Gut microbiota of one-and-a-half-year-old food-allergic and healthy children.

BACKGROUND: Intestinal bacteria may play a role in the development of food allergies. This study aimed to analyze and compare the gut microbiota of food-allergic children with that of healthy children of the same age. METHODS: Stool samples were collected from one-and-a-half-year-old food-allergic (FA group, n = 29) and healthy controls (HC group, n = 19). A questionnaire was provided to examine the children's birth, dietary, medical, and social histories. The gut microbiota was profiled by 16S rRNA sequencing. Differences in taxonomic composition were assessed using linear discriminant analysis effect size (LEfSe), and microbial functional profiles were predicted with Tax4Fun2. RESULTS: No significant difference in the alpha diversity index between the two groups; however, a negative correlation was observed between the Shannon diversity index and the relative abundance of Bacteroides. A significant difference was observed in beta diversity (permutational multivariate analysis of variance) in the bacterial composition between the FA and HC groups (P < 0.05). The FA group had a higher abundance of Escherichia and Anaeromassilibacillus and a lower abundance of Bacteroides, Oscillibacter, Ruminococcus, Hungateiclostridium and Anaerotaenia than the HC group (LEfSe: linear discriminant analysis score >2). The FA group showed a predicted increase in the expression levels of genes associated with intestinal pathogenicity compared with that in the HC group. CONCLUSIONS: The gut microbiota of food-allergic children has a higher abundance of bacteria involved in intestinal inflammation and a lower abundance of bacteria involved in immune tolerance than that of healthy children. This dysbiosis may also be associated with food allergies.

The CXCL10-CXCR3 axis plays an important role in Kawasaki disease.

The precise pathogenesis of Kawasaki disease remains unknown. In an attempt to elucidate the pathogenesis of KD through the analysis of acquired immunity, we comprehensively examined the immunophenotypic changes in immune cells such as lymphocytes and monocytes along with various cytokines, focusing on differences between pre- and post- treatment samples. We found high levels of CXCL9 and CXCL10 chemokines that decreased with treatment, which coincided with a post-treatment expansion of Th1 cells expressing CXCR3. Our results show that the CXCL10-CXCR3 axis plays an important role in the pathogenesis of KD.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization.

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Activation of inflammasomes and mechanisms for intracellular recognition of Listeria monocytogenes.

The high mortality rate associated with Listeria monocytogenes can be attributed to its ability to invade the body systemically and to activate inflammasomes. Both of these processes are facilitated by expressing a major virulence factor known as listeriolysin O, a 56 kDa pore-forming protein encoded by the hly gene. Listeriolysin O plays a crucial role in the pathogenesis of the bacterium by facilitating the escape of the pathogen from the phagosome into the cytosol. This process is essential for the successful establishment of infection. In addition, listeriolysin O is known as an immunomodulator that activates host signal transduction. In addition to listeriolysin O, Listeria expresses a variety of bacterial ligands, such as lipoteichoic acid, nucleotide, and flagellin, that are recognized by host intracellular pattern-recognition receptors including Nod-like receptors, AIM2-like receptors, and RIG-I-like receptors. This review introduces intracellular recognition of Listeria monocytogenes since recent studies have revealed that the activation of inflammasome exacerbates Gram-positive bacteria 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.

Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

IL-1α serves as a pro-inflammatory cytokine. Although pro-IL-1α has cytokine activity, proteolytic maturation increases its potency and release from cells. IL-1α maturation occurs in a caspase-1-dependent manner following inflammasome activation. However, pro-IL-1α is not a substrate of caspase-1, and it remains unclear what mediates the maturation of this cytokine downstream of inflammasomes. Here, we show that gasdermin D (GSDMD), an executor of pyroptosis, is required for the rapid induction of IL-1α maturation by non-particulate inflammasome activators. Ablation of GSDMD abrogates the maturation of IL-1α, but not of IL-1ß. Inflammasome-induced maturation of IL-1α relies on extracellular Ca2+ and calpains. Ca2+ influx and calpain activation are induced in a GSDMD-dependent manner. Glycine, which inhibits cell lysis, but not GSDMD pore formation, does not affect IL-1α maturation. These results suggest that during inflammasome activation, GSDMD processed by caspase-1 forms plasma membrane pores that mediate Ca2+ influx, resulting in the calpain-dependent maturation of IL-1α.

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.

The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

IL-22 Controls Iron-Dependent Nutritional Immunity Against Systemic Bacterial Infections.

Host immunity limits iron availability to pathogenic bacteria, but whether immunity limits pathogenic bacteria from accessing host heme, the major source of iron in the body, remains unclear. Using Citrobacter rodentium, a mouse enteric pathogen and Escherichia coli, a major cause of sepsis in humans as models, we find that interleukin-22, a cytokine best known for its ability to promote epithelial barrier function, also suppresses the systemic growth of bacteria by limiting iron availability to the pathogen. Using an unbiased proteomic approach to understand the mechanistic basis of IL-22 dependent iron retention in the host, we have identified that IL-22 induces the production of the plasma hemoglobin scavenger haptoglobin and heme scavenger hemopexin. Moreover, the anti-microbial effect of IL-22 depends on the induction of hemopexin expression, while haptogloblin is dispensable. Impaired pathogen clearance in infected Il22-/- mice was restored by hemopexin administration and hemopexin-deficient mice had increased pathogen loads after infection. These studies reveal a previously unrecognized host defense mechanism regulated by IL-22 that relies on the induction of hemopexin to limit heme availability to bacteria leading to suppression of bacterial growth during systemic infections.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1ß. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1ß secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1ß release, was not essential for MLKL-dependent death or IL-1ß secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1ß cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Mechanism and Regulation of NLRP3 Inflammasome Activation.

Members of the nucleotide-binding domain and leucine-rich repeat (LRR)-containing (NLR) family and the pyrin and HIN domain (PYHIN) family can form multiprotein complexes termed 'inflammasomes'. The biochemical function of inflammasomes is to activate caspase-1, which leads to the maturation of interleukin 1 beta (IL-1ß) and IL-18 and the induction of pyroptosis, a form of cell death. Unlike other inflammasomes, the NLRP3 inflammasome can be activated by diverse stimuli. The importance of the NLRP3 inflammasome in immunity and human diseases has been well documented, but the mechanism and regulation of its activation remain unclear. In this review we summarize current understanding of the mechanism and regulation of NLRP3 inflammasome activation as well as recent advances in the noncanonical and alternative inflammasome pathways.

Inactivation of the MAPK signaling pathway by Listeria monocytogenes infection promotes trophoblast giant cell death.

Listeria monocytogenes has a well-characterized ability to cross the placental barrier, resulting in spontaneous abortion and fetal infections. However, the mechanisms resulting in infection-associated abortion are not fully understood. In this study, we demonstrate that the dephosphorylation of MAPK family proteins caused by L. monocytogenes infection of trophoblast giant (TG) cells, which are placental immune cells, contributes to infectious abortion. Dephosphorylation of c-Jun, p38, and ERK1/2 was observed in infected TG cells, causing the downregulation of cytoprotective heme oxygenase (HO)-1. Blocking the dephosphorylation of proteins, including MAPK family proteins, inhibited the decrease in HO-1 expression. Treatment with MAPK inhibitors inhibited bacterial internalization into TG cells. Moreover, Toll-like receptor 2 involved in the expression of MAPK family proteins. Infection with a listeriolysin O-deleted mutant impaired dephosphorylation of MAPK family proteins in TG cells and did not induce infectious abortion in a mouse model. These results suggest that inactivation of the MAPK pathway by L. monocytogenes induces TG cell death and causes infectious abortion.

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.

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.

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.