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.

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.

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.

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 Ca(2+) levels, and Ca(2+) 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 Ca(2+) followed by calpain activation.

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.

Two genetically-related multidrug-resistant Mycobacterium tuberculosis strains induce divergent outcomes of infection in two human macrophage models.

Mycobacterium tuberculosis has a considerable degree of genetic variability resulting in different epidemiology and disease outcomes. We evaluated the pathogen-host cell interaction of two genetically closely-related multidrug-resistant M. tuberculosis strains of the Haarlem family, namely the strain M, responsible for an extensive multidrug-resistant tuberculosis outbreak, and its kin strain 410 which caused a single case in two decades. Intracellular growth and cytokine responses were evaluated in human monocyte-derived macrophages and dU937 macrophage-like cells. In monocyte-derived macrophages, strain M grew more slowly and induced lower levels of TNF-α and IL-10 than 410, contrasting with previous studies with other strains, where a direct correlation was observed between increased intracellular growth and epidemiological success. On the other hand, in dU937 cells, no difference in growth was observed between both strains, and strain M induced significantly higher TNF-α levels than strain 410. We found that both cell models differed critically in the expression of receptors for M. tuberculosis entry, which might explain the different infection outcomes. Our results in monocyte-derived macrophages suggest that strain M relies on a modest replication rate and cytokine induction, keeping a state of quiescence and remaining rather unnoticed by the host. Collectively, our results underscore the impact of M. tuberculosis intra-species variations on the outcome of host cell infection and show that results can differ depending on the in vitro infection model.

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.

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.

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.

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.

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.

Expression of the Mycobacterium tuberculosis PPE37 protein in Mycobacterium smegmatis induces low tumour necrosis factor alpha and interleukin 6 production in murine macrophages.

PPE37 is a member of the Mycobacterium tuberculosis proline-proline-glutamic acid (PPE) multigene family. Its expression is upregulated in bacteria that are phagocytosed by macrophages and is enhanced even more in bacteria isolated from the lungs of infected mice. This raises the possibility that PPE37 may play a role in the virulence of M. tuberculosis and led to this investigation of the function of PPE37. Recombinant bacterial strains, one expressing the M. tuberculosis PPE37 protein (Ms_ppe37) and another harbouring the vector alone (Ms_vec) were generated from the non-pathogenic Mycobacterium smegmatis. These bacterial strains were used to infect peritoneal exudate and bone marrow-derived macrophages. It was found that, despite the comparable intracellular survival between the two recombinant M. smegmatis strains, Ms_ppe37 induced a significantly lower level of tumour necrosis factor alpha and interleukin 6 in the infected macrophages compared with Ms_vec. Western blot analyses revealed that the activation levels of nuclear factor kappa B, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase and MAPK/p38 were lower in macrophages infected with Ms_ppe37 than in macrophages infected with Ms_vec. These results suggest that PPE37 may have a potential role in interfering with the pro-inflammatory cytokine response of infected macrophages.

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.

[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.

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.

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.

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.

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.