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This study investigated olfactory/taste functions in older persons requiring nursing care to clarify whether these functions are associated with appetite or nutritional status. Seventy-two older persons requiring nursing care and 37 unassisted persons were surveyed for olfactory function, taste function, appetite, and nutritional status. Age-adjusted covariance analysis was conducted between the two groups. Both groups showed reduced olfactory and taste functions; these functions were more markedly reduced in participants requiring nursing care compared to those who did not. Both groups had similar appetite and nutritional status findings, suggesting that these factors are not associated with olfactory and taste functions.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aim: Although mucociliary clearance is important for preventing pneumonia, its association with the onset of pneumonia is unclear. The aim of this study is to examine the association between saccharin test results as a potential measure of mucociliary clearance and history of pneumonia in nursing home residents., Patients and Methods: Ninety elderly nursing home residents (elderly group) were selected, 35 of whom had a history of pneumonia. Twenty-five healthy adults (adult group) were also investigated to provide baseline values for this study. We conducted the saccharin test to evaluate mucociliary clearance and compared the saccharin time (ST) between those with and without history of pneumonia., Results: Mean ST in the adult group was 12±6 min. The ST in the pneumonia group was significantly longer than that in the non-pneumonia group (32±23 min vs. 17±13 min) (p<0.05)., Conclusion: Impaired mucociliary clearance is a factor in the development of pneumonia among nursing home residents., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: To investigate olfactory function in elderly subjects requiring nursing care to clarify its association with appetite and nutritional status., Setting: Facility for the elderly requiring nursing care., Participants: The subjects were 158 elderly people requiring nursing care and 37 elderly people not requiring nursing care., Measurements: Experiment I: Olfactory function and factors (cognitive function, appetite, and nutritional status) that may be associated with it were compared between the elderly subjects requiring nursing care and those not requiring nursing care using covariance analysis in consideration of age. For evaluation, the OSIT-J was used for olfactory function, the HDS-R for cognitive function, the CNAQ for appetite, and BMI for nutritional status. Experiment II: The subjects were the same elderly subjects requiring nursing care in Experiment I, and food intake was surveyed in addition to the OSIT-J, HDS-R, CNAQ, and BMI. A univariate linear regression analysis was performed with OSIT-J as the response variable, and age, HDS-R, CNAQ, BMI, and food intake as the explanatory variables., Results: Experiment I: On covariance analysis, the OSIT-J score was significantly lower for the elderly subjects requiring nursing care than for those not requiring nursing care (p<0.01). The mean score was 8 or lower in both groups, demonstrating lower olfactory function in both groups. Regarding factors that may be associated with olfactory function, a significant difference was noted in the HDS-R (p<0.01), confirming significantly lower cognitive function in the elderly subjects requiring nursing care. No significant difference was noted in the CNAQ or BMI. Experiment II: On a univariate linear regression analysis, an association with the OSIT-J was noted for age and HDS-R. Age was inversely correlated and the HDS-R was positively correlated. Factors associated with lower olfactory function in the elderly subjects requiring nursing were age and cognitive function, whereas appetite, nutritional status, and food intake were not associated., Conclusion: Olfactory function in elderly subjects requiring nursing care was poorer than that in those not requiring nursing care, suggesting that aging and cognitive decline are associated with lower olfactory function. In addition, no association of lower olfactory function with appetite, nutritional status, or food intake was noted in the elderly subjects requiring nursing care., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

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.

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.

A large number of gamma delta T cells (γδ T cells) are located within epithelial tissues including the skin. In mice, epidermal and dermal γδ T cells consist of distinct subsets and have specific roles in cutaneous immune responses. A recent study demonstrated that γδ T cells and cutaneous dendritic cells migrate from the skin to the draining lymph nodes (LNs). However, it remains unclear whether they regulate the antigen-specific immune response within the LNs. Herein, we investigated their properties and role in the LNs using the Mycobacterium bovis bacille Calmette-Guérin (BCG) infection model. In vivo cell labeling analysis revealed that most of the migratory subset comprised dermal Vγ4(+) cells. This population transmigrated from the skin to the LNs in a Gi-coupled chemokine receptor-independent manner. By depleting Vγ4(+) cells, the intranodal expansion of CD8(+) T cell against BCG was significantly attenuated. In addition, in vitro analysis revealed that Vγ4(+) cells produced TNF-α and enhanced IL-12 production by dendritic cells. Taken together, these findings suggest that dermal Vγ4(+) cells are a unique subset that possesses a migratory potency to the skin-draining LNs and enhances the dendritic cell function therein.

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/β 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/β 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.

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., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation., (© 2013.)

Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.

Among a number of laboratory strains of Listeria monocytogenes used in experimental infection, strain LO28 is highly capable of inducing robust beta interferon (IFN-β) production in infected macrophages. In this study, we investigated the molecular mechanism of the IFN-β-inducing ability of LO28 by comparing it with that of strain EGD, a low-IFN-β-inducing strain. It was found that LO28 secretes a large amount of IFN-β-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-β 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-β-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.

Adult T-cell leukemia (ATL) patients and human T-cell leukemia virus-1 (HTLV-1) infected individuals succumb to opportunistic infections. Cell mediated immunity is impaired, yet the mechanism of this impairment has remained elusive. The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the viral DNA and is constitutively expressed in infected cells and ATL cells. To test the hypothesis that HBZ contributes to HTLV-1-associated immunodeficiency, we challenged transgenic mice that express the HBZ gene in CD4 T cells (HBZ-Tg mice) with herpes simplex virus type 2 or Listeria monocytogenes, and evaluated cellular immunity to these pathogens. HBZ-Tg mice were more vulnerable to both infections than non-Tg mice. The acquired immune response phase was specifically suppressed, indicating that cellular immunity was impaired in HBZ-Tg mice. In particular, production of IFN-γ by CD4 T cells was suppressed in HBZ-Tg mice. HBZ suppressed transcription from the IFN-γ gene promoter in a CD4 T cell-intrinsic manner by inhibiting nuclear factor of activated T cells and the activator protein 1 signaling pathway. This study shows that HBZ inhibits CD4 T-cell responses by directly interfering with the host cell-signaling pathway, resulting in impaired cell-mediated immunity in vivo.

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&#95;ppe37) and another harbouring the vector alone (Ms&#95;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&#95;ppe37 induced a significantly lower level of tumour necrosis factor alpha and interleukin 6 in the infected macrophages compared with Ms&#95;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&#95;ppe37 than in macrophages infected with Ms&#95;vec. These results suggest that PPE37 may have a potential role in interfering with the pro-inflammatory cytokine response of infected macrophages.

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.

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 (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 (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.

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.

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.

Listeria monocytogenes evades the antimicrobial mechanisms of macrophages by escaping from the phagosome into the cytosolic space via a unique cytolysin that targets the phagosomal membrane, listeriolysin O (LLO), encoded by hly. Gamma interferon (IFN-gamma), which is known to play a pivotal role in the induction of Th1-dependent protective immunity in mice, appears to be produced, depending on the bacterial virulence factor. To determine whether the LLO molecule (the major virulence factor of L. monocytogenes) is indispensable or the escape of bacteria from the phagosome is sufficient to induce IFN-gamma production, we first constructed an hly-deleted mutant of L. monocytogenes and then established isogenic L. monocytogenes mutants expressing LLO or ivanolysin O (ILO), encoded by ilo from Listeria ivanovii. LLO-expressing L. monocytogenes was highly capable of inducing IFN-gamma production and Listeria-specific protective immunity, while the hly-deleted mutant was not. In contrast, the level of IFN-gamma induced by ILO-expressing L. monocytogenes was significantly lower both in vitro and in vivo, despite the ability of this strain to escape the phagosome and the intracellular multiplication at a level equivalent to that of LLO-expressing L. monocytogenes. Only a negligible level of protective immunity was induced in mice against challenge with LLO- and ILO-expressing L. monocytogenes. These results clearly show that escape of the bacterium from the phagosome is a prerequisite but is not sufficient for the IFN-gamma-dependent Th1 response against L. monocytogenes, and some distinct molecular nature of LLO is indispensable for the final induction of IFN-gamma that is essentially required to generate a Th1-dependent immune response.

Listeriolysin O (LLO), a member of the cholesterol-dependent cytolysin (CDC) family, is a major virulence factor of Listeria monocytogenes and contributes to bacterial escape from intracellular killing of macrophages. LLO is activated under weakly acidic conditions; however, the molecular mechanism of this pH-dependent expression of cytolytic activity of LLO is poorly understood. In this study, CDCs including LLO, ivanolysin O (ILO), seeligeriolysin O (LSO), pneumolysin (PLY), streptolysin O (SLO) and perfringolysin O (PFO) were prepared as recombinant proteins and examined for their functional changes after treatment under various pH conditions. Haemolytic and membrane cholesterol-binding activities were not affected in PLY, SLO and PFO at any pH examined. By contrast, all the Listeria-derived cytolysins, LLO, ILO and LSO, were active only at an acidic pH and rapidly inactivated under neutral or alkaline conditions. Once inactivated, LLO could not be reactivated even by a downward pH shift. The hydrophobicity of LLO treated at neutral or alkaline pH was increased. These data suggested that the pH-dependent loss of cytolytic activity appeared to be due to irreversible structural changes of domain 4 that resulted in the loss of target membrane cholesterol binding.

In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.

Objective: The purpose of this study was to evaluate the effectiveness of a sustained-release sheet with vancomycin (VCM) to prevent prosthetic graft infection., Methods: VCM was incorporated into a poly-L-lactide-co-caprolactone (PLCA) sheet (VCM-PLCA). The release profile of VCM from the VCM-PLCA sheet and the tissue concentration of VCM released in vivo were examined. To assess the antibacterial effect of the VCM-PLCA sheet, a sterile Dacron sheet was implanted into 96 male mice (C57BL/6), who were randomly divided into four groups of 24 each and treated as follows: no treatment (group A, control group), a local bolus injection of an aqueous solution of VCM (group B), a plain PLCA sheet (group C), and a VCM-PLCA sheet (group D). After the treatment, methicillin-resistant Staphylococcus aureus (MRSA) (1 x 10(6) colony forming units) was inoculated onto the Dacron graft surface. The Dacron grafts were retrieved on days 3, 7, 10, and 14 after the implantation, and the number of MRSA in the Dacron grafts was counted., Results: VCM was slowly released from the VCM-PLCA sheet over 2 weeks in vivo, and the mean in vivo concentrations of VCM in the tissue around a VCM-PLCA sheet were 7.95, 26.39, 13.87, 12.51, 8.36, and 10.33 mug/mL (the minimum inhibitory concentration of VCM against MRSA is 2.0 mug/ml), at 1, 2, 5, 7, 10, and 14 days after the implantation, respectively. MRSA colonization on the cultivated agar plates was detected in all samples from groups A, B, and C at any postoperative time points. In contrast, some samples were negative for bacterial cultures in group D (2, 3, 1, and 2 samples out of 6 samples each on days 3, 7, 10, and 14 after the implantation, respectively). At all time points, the number of MRSA bacteria in the implanted Dacron graft in group D was by far the lowest (P < .01 at each time point)., Conclusions: The sustained-release sheet with VCM appears to be effective for the reduction of subcutaneous prosthetic graft infection.

Listeriolysin O (LLO) derived from Listeria monocytogenes is highly capable of inducing interleukin (IL)-12, IL-18 and interferon (IFN)-gamma, and facilitates the generation of Th1 cells. We have recently shown that recombinant LLO (rLLO) inhibits generation of ovalbumin (OVA)-specific Th2 immune response by skewing maturation of antigen-specific T cells into Th1 cells. In the present study, we investigated the effect of rLLO on the effector phase of Th2-dependent allergic rhinitis in BALB/c mice sensitized with OVA. In mice sensitized intraperitoneally and challenged intranasally with OVA, nasal allergic symptoms such as sneezing and nose-scratching were observed at a high frequency. A high titre of anti-OVA IgE antibody was detected in sera and a large number of eosinophils migrated into the nasal tissue. However, rLLO treatment during the intranasal challenge inhibited the allergic symptoms, production of anti-OVA IgE antibody and eosinophil infiltration. Though rLLO did not affect antigen-specific cytokine production from splenic CD4(+) T cells, rLLO significantly suppressed OVA-specific IL-4 and IL-5 production from nasal mononuclear cells. We further found that rLLO inhibited the recruitment of CD4(+) T cells in nasal mucosa, and diminished the transcription and cell surface expression of CCR4 on splenic CD4(+) T cells. Moreover, rLLO was able to inhibit the passive cutaneous anaphylaxis reaction mediated by anaphylactic antibodies (IgE and IgG(1)) and mast cells. Taken together, these data showed that rLLO suppresses the effector phase of allergic rhinitis by inhibition of Th2 cell recruitment to nasal mucosa and type I allergic reaction.

Among bacterial haemolysins, cholesterol-dependent cytolysins (CDCs) produced by various Gram-positive bacteria are known to exhibit a lethal activity in mice. In this study, recombinant CDCs of streptolysin O, pneumolysin, ivanolysin O, listeriolysin O and several listeriolysin O mutants were constructed and the relationship between cytolytic activity and the lethal activity of each recombinant protein in mice was examined. Specific activity for cytolysis was determined by a quantitative haemolytic assay. Each protein was injected intravenously into mice and the lethal activity was evaluated by measuring the time until death of the mice. The four full-length CDC proteins exhibited lethal activity and their activities were highly proportional to their cytolytic activities. Inhibition of haemolytic activity resulted in the loss of lethal activity and non-haemolytic mutants of listeriolysin O did not exhibit any lethal activity. These data clearly indicate that the lethal effect of CDC proteins is dependent on the cytolytic activity.

Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-beta and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-beta, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.

Catheter-related infection is a frequent and serious complication. One factor responsible for catheter-related infection is bacterial invasion at the catheter-insertion site. We have shown that the sustained-release form of basic fibroblast growth factor (bFGF) enhances tissue regeneration and angiogenesis in various pathological conditions. The purpose of this study was to determine whether topical use of sustained-release form of bFGF promotes tissue regeneration around the wound and prevents catheter-related bacterial invasion. Fifty-four male mice (C57BL/6) were divided into three groups according to what was implanted subcutaneously on the back (each group, n=18): a Dacron sheet alone (group A), a Dacron sheet and a plain gelatin sheet (group B), and a Dacron sheet and sustained-release of bFGF (50 microg) (group C). Seven days after the implantation, the tissue immediately above the Dacron sheet was inoculated with methicillin-resistant Staphylococcus aureus (MRSA). In histological examinations, group C had a larger granulation tissue area containing a larger amount of collagen tissue and vessels than the other groups. Two days after the MRSA inoculation, the number of MRSA in the Dacron sheet of group C was significantly smaller than the other groups (P<0.01). Pretreatment with sustained-release form of bFGF may prevent catheter-related bacterial invasion.

Listeriolysin O (LLO), a cholesterol-dependent cytolysin derived from Listeria monocytogenes, is a potent inducer of interleukin (IL)-12, IL-18 and interferon (IFN)-gamma. We have shown that LLO facilitates development of T cells mediating protective immunity against L. monocytogenes through the induction of IFN-gamma production at an early stage. Based on this finding, it is postulated that LLO inhibits differentiation of Th2 cells and the Th2 immune response. By using a murine model of ovalbumin (OVA)-induced allergic rhinitis, we investigated whether LLO has an ability to modulate the Th2-type immune disorder. In mice sensitized intraperitoneally with ovalbumin (OVA)/alum and challenged intranasally with OVA, a large number of eosinophils migrated into the nasal tissue, and high titres of anti-OVA IgE and IgG(1) antibodies were detected in sera. However, LLO treatment during sensitization markedly inhibited the eosinophil infiltration and production of these anti-OVA antibodies. A large number of T cells from mice sensitized and challenged with OVA produced high level of IL-4 and IL-5 but not IFN-gamma after stimulation with OVA. In contrast, OVA-specific IFN-gamma-producing T cells were preferentially induced in mice treated with LLO at the time of sensitization. In the absence of LLO administration, the expression level of GATA-3 and SOCS-3 in CD4(+) T cells was enhanced after sensitization with OVA. LLO treatment resulted in a reduction of GATA-3 and SOCS-3 expressions but induced the transcription of T-bet instead. Taken together, these data show clearly that LLO is capable of inhibiting Th2 immune response by skewing differentiation of antigen-specific T cells into Th1 cells.

Peritoneal exudate cells of mice were stimulated with a streptomycin-dependent Mycobacterium tuberculosis strain, 18b. Gamma interferon production by natural killer cells depending on interleukin-12 and interleukin-18 was induced only in the presence of a high dose of streptomycin. This study suggested the requirement of active bacterial metabolism for this host response.

Listeriolysin O (LLO), a major virulence factor of Listeria monocytogenes, is a member of the cholesterol-dependent cytolysin family and plays important roles not only in survival of this bacterium in phagocytes but also in induction of various cellular responses, including cytokine production. In this work, we examined the involvement of LLO in induction of the cytokine response in intestinal epithelial cells, the front line of host defense against food-borne listeriosis. Infection of Caco-2 cells with wild-type L. monocytogenes induced persistent expression of interleukin-6 (IL-6) mRNA. In contrast, IL-6 expression was observed only transiently during infection with non-LLO-producing strains. A sublytic dose of recombinant LLO (rLLO) induced the expression of IL-6 via formation of membrane pores. Under conditions of LLO-induced pore formation without extensive cell lysis, Ca2+ influx was observed, and the IL-6 expression induced by rLLO was inhibited by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), an intracellular Ca2+ chelator. LLO secreted by cytoplasmic L. monocytogenes appeared to induce pore formation in the membrane and to enable the trafficking of intracellular and extracellular molecules. Pretreatment with BAPTA-AM inhibited persistent IL-6 expression in Caco-2 cells infected with wild-type L. monocytogenes. These results suggest that LLO is involved in IL-6 production in the late phase of infection through the formation of Ca2+-permeable pores and subsequent Ca2+-dependent modulation of signaling and gene expression.