Jun 26, 2015 - The Influence of Glycated Hemoglobin on the Cross Susceptibility Between. Type 1 Diabetes Mellitus and Periodontal. Disease. David F.
J Periodontol • November 2015
The Influence of Glycated Hemoglobin on the Cross Susceptibility Between Type 1 Diabetes Mellitus and Periodontal Disease David F. Lappin,* Douglas Robertson,* Penny Hodge,* David Treagus,* Raja A. Awang,† Gordon Ramage,* and Christopher J. Nile*
Background: Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)-6, IL-8, and C-X-C motif chemokine ligand 5 (CXCL5) in non-smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL-6, IL-8, and CXCL5 expression by THP-1 monocytes and OKF6/TERT-2 cells. Methods: There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high-performance liquid chromatography. Levels of IL-6, IL-8, and CXCL5 in plasma were determined by enzyme-linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT-2 cells and THP-1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL-6, IL-8, and CXCL5 were monitored by ELISA and real-time polymerase chain reaction. Results: Patients with diabetes and periodontitis had higher plasma levels of IL-8 than patients with periodontitis alone. Plasma levels of IL-8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL-6, IL-8, and CXCL5 expression in both OKF6/TERT-2 cells and THP-1 monocytes. Conclusions: Elevated plasma levels of IL-8 potentially contribute to the cross-susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL-6, IL-8, and CXCL5 from THP-1 monocytes and OKF6/TERT-2 cells than LPS alone. J Periodontol 2015;86:1249-1259. KEY WORDS Chemokines; cytokines; diabetes mellitus, type 1; glycosylation end products, advanced; periodontitis. * Infection and Immunity Research Group; University of Glasgow Dental School; School of Medical, Veterinary, and Life Sciences; School of Medicine; University of Glasgow, Glasgow, UK. † School of Dental Sciences, University of Science Malaysia, Kubang Kerian, Kelantan, Malaysia.
doi: 10.1902/jop.2015.150149
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Type 1 Diabetes Mellitus and Periodontal Disease
I
t has been established by a large body of evidence that the prevalence, severity, and extent of periodontitis is increased in patients suffering from type 2 diabetes mellitus (T2DM).1-4 However, understanding of the biologic mechanisms behind this cross-susceptibility is currently lacking.1,2,5 One hypothesis is that chronic hyperglycemia leads to the formation of advanced glycation end products (AGE) through the irreversible non-enzymatic glycation of proteins and lipids.3 This results in a loss of protein functionality and creates molecules that stimulate proinflammatory mediator release through interaction with the receptor for AGE (RAGE) found on the surfaces of immune and resident tissue cells.3,6,7 Elevated serum and tissue levels of AGE are associated with immune and non-immune diabetes (type 1 diabetes mellitus [T1DM] and T2DM).2,3 Once in circulation, AGE can initiate chronic, low-level, inflammatory responses at sites throughout the body, including the periodontium, via the AGE/RAGE axis.8-11 Within the periodontium, numerous cells express RAGE, including oral keratinocytes, fibroblasts, and resident and invading immune cells, such as macrophages and monocytes.8,12,13 Interactions between AGE and RAGE perpetuate an inflammatory response through the upregulation of expression of molecules, such as matrix metalloproteinases10 and osteolytic activators,11 potentially damaging the periodontal ligament and alveolar bone and resulting in periodontal disease. There have been many clinical case and cohort studies investigating the link between T2DM and periodontal disease.4 Although cytokine levels are generally greater in the tissue fluids of patients with T2DM than systemically healthy individuals, studies are contradictory regarding the differences in tissue fluid levels of interleukin (IL)-6, IL-8, and many other mediators in patients with T2DM, with or without periodontitis. Indeed, although some studies report increases in IL-6,14 other investigations do not.4,15 Furthermore, both increases16 and decreases17,18 in serum IL-6 levels have been reported in studies after periodontal treatment in patients with T2DM. In T1DM, clinical studies have demonstrated that the disease is associated with elevated circulating levels of inflammatory mediators.19,20 IL-6 is a pleiotropic proinflammatory cytokine, elevated circulating levels of which are implicated in poor clinical outcomes in patients with T1DM.20 Furthermore, serum IL-6 levels have been implicated in the cross-susceptibility between T1DM and periodontal disease.21 The C-X-C motif chemokine ligand (CXCL) 8 (IL-8) is a major neutrophil chemoattractant and plays important roles in the induction and maintenance of inflammation. Like IL-6, elevated circulating levels of IL-8 are implicated in poor clinical outcomes in patients with T1DM.20 In addition, salivary22 and gingival 1250
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crevicular fluid23 levels of IL-8 have also been implicated in the cross-susceptibility between T1DM and periodontal disease. Another chemokine that is a major neutrophil chemoattractant and activator is CXCL5. Circulating levels of CXCL5 have been found to be a good systemic indicator of the inflammatory process and marker of disease severity in individuals with periodontitis.24 However, little is known about the role of this chemokine in the pathogenesis of T1DM, periodontal disease, and the cross-susceptibility between the two conditions. Despite a number of investigations into AGE and their role in the pathogenesis of T2DM-associated periodontitis,3,4 there is a paucity of data on T1DM. Indeed, to the best of the authors’ knowledge, there is only one clinical study that demonstrates that AGE accumulation in the gingivae of patients with periodontitis was common to both T1DM and T2DM, although the key factors associated with AGE accumulation were not clear.25 Stimulation of monocytes in vitro with AGE has been found to induce expression of IL-626 and IL-8.27 However, stimulation of monocytes with Escherichia coli lipopolysaccharide (LPS) in combination with AGE had no cumulative effects on IL-6 or IL-8 expression.28 In contrast, similar studies in endothelial cells demonstrated that E. coli LPS and AGE had a synergistic effect on the expression of IL-6 and IL-8.29 Despite evidence for AGE accumulation in gingival tissues of patients with periodontitis,25 to date, the effect of LPS from known periodontal pathogens, such as Porphyromonas gingivalis, on AGE-induced expression of cytokines and chemokines by oral epithelial cells and monocytes is unknown. The aims of this study are as follows: 1) to compare circulating levels of IL-6, IL-8, and CXCL5 in patients with T1DM, with and without periodontitis, to control groups consisting of systemically healthy, nonsmoking individuals with and without periodontitis; 2) to correlate glycated hemoglobin and biomarker levels in these patients; and 3) begin to determine the effect of AGE in the presence and absence of P. gingivalis LPS on IL-6, IL-8, and CXCL5 expression by THP-1 monocytes and OKF6/TERT-2 cells. MATERIALS AND METHODS In total, 104 patients and volunteers (49 males and 55 females, aged 20 to 56 years; mean age: 36.4 – 9.9 years) participated in this study: 1) 19 healthy volunteers (H group); 2) 23 patients with periodontitis (P group); 3) 28 patients with T1DM (T1DM group); and 4) 34 patients with T1DM and periodontitis (T1DM + P group). Ethical approval was obtained from the Glasgow Royal Infirmary Ethics Committee (Reference 05/ S0705/70). The study was conducted in accordance
Lappin, Robertson, Hodge, et al.
J Periodontol • November 2015
with the ethical principles described in the Declaration of Helsinki 2008. Written Informed consent was obtained from all participants. Patients were recruited from diabetic outpatient clinics at Glasgow Royal Infirmary, Stobhill Hospital, and Glasgow Dental Hospital and School (all in Glasgow, UK) between 2007 and 2010. All patients with diabetes were diagnosed by trained clinicians (PH and DR), were currently on insulin, and had been attending the outpatient clinic for monitoring of glycated hemoglobin for >2 years. To be included in the periodontitis group, participants had to have a minimum of two sites with probing depth (PD) and attachment loss (AL) ‡5 mm. However, in all cases, the newly diagnosed patients with periodontitis had many sites with PD or AL >3 mm. None of the participants were receiving treatment for periodontitis at the time of diagnosis but were offered treatment whether they agreed, declined to participate, or were excluded from the study. The exclusion criteria for this study were: 1) no history of smoking within the past 5 years; 2) pregnancy at the time of recruitment; 3) immunosuppression attributable to medication or concurrent illness; 4) use of antibiotics or anti-inflammatory drugs within 6 weeks of recruitment; 5)
