|Year : 2013 | Volume
| Issue : 1 | Page : 21-26
Interleukin-17 in sera from patients with acute myocardial infarction and chronic periodontitis
N Ravindra Reddy1, DS Madhu Babu2, Vinay Chavan1, N Sarath1, C Venkat Subba Reddy1, A Kishore Kumar1
1 Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, India
2 Department of Periodontics, Government Dental College, RIMS, Kadapa, Andhra Pradesh, India
|Date of Web Publication||20-Jun-2013|
N Ravindra Reddy
Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: Interleukin-17 (IL-17) is a cytokine that modulate the inflammatory process in acute myocardial infarction (AMI) and chronic periodontitis (CP) and has not been assessed in patients with AMI and CP. The aim of this study was to investigate possible association between AMI and CP by estimating and comparing serum concentrations of IL-17 between the group of patients with AMI, periodontal disease and health. Materials and Methods: Total 37 subjects divided into three groups were included in this study, 16 patients with AMI and CP was termed as Group-I, 16 patients with AMI were termed as Group-II and 5 healthy subjects were termed as Group-III. Gingival index, russel's periodontal index, probing pocket depth were assessed in all groups. Serum samples were collected from all participants and tested for lipid profile and were quantified for IL-17 levels using enzyme-linked immunosorbent assay. Results: IL-17 was detected in sera from all three grouped patients; serum concentrations of IL-17 were significantly higher in Group-I when compared to Group-II and III. IL-17 concentrations correlated positively with clinical and lipid parameters in Group-I except for high-density lipoprotein, and with all lipid profile parameters in Group-II and lipid triglycerides correlated positively with IL-17 in Group-III. Conclusions: It can be stated that poor periodontal health which produces higher levels of IL-17, which in turn produces many pro-inflammatory cytokines can place an individual at higher risk for AMI even in the absence of certain traditional risk factors.
Keywords: Acute myocardial infarction, chronic periodontitis, cytokines, enzyme linked immunosorbent assay, interleukin-17, serum
|How to cite this article:|
Reddy N R, Madhu Babu D S, Chavan V, Sarath N, Reddy C V, Kumar A K. Interleukin-17 in sera from patients with acute myocardial infarction and chronic periodontitis. J Orofac Sci 2013;5:21-6
|How to cite this URL:|
Reddy N R, Madhu Babu D S, Chavan V, Sarath N, Reddy C V, Kumar A K. Interleukin-17 in sera from patients with acute myocardial infarction and chronic periodontitis. J Orofac Sci [serial online] 2013 [cited 2020 Feb 25];5:21-6. Available from: http://www.jofs.in/text.asp?2013/5/1/21/113687
| Introduction|| |
Chronic periodontitis (CP) is an infectious disease caused by a small group of predominantly anaerobic gram-negative bacteria present on the tooth surface as bioﬁlms. Chronic periodontitis (CP) affects approximately 30% of adult US population, 5-15% of any given population globally and severity is generally more prevalent in males than in females.  There is increasing evidence that poor dental health, especially the presence of periodontal disease increases the risk of coronary heart disease. Many studies conducted on different populations have suggested that atherosclerosis and occurrence of acute thromboembolic events associated with acute myocardial infarction (AMI) could be linked with chronic oral infections.  In periodontal disease, lipopolysaccharides and other microbial substances gain access to the gingival tissues, initiate and perpetuate inﬂammation, resulting in production of high levels of pro-inﬂammatory cytokines, which lead to the destruction of the periodontal ligament and alveolar bone.  Interleukin-17 (IL-17) is one such pro-inflammatory cytokine and the prototype cytokine for a subset of T-helper (Th17) cells which stimulates a variety of cell types to produce inflammatory mediators such as IL-1, IL-6, tumor necrosis factor-α, metallo proteinases, and chemokines.  Elevated circulating levels of IL-17, has been related to an increased risk for the development of cardio vascular events and other systemic disorders. 
Atherosclerosis is a lipid-driven, chronic inflammatory disease of the vessel wall in which both innate and adaptive immune responses play a role.  Macrophages, T lymphocytes and to a lesser extent, mast cells contribute to the smouldering inflammatory response in the vessel wall. , In the later stages of atherogenesis, B cells and plasma cells also appear in the deeper layers of the plaque and in the adventitia.  At present TH1 cytokines are regarded as important pro-inflammatory regulators during atherogenesis and in addition, TH2 cytokines are present in the plaque and may contribute to the local inflammatory response as well. ,
So far, the causality and possible pathways of the association between periodontal disease and cardiovascular disease are not clearly understood. Factors that place individuals at risk for periodontitis may also place them at risk for cardiovascular disease since both share common risk factors. It has been speculated that periodontal disease as a chronic infection may be related with cardiovascular disease through infection-related mediators and hyperactivity of white blood cells and of blood platelets, thus promoting the development of disease.  Although the precise relationship between systemic and periodontal diseases has not been completely elucidated, a low-grade inﬂammatory burden was proposed as possible biologic mechanism linking periodontitis and systemic disorders. Infected and inﬂammed periodontal tissues may act as sources of pathogens, virulence factors, and inﬂammatory mediators that spread into the circulation, creating and sustaining a chronic systemic inﬂammatory burden. , Low-grade inﬂammation (i.e., a slight elevation of the levels of inﬂammatory markers) has been in the focus of studies, , because it is recognized as an important condition in the pathogenesis of systemic diseases, including cardio vascular diseases (i.e., atherosclerosis, acute myocardial infarction, and stroke). 
We hypothesize that IL-17 may play a similar role in periodontal disease and AMI pathogenesis, and the aim of this study was to clarify the association between AMI and periodontal disease by estimating and comparing the serum concentrations of IL-17 between a group of patients with acute myocardial infarction, periodontal disease and health.
| Materials and Methods|| |
A total of 110 patients with age group of 40-67 years, including both males and females who had recent AMI and admitted at inpatient section of our hospital were screened for the presence or absence of periodontitis. Diagnosis for AMI was established by typical changes in the initial electrocardiogram with elevation of ST not required segment of 2 mm or more in a chest lead, or elevation of ST segment of 1 mm or more in a limb lead, broad and deep Q wave, tall and pointed T wave with elevation of serum enzymes (Serum Glutamicoxaloacetic Transaminase, Creatinine Phosphokinase [CPK], and Creatinine Phosphokinase MB-isoenzyme [CPK-MB]) together with or without chest discomfort consistent with myocardial infarction  and diagnosis for periodontitis was established by presence of least eight teeth with probing pocket depth (PPD) ≥5 mm, gingival index (GI)  >1.30 Subjects with age group of 35-55 years, admitted to the Department of Periodontics, CKS Teja Institute of Dental Sciences and Research, were clinically screened for clinically healthy periodontium who had at least 16 natural teeth and PPD <3 mm. patients who received scaling and root planning, periodontal surgery, or had used antibiotics within the preceding 6 months, also those suffering from diabetes, hypertension, diseases of immune system, respiratory system and had smoking habit were excluded.
Groups were assigned based on the clinical features of AMI, periodontitis and healthy periodontium as:
Group-I: 16 patients with clinical features of AMI and periodontitis
Group-II: 16 patients with clinical features of AMI only
Group-III: Five patients with clinically healthy periodontium.
The institutional ethical committee approved the study and informed consent was obtained from all the participants.
All teeth except third molars were examined. GI,  PPD, and Russels periodontal index (RI)  were recorded for all the patients. Probing depths were measured by using a University of North Carolina UNC-15 Probe (Hu-Friedy, Chicago, IL, USA).
Blood sample collection
Five milliliters of venous blood was collected from the ante-cubital fossa by venipuncture using the 20-gauge needle with 5 ml syringe, three milliliters of which was transferred to a sterile vial and was sent immediately to the clinical biochemistry lab for analyzing Serum total cholesterol (CHL), high-density lipoprotein (HDL), CHL, low-density lipoprotein (LDL), Triglyceride (TGY) concentrations, rest two milliliters transferred to the vacutainer containing clot activation factors and was immediately transferred to a laboratory for serum analysis. Blood sample was allowed to clot at room temperature and after 1 h, serum was separated from blood by centrifuging at 3000 rpm for 5 min. The separated serum was immediately transferred to plastic vial (eppendorf tube) and stored at –70°C until the time of an assay.
The serum samples were assayed for IL-17 using a Quantikine® Human IL-17 Immunoassay kit obtained from R and D systems, USA (Catalog No. D1700). 100 μl of assay diluent was added to each well. 100 μl of Standard, control, or sample was added per well and reagent addition is uninterrupted, completed within 15 min and incubated 3 h at room temperature. Each well aspirated and washed with wash buffer (400 μl), repeating the process twice for a total of three washes. After the last wash, any remaining wash buffer was removed by aspiration. 200 μl of IL-17 conjugate was added to each well, covered with a new adhesive strip, incubated for 1 h at room temperature. Wash step was repeated as stated above and 200 μl of substrate solution was added to each well, incubated for 30 min at room temperature. Enzyme substrate reaction produces a blue color, and then 50 μl of Stop Solution was added, which produces yellow color. Using a Micro titer plate reader, the plate was read at the wavelength of 450 nm that is appropriate for the Substrate solution used. Absorbance of each well was read on the enzyme linked immunosorbent assay ELISA reader using 450 nm as the primary wavelength for IL-17. The concentrations of IL-17 in the test samples were estimated using the standard curve.
All data were analyzed using a software program SPSS not required 17.1 (IBM not required, Chicago, IL). Descriptive statistics (means ± SDs) were calculated for each parameter. Parametric test were carried out for assessing significance of difference within groups. The group comparisons for non-parametric variables, i.e. concentration of IL-17 were performed by the Kruskal-Wallis test. In addition, pair-wise comparisons using the Mann-Whitney U-test were carried out to explore which pair or pairs differed. The correlation between Concentration of IL-17 with clinical periodontal parameters was analyzed using the Spearman rank correlation coefficient. P < 0.05 is indicated as statistically significant.
| Results|| |
Descriptive statistics of baseline parameters of the study population are shown in [Table 1]. The results of the present study indicate that the mean IL-17 concentration was highest in Group-I (89.8 pg/ml). The mean IL-17 concentration increased from healthy to AMI groups and then from AMI to AMI-GCP not required groups (24.4 > 49.4 > 89.8 pg/ml).
|Table 1: Descriptive statistics of baseline parameters in the study population (mean±SD)|
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To test the hypothesis of equality of means among the three groups, the Kruskal-Wallis test was carried out, which indicated that the means differed significantly (P < 0.05) among groups for IL-17 [Table 2].
|Table 2: Results of Kruskal-Wallis test comparing mean interleukin-17 concentrations among groups|
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However, further multiple comparisons using the Mann-Whitney U-test were carried out to find which pair or pairs differed significantly. The results showed that the differences were statistically significant among three groups for IL-17 (P < 0.05) [Table 3].
|Table 3: Mann-Whitney U test for pair wise comparison of interleukin - 17 concentrations|
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The Spearman rank correlation coefficient test was carried out to find correlations among parameters (i.e., GI, RI, PPD, HDL, LDL, CHL, TGY, CPK, CPK-MB and IL-17 concentration). The test showed a significant positive correlation between IL-17 concentration and GI, RI, PPD, LDL, CHL, TGY in Group-I, and IL-17, HDL, LDL, CHL, TGY in Group-II, and IL-17, TGY in Group-III [Table 4].
|Table 4: Spearman correlation coefficient test among groups with respect to interleukin-17 and parameters|
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| Discussion|| |
To the best of our knowledge this is the first study reporting serum levels of IL-17 in the systemic circulation of patients with AMI and chronic periodontitis. The results of the present study showed that the mean serum levels of IL-17 were significantly higher in AMI with CP group (Group-I) as compared with AMI alone group and healthy group (Group-II and III). Our results are in agreement with those of Jafarzadeh et al.  who also reported significantly high levels of IL-17 in AMI patients as compared with the control group, also with studies done by Takahashi et al.,  Vernal et al.,  Ohyama et al.,  Johnson et al.,  Lester et al.,  who reported elevated levels of IL-17 in chronic periodontitis patients. The observation of increased serum IL-17 in AMI with CP is similar to previously demonstrated data in Crohn's and ulcerative colitis diseases, in which it has been reported that there is increased expression of IL-17 in the intestinal mucosa and increased serum IL-17 concentrations in patients with these diseases. 
However, so far the levels of IL-17 was estimated individually either for AMI patients or CP patients. Since both AMI and CP are inflammatory lesions, we estimated the serum levels of IL-17 in patients with AMI and CP and compared them with levels in AMI and healthy patients. Our results regarding high serum levels of IL-17 in patients with AMI and CP implicates that the IL-17 dependent mechanisms may be related with both AMI and CP events.
When mean serum levels of LDL, total CHL levels and TGY were compared between groups in the present study, levels were higher in Group-I than in Group-II and Group-III. The results are in agreement with Kimmo J. Mattila et al.  who reported higher concentrations of total CHL, LDL and TGYs and lower concentrations of HDL, CHL in individuals with periodontitis before periodontal treatment and Emingil et al.  who reported higher concentrations of total CHL, LDL and TGYs in AMI group compared to chronic coronary heart disease. One possible reason for high levels of LDL, CHL, TGY in Group-I might be due to the fact that Periodontal disease is a microbial infection caused by gram-negative bacteria and acute infections are known to interfere with lipid metabolism, causing elevation of plasma TGYs. , These changes are thought to be mediated by cytokines, which may be produced at the inﬂamed periodontal tissue in high quantities. ,,
In our present study, IL-17 was also detected in low-levels in healthy group, the possible reason for this might be having subclinical inflammatory conditions associated with periodontitis in this group. Beklen et al.  also reported that IL-17 are produced by healthy gingival tissue at low levels, but are highly elevated in periodontitis.
In our present study, levels of IL-17 were significantly higher in AMI with CP and AMI groups as compared with the healthy group, even though patients with conventional systemic risk factors like hypertension, diabetis, smoking, which are common for both AMI and CP patients were excluded. It was stated in a study by Jafarzadeh et al.  that the mean serum levels of IL-17 in the patient groups with or without certain conventional risk factors were higher than that observed in healthy control group and suggested that levels of IL-17 may dependently (in the context of conventional risk factors) or independently (in the absence of conventional risk factors) associated with ischemic heart disease events. This finding of Jafarzadeh et al.  is in group of population without estimating periodontal status. It can be stated through results of present study that presence of periodontitis may be a possible reason for high levels of IL-17 even when traditional risk factors were absent. Even though IL-17 was detected in patients with AMI and without CP may be due to presence of hyperlipidemia, these levels were significantly higher in patients with AMI with CP, suggesting that presence of periodontitis with local risk factors (GI > 1, PPD > 5 mm) can possibly make AMI happen without presence of systemic risk factors for AMI, since high levels of serum IL-17 derived initially from gingival tissues, may mediate inflammatory responses in tissues distant from the oral cavity.  IL-17 is a pro-inﬂammatory cytokine, which promotes rapid recruitment of neutrophils, stimulates various types of cells to produce pro-inﬂammatory mediators such as IL-1 and 6, TNF not required-α.  This Elevated circulating levels of TNF-α, IL-6 have been related to an increased risk for the development of cardio vascular events. Furthermore, previously several studies ,,,,,, have indicated that subjects with periodontal disease may have a higher risk for cardiovascular diseases when compared to subjects with a healthy periodontium. Since IL-17 is speculated to stimulate these pro inflammatory mediators, and presence of high levels of this cytokine in periodontitis patients suggests a role of periodontal diseases as potential contributors to general inﬂammation and the development of systemic diseases like AMI. 
| Conclusion|| |
The present study for the first time demonstrates that serum IL-17 levels are significantly elevated in AMI and CP patients. Also, poor periodontal health may produce higher levels of IL-17 which in turn place an individual at risk for AMI. However further studies with large sample size and longer follow-up period are needed to determine whether serum IL-17 levels in patients with chronic periodontitis can be used as biomarker in estimating the risk of AMI.
| Acknowledgments|| |
The authors acknowledge Krishnaiya, honorary statistician, Tirupathi, India, for helping prepare the statistics of the study and R. Ventakswamy, Technician, Department of Microbiology, Kempegowda Institute of Medical Sciences, Bangalore, India, for helping run the ELISA and Dr. P. Srinivasulu, Principal Scientist, Watson Life Sciences, Tirupathi for providing laboratory facilities.
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[Table 1], [Table 2], [Table 3], [Table 4]