Augmentation of host defense against bacterial infection pretreated intraperitoneally with an alpha-glucan RBS in mice

Protection against Listeria monocytogenes and Escherichia coli in mice was enhanced by an intraperitoneal (i.p.) administration of polysaccharide "RBS". Peritoneal macrophages from mice administered i.p. with 30 mg/kg doses of RBS 4 days earlier exhibited increased scavenger functions as assessed by in vivo phagocytosis, in vitro intracellular killing and generation of superoxide anion. When cytokine production of the macrophages was assessed by biological assay and Northern blotting analysis, interleukin (IL)-1 production and IL-1 alpha gene expression were significantly increased in macrophages from RBS-treated mice. On the other hand, tumor necrosis factor (TNF) alpha gene was expressed in macrophages from RBS-treated mice at a much reduced level as compared with those in mice treated i.p. with Corynebacterium parvum on 4 days earlier. In correlation with expression of TNF gene in the macrophages, RBS-treated mice were less susceptible to the lethal toxicity of LPS than C.parvum-treated mice. In RBS-treated mice, in vivo elimination of bacteria was enhanced at the early phase of infection with L.monocytogenes or E.coli, resulting in augmentation of host defense against these bacterial infection. These results suggest that adequately enhanced activities of macrophages acting as scavenger phagocytes play important roles in the enhanced resistance against bacteria in mice treated i.p. with RBS.     

Suppression of host resistance against Listeria monocytogenes infection by 15-deoxyspergualin in mice

The effects of 15-deoxyspergualin (DSG), an immunosuppressive agent, on host resistance against Listeria monocytogenes were studied in mice. Administration of DSG in the early phase of infection resulted in fatal listeriosis by preventing acquired anti-listerial resistance, even though the infectious dose was sublethal for the untreated controls. In contrast, DSG treatment started after development of the acquired immunity was ineffective. Endogenous production of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) in the bloodstreams induced by the infection was normal in DSG-treated mice. Nevertheless, augmentation of macrophage functions such as expression of major histocompatibility complex (MHC) class II antigens, phagocytic activity and listericidal activity induced by the infection was abrogated by DSG treatment. These results suggest that the inhibitory effect of DSG on anti-listerial resistance might be different from cyclosporine A (CsA).     

Endogenous tumor necrosis factor (cachectin) is essential to host resistance against Listeria monocytogenes infection.

During a sublethal murine infection with Listeria monocytogenes cells, tumor necrosis factor (TNF) activity was detectable in neither sera nor spleen homogenates at any stage of the infection when a bioassay with L-929 cells (less than 4 U/ml) was used. However, injecting the mice with an immunoglobulin fraction obtained from a rabbit hyperimmunized with recombinant murine TNF-alpha resulted in acceleration of listeriosis. When 1 mg of anti-TNF antibody was injected per mouse, all the mice died from listeriosis, even though the infectious dose was sublethal for the untreated controls. The antigen-specific elimination of the bacterium from the spleens and livers of anti-TNF antibody-treated mice was delayed, depending on the dose of the antibody injected. Endogenous TNF seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-TNF antibody was given between day zero and day 2 of infection. The effect of endogenous TNF on antilisterial resistance was due to neither regulation of alpha interferon (IFN-alpha) and IFN-gamma production nor induction of IFN-beta subtype 1 (IFN-beta 1), because anti-TNF antibody treated-mice produced normal levels of IFN-alpha and IFN-gamma in the bloodstream during infection and administration of monoclonal anti-murine IFN-beta 1 antibody had no effect on the development of listeriosis. Alternatively, the listericidal activity of peritoneal macrophages of L. monocytogenes-infected mice could be abrogated by injection of anti-TNF antibody in vivo. These results suggest that the lower level of TNF is produced endogenously in mice that received L. monocytogenes infection and that it plays an essential role in the host defense against L. monocytogenes infection.     

Augmentation of host resistance to Listeria monocytogenes infection by a traditional Chinese medicine, ren-shen-yang-rong-tang (Japanese name: ninjin-youei-to).

Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NIN), a traditional Chinese medicine, is a drug made of spray-dried powder of hot water extract obtained from twelve species of medical plants. An intraperitoneal (ip) injection with NIN 2 days before intravenous (iv) infection with Listeria monocytogenes (L. monocytogenes) accelerated elimination of viable bacteria in the spleen in the early stage of infection (from day 1) and protected mice from the lethal infection. It was suggested that the protective effect of NIN was mediated by the activation of nonimmune macrophages playing a principle role in resistance in the early stage of infection. Two days after ip injection with NIN just before infection, significantly increment in the number of monocytes in the peripheral blood was observed, though macrophage number in the spleen and their intracellular killing activity were unchanged. At 12 hours after infection with L. monocytogenes, a significantly enhanced increase of splenic macrophage number was observed in NIN-treated mice, compared to controls. After ip injection of NIN, interleukin-1 (IL-1), IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) became detectable in the serum or peritoneal cavity. These results suggested that NIN stimulated macrophage-precursor cells in the bone marrow via the production of IL-1, IL-6, GM-CSF by macrophages, accelerated the supply of peripheral macrophages, and such macrophages accumulated into the site of infection in the very early stage of infection. Similar protective effects of NIN were observed by oral administration for 7 days till 1 day before iv infection with L. monocytogenes.     

Augmentation of mitogen-induced interleukin 2 production by oral administration of polysaccharide SPR-901.

The effects of oral administration of antitumor polysaccharide SPR-901 (RBS), an alpha-1,3 branched alpha-1,6 glucan, on concanavalin A (Con A)-induced interleukin 2 (IL-2) production of splenocytes were studied. The augmentation effect on Con A-induced IL-2 production was evident when more than 30 mg/kg of SPR-901 was administered orally to mice. On the other hand, oral administration of B512 dextran, an analogous alpha-1,6 glucan, did not show any augmentation effects on IL-2 production. The augmentation effect of SPR-901 on IL-2 production seemed to be mediated mainly by macrophages stimulated with SPR-901.     

Transforming growth factor beta is protective in host resistance against Listeria monocytogenes infection in mice.

The role of transforming growth factor beta (TGF-beta) in host resistance against Listeria monocytogenes infection was studied with mice. The constitutive expression of TGF-beta 1 mRNA was observed in the spleens and livers of mice before and after infection. Injecting the mice with anti-TGF-beta 1 peptide serum resulted in diminished antilisterial resistance, whereas the administration of human platelet-derived TGF-beta 1 enhanced the resistance. Moreover, mice were protected against lethal infection when treated with TGF-beta 1. These results suggest the TGF-beta 1 might be involved in antilisterial resistance. On the other hand, injecting the mice with TGF-beta 1 resulted in a decrease in the titers of endogenous gamma interferon, tumor necrosis factor alpha, and interleukin-6, which are crucial in antilisterial resistance, in sera and in extracts of spleen and liver. Thus, a complicated mechanism might be involved in the role of TGF-beta 1 in host resistance against L. monocytogenes infection.     

Augmentation of protective immune responses against sendai virus infection by fungal polysaccharide schizophyllan

When treated with fungal polysaccharide schizophyllan, mice survived otherwise lethal Sendai virus infection. Both intraperitoneal and oral administrations were effective when sonicated schizophyllan with a relative molecular mass (M_r) of 4.6 × 10⁵ was used. Antiviral antibody in the serum could be detected at an earlier time after virus infection and virus spread in the lung was more efficiently inhibited in schizophyllan-treated mice than in untreated controls. Schizophyllan also augmented protective immune responses induced by low doses of a live Sendai virus vaccine that were insufficient to confer complete protection against challenge infection with a virulent strain. On the other hand, schizophyllan did not influence interferon production in mice whether or not infected with Sendai virus. The present results suggest that schizophyllan confers better protection against virus infection through augmentation of antiviral immune responses and can be used as an immune enhancer.     

Riboflavin (vitamin B2) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes

Riboflavin, also known as vitamin B₂, is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte‐specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK‐deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF‐induced priming of Nox2, which is needed for oxidative burst, was defective in RFK‐deficient phagocytes. Lack of riboflavin in wild‐type macrophages for only 6 h shut down TNF‐induced, RFK‐mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti‐listerial activity. Vice versa, ROS production by riboflavin‐deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.     

Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes

Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.     

Host resistance of CD18 knockout mice against systemic infection with Listeria monocytogenes

Mice with targeted mutations of CD18, the common beta2 subunit of CD11/CD18 integrins, have leukocytosis, impaired transendothelial neutrophil emigration, and reduced host defense to Streptococcus pneumoniae, a gram-positive extracellular bacterium. Previous studies using blocking monoclonal antibodies suggested roles for CD18 and CD11b in hepatic neutrophil recruitment and host innate response to Listeria monocytogenes, a gram-positive intracellular bacterium. We induced systemic listeriosis in CD18 knockout (CD18-ko) and wild-type (WT) mice by tail vein injection with Listeria. By 14 days postinjection (dpi), 8 of 10 WT mice died, compared with 2 of 10 CD18-ko mice (P < 0.01). Quantitative organ culture showed that numbers of Listeria organisms in livers and spleens were similar in both groups at 20 min postinfection. By 3, 5, and 7 dpi, however, numbers of Listeria organisms were significantly lower in livers and spleens of CD18-ko mice than in WT mice. Histopathology showed that following Listeria infection, CD18-ko mice had milder inflammatory and necrotizing lesions in both spleens and livers than did WT mice. Cytokine assays indicated that baseline interleukin-1beta and granulocyte colony-stimulating factor (G-CSF) levels were higher in CD18-ko mice than in WT mice and that CD18-ko splenocytes produced higher levels of interleukin-1beta and G-CSF than WT splenocytes under the same amount of Listeria stimulation. These findings show that CD18 is not an absolute requirement for antilisterial innate immunity or hepatic neutrophil recruitment. We propose that the absence of CD18 in the mice results in the priming of innate immunity, as evidenced by elevated cytokine expression, and neutrophilic leukocytosis, which augments antilisterial defense.     

Changes in host resistance caused by Nocardia brasiliensis in mice: cross-protection against Listeria monocytogenes

Listeria monocytogenes was used to study the rate of development, magnitude, and persistence of the antimicrobial resistance engendered by Nocardia brasiliensis infection in mice. The growth of Listeria in the liver and spleen was more effectively restricted in Nocardia-infected mice than in noninfected animals. The development of delayed-type hypersensitivity to the Nocardia antigen was closely correlated to the increased resistance to Listeria, suggesting that both properties are the consequence of a single immunological event. The antibacterial resistance was also demonstrated in vitro. The results of the foregoing studies indicate that the microbicidal ability of macrophages, very likely activated by cell-mediated immunity, in enhanced in mice infected with Nocardia.     

Human HLA-B27 gene enhances susceptibility of rats to oral infection by Listeria monocytogenes.

Germfree rats transgenic for the human genes HLA-B27 and beta 2-microglobulin were colonized with hemolysin-positive (Hly+) or hemolysin-negative (Hly-) strains of Listeria monocytogenes. HLA-B27 rats were very susceptible to infection with Hly+ L monocytogenes none survived beyond 6 days. Conversely, nontransgenic control rats survived alimentary tract colonization with the Hly+ strain, and both transgenic and nontransgenic rats survived colonization with the Hly- strain of L monocytogenes. After colonization with Hly+ L monocytogenes, both transgenic and nontransgenic rats developed severe bowel inflammation which consisted histologically of microab scesses, granulomatous lesions, and ulcers; however, whereas the transgenic rats died within 6 days, only very mild intestinal lesions were seen in nontransgenic rats 10 to 42 days after colonization. Liver and splenic lesions were small and transient in nontransgenic rats. Transgenic and nontransgenic control rats infected with Hly- Listeria developed mild transient diarrhea but showed no histological changes in the intestine. This study thus documents an association between a particular bacterial product (hemolysin produced by L monocytogenes) and the induction of severe inflammatory disease and death in rats expressing HLA-B27 and beta 2-microglobulin.     

Mouse peptidoglycan recognition protein PGLYRP-1 plays a role in the host innate immune response against Listeria monocytogenes infection

The role of mouse peptidoglycan recognition protein PGLYRP-1 in innate immunity against Listeria monocytogenes infection was studied. The recombinant mouse PGLYRP-1 and a polyclonal antibody specific to PGLYRP-1 were prepared. The mouse PGLYRP-1 showed antibacterial activities against L. monocytogenes and other Gram-positive bacteria. PGLYRP-1 mRNA expression was induced in the spleens and livers of mice infected with L. monocytogenes. The viable bacterial number increased, and the production of cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was reduced in mice when mice had been injected with anti-PGLYRP-1 antibody before infection. The levels of IFN-γ and TNF-α titers in the organs were higher and the viable bacterial number was reduced in mice injected with recombinant mouse PGLYRP-1 (rmPGLYRP-1) before infection. PGLYRP-1 could directly induce these cytokines in spleen cell cultures. The elimination of intracellular bacteria was upregulated in NMuLi hepatocyte cells overexpressing PGLYRP-1. The enhancement of the elimination of L. monocytogenes from the organs was observed in IFN-γ(-/-) mice by rmPGLYRP-1 administration but not in TNF-α(-/-) mice. These results suggest that PGLYRP-1 plays a role in innate immunity against L. monocytogenes infection by inducing TNF-α.     

The protective activity of immunostimulants against Listeria monocytogenes infection in mice.

The function of peritoneal macrophages induced by intraperitoneal (i.p.) injection of attenuated Streptococcus pyogenes (OK-432), Bacillus Calmette Guérin (BCG), protein-bound polysaccharide preparation isolated from Coriolus vesicolor (PSK) or Lactobacillus casei was examined. The PMA-triggered respiratory burst (production of O2- and H2O2) of macrophages induced by OK-432, BCG or Lac. casei was greater than that of resident or thioglycollate-stimulated macrophages and was similar to that of BCG-activated macrophages. PSK failed to enhance the production of O2- or H2O2 by macrophages. Alkaline phosphodiesterase (APD) activity was reduced in macrophages induced by OK-432, BCG or Lac. casei injection and in BCG-activated macrophages. The APD activity of macrophages obtained 7 and 13 days after i.p. injection of PSK was elevated, as with thioglycollate-stimulated macrophages. Listericidal activity in vitro was enhanced in macrophages obtained in 13 and 7 days, but suppressed in macrophages obtained 2 days after OK-432, BCG or Lac. casei injection. Lac. casei administered either 2 or 13 days before infection with Listeria monocytogenes was protective but OK-432, BCG (0.1 mg) and PSK were not, even though they were able to stimulate macrophage function.     

Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha.

Recombinant human tumor necrosis factor alpha (rHuTNF-alpha) administered intravenously to mice resulted in enhanced resistance to a lethal challenge infection of Listeria monocytogenes given 24 h later. The observed protection was lost following treatment of the rHuTNF-alpha preparations with rabbit polyclonal antibody rHuTNF-alpha but not with normal rabbit immunoglobulin G.     

Kinetics of interleukin-1 production by macrophages during infection with Listeria monocytogenes

Production of interleukin-1 (IL-1) by peritoneal macrophages from mice inoculated intravenously with Listeria monocytogenes was measured at increasing intervals of infection. IL-1 activity in the 24 h macrophage supernatants was determined by using the thymocyte PHA co-mitogenesis assay. IL-1 production increased as the infection progressed, reached a peak on the 9th or 10th day and then declined progressively to approach normal values by the 20th day. Our data on the kinetics of IL-1 levels during an acute infection with L.monocytogenes are discussed in relationship to the development of cell-mediated immunity and its regulation by macrophages.     

Case of stillbirth due to infection with Listeria monocytogenes

A case of listeriosis in a stillborn baby is described. The causative organism was isolated from lung, placenta, and meconium. The only manifestation of infection of the baby's mother was a slight pyrexia three days before delivery which subsided quickly after treatment with ampicillin. Examination of the mother 11 days after delivery failed to produce evidence of listerial infection.