Tomomi Hashizume

Peyer's Patches Are Required for Intestinal Immunoglobulin A Responses to Salmonella spp.

ABSTRACT Previous studies have shown that Peyers patches (PP) are not required for intestinal immunoglobulin A (IgA) responses to orally administered soluble protein. However, the roles of PP in regulation of mucosal immune responses against bacterial antigen remain to be clarified. In the present study, we generated several gut-associated lymphoreticular tissue-null mice by treatment with anti-interleukin-7 receptor antibody, the fusion protein of lymphotoxin β receptor and IgG Fc, and/or tumor necrosis factor receptor p55 and IgG Fc. These mice were then immunized with recombinant Salmonella expressing the C fragment of the tetanus toxin (rSalmonella-Tox C). Orally immunized PP-null mice as well as isolated lymphoid follicle (ILF)-null, PP/ILF-null, and PP/ILF/mesenteric lymph node-null mice induced identical levels of tetanus toxoid (TT)-specific systemic IgG responses to those of control mice. However, PP-null mice, but not ILF-null mice, failed to induce TT-specific intestinal IgA antibodies. Analysis of TT-specific CD4+ T-cell responses showed a reduction of gamma interferon (IFN-γ) synthesis in the intestinal lamina propriae of PP-null mice given oral rSalmonella-Tox C. In contrast, TT-specific IFN-γ responses in the spleen and delayed-type hypersensitivity responses were intact in those immunized mice. Interestingly, Salmonella lipopolysaccharide (LPS)-specific fecal IgA responses were not elicited in PP-null mice, while serum IgG anti-LPS antibodies were identical to those of control mice. These results suggest that while none of the gut-associated lymphoreticular tissues are required for the induction of systemic immune responses, PP are an essential lymphoid tissue for induction and regulation of intestinal IgA immunity against orally administered rSalmonella.

Aggregatibacter actinomycetemcomitans accelerates atherosclerosis with an increase in atherogenic factors in spontaneously hyperlipidemic mice.

Cariogenic and periodontal pathogens are thought to be etiological factors in the development of cardiovascular disease. We assessed the involvement of the periodontal pathogen Aggregatibacter actinomycetemcomitans and cariogenic pathogen Streptococcus mutans in the development of atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. The mice were treated intravenously with A. actinomycetemcomitans HK1651, S. mutans GS-5, or phosphate-buffered saline three times a week for 3 weeks and killed at 15 weeks of age. The areas of the aortic sinus that were covered with atherosclerotic plaque were significantly larger in Apoe(shl) mice challenged with A. actinomycetemcomitans compared with S. mutans- or vehicle-challenged mice. Aggregatibacter actinomycetemcomitans challenge increased serum high-sensitive C-reactive protein and lipopolysaccharide levels. Bacterial DNA was detected in the blood, heart, and spleen, but not in the liver. Furthermore, serum interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, and MCP-1 levels and Toll-like receptor (TLR)2, TLR4, ICAM-1, E-selectin, P-selectin, LOX-1, HSP60, CCL19, CCL21, CCR7, and MCP-1 expressions in the aorta were significantly increased in mice challenged with A. actinomycetemcomitans. These results suggest that systemic infection with A. actinomycetemcomitans accelerates atherosclerosis in Apoe(shl) mice by exposing the whole microorganisms or their products, followed by initiating inflammation. Increases in proatherogenic factors may explain the aggravation of atherosclerosis by A. actinomycetemcomitans infection.

Nasal Vaccination with the 40-Kilodalton Outer Membrane Protein of Porphyromonas gingivalis and a Nontoxic Chimeric Enterotoxin Adjuvant Induces Long-Term Protective Immunity with Reduced Levels of Immunoglobulin E Antibodies

ABSTRACT In this study, we demonstrated that the 40-kDa outer membrane protein of Porphyromonas gingivalis (40-kDa OMP) nasally administered with a nontoxic chimeric adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli (mCTA/LTB) elicited a long-term protective immune response. Immunization with the 40-kDa OMP and mCTA/LTB induced high levels of 40-kDa-OMP-specific immunoglobulin G (IgG) and IgA antibodies (Abs) in sera and elicited a significant IgA anti-40-kDa OMP Ab response in saliva. These Ab responses were maintained for at least 1 year after the immunization. Although using adjuvant mCTA/LTB gave Ab responses in the saliva comparable to those obtained using native cholera toxin (nCT) as the adjuvant, the levels of total IgE and 40-kDa-OMP-specific IgE Abs as well as interleukin-4 levels induced by the immunization with mCTA/LTB were lower than those induced by the immunization with nCT. Importantly, IgG Abs generated by nasal immunization with the 40-kDa OMP plus mCTA/LTB inhibited the coaggregation and hemagglutinin activities of P. gingivalis. Furthermore, the mice given nasal 40-kDa OMP plus mCTA/LTB showed a significant reduction of alveolar bone loss caused by oral infection with P. gingivalis even 1 year after the immunization compared to the loss in unimmunized mice. Because mCTA/LTB is nontoxic, nasally administered 40-kDa OMP together with mCTA/LTB should be an effective and safe mucosal vaccine against P. gingivalis infection in humans and may be an important tool for the prevention of chronic periodontitis.

Sublingual vaccination with outer membrane protein of Porphyromonas gingivalis and Flt3 ligand elicits protective immunity in the oral cavity

In this study, we demonstrated that the 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) sublingually administered with a cDNA vector plasmid encoding Flt3 ligand (pFL) elicited a protective immune response. Sublingual immunization of mice with 40k-OMP plus pFL induced significant serum IgG and IgA, as well as salivary IgA, antibody responses that were comparable to those induced by 40k-OMP plus cholera toxin as adjuvant. When the subclasses of 40k-OMP-specific IgG were evaluated, sublingual immunization with 40k-OMP plus pFL induced both IgG1 and IgG2a antibody responses. Sublingual delivery of pFL resulted in FL expression in submandibular glands, but not in other oral tissues. Furthermore, marked increases in FL protein occurred in saliva and serum, and the frequencies of both CD11c(+)CD11b(+) and CD11c(+)CD8alpha(+) dendritic cells with up-regulated expression of CD80, CD86 and CD40 molecules significantly increased in submandibular lymph nodes and spleen. Importantly, the mice given sublingual 40k-OMP plus pFL showed a significant reduction of alveolar bone loss caused by oral infection with P. gingivalis. These findings suggest that sublingual administration of 40k-OMP with pFL acts as an effective and safe mucosal vaccine against oral P. gingivalis infection, and may be a useful tool in the prevention of chronic periodontitis.

Green tea epigallocatechin-3-gallate attenuates Porphyromonas gingivalis-induced atherosclerosis

The purpose of this study was to determine whether epigallocatechin-3-gallate (EGCG) ameliorates Porphyromonas gingivalis-induced atherosclerosis. EGCG is a polyphenol extract from green tea with health benefits and P. gingivalis is shown here to accelerate atheroma formation in a murine model. Apolipoprotein E knockout mice were administered EGCG or vehicle in drinking water; they were then fed high-fat diets and injected with P. gingivalis three times a week for 3 weeks. Mice were then killed at 15 weeks. Atherosclerotic plaques in the proximal aorta were determined by Oil Red O staining. Atherosclerosis risk factors in serum, liver or aorta were analysed using cytokine antibody arrays, enzyme-linked immunosorbent assay and real-time PCR. Atherosclerotic lesion areas of the aortic sinus caused by P. gingivalis infection decreased in EGCG-treated groups, wherein EGCG reduced the production of C-reactive protein, monocyte chemoattractant protein-1, and oxidized low-density lipoprotein (LDL), and slightly lowered LDL/very LDL cholesterol in P. gingivalis-challenged mice serum. Furthermore, the increase in CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22phox and iNOS gene expression levels in the aorta of P. gingivalis-challenged mice were reduced in EGCG-treated mice. However, HO-1 mRNA levels were elevated by EGCG treatment, suggesting that EGCG, as a natural substance, inhibits P. gingivalis-induced atherosclerosis through anti-inflammatory and antioxidative effects.

Porphyromonas gingivalis stimulates monocyte adhesion to human umbilical vein endothelial cells

Monocyte recruitment to the endothelium is a crucial step in the inflammatory response that precedes the development of atherosclerosis. We assessed the effect of Porphyromonas gingivalis on monocyte adhesion to endothelial cells, cytokine production during monocyte/endothelial cell co-culture, and the expression of cell adhesion molecules on human umbilical vein endothelial cells (HUVEC) and their ligands on monocytes. Porphyromonas gingivalis challenge significantly increased the adhesion of THP-1 monocytes to HUVEC, the production of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein 1 (MCP-1) in co-cultures of HUVEC and THP-1 cells, and the transcription and translation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HUVEC. The transcription of tumor necrosis factor receptor-associated factor 1 was also increased in HUVEC and THP-1 cells by P. gingivalis infection. Moreover, the stimulation of monocyte adhesion to HUVEC by P. gingivalis infection was partially inhibited by pretreatment with a mixture of anti-ICAM-1, -VCAM, and -E-selectin monoclonal antibodies. These data suggest that adherence between HUVEC and THP-1 cells, followed by the production of cytokines and chemokines, was enhanced by increased expression of cell adhesion molecules on P. gingivalis-sensitized HUVEC, which in turn led to inflammatory atherogenesis.

Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product

Periodontitis and dental caries are common oral diseases, in these days, and the passive immunization is one of the most effective approaches for prevention. For this purpose, we have constructed mouse and human monoclonal antibodies to inhibit the Porphyromonas gingivalis-associated hemagglutination and coaggregation. In addition, an artificial antibody, single-chain variable fraction, or scFv, which also inhibited the hemagglutination, was constructed. Specifically for dental caries, mouse and human monoclonal antibodies that inhibited the glucosyltransferase (GTF) activity, responsible for biofilm formation, were also constructed. The advantage of scFv over the native antibody is that the former molecule does not induce possible side-effects due to Fc, such as autoimmune disease, because it consists only of variable regions originating from both heavy and light chains. To increase the abilities of the antibody preparations, we attempted to construct an additional scFv using Bacillus brevis, a secretion-proficient gram-positive bacterium, as a host cell. An scFv protein possessing the same biological activity as that of the parental antibody was successfully secreted from a B. brevis transformant following the construction of a chimeric shuttle plasmid, which was accomplished by employing a new heterodimer system.

Porphyromonas gingivalis accelerates atherosclerosis in C57BL/6 mice fed a high-fat diet

Objective: Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in atherosclerotic apo E-deficient mice. Here, we investigated whether repeated P. gingivalis injection affected the inflammatory and atherosclerotic responses of C57BL/6 mice fed a high-fat diet (HFD). Materials and methods: Eight-week-old C57BL/6 mice fed either HFD or a regular chow diet (RD) were inoculated intravenously with P. gingivalis or phosphate-buffered saline three times per week for 10 weeks and sacrificed at 19 weeks of age. Atheromatous lesions in the proximal aorta of each animal were analyzed histomorphometrically, and the serum cytokine and C-reactive protein (CRP) levels were determined. Results: Long-term HFD feeding as compared to RD feeding led to a slight increase in atheromatous lesions in the aortic sinus as well as increases in the levels of serum monocyte chemoattractant protein 1. Further, P. gingivalis injection significantly enhanced the formation of atherosclerotic plaque, and increased CRP and inflammatory cytokine levels, in mice fed the HFD, although no further increase in LDL was observed. Conclusion: These results suggest that bacteremia-induced by repeated injection with P. gingivalis accelerates atherosclerosis in normal C57BL/6 mice by initiating inflammation, and is therefore implicated in chronic infection-related pathogenicity.

Oral immunization with Porphyromonas gingivalis outer membrane protein and CpG oligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity

The aim of this study was to evaluate the efficacy of an oral vaccine containing the 40-kDa outer membrane protein of Porphyromonas gingivalis (40K OMP) and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) to control oral infection by P. gingivalis. [run on]40K-OMP40K-OMP40K-OMPOral immunization with 40K-OMP plus CpG ODN induced significant 40K-OMP-specific serum IgG, IgA and saliva IgA antibody responses. The 40K-OMP-specific CD4(+) T cells induced by oral 40K-OMP plus CpG ODN produced both Th1 (IFN-gamma) and Th2 (IL-4) cytokines. Furthermore, increased frequencies of CD11c(+)B220(+) DCs and CD11c(+)CD11b(+) DCs with up-regulated expression of CD80, CD86, CD40 and MHC II molecules were noted in spleen, Peyers patches and cervical lymph nodes. Immunized mice were then infected orally with P. gingivalis to determine whether the immune responses induced by oral 40K-OMP plus CpG ODN were capable of suppressing bone resorption caused by P. gingivalis infection. Mice given 40K-OMP plus CpG ODN showed significantly reduced bone loss associated with oral infection by P. gingivalis.Thus, oral administration of 40K-OMP together with CpG ODN induces Th1- and Th2-type cells, which provide help for protective immunity against P. gingivalis infection. This may be an important tool for prevention of chronic periodontitis.

Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

ABSTRACT We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4+ T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-γ), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c+ CD8α+, but not CD11c+ CD11b+ or CD11c+ B220+, dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans.