Hypertension favors the inflammatory process in rats with experimentally induced periodontitis

Bonato CF, do‐Amaral CCF, Belini L, Salzedas LMP, Oliveira SHP. Hypertension favors the inflammatory process in rats with experimentally induced periodontitis. J Periodont Res 2012; 47: 783–792. © 2012 John Wiley & Sons A/S Background and Objective: Cardiovascular diseases are significantly correlated with chronic periodontitis. The aim of this study was to evaluate bone‐loss level, neutrophil migration, CXCL2/CINC‐2α, CXCL5/LIX, CCL20/MIP‐3α and tumor necrosis factor‐α (TNF‐α) production, inducible nitric oxide synthase (iNOS) expression and C‐reactive protein (CRP) release in spontaneously hypertensive rats (SHRs) and normotensive (WTK) rats after experimental induction of periodontal disease. Material and Methods: Periodontitis was induced by placement of silk yarn ligatures around the first molar counterparts. The levels of CRP, CCL20/MIP‐3α and CXCL5/LIX were evaluated in the peripheral blood, and bone‐loss level, neutrophil recruitment, the production of myeloperoxidase, CXCL2, CXCL5, CCL20 and TNF‐α, and the expression of iNOS were evaluated in the gingival tissue. Histological sections were taken to evaluate and measure bone resorption and neutrophil recruitment in the furcation region. Results: Rats with periodontitis had alveolar bone resorption. SHRs with periodontitis showed marked bone loss and increased neutrophil infiltration in comparison with WTK rats. SHRs with periodontitis showed increased levels of TNF‐α and CXCL2, and a slight tendency for increased levels of CXCL5, in the gingival tissue but no increase in the level of CCL20. In SHRs, even without periodontitis, the levels of TNF‐α, CXCL2, CXCL5 and CCL20 showed a slight tendency to increase. In the WTK rats, TNF‐α, CXCL2 and CXCL5 levels were increased with periodontitis, but the level of CCL20 was not. iNOS was expressed in the gingival tissue of WTK rats and SHRs with periodontitis; however, SHRs appeared to express a higher level of iNOS than did WKT rats. The CRP level was elevated in both types of rats with periodontitis; however, the CRP level was higher in SHRs with periodontitis than in WTK rats with periodontitis. Conclusion: In SHRs, the hypertensive condition per se seems to favor the inflammatory processes that become potentiated with periodontitis, when compared with WKT rats.     

Effect of Hydrosoluble Chlorine–Mediated Antimicrobial Photodynamic Therapy on Clinical Parameters and Cytokine Profile in Ligature‐Induced Periodontitis in Dogs

Background: Recently, a hydrosoluble chlorine composed of sodium salts chlorine e6, chlorine p6, and purpurine‐5 has been shown to be a promising photosensitizer in antimicrobial photodynamic therapy (aPDT). The aim of this study is to evaluate the effects of adjunctive application of hydrosoluble chlorine–mediated aPDT compared with scaling and root planing (SRP) alone on clinical parameters and cytokine levels in gingival crevicular fluid of dogs with experimental periodontitis. Methods: Periodontal disease was induced by placing silk ligatures around both maxillary and mandibular teeth. After establishment of attachment loss, full‐mouth SRP was performed in all dogs. One day after SRP, each quadrant randomly received one of the following treatment modalities: hydrosoluble chlorine plus diode laser (wavelength 662 nm, power 100 mW, continuous mode, time of irradiation 20 seconds), hydrosoluble chlorine alone, laser alone, or no adjunctive treatment. The same adjunctive procedure was repeated 1 week later. Clinical parameters including periodontal probing depth, clinical attachment level, and bleeding on probing, as well as crevicular levels of interleukin‐1β and tumor necrosis factor‐α, were evaluated at baseline, at 3 weeks, and at 3 months after treatment. Results: After both 3 weeks and 3 months, all treatment groups showed significant improvement in all clinical and immunologic parameters (P <0.001). No significant differences were found between the four groups with regard to the measured parameters (P >0.05). Conclusion: Based on the results of this study, adjunctive use of hydrosoluble chlorine–mediated aPDT with the current setting has no additional effect on the clinical parameters or proinflammatory cytokine levels in ligature‐induced periodontitis.     

Osteocytic Sclerostin Expression in Alveolar Bone in Rats With Diabetes Mellitus and Ligature‐Induced Periodontitis

Background: Osteocytic sclerostin inhibits bone formation, and its expression is stimulated by tumor necrosis factor (TNF)‐α. This study investigates sclerostin and TNF‐α expression in rats with diabetes mellitus (DM) and periodontitis. Methods: Rats were divided into control (C), periodontitis (P), and DM + periodontitis (DP) groups. After induction of DM by streptozotocin, periodontitis was induced by ligature. At day 0 (control) and at days 3 and 20 after induction of periodontitis, alveolar bone, osteoclasts, osteoid area, and TNF‐α and sclerostin expression were evaluated. Results: The distance between the cemento‐enamel junction and the alveolar bone crest of the DP group was longer than that of the P group at day 20 after induction of periodontitis, but the number of osteoclasts was not different. Osteoid area decreased in both the P and DP groups by day 3, but whereas sustained osteoid suppression was observed in the DP group at day 20, osteoid formation was increased in the P group. The number of sclerostin‐positive osteocytes increased in both groups at day 3, but the increased number of sclerostin‐positive osteocytes was maintained only in the DP group through day 20. The number of TNF‐α–positive cells increased more in the DP group than in the P group. Conclusions: Enhanced alveolar bone loss, suppressed bone formation, and prevalent TNF‐α expression were characteristic of the DP group compared with the P group. Suppressed bone formation in the DP group was observed simultaneously with increased sclerostin and TNF‐α expression. These results suggest that upregulated osteocytic sclerostin expression in periodontitis accompanied by DM may play a role in suppressed bone formation.     

The effects of tumour necrosis factor‐α on bone cells involved in periodontal alveolar bone loss; osteoclasts,osteoblasts and osteocytes

Periodontitis is the most common bone loss pathology in adults and if left untreated is responsible for premature tooth loss. Cytokines, such as tumour necrosis factor‐α (TNFα), involved in the chronic inflammatory response within the periodontal gingiva, significantly influence the normal bone remodelling processes. In this review, the effects of TNFα on bone metabolism in periodontitis are evaluated in relation to its direct and indirect actions on bone cells including osteoclasts, osteoblasts and osteocytes. Evidence published to date suggests a potent catabolic role for TNFα through the stimulation of osteoclastic bone resorption as well as the suppression of osteoblastic bone formation and osteocytic survival. However, the extent and timing of TNFα exposure in vitro and in vivo greatly influences its effect on skeletal cells, with contradictory anabolic activity observed with TNFα in a number of studies. None the less, it is evident that managing the chronic inflammatory response in addition to the deregulated bone metabolism is required to improve periodontal and inflammatory bone loss treatments???????????????????????????????????????.     

Quantitative messenger RNA expression of Toll‐like receptors and interferon‐α1 in gingivitis and periodontitis

Introduction: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll‐like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, ‐7, and ‐9 messenger RNAs (mRNAs) in addition to those of TLR2 and ‐4, and compared gingivitis and periodontitis. Interferon‐α1 (IFN‐α1), which is important for the antiviral response, was also compared. Methods: Gene expression was analyzed using quantitative real‐time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN‐α, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. Results: The expression levels of TLR2, ‐4, ‐7, and ‐9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and ‐4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN‐α1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody‐positivity between gingivitis and periodontitis. Conclusion: This is the first study to show that a variety of TLRs are up‐regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.     

Lethal outcome caused by Porphyromonas gingivalis A7436 in a mouse chamber model is associated with elevated titers of host serum interferon‐γ

Background/aims: Septic shock caused by gram‐negative bacteria has been associated with cytokines produced by hosts. Porphyromonas gingivalis A7436, a disseminating strain, caused septic shock‐like symptoms and even animal death in a mouse chamber model. However, P. gingivalis exhibits lower endotoxin activities in its lipopolysaccharide than other typical gram‐negative bacteria. In this study, we examined the effects of P. gingivalis lethal infection on host pro‐inflammatory cytokines production. Methods: Nude and normal BALB/c mice were infected with a lethal dose of P. gingivalis A7436 using a mouse chamber model. Serum levels of tumor necrosis factor, interleukin (IL)‐1β, IL‐12 and interferon‐γ were evaluated. The effects of tumor necrosis factor inhibitor (thalidomide) and anti‐interferon‐γ antibody on infection outcomes were examined. Results: All nude mice survived infectious challenge, whereas 100% of normal mice died with abdominal lesions. Bacterial cultures indicated P. gingivalis dissemination to the circulation. Serum levels of tumor necrosis factor, IL‐1β and IL‐12 showed no significant differences between nude and normal mice. Thalidomide treatment did not protect normal mice from death but decreased remote lesion occurrence, with concurrent reduced bacterial counts recoverable from blood. There was a 3.5‐fold elevation in normal mice serum interferon‐γ titers compared to those of nude mice and anti‐interferon‐γ antibody treatment resulted in 100% protection from lethal outcome. Conclusion: Lethal outcome following P. gingivalis A7436 infection is T‐lymphocyte dependent and involves an increase in systemic interferon‐γ levels. The data further indicate that P. gingivalis transvascular dissemination (bacteremia) alone is not sufficient for lethal outcome.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Effects of treatment on soluble tumour necrosis factor receptor type 1 and 2 in chronic periodontitis

Aim: We reported that soluble tumour necrosis factor receptor type 2 (sTNFR2)/type 1 (sTNFR1) ratios in gingival crevicular fluid (GCF) decreased as the severity of chronic periodontitis (CP) increased. This study investigated the effects of the periodontal treatment on TNF‐α, sTNFR1 and R2 in GCF and serum of CP patients. Material and Methods: Thirty‐five serum and 90 GCF samples were obtained from 35 CP patients (23 non‐smokers and 12 smokers) at baseline and after treatment. The levels of TNF‐α, sTNFR1 and R2 in serum and GCF were quantified by enzyme‐linked immunosorbant assay. Results: No significant differences were found in the serum levels of TNF‐α, sTNFR1 and R2 and the ratio of sTNFR2/R1 between baseline and after treatment. After treatment, sTNFR1 and R2 levels in GCF of non‐smokers and smokers were significantly decreased compared with baseline. However, the sTNFR2/R1 ratio was significantly increased (non‐smoker: 0.56±0.03–0.84±0.03, p<0.0001; smoker: 0.59±0.06–0.85±0.04, p=0.0019). There were no significant differences between non‐smoking and smoking CP groups in serum and GCF. Conclusion: The ratio of sTNFR2/R1 in GCF significantly increased after treatment, and could be related to the clinical state of CP.     

Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease

Da? A, F?rat ET, Kadiro?lu AK, Kale E, Y?lmaz ME. Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease. J Periodont Res 2010; 45: 445–450. © 2010 John Wiley & Sons A/S Background and Objective: The prevalence of chronic renal disease in industrialized countries is increasing, and chronic renal disease and periodontitis can have significant, reciprocal effects. The aim of this study was to evaluate the associations between specific clinical parameters and the levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) in the gingival crevicular fluid of hemodialysis (HD) patients with periodontal disease. Material and Methods: Forty‐three HD patients and 43 systemically healthy subjects were enrolled in this study. Plaque index (PI), gingival index (GI) and pocket depth were used to determine periodontal status. Venous blood samples were obtained from each patient in the morning before the dialysis session and analyzed to determine the levels of inflammatory, biochemical and hematological parameters. Gingival crevicular fluid was collected from all subjects, and the levels of TNF‐α and IL‐8 were determined in the gingival crevicular fluid samples. Results: The following results were obtained from HD patients and controls: TNF‐α (pg/mL), 31.40 ± 1.46 and 3.06 ± 0.15 (p < 0.001); IL‐8 (pg/mL), 90.98 ± 94.03 and 35.05 ± 16.86 (p < 0.001); PI, 1.69 ± 1.02 and 0.04 ± 0.02 (p < 0.001); GI, 0.82 ± 0.06 and 0.04 ± 0.02 (p < 0.001); and pocket depth, 2.23 ± 0.63 and 1.51 ± 0.05 (p < 0.001), respectively. In addition, there were positive correlations between TNF‐α and PI (r = 0.642, p < 0.001), between TNF‐α and GI (r = 0.565, p < 0.001), between TNF‐α and pocket depth (r = 0.522, p < 0.001), between IL‐8 and PI (r = 0.402, p = 0.002), between IL‐8 and GI (r = 0.396, p = 0.002), and between IL‐8 and pocket depth (r = 0.326, p = 0.012). There were negative correlations between albumin and PI (r = ?0.491, p < 0.001), albumin and GI (r = ?0.406, p < 0.001), albumin and pocket depth (r = ?0.464, p < 0.001) and albumin and CRP (r = ?0.467, p = 0.002) and between the gingival crevicular fluid levels of TNF‐α and IL‐8, TNF‐α and hemoglobin (r = ?0.745, p < 0.001; r = ?0.285, p < 0.05) (respectively). Conclusion: The levels of TNF‐α and IL‐8 in gingival crevicular fluid were significantly higher in HD patients than in controls. There were strong, positive correlations between clinical periodontal parameters and the levels of inflammatory cytokines in gingival crevicular fluid from the HD patients.     

A pathogenic trace of Tannerella forsythia – shedding of soluble fully active tumor necrosis factor α from the macrophage surface by karilysin

Tannerella forsythia is implicated as a pathogen causing chronic and aggressive periodontitis. However, its virulence factors, including numerous putative proteases, are mostly uncharacterized. Karilysin is a newly described matrix metalloprotease‐like enzyme of T. forsythia. Since pathogen‐derived proteases may affect the host defense system via modulation of the cytokine network, the aim of this study was to determine the influence of karilysin on tumor necrosis factor‐α (TNF‐α). The results showed that karilysin cleaved the membrane form of TNF‐α on the surface of macrophages, and that this led to an increased concentration of soluble TNF‐α in the conditioned medium. Importantly, despite partial degradation of soluble TNF‐α by karilysin, the released cytokine retained its biological activity, inducing apoptosis and stimulating autocrine pathway of pro‐inflammatory gene expression. Notably, the observed effect required proteolytic activity by karilysin, since a catalytically inactive mutant of the enzyme did not affect TNF‐α secretion. The shedding was independent of the activity of ADAM17, a major endogenous TNF‐α converting enzyme. Karilysin‐dependent TNF‐α release from the cell surface is likely to occur in vivo because human plasma, the main constituent of gingival crevicular fluid, only slightly affected the sheddase activity of karilysin. Taken together, these results indicate that karilysin modulates the host immune response through regulation of TNF‐α secretion, and should therefore be considered as a new virulence factor of T. forsythia.