IL-8 as a Potential Diagnostic Salivary Biomarker in Periodontal Diseases- A Systematic Review

IL-8 (chemokine) stimulate the recruitment of inflammatory cells and thus known to play a major role in the initiation and progression of any inflammation, Current systematic review aimed in finding role of salivary IL-8 as a biomarker in Periodontal Diseases, despite of having an important role in the pathogenesis of Periodontal diseases only few studies were done till date to find its co-relation with Periodontitis, Electronic searches of the PubMed, and Scopus databases were conducted from 2000 to 2016 for literature detecting IL-8 levels in individuals with CP compared with healthy individuals in the saliva of the patients. Only 5 Publications till date reported with levels of salivary Il-8 in CP, overall systematic review reported contradictory findings regarding IL-8 levels in patients with Chronic Periodontitis when compared with healthy controls, thus more number of longitudinal studies are required to conform the exact co-relation. Key wordsGingivitis, Periodontitis, ELISA, IL-8, Saliva, Oral health, Biochemical analysis, Inflammation INTRODUCTION Periodontitis is a chronic inflammatory disease of the oral cavity that affects tooth-supporting tissues and alveolar bone, leading to progressive tooth loss 1-3 . The pathogenesis of chronic periodontitis is multifactorial, resulting from a complex interaction between pathogenic microbes, host immune responses, environmental and genetic factors. 3-5 Although bacterial infection is the primary cause in triggering periodontal disease, it’s progression depends on the production of host mediators in response to bacteria and it’s metabolic products. 4,6 Innate and adaptive immune reactivity control the response to periodontal pathogens 7 . The numbers of systemic inflammatory markers such as cytokines play an important role in the pathogenesis and progression of the periodontal disease and determine the nature and duration of the immune response 8 . Cytokines in Plasma, Serum, GCF and Saliva have been identified as inflammatory indicator of periodontal disease and can be used as a potential diagnostic biomarker. 9 The requirement of reliable biomarkers helps to distinguish progressive periodontitis from normal biological processes and is considered fundamental to identify periodontitis at an earlier or even preclinical stage, to initiate preventive treatment, and also to conduct Epidemiological studies. 10,11 Interleukin-8 (IL-8), a member of the chemokine family, has been identified as Jyoti Chand et.al. IL-8 as a potential diagnostic salivary biomarker in periodontal diseasesa systematic review International Journal of Science and Healthcare Research (www.ijshr.com) 56 Vol.5; Issue: 1; January-March 2020 a neutrophil chemotactic polypeptide, produced in response to inflammation by macrophages, epithelial and endothelial cells, IL-8 functions primarily to activate neutrophils, and plays a role in PMN recruitment in the inflammatory sites 12 . The protein, consisting of 72 amino acids in its mature form, is identified as a basic and heparin-binding protein. The unique coordinated expression of IL-8 facilitates the transmission of neutrophils from the highly vascularized gingival tissue to the gingival crevice and subsequently to the GCF and saliva. 13,14 The idea of using saliva in diagnostics was made in the second half of the 20th century 15 . Its main advantage is that it is easy and non-invasive sample taking procedures compared to peripheral blood. The clinical applications of saliva range from the forensic field to drug monitoring and diagnosis of systemic and local conditions. 15 Hence an attempt is made to review the literature on the diagnostic applications of saliva for monitoring the levels of biomarker IL-8 in Periodontal Diseases. The relationship of interleukins in saliva and GCF in patients with Gingivitis was initially analyzed in 2003 by Faizuddin et al 16 , who undertook a case-control study in which IL-1β in the crevicular fluid of patients was analyzed. In this study their results showed that the levels of IL-1β in the crevicular fluid of patients with periodontitis were higher than those in patients with gingivitis and also that the levels of this interleukin in Gingivitis and Periodontitis were much higher than in healthy patients 16 . METHODOLOGY A Systematic review methodology was followed and database searching was done which yields 312 records and 10 from additional sources 55 duplicate records were removed from total of 267, Records from year 2000-2016 were taken into consideration, 31 records were found out to be from years before 2000, records were further reviewed and 94 records were excluded on the basis of not having full text available. 142 articles were reviewed, 15 articles were excluded on the basis of language other than English. From total of 127 records, 5 animal studies, 12 in vitro studies and 85 studies which used methodology not appropriate for this review and 20 case reports were excluded. A total studies to be systematically reviewed came out to be 5. RESULTS In spite of the importance of IL-8 in the course of Periodontal diseases, a variety of studies in the present systematic review revealed contradictory findings regarding IL-8 levels in the Saliva of patients with CP used for measuring the disease progression and the effectiveness of therapy. While some studies demonstrate higher levels of IL-8 in the saliva of patients with CP, others suggested the opposite, table 1 showed that the electronic search generated 5 studies between 2009-2015 assessing cytokine profile (including IL-8 levels) in the saliva of patients with and without CP. Only 2 study used the Luminex (Multiplex bead assay) evaluation method and one while the other 3 used the ELISA method. The number of subjects sampled ranged from 31 to 160 patients. Three studies presented higher 21,22,25 , while the other two studies detected lower IL-8 23,24 levels in the diseased group. Interestingly, significant differences were found only in 1 study, which reported higher levels of IL-8 in control subjects; however, this study did not report the numerical data (mean/standard deviation) of IL-8 levels. Only 2 other studies reported the numerical data (mean/standard deviation) for IL-8 levels. Jyoti Chand et.al. IL-8 as a potential diagnostic salivary biomarker in periodontal diseasesa systematic review International Journal of Science and Healthcare Research (www.ijshr.com) 57 Vol.5; Issue: 1; January-March 2020 METHODOLOGY Table 1: Search of Literature


INTRODUCTION
Periodontitis is a chronic inflammatory disease of the oral cavity that affects tooth-supporting tissues and alveolar bone, leading to progressive tooth loss [1][2][3] . The pathogenesis of chronic periodontitis is multifactorial, resulting from a complex interaction between pathogenic microbes, host immune responses, environmental and genetic factors. [3][4][5] Although bacterial infection is the primary cause in triggering periodontal disease, it's progression depends on the production of host mediators in response to bacteria and it's metabolic products. 4,6 Innate and adaptive immune reactivity control the response to periodontal pathogens 7 .
The numbers of systemic inflammatory markers such as cytokines play an important role in the pathogenesis and progression of the periodontal disease and determine the nature and duration of the immune response 8 .
Cytokines in Plasma, Serum, GCF and Saliva have been identified as inflammatory indicator of periodontal disease and can be used as a potential diagnostic biomarker. 9 The requirement of reliable biomarkers helps to distinguish progressive periodontitis from normal biological processes and is considered fundamental to identify periodontitis at an earlier or even preclinical stage, to initiate preventive treatment, and also to conduct Epidemiological studies. 10,11 Interleukin-8 (IL-8), a member of the chemokine family, has been identified as a neutrophil chemotactic polypeptide, produced in response to inflammation by macrophages, epithelial and endothelial cells, IL-8 functions primarily to activate neutrophils, and plays a role in PMN recruitment in the inflammatory sites 12 . The protein, consisting of 72 amino acids in its mature form, is identified as a basic and heparin-binding protein.
The unique coordinated expression of IL-8 facilitates the transmission of neutrophils from the highly vascularized gingival tissue to the gingival crevice and subsequently to the GCF and saliva. 13,14 The idea of using saliva in diagnostics was made in the second half of the 20th century 15 . Its main advantage is that it is easy and non-invasive sample taking procedures compared to peripheral blood. The clinical applications of saliva range from the forensic field to drug monitoring and diagnosis of systemic and local conditions. 15 Hence an attempt is made to review the literature on the diagnostic applications of saliva for monitoring the levels of biomarker IL-8 in Periodontal Diseases.
The relationship of interleukins in saliva and GCF in patients with Gingivitis was initially analyzed in 2003 by Faizuddin et al 16 , who undertook a case-control study in which IL-1β in the crevicular fluid of patients was analyzed. In this study their results showed that the levels of IL-1β in the crevicular fluid of patients with periodontitis were higher than those in patients with gingivitis and also that the levels of this interleukin in Gingivitis and Periodontitis were much higher than in healthy patients 16 .

METHODOLOGY
A Systematic review methodology was followed and database searching was done which yields 312 records and 10 from additional sources 55 duplicate records were removed from total of 267, Records from year 2000-2016 were taken into consideration, 31 records were found out to be from years before 2000, records were further reviewed and 94 records were excluded on the basis of not having full text available. 142 articles were reviewed, 15 articles were excluded on the basis of language other than English. From total of 127 records, 5 animal studies, 12 in vitro studies and 85 studies which used methodology not appropriate for this review and 20 case reports were excluded. A total studies to be systematically reviewed came out to be 5.

RESULTS
In spite of the importance of IL-8 in the course of Periodontal diseases, a variety of studies in the present systematic review revealed contradictory findings regarding IL-8 levels in the Saliva of patients with CP used for measuring the disease progression and the effectiveness of therapy. While some studies demonstrate higher levels of IL-8 in the saliva of patients with CP, others suggested the opposite, table 1 showed that the electronic search generated 5 studies between 2009-2015 assessing cytokine profile (including IL-8 levels) in the saliva of patients with and without CP. Only 2 study used the Luminex (Multiplex bead assay) evaluation method and one while the other 3 used the ELISA method. The number of subjects sampled ranged from 31 to 160 patients.
Three studies presented higher 21,22,25 , while the other two studies detected lower IL-8 23,24 levels in the diseased group. Interestingly, significant differences were found only in 1 study, which reported higher levels of IL-8 in control subjects; however, this study did not report the numerical data (mean/standard deviation) of IL-8 levels.
Only 2 other studies reported the numerical data (mean/standard deviation) for IL-8 levels.

DISCUSSION
In the studies analyzing cytokine levels in periodontitis health and disease, it is important to mention that inflammatory cytokines detected in whole saliva do not originate from major salivary glands secretions instead, the GCF is the probable source 17 . Cytokines are present at significantly lower concentrations in saliva than are other mediators (e.g. enzymes such as matrix metalloproteinases) and therefore subtle changes may not always be detected. Indeed, there is some evidence that analytical technique with greater sensitivity periodontitis-associated biomarkers not detected by conventional ELISAs, which are almost universally used in research in this field. 18 Zhang et al 19 demonstrated that both gingival and oral epithelial cells infected with P.gingivalis produce IL-8, and after infection these cells continued to express IL-8 mRNA, although the accumulation of the secreted protein could not be detected. This study suggested that IL-8 could be degraded locally by P. gingivalis proteinases, and the finding might explain the results of the present systematic review, but further research should confirm or negate this hypothesis.
Moreover, a previous study indicated that the levels of IL-1b, IL-8, IL-10, and TNF-a were higher and the levels of other cytokines were almost equal in saliva compared to plasma or urine (Khan 2012). Deinzer et al., in the year 2007 20 , published a case-control study which compared a group of gingivitis patients with another of experimental gingivitis. Outstanding among their results were that the individuals who were subject to experimental gingivitis presented higher IL-1β and lower IL-8 levels at 4 weeks than individuals with persistent gingivitis 26 .
The only study which used LUMINAX method for evaluation of IL-8 in saliva is done by Teles et al in 2009 21 , examined 20 teeth in both controls (CAL<3mm) and diseased subjects( CAL >3mm), and found salivary IL-8 levels to be higher in diseased subjects. 27 Michiels et al 22 in 2009 studied 31 subjects and found salivary IL-8 levels to be higher in diseased subjects.
Contrary to this, Chen et al 23 in 2014 studied salivary IL-8 levels in 21 subjects and his results showed that IL-8 levels in patients with Periodontitis were only marginally significantly higher in healthy controls (P = 0.014).
Khalaf et al 24 in 2014 reported significantly higher IL-8 levels in saliva from Periodontally healthy individuals when compared with those affected by CP, Interestingly, this result is in agreement with IL-8 in GCF, which showed higher levels of IL-8 in the GCF of control subjects.
In another study by Tamaki et al in 2015 25 the concentrations of eight cytokines including IL-8 were measured using multiplex bead assays and significant differences were found in salivary IL-8 levels between periodontitis classifications.
The percentage of sites exhibiting severe periodontitis (deep pocket sites) in subjects decides the final dilution of the saliva. 26 Therefore, more severe periodontal disease, exemplified by the higher mean percentage of sites with pocket depth, might be associated with higher IL-8 levels. 27,28 In another important study by R. P. Teles, V. Likhari, S. Socransky, and A. D. Haffajee in the year 2008 28 74 subjects with chronic periodontitis and 44 periodontaly healthy individuals were examined and the levels of IL-8 along with other cytokines such as Granulocyte-macrophage colonystimulating factor, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, etc. were measured in whole saliva using a multiplexed bead immunoassay (Luminex). In his results he could not discriminate between the levels of cytokines in periodontal health and disease.
The lack of association between the levels of salivary biomarkers and periodontal disease could be explained by differences in the methods of saliva collection (stimulated or unstimulated), processing (speed and time of