Chikara Kohda

Essential Role of Interleukin-12 (IL-12) and IL-18 for Gamma Interferon Production Induced by Listeriolysin O in Mouse Spleen Cells

ABSTRACT The mechanism of gamma interferon (IFN-γ) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-γ when its cytolytic activity was blocked by cholesterol treatment. The IFN-γ-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b+ cells resulted in a profound decrease in IFN-γ production in response to LLO stimulation. Negative selection also suggested the contribution of DX5+ cells in IFN-γ production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b+ fraction of spleen cells was unchanged. There was no change in the expression of the IFN-γ-inducing cytokine genes in the CD11b− fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-γ production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-γ after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-γ from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.

Dissociated Linkage of Cytokine-Inducing Activity and Cytotoxicity to Different Domains of Listeriolysin O from Listeria monocytogenes

ABSTRACT Listeriolysin O (LLO), a cholesterol-binding cytolysin of Listeria monocytogenes, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-γ)-inducing activities. rLLO529 exhibited both IFN-γ-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-γ production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-γ-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-γ production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-γ-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.

Listeriolysin O-induced membrane permeation mediates persistent interleukin-6 production in Caco-2 cells during Listeria monocytogenes infection in vitro.

ABSTRACT 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.

Induction of gamma interferon and nitric oxide by truncated pneumolysin that lacks pore-forming activity.

ABSTRACT Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-γ) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-γ induction. Therefore, the IFN-γ-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-γ-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-γ abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-γ production in spleen cells by a mechanism different from pore formation and that the induced IFN-γ stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.

Listeriolysin O, a cytolysin derived from Listeria monocytogenes, inhibits generation of ovalbumin-specific Th2 immune response by skewing maturation of antigen-specific T cells into Th1 cells

Listeriolysin O (LLO), a cholesterol‐dependent cytolysin derived from Listeria monocytogenes, is a potent inducer of interleukin (IL)‐12, IL‐18 and interferon (IFN)‐γ. We have shown that LLO facilitates development of T cells mediating protective immunity against L. monocytogenes through the induction of IFN‐γ 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 IgG1 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‐γ after stimulation with OVA. In contrast, OVA‐specific IFN‐γ‐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.

Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii.

ABSTRACT Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-γ) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD50) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD50 was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-γ in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-γ-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-γ-inducing activity than LLO. By comparing the IFN-γ-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-γ-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-γ production is responsible for the failure of L. ivanovii to generate effective protective immunity.

Seeligeriolysin O, a cholesterol-dependent cytolysin of Listeria seeligeri, induces gamma interferon from spleen cells of mice

ABSTRACT Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-γ) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-γ production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-γ production but also LSO-induced IFN-γ production. Both NK cells and CD11b+ cells were required for LSO-induced IFN-γ production. Among the various cytokines expressed in CD11b+ cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1β but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Background: We previously reported that Kgp, a lysine gingipain, degraded osteoprotegerin, an osteoclastogenesis inhibitory factor, to enhance lipopolysaccharide-induced osteoclastogenesis. Results: Kgp enhanced tumor necrosis factor-α- and interleukin-1β-induced osteoclastogenesis. Conclusion: Kgp degraded osteoprotegerin more efficiently than other cytokines, which might be related to enhancement of osteoclastogenesis by Kgp. Significance: Degradation of osteoprotegerin may be a crucial event in periodontal osteolysis. Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159–166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

Low HCMV DNA copies can establish infection and result in significant symptoms in extremely preterm infants: a prospective study.

Breast milk (BM) is the main source of human cytomegalovirus (HCMV) infection. We examined whether the number of HCMV DNA copies in BM is related to HCMV infection in very low birth weight (VLBW) infants. We identified 11 pairs of VLBW infants and mothers. BM samples were collected every week until 10 weeks postpartum. Urine samples were collected from the infants within 1 week, at 6 to 8 weeks, at discharge, and whenever HCMV infection was suspected. HCMV DNA in BM was positive in 7 of 11 mothers and reached a peak at 4 to 5 weeks postpartum. Of the 11, 5 infants were determined to be infected from positive HCMV DNA in the urine, despite the fact that BM was used after being frozen. Of the five, four infected infants exhibited symptoms between 35 and 60 days of age. Symptomatic infants had longer stays and slower weight gain. The HCMV infection rate is high in very preterm infants. A new strategy to prevent HCMV infection other than freezing should therefore be established.

A simple smart amplification assay for the rapid detection of human cytomegalovirus in the urine of neonates.

Human cytomegalovirus (CMV) is the most common cause of infection-related congenital abnormalities in neonates and the leading cause of non-hereditary sensorineural hearing loss in childhood. In addition, the number of low-birth-weight infants has recently increased, especially in Japan, in association with an increasing frequency of postnatal CMV infections transferred through raw breast milk. The increase in the number of congenital CMV and postnatal CMV infections in low-birth-weight infants requires rapid detection at the bedside in order to ensure a correct diagnosis and provide early anti-viral therapy. In this report, a simplified smart amplification (SMAP) method was developed to detect CMV in the urine of neonates. This method does not require DNA extraction, and the DNA amplification procedure is performed under isothermal conditions. Therefore, it takes only 60 min to detect CMV in a urine sample, and CMV DNA was rapidly detectable in symptomatic infants. In brief, this SMAP-based assay provides a simple, rapid and efficient method for detecting human CMV at the bedside.