Systemic Immunologic Consequences of Chronic Periodontitis

Chronic periodontitis (ChP) is a prevalent inflammatory disease affecting 46% of the US population. ChP produces a profound local inflammatory response to dysbiotic oral microbiota that leads to destruction of alveolar bone and tooth loss. ChP is also associated with systemic illnesses, including cardiovascular diseases, malignancies, and adverse pregnancy outcomes. However, the mechanisms underlying these adverse health outcomes are poorly understood. In this prospective cohort study, we used a highly multiplex mass cytometry immunoassay to perform an in-depth analysis of the systemic consequences of ChP in patients before (n = 28) and after (n = 16) periodontal treatment. A high-dimensional analysis of intracellular signaling networks revealed immune system–wide dysfunctions differentiating patients with ChP from healthy controls. Notably, we observed exaggerated proinflammatory responses to Porphyromonas gingivalis–derived lipopolysaccharide in circulating neutrophils and monocytes from patients with ChP. Simultaneously, natural killer cell responses to inflammatory cytokines were attenuated. Importantly, the immune alterations associated with ChP were no longer detectable 3 wk after periodontal treatment. Our findings demarcate systemic and cell-specific immune dysfunctions in patients with ChP, which can be temporarily reversed by the local treatment of ChP. Future studies in larger cohorts are needed to test the boundaries of generalizability of our results

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

BACKGROUND: Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease

Epithelial cell type affects interleukin-6 response to Porphyromonas gingivalis

Objectives: Interleukin-6 (IL-6) is a pleiotropic cytokine that may mediate both beneficial and harmful effects in periodontal disease. IL-6 stimulates immunoglobulin secretion and plays an important role in regulating immune responses to periodontal pathogens. Since IL-6 promotes osteoclastogenesis and induces bone resorption, excessive secretion of IL-6 in response to Porphyromonas gingivalis (Pg) may play a role in inducing alveolar bone loss. Previous studies on the effect of Pg on IL-6 production by oral epithelial cells have shown inconsistent results. The objective of the study was to compare IL-6 responses of three human oral epithelial cell lines to virulent and avirulent strains of Pg. Methods: Two Pg strains, avirulent 2561 and highly virulent W83, were sub-cultivated on blood agar plates and suspended in Medium 199 (4x108 Pg/ml). Non-tumor-derived immortalized oral epithelial GMSM-K cells, and HSC-3 and H413 cells, derived from oral squamous cell carcinoma (OSCC) were exposed to live Pg at 107 bacteria/well, and incubated at 37°C for 6 and 24 h. IL-6 was determined by ELISA. Results: Control, uninfected H413 cells produced higher levels of IL-6 than HSC-3 and GMSM-K cells. Exposure of HSC-3 and GMSM-K cells to Pg-2561 and Pg-W83 for 24 h resulted in a 6- and 8-fold increase in IL-6 secretion, respectively. In H413 cells, Pg-2561 down-regulated IL-6 by 30%, while Pg-W83 up-regulated IL-6 by only 50%. Conclusions: The amount of IL-6 secreted by uninfected cells was strongly dependent on the cell type. Both Pg strains induced IL-6 secretion at similar levels in HSC-3 and GMSM-K cells. However, H413 cells were not highly responsive to Pg. Our results indicate that conclusions on cytokine responses to Pg should not be based on studies with a single cell type

The relationship between TGF-beta and periodontal status in HIV+ patients

Objectives: The purpose of this study was to investigate the relationship between transforming growth factor-beta (TGF-beta) in gingival crevicular fluid (GCF) and periodontal status in HIV+ patients. Methods: Medical and demographic variables including age, race, cigarette smoking, oral hygiene practices, current CD4 cell count and viral load values were recorded. Clinical measurements including gingival index, plaque index, bleeding index, probing depth (PD), attachment loss (AL) and GCF samples were taken from 3 periodontitis sites (GI\u3e0, PD\u3e4mm, AL\u3e2mm), 3 gingivitis sites (GI\u3e0, PD\u3c4mm, AL=0), 2 healthy sites (including sites with gingival recession, GI=0, PD\u3c4mm, AL\u3e2mm) of each of the 11 HIV+ patients at baseline and 6-month visits by means of paper strips. GCF TGF-beta levels were determined by sandwich ELISA assays. SAS statistical package was used to analyze the data. Results: The mean amounts of GCF TGF-beta in diseased sites were significantly higher in gingivitis and periodontitis sites than in healthy sites (p \u3c 0.0001). An active site was defined as a site that had 2mm or more attachment loss during 6-month study period. GCF levels of TGF-beta were highly correlated with probing depth, attachment loss, age, pack years, CD4, viral load at baseline and 6-month visits (0.0001 \u3c p \u3c 0.05). Repeated measures analysis of 9 active sites versus 79 inactive sites indicated that GCF TGF-beta levels were significantly higher in active sites than in inactive sites (p \u3c 0.0001). Conclusion: These data indicate that sites with high GCF levels of TGF-beta in HIV+ patients are at significantly greater risk for progression of periodontitis. This study was supported by NIDCR grant DE12417 and University of the Pacific Arthur A. Dugoni School of Dentistry

Epithelial cell type affects interleukin-6 response to Porphyromonas gingivalis

Objectives: Interleukin-6 (IL-6) is a pleiotropic cytokine that may mediate both beneficial and harmful effects in periodontal disease. IL-6 stimulates immunoglobulin secretion and plays an important role in regulating immune responses to periodontal pathogens. Since IL-6 promotes osteoclastogenesis and induces bone resorption, excessive secretion of IL-6 in response to Porphyromonas gingivalis (Pg) may play a role in inducing alveolar bone loss. Previous studies on the effect of Pg on IL-6 production by oral epithelial cells have shown inconsistent results. The objective of the study was to compare IL-6 responses of three human oral epithelial cell lines to virulent and avirulent strains of Pg. Methods: Two Pg strains, avirulent 2561 and highly virulent W83, were sub-cultivated on blood agar plates and suspended in Medium 199 (4x108 Pg/ml). Non-tumor-derived immortalized oral epithelial GMSM-K cells, and HSC-3 and H413 cells, derived from oral squamous cell carcinoma (OSCC) were exposed to live Pg at 107 bacteria/well, and incubated at 37°C for 6 and 24 h. IL-6 was determined by ELISA. Results: Control, uninfected H413 cells produced higher levels of IL-6 than HSC-3 and GMSM-K cells. Exposure of HSC-3 and GMSM-K cells to Pg-2561 and Pg-W83 for 24 h resulted in a 6- and 8-fold increase in IL-6 secretion, respectively. In H413 cells, Pg-2561 down-regulated IL-6 by 30%, while Pg-W83 up-regulated IL-6 by only 50%. Conclusions: The amount of IL-6 secreted by uninfected cells was strongly dependent on the cell type. Both Pg strains induced IL-6 secretion at similar levels in HSC-3 and GMSM-K cells. However, H413 cells were not highly responsive to Pg. Our results indicate that conclusions on cytokine responses to Pg should not be based on studies with a single cell type

Porphyromonas gingivalis stimulates IL-18 secretion in monocytic THP-1 cells

Objectives: Porphyromonas gingivalis (Pg) is one of the most important bacteria that contribute to the pathogenesis of chronic periodontitis. Interleukin-18 (IL-18), a potent pro-inflammatory cytokine that mediates the Th1 immune response is considered to be a key factor in the initiation and progression of periodontal disease. We examined the IL-18 levels secreted by differentiated human macrophage-like THP-1 cells after exposure to live and heat-inactivated Pg, and to lipopolysaccharide (LPS) from E. coli and Pg. Methods: THP-1 cells were differentiated with 1.6 nM phorbol 12-myristate 13-acetate (PMA) for 2 days at 37°C. Two Pg strains, the avirulent 2561 (ATCC 33277) and highly virulent W83 (ATCC BAA-308) were sub-cultivated on blood agar plates and suspended in Medium 199 (4 x 108 Pg/ml). The bacterial suspension was mixed 1:4 with RPMI/10% FBS and added to differentiated THP-1 cells, at ratios of 2-100 bacteria/cell, and incubated at 37°C for 24 h. IL-18 was determined by ELISA. LPS from E. coli and Pg were also tested at 100 ng/ml and 5 µg/ml. Results: IL-18 secretion depended on the number of bacteria/cell. For example, treatment with live Pg 2561 and W83 at 100 Pg/cell produced 388.3±2.3 and 293.0±95.5 pg IL-18/ml, respectively. IL-18 up-regulation was reduced significantly by heat-inactivation of Pg. Exposure to heat-inactivated Pg 2561 and W83 at 100 Pg/cell produced 56.4±19.4 and 122.0±21.5 pg IL-18/ml, respectively. Treatment with LPS from Pg and E. coli at 100 ng/ml resulted in the production of 32.9±5.6 and 75.9±6.2 pg IL-18/ml, respectively. Conclusion: Pg induced significant IL-18 secretion by differentiated macrophage-like THP-1 cells. The more virulent Pg W83 strain did not induce higher production of IL-18. Induction of IL-18 by heat-inactivated Pg is probably caused by LPS

Cytokine responses of oral epithelial cells exposed to Porphyromonas gingivalis

Objectives: The periodontopathogen Porphyromonas gingivalis (Pg) adheres to, invades and replicates within human oral epithelial cells. The pro-inflammatory cytokine interleukin-8 (IL-8) is a potent chemoattractant inducing the influx of neutrophils into periodontal lesions. We quantified the IL-8 secreted by human oral squamous HSC-3 cells after exposure to live and heat-inactivated Pg, and to lipopolysaccharide (LPS) from Pg and E. coli. Methods: The Pg strain 2561 (ATCC 33277) was sub-cultivated on blood agar plates and suspended in Medium 199 (4x108 Pg/ml). HSC-3 cells were challenged with live or heat-killed Pg at 107 or 108 bacteria/well, and incubated at 37°C for 6, 24 and 48 h. The Multi-Analyte Profiler ELISArray kit was used to profile pro-inflammatory cytokines and chemokines. IL-8 was determined by ELISA. LPS from E. coli and Pg were tested at 5 µg/ml. Results: Analysis by the Profiler ELISA indicated the stimulation of IL-6 and IL-8. Secretion of IL-8 was affected by the number and heat killing of bacteria, and the time of incubation. Untreated HSC-3 cells produced 5.2±0.9 ng IL-8/ml within 24 h, while cells incubated with 107 and 108 live Pg secreted 9.6±2.3 and 12.9±2.8 ng IL-8/ml, respectively. Cells stimulated with heat-killed Pg produced higher levels of IL-8: 12.0±2.1 and 18.1±1.9 ng IL-8/ml. Higher IL-8 stimulation by heat-killed Pg was also evident at 48 h. Treatment with LPS from Pg and E. coli at 5 µg/ml resulted in the production of 11.8±6.1 and 8.6±3.2 ng IL-8/ml, respectively in 24 h. Conclusion: Pg induced significant IL-8 secretion by HSC-3 cells. Degradation of IL-8 by cysteine proteinases (gingipains) produced by live Pg may be responsible for the higher up-regulation of IL-8 observed with heat-killed bacteria. Supported by Research Pilot Project Award 03-Activity 069 from the University of the Pacific, School of Dentistry (K. Konopka)

Porphyromonas gingivalis stimulates interleukin-8 secretion in human oral epithelial cells

Objectives: Infection of epithelial cells with Porphyromonas gingivalis (Pg) results in production of pro-inflammatory cytokines that are involved in the initiation and progression of periodontal disease. The pro-inflammatory cytokine interleukin-8 (IL-8) is a potent chemoattractant inducing the influx of neutrophils into periodontal lesions. IL-8 is the primary focus of this project, since conflicting results have been reported on Pg-induced stimulation of IL-8 in human epithelial cells. Methods: Two Pg strains, avirulent 2561 and highly virulent W83, were sub-cultivated on blood agar plates and suspended in Medium 199. HSC-3 and H413 cells, derived from oral squamous cell carcinoma (OSCC), and non-tumor-derived immortalized oral epithelial GMSM-K cells were challenged with live Pg at 107bacteria/well, and incubated at 37°C for 6 and 24 h. IL-8 was determined by ELISA. Results: Control non-infected HSC-3, H413 and GMSM-K cells produced 920, 2624 and 11 pg IL-8/ml, respectively within 6 h, and 2687, 6564, and 22 pg IL-8/ml, within 24 h. Exposure of HSC-3 to Pg-2561 and Pg-W83 resulted in a 6- and 3-fold increase in IL-8 secretion, respectively. Incubation of GMSM-K cells with these strains increased IL-8 by 21- and 12-fold. In H413 cells, Pg-2561 down-regulated IL-8 by 25%, while Pg-W83 did not modify IL-8. Conclusions: The amount of IL-8 secreted by control cells and their response to Pg were strongly dependent on the cell type. GMSM-K cells secreted significantly less IL-8 than OSCC cells. Both Pg strains induced IL-8 secretion in HSC-3 and GMSM-K cells. Degradation of IL-8 by Pg gingipains may be responsible for the down-regulation of IL-8 observed with H413 cells. The etiology of different epithelial cell responses to Pg is not well known. Supported by Research Pilot Project Award 03-Activity 074 from the University of the Pacific, School of Dentistry (S. Kim)

Inflammatory cytokine secretion by THP-1 cells exposed to Porphyromonas gingivalis

Objectives: Interleukin-18 (IL-18) is a pro-inflammatory cytokine that plays an essential role in the T-cell response and is elevated in inflammatory diseases such as periodontal disease. Porphyromonas gingivalis (Pg) is one of the most important bacterial pathogens that contribute to pathogenesis of chronic periodontitis. Here we examined the IL-18 levels expressed by differentiated macrophage-like THP-1 cells after exposure to live and heat-killed Pg and to E.coli LPS. Methods: THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) for 5 days at 37°C. Pg grown in chopped meat medium under anaerobic conditions, was added to differentiated THP-1 cells, at ratios of 2-100 bacteria/cell, and incubated at 37°C for 24 h. IL-18 was determined by ELISA. The Multi-Analyte Profiler ELISArray kit was used to profile other pro-inflammatory cytokines and chemokines. Values were compared to that obtained with differentiated THP-1 cells treated with medium alone. Results: Exposure to Pg at ratios of 20, 50, and 100 bacteria/cell induced significant expression of IL-18 after 24 h. For example, treatment with live and heat-killed Pg at 100 Pg/cell produced 174.3 ± 41.3 and 118.0 ± 17.3 pg IL-18/ml, respectively. THP-1 cells treated with heated and regular conditioned Pg medium produced the same level of IL-18 (~124.5 pg/ml). Treatment with E. coli LPS at 50 and 100 ng/ml resulted in the production of 61.1 ± 3.6 pg/ml IL-18. Analysis by the Multi-Analyte Profiler ELISA indicated the stimulation of IL-1β, IL-12 and TNF-α. Conclusions: P. gingivalis significantly increased IL-18 secretion by differentiated THP-1 macrophage-like cells. Our results suggest that Pg factors other than LPS also contribute to the activation of THP-1 cells and production of IL-18. Supported by Research Pilot Project Award 03-Activity 064 from the University of the Pacific, Arthur A. Dugoni School of Dentistry (A. Kim)