Frank C. Nichols

Effect of calcium hydroxide on bacterial lipopolysaccharide

Apical periodontitis and its concomitant periapical osteolysis is caused by pulpal infection, and bacterial lipopolysaccharide (LPS) is known to play a major role in the bone resorption process. Little is known concerning the effect of root canal intervisit dressings on residual LPS in root canals after bacterial cell lysis. The purpose of this study was to evaluate the effects of calcium hydroxide on bacterial LPS. Free hydroxy fatty acids were quantified in samples of LPS treated with calcium hydroxide. Calcium hydroxide treatment of LPS was shown to release elevated quantities of hydroxy fatty acids. It was concluded that calcium hydroxide hydrolyzed the lipid moiety of bacterial LPS, resulting in the release of free hydroxy fatty acids. This result suggests that calcium hydroxide-mediated degradation of LPS may be an important reason for the beneficial effects obtained with calcium hydroxide use in clinical endodontics.

Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment

Bacterial lipopolysaccharide (LPS) plays a major role in the development of periapical bone resorption. Although the chemical properties of LPS are altered by treatment with an alkali such as calcium hydroxide, the effects of calcium hydroxide on the biological properties of LPS are not known. The purpose of this study was to investigate whether treatment of LPS with calcium hydroxide alters its biological action as measured by human monocyte secretion of prostaglandin E2. Monocyte cell cultures were stimulated with LPS or calcium hydroxide-treated LPS and culture supernatants were analyzed for prostaglandin E2 content using gas chromatography-mass spectrometry. Prostaglandin E2 was identified in supernatants of LPS-stimulated monocytes but not in those stimulated with calcium hydroxide-treated LPS. It was concluded that the treatment with calcium hydroxide may alter biological properties of bacterial LPS.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Immunoregulation: Enhancement of Regulatory T Cells through PPARγ-Dependent and -Independent Mechanisms

Peroxisome proliferator-activated receptor (PPAR)γ is a nuclear hormone receptor primarily characterized for its effect on insulin metabolism. PPARγ ligands, used to treat human type 2 diabetes, also down-regulate most immune system cells including APCs and pathogenic T cells. These effects putatively underlie the efficacy of PPARγ ligands in treating animal models of autoimmunity, leading to projections of therapeutic potential in human autoimmunity. However, the relationship between PPARγ ligands and CD4+CD25+ regulatory T cells (Tregs) has not been examined. Specifically, no studies have examined the role of Tregs in mediating the in vivo immunoregulatory effects of PPARγ ligands, and there have been no investigations of the use of PPARγ ligands to treat autoimmunity in the absence of Tregs. We now characterize the novel relationship between ciglitazone, a thiazolidinedione class of PPARγ ligand, and both murine natural Tregs (nTregs) and inducible Tregs (iTregs). In vitro, ciglitazone significantly enhances generation of iTregs in a PPARγ-independent manner. Surprisingly, and contrary to the current paradigm, we find that, in a model of graft-vs-host disease, the immunotherapeutic effect of ciglitazone requires the presence of nTregs that express PPARγ. Overall, our results indicate that, unlike its down-regulatory effect on other cells of the immune system, ciglitazone has an enhancing effect on both iTregs and nTregs, and this finding may have important implications for using PPARγ ligands in treating human autoimmune disease.

Natural but Not Inducible Regulatory T Cells Require TNF-α Signaling for In Vivo Function

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti–TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2−/− nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2−/− nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti–TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.

Unique Lipids from a Common Human Bacterium Represent a New Class of Toll-Like Receptor 2 Ligands Capable of Enhancing Autoimmunity

Recent reports suggest that commensal bacteria may play a down-regulatory role in autoimmune disease. In the present studies, we demonstrate that phosphorylated dihydroceramides, uniquely structured lipids derived from the common human oral bacterium Porphyromonas gingivalis and from bacteria commonly found in the gastrointestinal tract and other organs, are capable of enhancing autoimmunity. We have previously reported that these lipids have proinflammatory effects on human fibroblasts in vitro and, in preliminary studies, have recovered these lipids from surgically removed human carotid atheroma, suggesting that they may play a role in human inflammatory disease. To investigate whether these lipids have functional effects on autoimmunity, we administered phosphorylated dihydroceramides to mice with the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). We find that these lipids, and particularly the phosphoethanolamine dihydroceramide (PE DHC) fraction, significantly enhanced EAE. Mechanistically, PE DHC enhances EAE in mice lacking natural killer T cells, fails to enhance EAE in Toll-like receptor 2 (TLR2)-deficient mice and, in vitro, induces dendritic cell interleukin-6 secretion in a TLR2-dependent manner. Finally, PE DHC-treated mice with EAE demonstrate a decreased percentage of spinal cord Foxp3+ T cells, suggesting that these lipids may affect regulatory aspects of adaptive immune responses. Overall, our results suggest that phosphorylated dihydroceramides derived from common human bacteria function as TLR2 ligands and may play a previously unrecognized role in human autoimmune diseases.

Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands

ABSTRACT The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2−/−) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2−/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Porphyromonas gingivalis Lipids Inhibit Osteoblastic Differentiation and Function

ABSTRACT Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo, as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.

Bacterial lipodipeptide, Lipid 654, is a microbiome-associated biomarker for multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria‐derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll‐like receptor 2 ligand. Using multiple‐reaction‐monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimers disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.

PPARγ regulates retinoic acid‐mediated DC induction of Tregs

CD4+ CD25+ Foxp3+ Tregs are critical regulators of immune responses and autoimmune diseases. nTregs are thymically derived; iTregs are converted in the periphery from CD4+ CD25– Foxp3– Teffs. Recent studies reported that GALT CD103+ DCs mediated enhanced iTreg conversion via the secretion of RA. However, the factors regulating RA secretion and hence, the induction of iTregs by DCs are not yet clear. Activation of the nuclear hormone receptor PPARγ has been shown to induce RA expression in human DCs, and thus, we postulated that PPARγ activation in DCs may be an important regulator of RA secretion and iTreg generation. Using in vitro and in vivo approaches, we now demonstrate that PPARγ activation enhances iTreg generation through increased RA synthesis from murine splenic DCs. In addition, we demonstrate that inhibition of DC PPARγ decreases iTreg generation, suggesting a role for endogenous PPARγ ligands in this process. Overall, our findings suggest that PPARγ may be important as a factor that stimulates DCs to produce RA and as a potential mechanism by which PPARγ ligands ameliorate autoimmunity.

Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis

The Gram-negative periodontal pathogen Porphyromonas gingivalis synthesizes several classes of novel phosphorylated complex lipids, including the recently characterized phosphorylated dihydroceramides. These sphingolipids promote the interleukin-1 (IL-1)-mediated secretion of inflammatory mediators from fibroblasts, including prostaglandin E2 and 6-keto prostaglandin F2α, and alter gingival fibroblast morphology in culture. This report demonstrates that one additional class of phosphorylated complex lipids of P. gingivalis promotes IL-1-mediated secretory responses and morphological changes in cultured fibroblasts. Structural characterization identified the new phospholipid class as 1,2-diacyl phosphatidylethanolamine, which substituted predominantly with isobranched C15:0 and C13:0 fatty acids. The isobranched fatty acids, rather than unbranched fatty acids, and the phosphoethanolamine head group were identified as the essential structural elements required for the promotion of IL-1-mediated secretory responses. These structural components are also observed in specific phosphorylated sphingolipids of P. gingivalis and likely contribute to the biological activity of these substances, in addition to the phosphatidylethanolamine lipids described in this report.