Jeom-Il Choi

Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

Melatonin inhibits Prevotella intermedia lipopolysaccharide‐induced production of nitric oxide and interleukin‐6 in murine macrophages by suppressing NF‐κB and STAT1 activity

Abstract:  Although a range of biological and pharmacological activities of melatonin have been reported, little is known about its potential anti‐inflammatory efficacy in periodontal disease. In this study, we investigated the effects of melatonin on the production of inflammatory mediators by murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory reactions in the periodontium, and sought to determine the underlying mechanisms of action. Melatonin suppressed the production of nitric oxide (NO) and interleukin‐6 (IL‐6) at both gene transcription and translation levels in P. intermedia LPS‐activated RAW264.7 cells. P. intermedia LPS‐induced NF‐κB‐dependent luciferase activity was significantly inhibited by melatonin. Melatonin did not reduce NF‐κB transcriptional activity at the level of IκB‐α degradation. Melatonin blocked NF‐κB signaling through the inhibition of nuclear translocation and DNA‐binding activity of NF‐κB p50 subunit and suppressed STAT1 signaling. Although further research is required to clarify the detailed mechanism of action, we conclude that melatonin may contribute to blockade of the host‐destructive processes mediated by these two proinflammatory mediators and could be a highly efficient modulator of host response in the treatment of inflammatory periodontal disease.

Establishment of Porphyromonas gingivalis heat-shock-protein-specific T-cell lines from atherosclerosis patients.

Human atherosclerotic plaques contain heat-shock proteins which may serve as potential targets of the immune response in atherosclerosis. Since periodontal infections are suggested as risk factors for the development of cardiovascular diseases, we undertook the present study to evaluate the T-cell immune responses specific to Porphyromonas gingivalis (P. gingivalis) heat-shock protein (hsp)60 in patients suffering from atherosclerosis. Anti-P. gingivalis hsp60 IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T-cell lines from the atheroma lesions and the peripheral blood. The T-cell lines were a mixture of CD4+ and CD8+ cells producing the cytokines characteristic of both Th1 and Th2 subsets. The present findings suggest that the T-cell immune response specific to P. gingivalis hsp60 may be involved in the immunopathologic process of atherosclerotic diseases.

Epitope Mapping of Porphyromonas gingivalis Heat-shock Protein and Human Heat-shock Protein in Human Atherosclerosis

To identify T- and/or cross-reactive B-cell epitopes of P. gingivalis and human heat-shock protein (HSP)60 in atherosclerosis patients, we synthesized 104 overlapping synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. T-cell epitopes of P. gingivalis HSP were identified with the use of previously established P. gingivalis HSP-reactive T-cell lines. B-cell epitopes of P. gingivalis HSP60 and human HSP60 were identified by the use of patients’ sera. Anti-P. gingivalis, anti-P. gingivalis HSP60, or anti-human HSP60 IgG antibody titers were higher in the atherosclerosis patients compared with the healthy subjects. Five immunodominant peptides of P. gingivalis HSP60, identified as T-cell epitopes, were also found to be B-cell epitopes. Moreover, 6 cross-reactive B-cell epitopes of human HSP60 were identified. It was concluded that P. gingivalis HSP60 might be involved in the immunoregulatory process of atherosclerosis, with common T- and/or B-cell epitope specificities and with cross-reactivity with human HSP60.

Identification of immunoreactive epitopes of the Porphyromonas gingivalis heat shock protein in periodontitis and atherosclerosis

BACKGROUND AND OBJECTIVE Heat shock protein 60 (HSP60) of Porphyromonas gingivalis, a major periodontal pathogen, might be a trigger molecule linking infectious periodontitis and autoimmune atherosclerosis. The aim of this study was to identify the peptide specificity of anti-P. gingivalis HSP60 monoclonal antibodies and their cross-reactivity with bacterial and human HSPs. Their specific immunoreactivity to periodontal or atherosclerotic lesions was also investigated. METHODS Twenty patients with chronic periodontitis and 20 atherosclerosis patients who had undergone surgical intervention for atheromatous plaques with evidence of ongoing periodontal disease, were selected. Synthetic peptide 19 ((TLVVNRLRGSLKICAVKAPG)-specific T-cell lines were established from inflamed gingiva and atheromatous plaque and the phenotypes and cytokine profiles were characterized. RESULTS Thirty per cent of periodontitis patients and 100% of atherosclerosis patients reacted positively to cross-reactive peptide 19 from both P. gingivalis and human HSP60. The peptide 19-specific T-cell lines demonstrated the phenotype characteristic of helper T cells (CD4(+)) but did not express CD25 or FOXP3. The interleukin-10 levels were elevated significantly in the peptide 19 T-cell line. CONCLUSION Synthetic peptide 19 of P. gingivalis HSP60 is an immunoreactive epitope in the periodontitis-atherosclerosis axis.

Predominant immunoreactivity of Porphyromonas gingivalis heat shock protein in autoimmune diseases.

BACKGROUND AND OBJECTIVE Autoimmune diseases, including atherosclerosis, diabetes mellitus and rheumatoid arthritis, can be triggered and aggravated by the pathogen-driven antigenic peptide from Porphyromonas gingivalis HSP60. P. gingivalis is the major pathogen of chronic periodontitis, which is a global epidemic prevalent in two-thirds of the adult population. The monoclonal antibody raised against peptide 19 (Pep19: TLVVNRLRGSLKICAVKAPG) from P. gingivalis HSP60 was polyreactive to the human homolog. The aim of this study was to determine if Pep19 from P. gingivalis HSP60 manifests itself as a predominant antigen in infection-triggered autoimmune diseases. MATERIAL AND METHODS  Pep19 from P. gingivalis HSP60, Mycobacterium tuberculosis HSP60 and Chlamydia pneumoniae HSP60 was synthesized for comparative recognition by the sera from patients with atherosclerosis, type 2 diabetes and rheumatoid arthritis, all with ongoing periodontal disease, and by the sera of a control group of patients with periodontal disease but with no history of atherosclerosis, type 2 diabetes or rheumatoid arthritis. RESULTS Of the Pep19 peptides from P. gingivalis HSP60, M. tuberculosis HSP60 and C. pneumoniae HSP60, Pep19 from P. gingivalis HSP60 was the peptide epitope predominantly and most consistently recognized by the serum samples of the four disease groups (chronic periodontitis, atherosclerosis, type 2 diabetes mellitus and rheumatoid arthritis). CONCLUSION Seroreactivity to Pep19 of P. gingivalis HSP60, an oral pathogen, was predominant in patients with autoimmune disease with ongoing periodontal disease.

Anti-inflammatory effects and the underlying mechanisms of action of daidzein in murine macrophages stimulated with Prevotella intermedia lipopolysaccharide

BACKGROUND AND OBJECTIVE Host modulatory agents directed at inhibiting specific proinflammatory mediators could be beneficial in terms of attenuating periodontal disease progression and potentially enhancing therapeutic responses. The aim of this study was to investigate whether daidzein could modulate the production inflammatory mediators in macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and to delineate underlying mechanisms of action. MATERIAL AND METHODS LPS was extracted from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The amounts of nitric oxide (NO) and interleukin-6 (IL-6) secreted into the culture medium were assayed. A real-time PCR was performed to quantify inducible nitric oxide synthase (iNOS) and IL-6 mRNA expression. We used immunoblot analysis to characterize iNOS protein expression, phosphrylation of c-Jun N-terminal kinase (JNK) and p38, degradation of inhibitory κB-α (IκB-α), nuclear translocation of nuclear factor-κB (NF-κB) subunits and phosphorylation of signal transducer and activator of transcription 1 (STAT1). The DNA-binding activity of NF-κB was assessed by using ELISA-based kits. RESULTS Daidzein significantly inhibited the production of NO and IL-6, as well as their mRNA expression, in P. intermedia LPS-treated RAW264.7 cells. The JNK and p38 pathways were not involved in the regulation of LPS-induced NO and IL-6 release by daidzein. Daidzein inhibited the degradation of IκB-α induced by P. intermedia LPS. In addition, daidzein suppressed NF-κB transcriptional activity via regulation of the nuclear translocation and DNA-binding activity of NF-κB p50 subunit and blocked STAT1 phosphorylation. CONCLUSION Although additional studies are required to dissect the molecular mechanism of action, our results suggest that daidzein could be a promising agent for treating inflammatory periodontal disease. Further research in animal models of periodontitis is necessary to better evaluate the potential of daidzein as a novel therapeutic agent to treat periodontal disease.

DHA suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

Several reports have indicated that dietary intake of DHA is associated with lower prevalence of periodontitis. In the present study, we investigated the effect of DHA on the production of proinflammatory mediators in murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. LPS was isolated from lyophilised P. intermedia ATCC 25,611 cells using the standard hot-phenol-water protocol. Culture supernatants were collected and assayed for NO, IL-1β and IL-6. Real-time PCR analysis was carried out to detect the expression of inducible NO synthase (iNOS), IL-1β, IL-6 and haeme oxygenase-1 (HO-1) mRNA. Immunoblot analysis was carried out to quantify the expression of iNOS and HO-1 protein and concentrations of signalling proteins. DNA-binding activities of NF-κB subunits were determined using an ELISA-based assay kit. DHA significantly attenuated the production of NO, IL-1β and IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. DHA induced the expression of HO-1 in cells treated with P. intermedia LPS. Selective inhibition of HO-1 activity by tin protoporphyrin IX significantly mitigated the inhibitory effects of DHA on LPS-induced NO production. DHA significantly attenuated the phosphorylation of c-Jun N-terminal kinase induced by LPS. In addition, DHA suppressed the transcriptional activity of NF-κB by regulating the nuclear translocation and DNA-binding activity of NF-κB p50 subunit and inhibited the phosphorylation of signal transducer and activator of transcription 1. Further in vivo studies are needed to better evaluate the potential of DHA in humans as a therapeutic agent to treat periodontal disease.

Vaccines against periodontitis: a forward-looking review.

Periodontal disease, as a polymicrobial disease, is globally endemic as well as being a global epidemic. It is the leading cause for tooth loss in the adult population and has been positively related to life-threatening systemic diseases such as atherosclerosis and diabetes. As a result, it is clear that more sophisticated therapeutic modalities need to be developed, which may include vaccines. Up to now, however, no periodontal vaccine trial has been successful in satisfying all the requirements; to prevent the colonization of a multiple pathogenic biofilm in the subgingival area, to elicit a high level of effector molecules such as immunoglobulin sufficient to opsonize and phagocytose the invading organisms, to suppress the induced alveolar bone loss, or to stimulate helper T-cell polarization that exerts cytokine functions optimal for protection against bacteria and tissue destruction. This article reviews all the vaccine trials so as to construct a more sophisticated strategy which may be relevant in the future. As an innovative strategy to circumvent these barriers, vaccine trials to stimulate antigen-specific T-cells polarized toward helper T-cells with a regulatory phenotype (Tregs, CD4+, CD25+, FoxP3+) have also been introduced. Targeting not only a single pathogen, but polymicrobial organisms, and targeting not only periodontal disease, but also periodontal disease-triggered systemic disease could be a feasible goal.

Effects of Luteolin on the Release of Nitric Oxide and Interleukin-6 by Macrophages Stimulated With Lipopolysaccharide From Prevotella Intermedia

BACKGROUND Although a range of biologic and pharmacologic activities of luteolin has been reported, little is known about its potential as an agent to treat periodontal disease. In the present study, we investigated whether luteolin could downregulate the production of proinflammatory mediators in murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia (Pi), a major cause of inflammatory periodontal disease, and we attempted to elucidate the possible mechanisms of action. METHODS LPS was prepared from lyophilized Pi ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for nitric oxide (NO) and interleukin (IL)-6. We used real-time polymerase chain reaction to detect inducible NO synthase (iNOS) and IL-6 mRNA expression. iNOS expression, phosphorylation of JNK and p38, IκB-α degradation, nuclear translocation of nuclear factor-kappaB (NF-κB) subunits, and signal transducer and activator of transcription-1 (STAT1) phosphorylation were characterized via immunoblotting. DNA-binding of NF-κB was also analyzed. RESULTS Luteolin strongly suppressed the production of NO and IL-6 at both gene transcription and translation levels in Pi LPS-activated RAW264.7 cells. Mitogen-activated protein kinase pathways were not involved in the inhibition of Pi LPS-induced NO and IL-6 release by luteolin. Luteolin did not reduce NF-κB transcriptional activity at the level of IκB-α degradation. Luteolin blocked NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and suppressed STAT1 signaling. CONCLUSIONS Although further research is encouraged to clarify the detailed mechanism of action, flavonoid luteolin may contribute to blockade of the host-destructive processes mediated by these two proinflammatory mediators and could have potential use in the treatment of inflammatory periodontal disease.