from each strain was replaced, it was confirmed that the distinct ability of LO28 is attributable mostly to tetR mutation. We concluded that the strong IFN-ß-inducing ability of LO28 is due to a genetic defect in tetR resulting in the overexpression of mdrT and a concomitant increase in the secretion of cyclic di-AMP through MdrT.

Involvement of absent in melanoma 2 in inflammasome activation in macrophages infected with Listeria monocytogenes.

Listeria monocytogenes invades the cytoplasm of macrophages and induces the activation of caspase-1 and the subsequent maturation of IL-1beta and IL-18. Although apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain (ASC), an adaptor protein of nucleotide-binding oligomerization domain (Nod)-like receptors, has been shown to play an essential role in inducing this cellular response to L. monocytogenes, the mechanism has not been fully elucidated. In this study, we demonstrate the role of absent in melanoma 2 (AIM2), a recently described receptor of cytosolic DNA, in the activation of caspase-1 upon infection with L. monocytogenes. Secretion of IL-1beta and IL-18 from Nod-like receptor family, pyrin domain containing 3 (NLRP3) and Nod-like receptor family, caspase-activating and recruitment domain containing 4 (NLRC4) knockout macrophages in response to L. monocytogenes was only slightly decreased compared with the levels secreted from wild-type macrophages, whereas secretion from ASC knockout macrophages was completely impaired, suggesting that receptors other than NLRP3 and NLRC4 also take part in inflammasome activation in an ASC-dependent manner. To identify such receptors, the abilities of several receptor candidates (NLRP2, NLRP6, NLRP12, and AIM2) to induce the secretion of IL-1beta in response to L. monocytogenes were compared using the inflammasome system reconstructed in HEK293 cells. Among these receptor candidates, AIM2 conferred the highest responsiveness to the bacterium on HEK293 cells. Knockdown of AIM2 significantly decreased the secretion of IL-1beta and IL-18 from L. monocytogenes-infected macrophages. These results suggest that AIM2, in cooperation with NLRP3 and NLRC4, plays an important role in the activation of caspase-1 during L. monocytogenes infection.

PD-1-PD-L1 pathway impairs T(h)1 immune response in the late stage of infection with Mycobacterium bovis bacillus Calmette-Guérin.

A major concern still prevails as to the reason why various mycobacteria are able to persist within infected host in which protective immunity is generated. To address this question, we monitored the generation of protective T cells during infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). CD4(+) T cells obtained 3 weeks after infection conferred protection against Mycobacterium tuberculosis challenge and produced IFN-γ and tumor necrosis factor (TNF)-α upon antigen stimulation. However, these abilities were decreased after 6 weeks of infection even though BCG was not thoroughly eliminated from the host. We analyzed the expression of ligands for the CD28/CTLA-4 family receptors on antigen-presenting cells and found that the expression of PD-L1, a ligand for programmed cell death-1 (PD-1), was up-regulated later than 3 weeks of infection. We also found that bacterial numbers in the spleen of PD-1-deficient mice were significantly reduced compared with wild-type mice at 6 and 12 weeks after BCG infection. Furthermore, CD4(+) T cells of PD-1-deficient mice showed a higher ability to confer protection and produce IFN-γ and TNF-α even at 12 weeks after infection. These results indicate that the PD-1-PD-L1 pathway impairs T(h)1 immunity in the late stage of BCG infection, thereby facilitating the bacterial persistence in the host.

Evaluation of the safety and efficacy of micafungin in Japanese patients with deep mycosis: a post-marketing survey report.

The safety and efficacy of micafungin were evaluated in a Japanese post-marketing survey involving 1,142 patients with deep mycosis caused by Candida or Aspergillus. The overall clinical response was 83.0%, and the respective responses for patients with candidiasis or aspergillosis were 86.3 and 70.8%. With regard to drug reactions, 562 adverse reactions were observed in 28.5% of patients. Among the 83 serious adverse drug reactions reported by 53 patients, a causal relationship with micafungin was assessed as definite or probable for 6 reactions in 5 patients. Age and baseline hepatic and renal function status did not affect the incidence of adverse reactions, although incidence increased significantly in proportion to the severity of mycosis and daily dose (p < 0.01). In multiple logistic regression analysis, neither baseline hepatic impairment nor increased daily dose of micafungin affected the incidence of hepatobiliary disorders, however, the severity of mycosis was found to correlate significantly with hepatobiliary disorders (p = 0.031). Taken together, our post-marketing findings show that micafungin is effective against deep mycosis caused by Candida or Aspergillus in patients across a range of backgrounds.

Alpha-galactosylceramide promotes killing of Listeria monocytogenes within the macrophage phagosome through invariant NKT-cell activation.

alpha-Galactosylceramide (alpha-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by alpha-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mphi) participate in the control of microbial pathogens. In the present study, we examined the participation of Mphi in immune intervention in infection by alpha-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mphi from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of alpha-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-gamma) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with alpha-GalCer and in culture supernatants of peritoneal Mphi from mice treated with alpha-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mphi by alpha-GalCer was abrogated by endogenous IFN-gamma neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mphi was increased after alpha-GalCer treatment. Our results suggest that amelioration of listeriosis by alpha-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mphi activated by IFN-gamma from iNKT cells residing in an organ(s) other than the peritoneal cavity.

Toll-like receptor 2- and MyD88-dependent phosphatidylinositol 3-kinase and Rac1 activation facilitates the phagocytosis of Listeria monocytogenes by murine macrophages.

Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2(-/-) macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2(-/-) and MyD88(-/-) macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2(-/-) macrophages but did not enhance the activity of MyD88(-/-) macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2(-/-) and MyD88(-/-) macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2(-/-) and MyD88(-/-) mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2(-/-) mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.

Listeriolysin O-dependent bacterial entry into the cytoplasm is required for calpain activation and interleukin-1 alpha secretion in macrophages infected with Listeria monocytogenes.

Listeriolysin O (LLO), an hly-encoded cytolysin of Listeria monocytogenes, plays an essential role in the entry of L. monocytogenes into the host cell cytoplasm. L. monocytogenes-infected macrophages produce various proinflammatory cytokines, including interleukin-1 alpha (IL-1 alpha), that contribute to the host immune response. In this study, we have examined IL-1 alpha production in macrophages infected with wild-type L. monocytogenes or a nonescaping mutant strain deficient for LLO (Delta hly). Expression of IL-1 alpha mRNA and accumulation of pro-IL-1 alpha in the cytoplasm were induced by both strains. In contrast, the secretion of the mature form of IL-1 alpha from infected macrophages was observed in infection with wild-type L. monocytogenes but not with the Delta hly mutant. A recovery of the ability to induce IL-1 alpha secretion was shown in a mutant strain complemented with the hly gene. The Toll-like receptor (TLR)/MyD88 signaling pathway was exclusively required for the expression of pro-IL-1 alpha, independently of LLO-mediated cytoplasmic entry of L. monocytogenes. The LLO-dependent secretion of mature IL-1 alpha was abolished by addition of calcium chelators, and only LLO-producing L. monocytogenes strains were able to induce elevation of the intracellular calcium level in infected macrophages. A calcium-dependent protease, calpain, was implicated in the maturation and secretion of IL-1 alpha induced by LLO-producing L. monocytogenes strains based on the effect of calpain inhibitor. Functional activation of calpain was detected in macrophages infected with LLO-producing L. monocytogenes strains but not with a mutant strain lacking LLO. These results clearly indicated that LLO-mediated cytoplasmic entry of bacteria could induce the activation of intracellular calcium signaling, which is essential for maturation and secretion of IL-1 alpha in macrophages during L. monocytogenes infection through activation of a calcium-dependent calpain protease. In addition, recombinant LLO, when added to macrophages infected with the Delta hly strain, could induce calcium influx and IL-1 alpha secretion at doses exhibiting cytolytic activity, suggesting that LLO produced by intracellular L. monocytogenes may be implicated in induction of calcium influx through pore formation.

[Protective immunity against Mycobacterium tuberculosis].

Mycobacterium tuberculosis (MTB) is an intracellular pathogen that has evolved strategies to enable growth in macrophages. The bacterium is able to inhibit fusion of phagosome with lysosome through secretion of some bacterial components and modulation of host cell intracellular signaling pathways. On the other hand, the complex system of protective immunity is expressed to control bacterial burden in host upon MTB infection. However, virulent MTB is capable of surviving in macrophages in vivo and persists in host even after acquired immunity has developed. These data suggest that MTB has developed a sophisticated immune evasion mechanism. In this issue, host protective response and the strategies of MTB for intracellular survival and immune evasion, which have been unraveled so far, are shown and the mechanisms are discussed.

The RD1 locus in the Mycobacterium tuberculosis genome contributes to activation of caspase-1 via induction of potassium ion efflux in infected macrophages.

A genomic locus called "region of difference 1" (RD1) in Mycobacterium tuberculosis has been shown to contribute to the generation of host protective immunity as well as to the virulence of the bacterium. To gain insight into the molecular mechanism, we investigated the difference in the cytokine-inducing ability between H37Rv and a mutant strain deficient for RD1 (DeltaRD1). We found that RD1 is implicated in the production of caspase-1-dependent cytokines, interleukin-18 (IL-18) and IL-1beta, from infected macrophages. The expression of these cytokines was similarly induced after infection with H37Rv and DeltaRD1. However, the activation of caspase-1 was observed only in H37Rv-infected macrophages. The cytokine production and caspase-1 activation were induced independently of type I interferon receptor signaling events. We also found that the activation of caspase-1 was markedly inhibited with increasing concentrations of extracellular KCl. Furthermore, the production of IL-18 and IL-1beta and caspase-1 activation were induced independently of a P2X7 purinergic receptor, and the inability of DeltaRD1 in caspase-1 activation was compensated for by nigericin, an agent inducing the potassium ion efflux. Based on these results, we concluded that RD1 participates in caspase-1-dependent cytokine production via induction of the potassium ion efflux in infected macrophages.

ASC and NLRP3 maintain innate immune homeostasis in the airway through an inflammasome-independent mechanism.

It is widely accepted that inflammasomes protect the host from microbial pathogens by inducing inflammatory responses through caspase-1 activation. Here, we show that the inflammasome components ASC and NLRP3 are required for resistance to pneumococcal pneumonia, whereas caspase-1 and caspase-11 are dispensable. In the lung of S. pneumoniae-infected mice, ASC and NLRP3, but not caspase-1/11, were required for optimal expression of several mucosal innate immune proteins. Among them, TFF2 and intelectin-1 appeared to be protective against pneumococcal pneumonia. During infection, ASC and NLRP3 maintained the expression of the transcription factor SPDEF, which can facilitate the expression of the mucosal defense factor genes. Moreover, activation of STAT6, a key regulator of Spdef expression, depended on ASC and NLRP3. Overexpression of these inflammasome proteins sustained STAT6 phosphorylation induced by type 2 cytokines. Collectively, this study suggests that ASC and NLRP3 promote airway mucosal innate immunity by an inflammasome-independent mechanism involving the STAT6-SPDEF pathway.

Critical involvement of pneumolysin in production of interleukin-1alpha and caspase-1-dependent cytokines in infection with Streptococcus pneumoniae in vitro: a novel function of pneumolysin in caspase-1 activation.

Pneumolysin is a pore-forming cytolysin known as a major virulence determinant of Streptococcus pneumoniae. This protein toxin has also been shown to activate the Toll-like receptor 4 (TLR4) signaling pathway. In this study, a mutant S. pneumoniae strain deficient in pneumolysin (Deltaply) and a recombinant pneumolysin protein (rPLY) were constructed. Upon infection of macrophages in vitro, the ability to induce the production of interleukin-1alpha (IL-1alpha), IL-1beta, and IL-18 was severely impaired in the Deltaply mutant, whereas there was no marked difference in the induction of tumor necrosis factor alpha (TNF-alpha) and IL-12p40 between the wild type and the Deltaply mutant of S. pneumoniae. When macrophages were stimulated with rPLY, the production of IL-1alpha, IL-1beta, and IL-18 was strongly induced in a TLR4-dependent manner, whereas lipopolysaccharide, a canonical TLR4 agonist, hardly induced these cytokines. In contrast, lipopolysaccharide was more potent than rPLY in inducing the production of TNF-alpha, IL-6, and IL-12p40, the cytokines requiring no caspase activation. Activation of caspase-1 was observed in macrophages stimulated with rPLY but not in those stimulated with lipopolysaccharide, and the level of activation was higher in macrophages infected with wild-type S. pneumoniae than in those infected with the Deltaply mutant. These results clearly indicate that pneumolysin plays a key role in the host response to S. pneumoniae, particularly in the induction of caspase-1-dependent cytokines.

[Advances in understanding of the virulence mechanism of Mycobacterium tuberculosis].

Mycobacterium tuberculosis (MTB) is an intracellular pathogen that has evolved strategies to enable growth in macrophages. The bacterium is able to inhibit fusion of phagosomes with lysosomes through secretion of some bacterial components and modulation of host intracellular signaling pathways. Furthermore, it has been shown that phagositosed MTB is killed within macrophages after treatment with IFN-gamma in vitro. However, virulent MTB is capable of surviving in macrophages in vivo and persists in host even after acquired immunity has developed. These data suggest that MTB has developed a sophisticated immune evasion mechanism. In this issue, the strategies of MTB for intracellular survival and immune evasion, which have been unraveled so far, are shown and the mechanisms are discussed.

RD1 region in mycobacterial genome is involved in the induction of necrosis in infected RAW264 cells via mitochondrial membrane damage and ATP depletion.

It was shown that virulent Mycobacterium tuberculosis H37Rv induces necrosis of infected RAW264 cells at 24 h post infection while avirulent H37Ra and an attenuated H37Rv mutant that is deficient for RD1 region (H37RvDeltaRD1) cause less necrosis of the infected cells. While H37Rv caused damage of the mitochondrial inner membrane and decreased the level of intracellular ATP, H37RvDeltaRD1 did not exhibit these harmful effects in infected cells. On the other hand, there was no difference in the level of intracellular reactive oxygen species after infection with H37Rv or H37RvDeltaRD1, and the intracellular bacterial numbers of H37RvDeltaRD1 and H37Ra were comparable to that of H37Rv. These results suggested that some virulence factors of H37Rv may contribute to the necrosis of infected cells through induction of mitochondrial dysfunction and depletion of intracellular ATP. RD1 appeared to encode some components possibly playing a central role in the induction of host cell necrosis after M. tuberculosis infection.