Atheroprotective nasal immunization with a heat shock protein 60 peptide from Porphyromonas gingivalis.

PURPOSE: Immunization with Porphyromonas gingivalis heat shock protein 60 (PgHSP60) may have an immunoregulatory effect on atherogenesis. The aim of this study was to determine whether nasal immunization with a PgHSP60 peptide could reduce atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. METHODS: Seven-week-old male ApoE KO mice were assigned to receive a normal diet, a Western diet, a Western diet and challenge with PgHSP60-derived peptide 14 (Pep14) or peptide 19 (Pep19), or a Western diet and immunization with Pep14 or Pep19 before challenge with Pep14 or Pep19. RESULTS: Atherosclerotic plaques were significantly smaller in mice that received a Western diet with Pep14 nasal immunization than in mice that received a Western diet and no Pep14 immunization with or without Pep14 challenge. An immunoblot profile failed to detect serum reactivity to Pep14 in any of the study groups. Stimulation by either Pep14 or Pep19 strongly promoted the induction of CD4+CD25+forkhead box P3 (FoxP3)+ human regulatory T cells (Tregs) in vitro. However, the expression of mouse splenic CD4+CD25+FoxP3+ Tregs was lower in the Pep14-immunized mice than in the Pep14-challenged or Pep19-immunized mice. Levels of serum interferon gamma (IFN-γ) and transforming growth factor beta were higher and levels of interleukin (IL) 10 were lower in the Pep14-immunized mice than in the other groups. Induction of CD25- IL-17+ T helper 17 (Th17) cells was attenuated in the Pep14-immunized mice. CONCLUSIONS: Nasal immunization with Pep14 may be a mechanism for attenuating atherogenesis by promoting the secretion of IFN-γ and/or suppressing Th17-mediated immunity.

Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein.

PURPOSE: The aim of this study was to evaluate the ability of Porphyromonas gingivalis (P. gingivalis) to induce oxidation of high-density lipoprotein (HDL) and to determine whether the oxidized HDL induced by P. gingivalis exhibited altered antiatherogenic function or became proatherogenic. METHODS: P. gingivalis and THP-1 monocytes were cultured, and the extent of HDL oxidation induced by P. gingivalis was evaluated by a thiobarbituric acid-reactive substances (TBARS) assay. To evaluate the altered antiatherogenic and proatherogenic properties of P. gingivalis-treated HDL, lipid oxidation was quantified by the TBARS assay, and tumor necrosis factor alpha (TNF-α) levels and the gelatinolytic activity of matrix metalloproteinase (MMP)-9 were also measured. After incubating macrophages with HDL and P. gingivalis, Oil Red O staining was performed to examine foam cells. RESULTS: P. gingivalis induced HDL oxidation. The HDL treated by P. gingivalis did not reduce lipid oxidation and may have enhanced the formation of MMP-9 and TNF-α. P. gingivalis-treated macrophages exhibited more lipid aggregates than untreated macrophages. CONCLUSIONS: P. gingivalis induced HDL oxidation, impairing the atheroprotective function of HDL and making it proatherogenic by eliciting a proinflammatory response through its interaction with monocytes/macrophages.

Targeting the epitope spreader Pep19 by naïve human CD45RA+ regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy.

PURPOSE: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance. METHODS: Human polyclonal CD4+CD25+CD127lo- Tregs (127-Tregs) and naïve CD4+CD25+CD45RA+ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ-/- mouse model of collagen-induced arthritis. RESULTS: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4+CD25+ Tregs at the articular joints in a mechanistic and orchestrated way. CONCLUSIONS: We propose human naïve CD4+CD25+CD45RA+ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

Robust immunoreactivity of teenager sera against peptide 19 from Porphyromonas gingivalis HSP60.

PURPOSE: Epitope spreading is a phenomenon in which distinct subdominant epitopes become major targets of the immune response. Heat shock protein (HSP) 60 from Porphyromonas gingivalis (PgHSP60) and peptide 19 from PgHSP60 (Pep19) are immunodominant epitopes in autoimmune disease patients, including those with periodontitis. It remains unclear whether Pep19 is a dominant epitope in subjects without periodontitis or autoimmune disease. The purpose of this study was to determine the epitope spreading pattern and verify Pep19 as an immunodominant epitope in healthy teenagers using dot immunoblot analysis. The patterns of epitope spreading in age-matched patients with type 1 diabetes mellitus (type 1 DM) and healthy 20- to 29-year old subjects were compared with those of healthy teenagers. METHODS: Peptide from PgHSP60, Mycobacterium tuberculosis HSP60 (MtHSP60), and Chlamydia pneumoniae HSP60 (CpHSP60) was synthesized for comparative recognition by sera from healthy subjects and patients with autoimmune disease (type 1 DM). Dot immunoblot analysis against a panel of peptides of PgHSP60 and human HSP60 (HuHSP60) was performed to identify epitope spreading, and a densitometric image analysis was conducted. RESULTS: Of the peptide from PgHSP60, MtHSP60, and CpHSP60, PgHSP60 was the predominant epitope and was most consistently recognized by the serum samples of healthy teenagers. Most sera from healthy subjects and patients with type 1 DM reacted more strongly with PgHSP60 and Pep19 than the other peptides. The relative intensity of antibody reactivity to Pep19 was higher in the type 1 DM group than in the healthy groups. CONCLUSIONS: Pep19 is an immunodominant epitope, not only in autoimmune disease patients, but also in healthy young subjects, as evidenced by their robust immunoreactivity. This result suggests that the Pep19-specific immune response may be an initiator that triggers autoimmune diseases.

Platelet-activating Factor Mediates Endotoxin Tolerance by Regulating Indoleamine 2,3-Dioxygenase-dependent Expression of the Suppressor of Cytokine Signaling 3.

Indoleamine 2,3-dioxygenase (IDO) mediates immune tolerance, and suppressor of cytokine signaling 3 (SOCS3) negatively regulates the JAK/STAT signal transduction pathway. We determined previously that platelet-activating factor (PAF) protects mice against LPS-induced endotoxic shock, but its detailed mechanism of action was unknown. We performed survival experiments in IDO+/+ and IDO-/- mice using an LPS-induced endotoxemia model and rated organ injury (neutrophil infiltration and liver function). Using ELISA and Western blotting, we also investigated the mechanism of PAF-mediated endotoxin tolerance during endotoxemia. PAF-mediated endotoxin tolerance was dependent on IDO in vivo and in vitro and was not observed in IDO-/- mice. JAK/STAT signaling, crucial for SOCS3 expression, was also impaired in the absence of IDO. In an IDO- and STAT-dependent manner, PAF mediated a decrease in IL-12 and a dramatic increase in IL-10 and reduced mouse mortality. In addition, PAF attenuated LPS-mediated neutrophil infiltration into the lungs and interactions between neutrophil-like (THP-1) and endothelial cells (human umbilical vein endothelial cells). These results indicate that PAF-mediated endotoxin tolerance is initiated via IDO- and JAK/STAT-dependent expression of SOCS3. Our study has revealed a novel tolerogenic mechanism of IDO action and an important association between IDO and SOCS3 with respect to endotoxin tolerance.

Peptide 19 of Porphyromonas gingivalis Heat Shock Protein Is a Potent Inducer of Low-Density Lipoprotein Oxidation.

BACKGROUND: Although periodontal pathogens show a strong association with development of atherosclerosis, little is known about how a microorganism contributes to disease onset and progression. Oxidation of low-density lipoprotein (LDL) is a major risk factor of atherogenesis. The principal objective of this study is to evaluate the ability of peptide 19 (Pep19) of Porphyromonas gingivalis (Pg) heat shock protein (HSP) as a potent inducer of LDL oxidation, and a secondary objective is to compare this ability with that of Pep19 from different bacteria. METHODS: HSP60, Pep14, and Pep19 from Pg and THP-1 monocytes were cultured, and the extent of LDL oxidation induced by each peptide was evaluated by an assay for thiobarbituric acid-reactive substances (TBARS). Pep19 and HSP60 from Chlamydia pneumoniae and Mycobacterium tuberculosis were also cultured with THP-1 monocytes and evaluated by the TBARS assay. After incubation of macrophages with LDL and peptides from Pg, Oil Red O staining was performed for examination of foam cells, macrophages that took up the oxidized LDL. RESULTS: Monocyte-mediated native-LDL oxidation under the influence of Pep19 or HSP60 from Pg was significantly stronger than oxidation induced by the counterpart Pep19 or HSP60 from C. pneumoniae or M. tuberculosis. Pep19 from Pg HSP60 showed a stronger ability to induce LDL oxidation than did Pep14 from Pg HSP60. CONCLUSION: These results suggest Pep19 from Pg HSP60 has a distinct ability to induce native-LDL oxidation as a plausible mechanism by which this peptide may drive epitope spreading to the neoantigen, i.e., oxidized LDL, in the pathogenesis of atherosclerosis.

Enhancement of paclitaxel-induced breast cancer cell death via the glycogen synthase kinase-3ß-mediated B-cell lymphoma 2 regulation.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine protein kinase that is known to mediate cancer cell death. Here, we show that B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, is regulated by GSK-3ß and that GSK-3ß-mediated regulation of Bcl-2 is crucial for mitochondrial-dependent cell death in paclitaxel-stimulated cells. We demonstrate that MCF7 GSK-3ß siRNA cells are more sensitive to cell death than MCF7 GFP control cells and that in the absence of GSK-3ß, Bcl-2 levels are reduced, a result enhanced by paclitaxel. Paclitaxel-induced JNK (c-Jun N-terminal kinase) activation is critical for Bcl-2 modulation. In the absence of GSK-3ß, Bcl-2 was unstable in an ubiquitination-dependent manner in both basal- and paclitaxeltreated cells. Furthermore, we demonstrate that GSK-3ß-mediated regulation of Bcl-2 influences cytochrome C release and mitochondrial membrane potential. Taken together, our data suggest that GSK-3ß-dependent regulation of Bcl-2 is crucial for mitochondria-dependent cell death in paclitaxel-mediated breast cancer therapy.

Resveratrol regulates naïve CD 8+ T-cell proliferation by upregulating IFN-γ-induced tryptophanyl-tRNA synthetase expression.

We found that resveratrol enhances interferon (IFN)-γ-induced tryptophanyl-tRNA-synthetase (TTS) expression in bone marrow- derived dendritic cells (BMDCs). Resveratrol-induced TTS expression is associated with glycogen synthase kinase-3ß (GSK-3ß) activity. In addition, we found that resveratrol regulates naïve CD8+ T-cell polarization by modulating GSK-3ß activity in IFN-γ-stimulated BMDCs, and that resveratol induces upregulation of TTS in CD8+ T-cells in the in vivo tumor environment. Taken together, resveratrol upregulates IFN-γ-induced TTS expression in a GSK-3ß-dependent manner, and this TTS modulation is crucial for DC-mediated T-cell modulation.

Ellagic acid modulates LPS-induced maturation of dendritic cells through the regulation of JNK activity.

Ellagic acid (EA) is a well- known phytochemical that modulates various cellular processes. It is present in a variety of foods, including grapes, strawberries, and nuts. However, the influence of EA on immunological responses is not well defined. Here, we investigated the effects of EA on the lipopolysaccharide (LPS)-induced bone marrow-derived dendritic cells (BMDCs). EA was not cytotoxic to DCs. EA suppressed LPS-induced expression of costimulatory molecules (CD80 and CD86), but it did not suppress the expression of major histocompatibility complex (MHC) class I and MHC class II in BMDCs. In particular, EA blocked LPS-induced c-Jun N-terminal kinase (JNK) activation. LPS-mediated expression of proinflammatory cytokines (IL-12 and IFN-γ) was diminished by EA. We also confirmed that levels of IL-12 and IFN-γ were not influenced by EA in the presence of a JNK inhibitor. Taken together, these data demonstrate that EA regulates the immune response through the modulation of LPS-induced DC maturation.

Nanoparticle-based vaccine delivery for cancer immunotherapy.

Development of nano-sized carriers including nanoparticles, nanoemulsions or liposomes holds great potential for advanced delivery systems for cancer immunotherapy, as such nanostructures can be used to more effectively manipulate or deliver immunologically active components to specific target sites. Successful development of nanotechnology based platform in the field of immunotherapy will allow the application of vaccines, adjuvants and immunomodulatory drugs that improve clinical outcomes for immunological diseases. Here, we review current nanoparticle-based platforms in the efficacious delivery of vaccines in cancer immunotherapy.

Rhamnogalacturonan II is a Toll-like receptor 4 agonist that inhibits tumor growth by activating dendritic cell-mediated CD8+ T cells.

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1ß, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.