Immunomodulation Mediated by Azithromycin in Experimental Periapical Inflammation.

INTRODUCTION: The purpose of the present study was to compare the immunomodulatory effect of azithromycin (AZM), ampicillin (AMP), amoxicillin (AMX), and clindamycin (CLI) in vitro and AZM on preexisting periapical lesions compared with AMP. METHODS: The susceptibility of 4 common human endodontic pathogens (Parvimonas micra, Streptococcus intermedius, Prevotella intermedia, and Fusobacterium nucleatum) to AZM, AMP, AMX, and CLI was confirmed by agar disk diffusion assay. Preexisting periapical lesions in C57BL/6J mice were treated with AZM, AMP, or phosphate-buffered saline (PBS). Periapical bone healing and the pattern of inflammatory cell infiltration were evaluated after a 10-day treatment by micro-computed tomographic and histology, respectively. Besides, the effect of antibiotics in pathogen-stimulated nuclear factor kappa B activation and the production of interleukin 1 alpha and tumor necrosis factor alpha was assessed in vitro by luciferase assay and enzyme-linked immunosorbent assay. RESULTS: All examined endodontic pathogens were susceptible to AZM, AMP, AMX, and CLI. AZM significantly attenuated periapical bone loss versus PBS. PBS resulted in widely diffused infiltration of mixed inflammatory cells. By contrast, AZM brought about localized infiltration of neutrophils and M2 macrophages and advanced fibrosis. Although the effect of AMP on bone was uncertain, inflammatory cell infiltration was considerably milder than PBS. However, most macrophages observed seemed to be M1 macrophages. AZM suppressed pathogen-stimulated nuclear factor kappa B activation and cytokine production, whereas AMP, AMX, and CLI reduced only cytokine production moderately. CONCLUSIONS: This study showed that AZM led to the resolution of preexisting experimental periapical inflammation. Our data provide a perspective on host response in antibiotic selection for endodontic treatment. However, well-designed clinical trials are necessary to better elucidate the benefits of AZM as an adjunctive therapy for endodontic treatment when antibiotic therapy is recommended. Although both AZM and AMP were effective on preexisting periapical lesions, AZM led to advanced wound healing, probably depending on its immunomodulatory effect.

Identification and Modification of Porphyromonas gingivalis Cysteine Protease, Gingipain, Ideal for Screening Periodontitis.

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

The fungal metabolite (+)-terrein abrogates osteoclast differentiation via suppression of the RANKL signaling pathway through NFATc1.

Pathophysiological bone resorption is commonly associated with periodontal disease and involves the excessive resorption of bone matrix by activated osteoclasts. Receptor activator of nuclear factor (NF)-κB ligand (RANKL) signaling pathways have been proposed as targets for inhibiting osteoclast differentiation and bone resorption. The fungal secondary metabolite (+)-terrein is a natural compound derived from Aspergillus terreus that has previously shown anti-interleukin-6 properties related to inflammatory bone resorption. However, its effects and molecular mechanism of action on osteoclastogenesis and bone resorption remain unclear. In the present study, we showed that 10 µM synthetic (+)-terrein inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner and without cytotoxicity. RANKL-induced messenger RNA expression of osteoclast-specific markers including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), the master regulator of osteoclastogenesis, cathepsin K, tartrate-resistant acid phosphatase (Trap) was completely inhibited by synthetic (+)-terrein treatment. Furthermore, synthetic (+)-terrein decreased RANKL-induced NFATc1 protein expression. This study revealed that synthetic (+)-terrein attenuated osteoclast formation and bone resorption by mediating RANKL signaling pathways, especially NFATc1, and indicated the potential effect of (+)-terrein on inflammatory bone resorption including periodontal disease.

Antimicrobial and antibiofilm effects of abietic acid on cariogenic Streptococcus mutans.

Dental caries is a type of oral microbiome dysbiosis and biofilm infection that affects oral and systemic conditions. For healthy life expectancy, natural bacteriostatic products are ideal for daily and lifetime use as anti-oral infection agents. This study aimed to evaluate the inhibitory effects of abietic acid, a diterpene derived from pine rosin, on the in vitro growth of cariogenic bacterial species, Streptococcus mutans. The effective minimum inhibitory concentration of abietic acid was determined through observation of S. mutans growth, acidification, and biofilm formation. The inhibitory effects of abietic acid on the bacterial membrane were investigated through the use of in situ viability analysis and scanning electron microscopic analysis. Cytotoxicity of abietic acid was also examined in the context of several human cell lines using tetrazolium reduction assay. Abietic acid was found to inhibit key bacterial growth hallmarks such as colony forming ability, adenosine triphosphate activity (both planktonic and biofilm), acid production, and biofilm formation. Abietic acid was identified as bacteriostatic, and this compound caused minimal damage to the bacterial membrane. This action was different from that of povidone-iodine or cetylpyridinium chloride. Additionally, abietic acid was significantly less cytotoxic compared to povidone-iodine, and it exerted lower toxicity towards epithelial cells and fibroblasts compared to that against monocytic cells. These data suggest that abietic acid may prove useful as an antibacterial and antibiofilm agent for controlling S. mutans infection.

Endodontic Infection-induced Inflammation Resembling Osteomyelitis of the Jaws in Toll-like Receptor 2/Interleukin 10 Double-knockout Mice.

INTRODUCTION: In general, mice develop chronic and nonhealing periapical lesions after endodontic infection. Surprisingly, we recently found that toll-like receptor 2 (TLR2)/interleukin 10 (IL-10) double-knockout (dKO) mice exhibited acute but resolving osteomyelitislike inflammation. In this study, we examined the kinetics of endodontic infection-induced inflammation in TLR2/IL-10 dKO mice and explored a potential mechanism of periapical wound healing mediated by the hypoxia-inducible factor 1 alpha (HIF-1α) subunit and arginase 1. METHODS: TLR2/IL-10 dKO and wild-type C57BL/6J mice were subjected to endodontic infection in the mandibular first molars. Mice were sacrificed on days 0 (noninfected), 10, and 21 postinfection. The extent of bone destruction, inflammation, bone deposition, and gene expression were determined by micro-computed tomographic imaging, histology, bone polychrome labeling, and microarray analysis. In addition, the effect of blocking endogenous HIF-1α was tested in infected TLR2/IL-10 dKO mice using the specific inhibitor YC-1. RESULTS: Infected TLR2/IL-10 dKO mice exhibited extensive bone destruction and inflammation on day 10 followed by spontaneous periapical wound healing including bone formation and resolution of inflammation by day 21 postinfection. In contrast, WT mice developed increasing chronic periapical inflammation over the 21-day observation period. Gene expression analyses and immunohistochemistry revealed that HIF-1α and arginase 1 were up-regulated in spontaneous wound healing in TLR2/IL-10 dKO mice. Blocking of HIF-1α in TLR2/IL-10 dKO mice using YC-1 resulted in significant inhibition of regenerative bone formation. CONCLUSIONS: The TLR2/IL-10 dKO mouse is a novel model resembling osteomyelitis of the jaws in which HIF-1α and arginase 1 appear to be crucial factors in spontaneous wound healing and bone repair.

Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss.

Hypoxia (low oxygen level) is an important feature during infections and affects the host defence mechanisms. The host has evolved specific responses to address hypoxia, which are strongly dependent on the activation of hypoxia-inducible factor 1 (HIF-1). Hypoxia interferes degradation of HIF-1 alpha subunit (HIF-1α), leading to stabilisation of HIF-1α, heterodimerization with HIF-1 beta subunit (HIF-1ß) and subsequent activation of HIF-1 pathway. Apical periodontitis (periapical lesion) is a consequence of endodontic infection and ultimately results in destruction of tooth-supporting tissue, including alveolar bone. Thus far, the role of HIF-1 in periapical lesions has not been systematically examined. In the present study, we determined the role of HIF-1 in a well-characterised mouse periapical lesion model using two HIF-1α-activating strategies, dimethyloxalylglycine (DMOG) and adenovirus-induced constitutively active HIF-1α (CA-HIF1A). Both DMOG and CA-HIF1A attenuated periapical inflammation and tissue destruction. The attenuation in vivo was associated with downregulation of nuclear factor-κappa B (NF-κB) and osteoclastic gene expressions. These two agents also suppressed NF-κB activation and subsequent production of proinflammatory cytokines by macrophages. Furthermore, activation of HIF-1α by DMOG specifically suppressed lipopolysaccharide-stimulated macrophage differentiation into M1 cells, increasing the ratio of M2 macrophages against M1 cells. Taken together, our data indicated that activation of HIF-1 plays a protective role in the development of apical periodontitis via downregulation of NF-κB, proinflammatory cytokines, M1 macrophages and osteoclastogenesis.

Relationship between hypertension and periapical lesion: an in vitro and in vivo study.

The aim of this study was to compare potential aspects of periapical lesion formation in hypertensive and normotensive conditions using hypertensive (BPH/2J) and wild-type control (BPN/3J) mice. The mandibular first molars of both strains had their dental pulp exposed. At day 21 the mice were euthanized and right mandibular molars were used to evaluate the size and phenotype of apical periodontitis by microCT. Proteins were extracted from periapical lesion on the left side and the expressions of IL1α, IL1ß and TNFα were analyzed by ELISA. Bone marrow stem cells were isolated from adult mice femurs from 2 strains and osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) in vitro. The amount of differentiated osteoclastic cells was nearly double in hypertensive mice when compared to the normotensive strain (p < 0.03). Periapical lesion size did not differ between hypertensive and normotensive strains (p > 0.7). IL1α, IL1ß and TNFα cytokines expressions were similar for both systemic conditions (p > 0.05). Despite the fact that no differences could be observed in periapical lesion size and cytokines expressions on the systemic conditions tested, hypertension showed an elevated number of osteoclast differentiation.

Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc.

Interrelationship Between Periapical Lesion and Systemic Metabolic Disorders.

BACKGROUND: Periapical periodontitis, also known as periapical lesion, is a common dental disease, along with periodontitis (gum disease). Periapical periodontitis is a chronic inflammatory disease, caused by endodontic infection, and its development is regulated by the host immune/inflammatory response. Metabolic disorders, which are largely dependent on life style such as eating habits, have been interpreted as a "metabolically-triggered" low-grade systemic inflammation and may interact with periapical periodontitis by triggering immune modulation. The host immune system is therefore considered the common fundamental mechanism of both disease conditions. METHOD: We have reviewed >200 articles to discuss the interrelationship between periapical lesions and metabolic disorders including type 2 diabetes mellitus, hypertension, and non-alcoholic fatty liver diseases (NAFLD), and their common pathological background in immunology/osteoimmunology and cytokine biology. RESULTS: An elevated inflammatory state caused by metabolic disorders can impact the clinical outcome of periapical lesions and interfere with wound healing after endodontic treatment. Although additional well-designed clinical studies are needed, periapical lesions appear to affect insulin sensitivity and exacerbate non-alcoholic steatohepatitis. CONCLUSION: Immune regulatory cytokines produced by various cell types, including immune cells and adipose tissue, play an important role in this interrelationship.

Relationship between hypertension and periapical lesion: an in vitro and in vivo study

Abstract The aim of this study was to compare potential aspects of periapical lesion formation in hypertensive and normotensive conditions using hypertensive (BPH/2J) and wild-type control (BPN/3J) mice. The mandibular first molars of both strains had their dental pulp exposed. At day 21 the mice were euthanized and right mandibular molars were used to evaluate the size and phenotype of apical periodontitis by microCT. Proteins were extracted from periapical lesion on the left side and the expressions of IL1α, IL1β and TNFα were analyzed by ELISA. Bone marrow stem cells were isolated from adult mice femurs from 2 strains and osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) in vitro. The amount of differentiated osteoclastic cells was nearly double in hypertensive mice when compared to the normotensive strain (p < 0.03). Periapical lesion size did not differ between hypertensive and normotensive strains (p > 0.7). IL1α, IL1β and TNFα cytokines expressions were similar for both systemic conditions (p > 0.05). Despite the fact that no differences could be observed in periapical lesion size and cytokines expressions on the systemic conditions tested, hypertension showed an elevated number of osteoclast differentiation.

Serum antibody response to group II chaperonin from Methanobrevibacter oralis and human chaperonin CCT.

Both group I (HSP60) and group II (CCT) chaperonins are targets of autoantibodies. Autoimmune reactions to HSP60 have been well characterized, while immune reactions to group II chaperonin have not been clarified. Methanobrevibacter oralis is a suspected periodontal pathogen with group II chaperonin. In this study, serum responses to M. oralis chaperonin, human HSP60, and CCT subunits were examined using sera from patients with periodontitis and autoimmune diseases. In comparison with healthy controls, periodontitis patients showed significantly higher responses to CCT4 and CCT8 on dot blot analysis. Signals for CCT3 and CCT8 in autoimmune disease patients were significantly higher than in controls. Significant differences were also demonstrated by Western blotting in anti-CCT4 response in both patient groups. All subjects showed strong reactivity to M. oralis chaperonin and faint signals to human HSP60. Autoantibodies were raised against CCT rather than HSP60; and CCT3, CCT4, and CCT8 were shown to be the main targets. Host immune systems may be frequently exposed to chaperonins of Archaea in various habitats. Although further studies of the cross-reactivity between M. oralis chaperonin and human CCT are required, anti-CCT autoantibodies may be involved in the pathogenesis of periodontitis and autoimmune diseases.