Immunization with gingipain A hemagglutinin domain of Porphyromonas gingivalis induces IgM antibodies binding to malondialdehyde-acetaldehyde modified low-density lipoprotein

Publisher Copyright: © 2018 Kyrklund et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pgand MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and to Pg in both Rgp44-and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1 alpha whereas whole Pg bacteria achieved through regulation of antiinflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/antiinflammatory processes in atherosclerosis.Peer reviewe

Vibrational spectroscopy and its future applications in microbiology

Abstract Vibrational spectroscopic techniques, namely Fourier transform infrared (FTIR) and Raman spectroscopy, are based on the study of molecular vibrations, and they are complementary techniques to each other. This review provides an overview of the vibrational spectroscopic techniques applied in microbiology during the past decade. In addition, future applications of the elaborated spectroscopic techniques will be highlighted. The results of this review show that both FTIR and Raman spectroscopy are promising alternatives to conventional diagnostic approaches because they provide label-free and noninvasive bacterial detection, identification, and antibiotic susceptibility testing in a single step. Cost-effective, accurate, and rapid tests are needed in order to improve diagnostics and patient care, to decrease the use of unnecessary antimicrobial agents, to prevent resistant microbials, and to decrease the overall burden of outbreaks. Prior to that, however, the presented approaches need to be validated in a clinical workflow against the conventional diagnostic approaches

Genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) reduces atherosclerotic plaques in mice

Abstract Objective: Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases). Approach and Results: We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor (P4h-tm−/−/Ldlr−/−) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm+/+/Ldlr−/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher lipoprotein lipase protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm−/−/Ldlr−/− livers as compared with P4h-tm+/+/Ldlr−/−. Conclusions: Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it

Immunization with gingipain A hemagglutinin domain of Porphyromonas gingivalis induces IgM antibodies binding to malondialdehyde-acetaldehyde modified low-density lipoprotein

Abstract Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pg and MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and toPg in both Rgp44- and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1α whereas whole Pg bacteria achieved through regulation of anti-inflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/anti-inflammatory processes in atherosclerosis

Mouse plasma IgM and IgG binding to MDA-LDL after immunization with <i>P. gingivalis</i>.

<p>C57BL/6 mice were immunized with heat-killed <i>P. gingivalis</i> ATCC33277 (Pg; n = 8) and controls received saline (Co; n = 8). Plasma IgM (A) and IgG (B) to MDA-LDL before (pre) and after immunization (post) were determined with chemiluminescence immunoassay. Each C57BL/6 plasma sample (1∶500) was measured in duplicate and an average for each individual was calculated. LDLR^−/− mice were immunized with killed <i>P. gingivalis</i> (3 strains mixed) (Pg; n = 7) and controls received PBS (Co; n = 8). Plasma IgM (C) and IgG (D) to MDA-LDL after the second booster immunization (imm) and after the HFD (end) were determined. Each LDLR^−/− plasma sample (1∶1000) was measured in duplicate and an average for each individual in two repeated assays was calculated. Additionally, mouse plasma IgM binding to CuOx-LDL (E, G) and PC-BSA (F, H) was determined. For C57BL/6 mice (E, F) this was done similarly as described for panel A. Plasma samples of LDLR^−/− mice (G, H) were pooled between three or four mice (1∶1000) for a single assay, in which the mean ± SD within a group is shown.</p

Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL.

During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr(-/-) mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDL-specific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects

Association between human serum IgM to <i>P. gingivalis</i> and MDA-LDL, and competitive binding with recombinant gingipain domains.

<p>Sera from 29 healthy adults were analyzed for IgM (A) and IgG (B) binding to Pg and MDA-LDL by chemiluminescence immunoassay. Associations between antibody levels were analyzed with Spearman rank correlation test. Human sera were pre-incubated with recombinant gingipain domains Rgp44, Rgp15–27, RgpCAT (C, D) in a competitive immunoassay detecting IgM binding to immobilized MDA-LDL. The ratio of serum IgM binding (B/B₀) to MDA-LDL with and without competitor (175 µg/ml) in 29 human serum samples (C) and dose-dependent competition assays of one sample (D). Reciprocal competition assay was performed to analyze human serum IgM binding to Pg antigen competed with MDA-LDL, nLDL and PC-BSA in a representative sample (E). RU, relative units.</p

Antibodies to recombinant gingipain in <i>P. gingivalis</i> immunized mice.

<p>Recombinant proteins of the arginine specific gingipain protease of <i>P. gingivalis</i> were produced in <i>E.coli</i>: two proteins in the hemagglutinin/adhesion domain, Rgp44 and Rgp15–27, and the catalytic domain, RgpCAT, which were used in chemiluminescence immunoassay to determine mouse plasma (1∶500) IgM and IgG binding in <i>P. gingivalis</i> immunized (Pg) and control (Co) groups in both A) C57BL/6 and B) LDLR^−/− mice. A) For C57BL/6 immunized and control mice the samples (n = 8 each) were determined as duplicate before (pre) and after (post) immunization. Box-plots represent the distribution of the means of the sample duplicates. B) Two plasma samples within the group were pooled for immunized (black bars) and control (hatched bars) LDLR^−/− mice and measured in duplicate. Samples were collected before (pre), after the second booster immunization (imm) and at the end of HFD (end). Columns represent the mean ± SD of pooled samples in each group. **P<0.01 and *P<0.05.</p

Mouse plasma IgM binding to apoptotic T lymphocytes after <i>P. gingivalis</i> immunization.

<p>C57BL/6 mice were immunized with heat-killed Pg and controls received sterile saline (n = 8 per group). Mouse plasma (1∶70) IgM binding to UV-irradiated Jurkat T cells was measured with flow cytometry. A, B) Apoptotic T cell population (R1) was verified with propidium iodide (PI) staining. C) Plasma IgM binding in gate R2 of preimmune (black) and postimmune (blue) plasma samples, and competition of IgM binding with 250 µg/ml MDA-LDL (green) or native LDL (red). Inset plots (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034910#pone-0034910-g006" target="_blank">Fig. 6C</a>) represent the secondary antibody control (2°Ab control) and plasma IgM binding to apoptotic cells in a Pg-immunized mouse (post). D) IgM binding to Jurkat cells was determined for each mouse in Pg-immunized and control group as geometric mean value in R2 subtracted by the 2°Ab control. Box-plot graphs represent the distribution of sample means calculated for two repeated assays. **P<0.01 and *P<0.05.</p

Cross-reactive saliva IgA antibodies to oxidized LDL and periodontal pathogens in humans

Abstract Aim: Oxidized low-density lipoproteins (oxLDL) are formed as a result of lipid peroxidation and are highly immunogenic and proatherogenic. In this study, saliva antibodies binding to oxLDL, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were characterized and their cross-reactivity was evaluated. Materials and Methods: Resting and stimulated saliva samples were collected from 36 healthy adults (mean age 26 years). Saliva IgA, IgG and IgM autoantibody levels to copper oxidized LDL (CuOx-LDL) and malondialdehyde acetaldehyde-modified LDL (MAA-LDL) were determined with chemiluminescence immunoassay. Results: Saliva IgA and IgG antibodies binding to MAA-LDL and CuOx-LDL were detected in all samples and they were associated with the saliva levels of IgA and IgG to P. gingivalis and A. actinomycetemcomitans. Competitive immunoassay showed that saliva antibodies to MAA-LDL cross-reacted specifically with P. gingivalis. The autoantibody levels to oxLDL in saliva were not associated with the autoantibody levels to oxLDL in plasma or with saliva apolipoprotein B 100 levels. Conclusions: Saliva contains IgA and IgG binding to oxLDL, which showed cross-reactive properties with the periodontal pathogens Porphyromonas gingivalis (P.g). The data suggest that secretory IgA to P.g may participate in immune reactions involved in LDL oxidation through molecular mimicry