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.

Potential biomarker proteins for aspiration pneumonia detected by shotgun proteomics using buccal mucosa samples: a cross-sectional case-control study.

BACKGROUND: Aspiration pneumonia (AP), which is a major cause of death in the elderly, does present with typical symptoms in the early stages of onset, thus it is difficult to detect and treat at an early stage. In this study, we identified biomarkers that are useful for the detection of AP and focused on salivary proteins, which may be collected non-invasively. Because expectorating saliva is often difficult for elderly people, we collected salivary proteins from the buccal mucosa. METHODS: We collected samples from the buccal mucosa of six patients with AP and six control patients (no AP) in an acute-care hospital. Following protein precipitation using trichloroacetic acid and washing with acetone, the samples were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We also determined the levels of cytokines and chemokines in non-precipitated samples from buccal mucosa. RESULTS: Comparative quantitative analysis of LC-MS/MS spectra revealed 55 highly (P values < 0.10) abundant proteins with high FDR confidence (q values < 0.01) and high coverage (> 50%) in the AP group compared with the control group. Among the 55 proteins, the protein abundances of four proteins (protein S100-A7A, eukaryotic translation initiation factor 1, Serpin B4, and peptidoglycan recognition protein 1) in the AP group showed a negative correlation with the time post-onset; these proteins are promising AP biomarker candidates. In addition, the abundance of C-reactive protein (CRP) in oral samples was highly correlated with serum CRP levels, suggesting that oral CRP levels may be used as a surrogate to predict serum CRP in AP patients. A multiplex cytokine/chemokine assay revealed that MCP-1 tended to be low, indicating unresponsiveness of MCP-1 and its downstream immune pathways in AP. CONCLUSION: Our findings suggest that oral salivary proteins, which are obtained non-invasively, can be utilized for the detection of AP.

Switching from Apoptosis to Pyroptosis: Gasdermin-Elicited Inflammation and Antitumor Immunity.

Pyroptosis is a necrotic form of regulated cell death. Gasdermines (GSDMs) are a family of intracellular proteins that execute pyroptosis. While GSDMs are expressed as inactive forms, certain proteases proteolytically activate them. The N-terminal fragments of GSDMs form pores in the plasma membrane, leading to osmotic cell lysis. Pyroptotic cells release pro-inflammatory molecules into the extracellular milieu, thereby eliciting inflammation and immune responses. Recent studies have significantly advanced our knowledge of the mechanisms and physiological roles of pyroptosis. GSDMs are activated by caspases and granzymes, most of which can also induce apoptosis in different situations, for example where the expression of GSDMs is too low to cause pyroptosis; that is, caspase/granzyme-induced apoptosis can be switched to pyroptosis by the expression of GSDMs. Pyroptosis appears to facilitate the killing of tumor cells by cytotoxic lymphocytes, and it may also reprogram the tumor microenvironment to an immunostimulatory state. Understanding pyroptosis may help the development of cancer immunotherapy. In this review article, recent findings on the mechanisms and roles of pyroptosis are introduced. The effectiveness and limitations of pyroptosis in inducing antitumor immunity are also discussed.

Gasdermin D-independent release of interleukin-1ß by living macrophages in response to mycoplasmal lipoproteins and lipopeptides.

Interleukin-1ß (IL-1ß) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1ß by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1ß. Living cells can also release IL-1ß via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M. salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1ß release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1ß release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1ß release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1ß was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1ß in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1ß release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability.

Inflammasome-associated cell death: Pyroptosis, apoptosis, and physiological implications.

Inflammasomes are innate immune mechanisms that promote inflammation by activating the protease caspase-1. Active caspase-1 induces pyroptosis, a necrotic form of regulated cell death, which facilitates the release of intracellular proinflammatory molecules, including IL-1 family cytokines. Recent studies identified mediators of inflammasome-associated cell death and suggested that inflammasomes induce not only pyroptosis, but also apoptosis. Caspase-1 has the potential to induce pyroptosis and apoptosis in a manner that is dependent on the expression of the pyroptosis mediator gasdermin D. Caspase-1-induced apoptosis is mediated by Bid and caspase-7. Caspase-8 is also activated following the formation of inflammasomes and may induce apoptosis. Because inflammasomes contribute to the pathogenesis of inflammatory disorders and host defenses against microbial pathogens, a more detailed understanding of the mechanisms underlying inflammasome-associated cell death may contribute to the development of novel therapeutic strategies for inflammasome-related diseases. Pyroptosis has been implicated in inflammasome-related diseases, and compounds that inhibit this process have been reported. The molecular mechanisms of inflammasome-associated cell death and its physiological implications are discussed herein.

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid.

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.

Vitamin B6 Prevents IL-1ß Protein Production by Inhibiting NLRP3 Inflammasome Activation.

Vitamin B6 includes six water-soluble vitamers: pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), and their phosphorylated forms. Pyridoxal 5'-phosphate (PLP) is an important cofactor for many metabolic enzymes. Several lines of evidence demonstrate that blood levels of PLP are significantly lower in patients with inflammation than in control subjects and that vitamin B6 has anti-inflammatory effects, with therapeutic potential for a variety of inflammatory diseases. Although one of our group previously demonstrated that PL inhibits the NF-κB pathway, the molecular mechanism by which vitamin B6 suppresses inflammation is not well understood. Here, we showed that both PL and PLP suppressed the expression of cytokine genes in macrophages by inhibiting Toll-like receptor (TLR)-mediated TAK1 phosphorylation and the subsequent NF-κB and JNK activation. Furthermore, PL and PLP abolished NLRP3-dependent caspase-1 processing and the subsequent secretion of mature IL-1ß and IL-18 in LPS-primed macrophages. In contrast, PM and PN had little effect on IL-1ß production. PLP, but not PL, markedly reduced the production of mitochondrial reactive oxygen species (ROS) in peritoneal macrophages. Importantly, PL and PLP reduced IL-1ß production induced by LPS and ATP, or by LPS alone, in mice. Moreover, PL and PLP protected mice from lethal endotoxic shock. Collectively, these findings reveal novel anti-inflammatory activities for vitamin B6 and suggest its potential for preventing inflammatory diseases driven by the NLRP3 inflammasome.

Pneumolysin-Dependent Calpain Activation and Interleukin-1α Secretion in Macrophages Infected with Streptococcus pneumoniae.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, is a pore-forming cytolysin that modulates host innate responses contributing to host defense against and pathogenesis of pneumococcal infections. Interleukin-1α (IL-1α) has been shown to be involved in tissue damage in a pneumococcal pneumonia model; however, the mechanism by which this cytokine is produced during S. pneumoniae infection remains unclear. In this study, we examined the role of PLY in IL-1α production. Although the strains induced similar levels of pro-IL-1α expression, wild-type S. pneumoniae D39, but not a deletion mutant of the ply gene (Δply), induced the secretion of mature IL-1α from host macrophages, suggesting that PLY is critical for the maturation and secretion of IL-1α during S. pneumoniae infection. Further experiments with calcium chelators and calpain inhibitors indicated that extracellular calcium ions and calpains (calcium-dependent proteases) facilitated the maturation and secretion of IL-1α from D39-infected macrophages. Moreover, we found that PLY plays a critical role in calcium influx and calpain activation, as elevated intracellular calcium levels and the degradation of the calpain substrate α-fodrin were detected in macrophages infected with D39 but not the Δply strain. These results suggested that PLY induces the influx of calcium in S. pneumoniae-infected macrophages, followed by calpain activation and subsequent IL-1α maturation and secretion.

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.

The inflammasome and its regulation.

Inflammasomes, multiprotein platforms of caspase-1 activation, are assembled in response to a number of exogenous and endogenous danger signals, leading to the production of pro-inflammatory cytokines and induction of inflammatory cell death through the activation of caspase-1. Inflammasomes have been implicated in a wide range of physiological and pathological processes, including host defense against microbial pathogens, maintenance of intestinal homeostasis, and even development of inflammatory disorders. Thus, inflammasomes can be both beneficial and detrimental, and understanding the mechanisms involved in inflammasome activation may provide a better approach to prevent the harmful effects of the inflammatory response. Although inflammasome complexes are formed via protein-protein interactions between their components, accumulating evidence suggests that inflammasome activation is positively and negatively regulated by ligand-binding receptors, accessory proteins, other caspases, cytokines, kinases/phosphatases, redox sensors, ion homeostasis, second messengers, organelles, cytoskeleton, and autophagy, among others. Moreover, inflammasome activation can result in the formation of another caspase-1-activating protein complex, the ASC speck/pyroptosome, which is also tightly controlled. In this review, we discuss how the assembly of inflammasomes and ASC speck is regulated by complex mechanisms. Recent findings on effector functions and biological roles of inflammasomes also are summarized.

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.

Differences in Biofilm Formation by Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains.

Staphylococcus aureus (S. aureus) is a common pathogen involved in community- and hospital-acquired infections. Its biofilm formation ability predisposes it to device-related infections. Methicillin-resistant S. aureus (MRSA) strains are associated with more serious infections and higher mortality rates and are more complex in terms of antibiotic resistance. It is still controversial whether MRSA are indeed more virulent than methicillin-susceptible S. aureus (MSSA) strains. A difference in biofilm formation by both types of bacteria has been suggested, but how only the presence of the SCCmec cassette or mecA influences this phenotype remains unclear. In this review, we have searched for literature studying the difference in biofilm formation by MRSA and MSSA. We highlighted the relevance of the icaADBC operon in the PIA-dependent biofilms generated by MSSA under osmotic stress conditions, and the role of extracellular DNA and surface proteins in the PIA-independent biofilms generated by MRSA. We described the prominent role of surface proteins with the LPXTG motif and hydrolases for the release of extracellular DNA in the MRSA biofilm formation. Finally, we explained the main regulatory systems in S. aureus involved in virulence and biofilm formation, such as the SarA and Agr systems. As most of the studies were in vitro using inert surfaces, it will be necessary in the future to focus on biofilm formation on extracellular matrix components and its relevance in the pathogenesis of infection by both types of strains using in vivo animal models.

The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice.

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

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.

Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.