Evaluation of the role of mitofusin-1 and mitofusin-2 in periodontal disease.

BACKGROUND: Mitochondria and endoplasmic reticulum are key cellular organelles and create contact sites (mitochondria-endoplasmic reticulum contact [MERC]), which plays a major role in calcium metabolism, apoptotic processes, and inflammation. Previously, proteins that have been associated with these MERC contact sites mitofusin-1 (MFN1) and mitofusin-2 (MFN2) have been found to be downregulated in periodontal disease in vitro. Therefore, the aim of the current study was to evaluate MFN1 and MFN2 in gingival crevicular fluid (GCF) of patients with periodontal disease compared with healthy controls clinically. METHODS: A total of 48 participants were divided into three groups including periodontally healthy (n = 16), patients with gingivitis (n = 16), and patients with stage 3 grade B periodontitis (n = 16). GCF levels of MFN1, MFN2, calcium (Ca), caspase-1, and tumor necrosis factor-alpha (TNF-α) were determined via enzyme-linked immunosorbent assay (ELISA). Results were calculated as total amount and concentration. RESULTS: MFN1 levels (total amount) were significantly higher in patients with periodontitis and gingivitis when compared with healthy controls (p < 0.05). However, concentration levels of MFN1, MFN2, Ca, caspase-1, TNF-α significantly decreased in periodontal disease groups compared with healthy controls (p < 0.05). A positive correlation was detected among all evaluated markers (p < 0.05). CONCLUSION: The MERC protein MFN1 may have a role in the pathogenesis of periodontal disease due to its increase in GCF of patients with periodontitis and gingivitis.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

Gene expression profiles of mitochondria-endoplasmic reticulum tethering in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: The current study aimed to elucidate the potential involvement of mitochondria-endoplasmic reticulum contact genes in the pathogenesis of periodontal disease by monitoring levels of contact associated genes including Mitofusion 1 (MFN1) and MFN2, inositol 1,4,5-trisphosphate receptor (IP3R), chaperone glucose-regulated protein 75 (GRP75), sigma non-opioid intracellular receptor 1 (SIGMAR1) and phosphate and tensin homolog induced putative kinase 1 (PINK1) in human gingival fibroblasts in response to periodontal pathogens Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) in vitro. DESIGN: Primary human gingival fibroblasts were exposed to live cultures of P. gingivalis (W83; ATCC BAA-308) and F. nucleatum (subsp. Polymorphum; ATCC 10953) alone or in combination for 4 h at a 50 or 200 multiplicity of infection. Escherichia coli lipopolysaccharide (10 µg/mL) exposure was used as a positive control. Gene expression levels of contact genes (MFN1, MFN2, IP3R, GRP75, SIGMAR1 and PINK1) as well as a proinflammatory cytokine, Tumor necrosis factor-α (TNF-α), and the apoptosis associated gene, Immediate early response 3 (IER3), were evaluated by reverse transcription polymerase chain reaction analysis. RESULTS: MFN1, GRP75, IP3R and PINK1 were significantly upregulated by P. gingivalis with or without F. nucleatum. Only P. gingivalis with F. nucleatum caused a significant upregulation of SIGMAR1. TNF-α and IER3 gene expression positively correlated with the contact-associated gene expression changes. CONCLUSION: F. nucleatum and P. gingivalis alone or in combination may differentially dysregulate the gene expression levels of contact-associated genes in human gingival fibroblasts. These host-microbiome interactions may mechanistically be important in the pathogenesis of periodontal disease.

Effects of Porphyromonas gingivalis and Fusobacterium nucleatum on inflammasomes and their regulators in H400 cells.

INTRODUCTION: Inflammasomes are multiprotein complexes that regulate immune processes in response to infections and tissue damage. They modulate Interleukin-1beta (IL-1ß) expression, a major proinflammatory cytokine. The inflammasome/IL-1ß pathway is involved in head and neck squamous cell carcinoma (HNSCC) progression and the periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) have been reported to cause chronic inflammation in HNSCC. The aim of this study was to characterise the role of these pathogens in regulating inflammasome activity and the IL-1ß response in HNSCC in vitro. METHODS: An HNSCC cell line (H400) was exposed to Fn and Pg individually or in combination for 24h, ± incubation for 30 min with 5 mM adenosine triphosphate (ATP). Transcript levels of inflammasomes, NLRP3 and AIM2; inflammasome-regulatory proteins, POP1, CARD16 and TRIM16; and inflammasome-component, ASC and caspase 1 and IL-1ß, were assayed by RT-PCR. Expression of IL-1ß was by immunocytochemistry and ELISA. RESULTS: NLRP3 expression was significantly upregulated in response to Pg, Fn + Pg, Pg + ATP and Fn + Pg + ATP. AIM2 was significantly upregulated by Fn, Pg and Fn + Pg + ATP exposure. All conditions significantly upregulated IL-1ß gene expression. POP1 expression was significantly downregulated by Pg or Fn exposure but not by Fn + Pg. Intracellular pro- and mature IL-1ß were significantly higher following Fn and Pg + ATP exposure. CONCLUSION: Pg alone increased IL-1ß by upregulating AIM2, NLRP3 and downregulating POP1. Fn promoted IL-1ß by increasing AIM2 and downregulating POP1. Pg + ATP with or without Fn upregulated NLRP3, IL-1ß by downregulating POP1. Periodontal pathogens may contribute to HNSCC pathogenesis by increasing the IL-1ß response due to inflammasome dysregulation.

Dysregulation of Inflammasomes in Human Dental Pulp Cells Exposed to Porphyromonas gingivalis and Fusobacterium nucleatum.

INTRODUCTION: Interleukin-1ß (IL-1ß) is a major proinflammatory cytokine that plays a significant role in pulpal inflammation. The regulation of IL-1ß as well as different cytokines and chemokines is controlled by multiprotein complexes named inflammasomes, which are known to be involved in pulpal inflammation. The goal of this study was to evaluate the effects of well-established endodontic bacteria and periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis on NLRP3 and AIM2 inflammasomes; the inflammasome regulatory proteins POP1, CARD16, and TRIM16; inflammasome components ASC and caspase-1; and IL-1ß levels in human dental pulp cells (HDPCs) in vitro. METHODS: HDPCs were exposed to either F. nucleatum or P. gingivalis or to the combination of both with an additional 30 minutes of 5 mmol/L adenosine triphosphate (ATP) incubation for 24 hours. Escherichia coli lipopolysaccharide exposure was used as a control. Gene expression of NLRP3, AIM2, POP1, CARD16, TRIM16, ASC and caspase-1, and IL-1ß were evaluated by reverse transcription polymerase chain reaction. The presence and levels of pro- and mature IL-1ß were monitored by immunocytochemistry and the release with enzyme-linked immunosorbent assay. RESULTS: Up-regulation of NLRP3 and AIM2 was detected in all exposure groups. IL-1ß was up-regulated in all groups, except for the F. nucleatum + ATP group. CARD16 was significantly down-regulated by F. nucleatum or P. gingivalis with or without ATP; however, POP1 was down-regulated only in P. gingivalis and E. coli LPS + ATP groups. P. gingivalis alone significantly increased intracellular pro- and mature IL-1ß levels. CONCLUSIONS: P. gingivalis and F. nucleatum in the presence of ATP may play a significant role in IL-1ß-induced pulpal inflammation by dysregulating inflammasomes and their regulators.

Oxidative stress, neutrophil elastase and IGFBP7 levels in patients with oropharyngeal cancer and chronic periodontitis.

OBJECTIVE: The present study focused on investigating levels of oxidative stress, neutrophil elastase (NE), and insulin-like growth factor-binding protein 7 (IGFBP7) in oropharyngeal cancers (OC) with the presence and absence of periodontitis. MATERIALS AND METHODS: A healthy non-periodontitis group (H-NP; n = 20), a systemically healthy chronic periodontitis group (H-P; n = 20), a non-periodontitis group with OC (OC-NP; n = 12), and a chronic periodontitis group with OC (OC-P; n = 16) formed the study groups. The levels of NE and IGFBP7 were measured in gingival crevicular fluid (GCF) and saliva. In addition, oxidative status was determined by evaluating total oxidant status (TOS), total antioxidant status (TAS), and OSI (TOS/TAS). RESULTS: Gingival crevicular fluid NE was higher in all the groups compared with the H-NP group (p < .01). Salivary NE was higher in the OC-P and H-P groups compared with the H-NP and OC-NP groups (p < .05). Salivary IGFBP7 was significantly higher in the OC-NP and OC-P groups compared with the H-NP and H-P groups (p < .001). GCF TOS and OSI levels were significantly higher in all groups compared with the H-NP group (p < .05). CONCLUSIONS: Gingival crevicular fluid NE levels were lower in healthy conditions compared with periodontal disease and OC. Salivary NE levels were higher in periodontal disease compared to states with no periodontal disease. Salivary IGFBP7 levels were higher in OC. Further analyses may help determine whether high salivary IGFBP7 levels distinguish OC from healthy conditions.

Inflammasomes and their regulation in periodontal disease: A review.

Interleukin-1ß (IL-1ß), which is secreted by host tissues leading to periodontal tissue inflammation, is a major pro-inflammatory cytokine in the pathogenesis of periodontal disease. The conversion of pro-IL-1ß into its biologically active form is controlled by multiprotein complexes named as inflammasomes, which are key regulator of host defense mechanisms and inflammasome involved diseases, including the periodontal diseases. Inflammasomes are regulated by different proteins and processes, including pyrin domain (PYD)-only proteins (POPs), CARD-only proteins (COPs), tripartite motif family proteins (TRIMs), autophagy, and interferons. A review of in vitro, in vivo, and clinical data from these publications revealed that several inflammasomes including (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) have been found to be involved in periodontal disease pathogenesis. To the best of our knowledge, the current article provides the first review of the literature focusing on studies that evaluated both inflammasomes and their regulators in periodontal disease. An upregulation for inflammasomes and a downregulation of inflammasome regulator proteins including POPs, COPs, and TRIMs have been reported in periodontal disease. Although interferons (types I and II) and autophagy have been found to be involved in periodontal disease, their possible role in inflammasome activation has not evaluated yet. Modulating the excessive inflammatory response by the use of inflammasome regulators may have potential in the management of periodontal disease.

Differential expression of inflammasome regulatory transcripts in periodontal disease.

BACKGROUND: The inflammasome modulates the release of key proinflammatory cytokines associated with periodontal disease pathogenesis. The aim of this study was to evaluate the expression of proteins that regulate the inflammasome, namely pyrin domain-only proteins (POPs), caspase activation recruitment domain (CARD)-only proteins, and tripartite motif-containing (TRIM) proteins, in periodontal diseases. METHODS: A total of 68 participants (34 males and 34 females) were divided into four groups, including periodontal health (H), gingivitis (G), chronic periodontitis (CP), and aggressive periodontitis (AgP) based on clinical parameters. Gingival tissue samples were obtained from all participants for reverse transcription polymerase chain reaction (RT-PCR)-based gene expression analyses of molecules that regulate the inflammasome, including apoptosis-associated speck-like protein (ASC) containing CARD, caspase-1, interleukin-1ß (IL-1ß), interleukin-18 (IL-18), nucleotide-binding domain, leucine rich family (NLR) pyrin domain containing 3 (NLRP3), NLR family pyrin domain containing 2 (NLRP2), AIM2 (absent in melanoma 2), POP1, POP2, CARD16, CARD18, TRIM16, and TRIM20 by RT-PCR. RESULTS: NLRP3 and IL-1ß were upregulated in the G, CP, and AgP groups compared with group H (P < 0.05). AIM2 was downregulated in the CP group compared with the H, G, and AgP groups (P < 0.05). TRIM20, TRIM16, and CARD18 were downregulated in the G, CP, and AgP groups compared with the H group (P < 0.05). POP1 and POP2 were downregulated in the CP and AgP, and AgP and G groups, respectively (P < 0.05). CONCLUSION: Active periodontal disease may result in downregulation of inflammasome regulators that may increase the activity of NLRP3 and IL-1ß in periodontal disease.

Six-month clinical outcomes of non-surgical periodontal treatment with antibiotics on apoptosis markers in aggressive periodontitis.

OBJECTIVE: Extrinsic and intrinsic pathways of apoptosis are involved in generalized aggressive periodontitis (GAgP). The aim of this study was to evaluate 6-month clinical outcomes relative to apoptosis of one-stage full-mouth disinfection (OSFMD) and systemic antibiotics (SA) in the treatment of GAgP. METHODS: Twenty-six patients with GAgP were included in this prospective follow-up intervention study. Gingival crevicular fluid (GCF) was collected from patients at baseline and 3 and 6 months after periodontal therapy, which consisted of OSFMD and SA (amoxicillin and metronidazole, 500 mg each for 7 days). The levels of p53, caspase-3, TNF-α, TRAIL, IL-1ß, and IL-10 in GCF were measured via ELISA. RESULTS: Periodontal parameters were improved at 3 and 6 months compared to baseline (p < 0.05). p53 was decreased up to 6 months (p < 0.05). TRAIL, TNF-α, and IL-10 were similar at baseline and 3 and 6 months. Caspase-3 and IL-1ß were decreased at 3 months (p < 0.05), but similar at 6 months compared to baseline (p > 0.05). CONCLUSION: Although OSFMD plus SA improves clinical periodontal parameters up to 6 months, this treatment protocol differentially regulates the apoptosis markers caspase-3 at 3 months and p53 at 6 months without influencing TRAIL in GCF.

The role of caspase-8, caspase-9, and apoptosis inducing factor in periodontal disease.

BACKGROUND: Caspases are key mediators of apoptosis. Caspase-8 mediates extrinsic, and caspase-9 initiates the intrinsic pathway of apoptosis. Apoptosis Inducing Factor (AIF), a mitochondrial proapoptotic protein, mediates cell death by a caspase-independent process. Because apoptosis is involved in periodontal disease, this study evaluated caspase-8, -9, and AIF in periodontal disease. METHODS: Twenty periodontally healthy volunteers (Group Healthy), 20 patients with generalized aggressive periodontitis (Group AgP), and 20 patients with generalized chronic periodontitis (Group CP) were included in this study. Levels of caspase-8, -9, and AIF were evaluated in gingival crevicular fluid (GCF) of all participants via enzyme-linked immunosorbent assays. RESULTS: AIF was significantly higher in the AgP (P = 0.07) and CP groups (P = 0.01) than the Healthy group, and similar to the CP and AgP groups (P > 0.05). Caspase-8 was significantly higher in the CP and Healthy groups than the AgP group (P = 0.00), and similar between Healthy and CP groups (P > 0.05). Caspase-9 was significantly higher in the AgP group than the Healthy group (P = 0.01), and similar between Healthy and CP groups (P > 0.05). CONCLUSIONS: The mitochondrial-centered intrinsic pathway involving caspase-9 and AIF, and the extrinsic pathway involving caspase-8 are significant for aggressive periodontitis. The intrinsic pathway involving caspase-independent AIF is also significant for chronic periodontitis.

Effects of colchicine on gingival inflammation, apoptosis, and alveolar bone loss in experimental periodontitis.

BACKGROUND: The aim of the study was to investigate the effects of colchicine on cytokine production, apoptosis, alveolar bone loss, and oxidative stress in an experimental model of periodontitis in rats. METHODS: Forty-eight rats were divided equally into four groups: healthy (H); periodontitis (P); periodontitis+colchicine low dose (CL, 30 µg/kg/day), and periodontitis+colchicine high dose (CH, 100 µg/kg/day). After 11 days, interleukin (IL) -1ß, IL-8, and IL-10 were analyzed in gingival samples using Enzyme-Linked ImmunoSorbent Assay. Receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), total oxidative stress (TOS), total antioxidant status (TAS), and oxidative stress index (OSI) were measured in gingiva and serum. Alveolar bone volume was evaluated via micro-CT. Apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in histological sections. RESULTS: Colchicine treatment significantly reduced IL-1ß, IL-8, RANKL, RANKL/OPG, TOS, OSI, and bone volume ratio levels, and increased TAS levels compared to group P (p < 0.05). High dose colchicine treatment (CH) significantly decreased TUNEL+ cell counts compared to group P (p < 0.05). CONCLUSIONS: These finding suggest that colchicine has a prophylactic potential for the prevention of periodontal tissue destruction through anti-inflammatory, anti-oxidative, anti-apoptotic, and bone-protective effects.

Metabolic control and periodontal treatment decreases elevated oxidative stress in the early phases of type 1 diabetes onset.

OBJECTIVE: Recently, increasing concern has been focused on the contribution of oxidative stress in the pathology of periodontal disease and diabetes mellitus. Firstly, the present study aimed to analyze gingival crevicular fluid (GCF), salivary, and serum oxidative status in children with type 1 diabetes mellitus (T1DM) at diagnosis and systemically healthy children with and without gingivitis. Additionally, the diabetic patients were reevaluated after diabetes and periodontal treatment. DESIGN: The study groups were composed of 32 T1DM patients at diagnosis, and age- and gender-matched thirty-six systemically healthy children with (G) and without (H) gingivitis. The diabetic patients who took insulin therapy (1.5 units/kg/day totally) and periodontal treatment (oral hygiene education with professional scaling) were reevaluated after 3 months. The levels of total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were recorded. RESULTS: GCF, salivary, and serum OSI were elevated in group T1DM compared to the other groups at baseline (p<0.05), and decreased in group T1DM at reevaluation compared to baseline (p<0.05). GCF OSI was positively correlated with periodontal clinical parameters (p<0.05). Glycated hemoglobin was positively correlated with GCF TOS (r=0.302, p=0.007), GCF OSI (r=0.346, p=0.002), salivary TOS (r=0.326, p=0.046), and serum TOS (r=0.239, p=0.044). CONCLUSION: The instability in the oxidative status that accompanies diabetes may be considered a significant pathogenic factor of diabetes-related periodontal inflammation.

Effects of bodybuilding and protein supplements in saliva, gingival crevicular fluid, and serum.

The effects of bodybuilding and protein supplements on periodontal tissues have not yet been evaluated. The present study aimed to examine the periodontal status and interleukin (IL)-1ß, apoptosis-associated speck-like protein containing C-terminal caspase-recruitment domain (ASC), and caspase 1 (CASP1) gene expression levels of body builders compared with those of controls. Twenty-five bodybuilders with gingivitis (BB-G) who used protein powder supplements were compared with 25 nonexercising males with (G) and 25 without (H) gingivitis. Saliva, gingival crevicular fluid (GCF), and serum were collected for gene expression analysis. Plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were recorded. GI and BOP were higher in group BB-G and G than in group H (P < 0.01), but PI, PD, and CAL were similar between groups (P > 0.05). In GCF, CASP1, ASC, and IL-1ß expression were upregulated in group G compared with groups BB-G and H (P < 0.01). In addition, ASC (P < 0.05) and IL-1ß (P < 0.01) were downregulated in group BB-G compared with group H. CASP1, IL-1ß (P < 0.01), and ASC in the saliva were downregulated in group BB-G compared with groups H and G (P < 0.05). CASP1, IL-1ß, and ASC may play a role in the pathogenesis of gingivitis. Bodybuilding and supplement usage may decrease gingival inflammation by downregulating CASP1, IL-1ß, and ASC.

Therapeutic effects of systemic vitamin k2 and vitamin d3 on gingival inflammation and alveolar bone in rats with experimentally induced periodontitis.

BACKGROUND: The synergistic effects of vitamin D3 and vitamin K2 on bone loss prevention have been reported. This study evaluates the effects of vitamin D3 and vitamin K2 supplementation in conjunction with conventional periodontal therapy (scaling and root planing [SRP]) on gingival interleukin (IL)-1ß and IL-10, serum bone alkaline phosphatase (B-ALP) and tartrate-resistant acid phosphatase 5b (TRAP-5b), and calcium and alveolar bone levels in rats with experimentally induced periodontitis. METHODS: Seventy-two rats were divided into the following groups: 1) healthy; 2) periodontitis; 3) SRP; 4) SRP + vitamin D3; 5) SRP + vitamin K2; and 6) SRP + vitamins K2 and D3. Periodontitis was induced by ligature placement for 7 days, and vitamin K2 (30 mg/kg) and/or vitamin D3 (2 µg/kg) were administered for 10 days in the SRP + vitamin D3, SRP + vitamin K2, and SRP + vitamins K2 and D3 groups by oral gavage. On day 18, the animals were sacrificed, serum B-ALP, TRAP-5b, and calcium levels were measured, gingiva specimens were extracted for IL-1ß and IL-10 analysis, and distances between the cemento-enamel junction and alveolar bone crest were evaluated. RESULTS: Alveolar bone levels in the periodontitis group were significantly greater than those in the other five groups. No significant differences were found in gingival IL-1ß and IL-10, serum B-ALP and TRAP-5b, and calcium and alveolar bone levels between the groups receiving SRP and vitamins and the group receiving SRP alone. CONCLUSION: Within the limitations of this study, vitamin D3 and K2 alone or in combination did not affect gingival IL-1ß and IL-10, serum B-ALP and TRAP-5b levels, or alveolar bone compared with conventional periodontal therapy alone.

Management of Cyclosporine and Nifedipine-Induced Gingival Hyperplasia.

Gingival enlargements modified by medications are becoming more common because of the increased use of inducing drugs, and may create speech, mastication, tooth eruption, periodontal, and aesthetic problems. We hereby present a case of a 54-year-old man with 12-month history of generalized gingival enlargement in the keratinized gingiva was referred to our clinic. The patient had a history of kidney transplant and was under medication of cyclosporine and nifedipine. After medical consultation, cyclosporine was changed to tacrolimus and nifedipine was changed to captopril. Gingivectomy was performed using a diode laser, and scaling and root planning were performed. At five months postoperative, the gingival enlargements relapsed and diode laser-assisted surgery was repeated. The patient was followed-up on second postoperatively at 18 months and no relapse was seen. Diode laser-assisted gingivectomy was found to be useful for coagulation during surgery and decreased postoperative bleeding. Recurrence risk of cyclosporine and nifedipine-induced gingival overgrowth is high, thus, there is a great need for prolonged care of patients following treatment and prosthetic restoration.