Cytokine Profile in Chronic Periodontitis Patients with and without Obesity: A Systematic Review and Meta-Analysis

To investigate the cytokine profile as biomarkers in the gingival crevicular fluid (GCF) of chronic periodontitis (CP) patients with and without obesity, MEDLINE/PubMed, EMBASE, ScienceDirect, and SCOPUS databases were combined with handsearching of articles published from 1977 up to May 2016 using relevant MeSH terms. Meta-analyses were conducted separately for each of the cytokines: resistin, adiponectin, TNF-α, leptin, IL-6, IL-8, and IL-1β. Forest plots were produced reporting standardized mean difference of outcomes and 95% confidence intervals. Eleven studies were included. Three studies showed comparable levels of leptin among obese and nonobese patients with CP. Four studies reported comparable levels of interleukin- (IL-) 6 and resistin whereas five studies reported comparable levels of adiponectin. Two studies reported similar levels of CRP in patients with periodontitis with and without obesity. One study showed higher levels of tumor necrosis factor-alpha in obese patients with CP. One study showed higher levels of IL-1β and IL-8 in obese patients with CP. The level of localized periodontal inflammation may have a greater influence on the GCF proinflammatory biomarker levels as compared to systemic obesity. Whether patients having chronic periodontitis with obesity have elevated proinflammatory GCF biomarkers levels compared to nonobese individuals remains debatable.


Introduction
Obesity is described as a condition associated with expansion in adipocytes and increased infiltration of macrophage cells in the adipose tissues, defining the inflammatory state [1,2]. Substantial evidence in the form of in vivo and in vitro studies has demonstrated a strong association between chronic periodontitis (CP) and obesity [3][4][5][6]. The underlying mechanisms of periodontitis in obesity are not well understood; however, it is suggested that the increased levels of proinflammatory cytokines as biomarkers (such as interleukin-(IL-) 6, IL-1 ) in the gingival crevicular fluids (GCF) of obese individuals are associated with poor periodontal health [7].
Cytokines are low molecular weight water-soluble glycoprotein biomarkers secreted by hematopoietic and nonhematopoietic cells in response to infection. Inflammatory biomarkers which are induced during inflammatory responses have been associated with the onset or progression of tissue insult [8,9]. It is suggested that proinflammatory biomarkers show pleiotropic effect and can target specific cells by controlling activation of cells, cell proliferation, and function in the periodontium [10,11]. As a result, raised proinflammatory biomarkers levels such as tumor necrosis factor-alpha (TNF-), IL-1 , IL-6, and IL-8 cause periodontal tissue destruction [12]. The reason for studying these proinflammatory cytokines in obesity is to validate the association of the same cytokines which are actively involved in jeopardizing periodontal tissues by mediating alveolar bone resorption and collagen destruction [12].
Obese patients are associated with a state of elevated systemic inflammatory burden due to increased serum proinflammatory cytokine levels [13,14]. In addition, studies have shown increased susceptibility of inflammatory periodontal 2 Disease Markers tissue destruction in overweight or obese patients as compared to healthy individuals [15]. Moreover, inflammatory cytokine mediated mechanisms are implicated in periodontal inflammatory conditions [11]. In this context, it may be hypothesized that the GCF cytokine profile in obese patients with CP should be elevated as compared to nonobese individuals with periodontal disease. In the study by Modéer et al. [16] GCF IL-1 and IL-8 levels among obese patient with CP were significantly higher as compared to nonobese CP patients. In contrast, Duzagac et al. [17] showed comparable GCF cytokine profile among obese and nonobese patients with CP. There appears to be a controversy with regard to GCF cytokine profile in chronic periodontitis patients with and without obesity. Therefore, the aim of this study was to systematically review the GCF cytokine profile in CP patients with and without obesity.

Protocol and
Registration. This review was registered at the National Institute for Health Research PROSPERO, International Prospective Register of Systematic Reviews (http://www.crd.york.ac.uk/PROSPERO/, registration number CRD42015029928). Based on the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines [18], a specific question was constructed. The addressed focused question was "Does the GCF cytokine profile of obese and nonobese patients with chronic periodontitis differ?" 2.2. Selection Criteria. The following eligibility criteria were entailed: (i) clinical trials (baseline data), cross-sectional and observational (prospective; baseline data) studies in humans (adults and adolescents only) using any type of assay method; (ii) cytokine profile in the GCF of patients with chronic periodontitis with and without obesity.
In vitro studies; animal studies; studies providing analyses of cytokines in fluids other than GCF; studies that reported cytokine profile among obese only with no normal weight controls; letters to the editor; and review papers and unpublished articles were excluded.

Screening and Selection.
Two reviewers (Z. A. and F. V.) independently screened titles and abstracts for eligible papers. If information relevant to the eligibility criteria was not available in the abstract or if the title was relevant but the abstract was not available, the paper was selected for full reading of the text. Next, full-text papers that fulfilled the eligibility criteria were identified and included in the review. Papers that fulfilled all of the selection criteria were processed for data extraction. Figure 1 describes the screening process according to PRISMA guidelines for flow diagram [18].

Data Extraction and Quality Assessment.
Two reviewers (Z. A. and F. V.) undertook this independently. The information from the accepted studies was tabulated according to the study designs, subject characteristics, sample characteristics, cytokines investigated, and main outcomes. Data collected were based on the focused question outlined for the present systematic review. Baseline data that compared the levels of cytokines among obese and nonobese patients in prospective studies were also included in the review. The reviewers crosschecked all extracted data. Any disagreement was resolved by discussion until consensus was reached. The quality of the included studies was assessed using the Newcastle-Ottawa Quality Assessment scale for Observational Studies [27] (Table 3), a validated scale for evaluating the quality of observational and nonrandomized studies. This scale uses a star system to evaluate the studies on three broad perspectives: the selection of the study groups; the comparability of the groups; and the ascertainment of either the exposure or outcome of interest for included studies respectively.

Statistical Analyses.
Meta-analyses were conducted separately for each of the cytokines: resistin, adiponectin, TNF-, leptin, IL-6, IL-8, and IL-1 . Heterogeneity among the included studies for each cytokine outcome was assessed using the -statistic and 2 statistic [28]. Outcome measures for each inflammatory mediator were combined with a random-effects model utilizing the DerSimonian-Laird method due to its robustness in comparison to fixed-effects models in the case of small sample sizes [29]. Forest plots were produced reporting standardized mean difference (SMD) of outcomes and 95% confidence intervals (CI). The pooled effect was considered significant if value was <0.05. All
The premise of increased proinflammatory cytokines in obesity is such that the metabolic cells such as adipocytes initiate inflammation by triggering inflammatory signalling pathways [33]. This mediates a modest, low-level induction of inflammatory cytokines such as TNF-, IL-1 , and IL-6 which occurs in response to excess nutrients. Overtime, this low-grade inflammation may induce the infiltration and activation of immune cells which is characterized by increase in the number of macrophages, mast cells, and T-lymphocytes that results in proinflammatory changes in the tissue environment and the inflammatory pathways and proinflammatory cytokines continue to reinforce. The inflammatory state therefore becomes maintained (chronic) and unresolved [33].
There are several explanations which can be posed regarding the similarity in cytokine levels in the GCF of CP patients with and without obesity. The studies [6,16,17,[19][20][21][22][23][24][25][26], which were included in this systematic review, were conducted with an aim to assess the level of cytokines in GCF as risk indicator for periodontal inflammation in obese patients. In the studies included [6,16,17,[19][20][21][22][23][24][25][26], the depth of periodontal pockets from which GCF was collected was not standardized among obese and nonobese patients. For instance, collection of GCF sampled sites from PD ≥ 5 mm was reported in some studies [6,19,22], whereas other studies did not report probing PD of GCF sampled sites [16,17,[23][24][25] (Table 1). This may characterize a bias as level of periodontal inflammation in diseased periodontal pockets is known to influence GCF cytokine levels [34,35]. Therefore it is hypothesized that the severity of localized periodontal inflammation on GCF cytokine levels in nonobese subjects could have exceeded the impact of obesity on GCF cytokine levels in obese CP patients. Moreover, in nearly half of the studies, the exclusion of patients with systemic diseases was not reported [6,16,6 Disease Markers    [17] ffff f fff 8 Gonçalves et al. [19] ffff ff ff 8 Pradeep et al. [20] fff ff ff 7 Pradeep et al. [21] fff ff ff 7 Ongöz Dede et al. [22] fff fff 6 Patel and Raju [23] ffff f f 6 Fadel et al. [24] fff ff f 6 Zimmermann et al. [6] ffff ff f 7 Pradeep et al. [25] fff ff ff 7 Pradeep et al. [26] fff ff ff 7 Modéer et al. [16] fff f ff 6 23,24]. It may therefore be speculated that the similarity in GCF cytokine profiles in obese and nonobese patients with CP could be associated with covert systemic conditions (such as diabetes) in the otherwise systemically healthy individuals [36,37]. It may also be proposed that the balance between proinflammatory and anti-inflammatory mediators in periodontal tissues of nonobese subjects may be shifted towards a hyperinflammatory state that could impair the host response against pathogens and periodontal deterioration [38]. It is reported that leptin (expressed from adipocytes) shows an inverse relation with periodontal inflammation demonstrating a protective role in periodontal disease [39][40][41]. Interestingly, in the present review, GCF leptin levels were found comparable among obese and nonobese subjects [6,19,24]. A possible explanation for this may be derived from the fact that leptins release is stimulated by TNF-, which is increased in CP patients as compared to healthy periodontium [42]. As all subjects included (obese and nonobese) in the studies reviewed had CP, the comparable stimulatory effect of TNF-on leptin could have resulted in its similar levels in obese and nonobese subjects with CP. Therefore it may be hypothesized that periodontal inflammation may have a greater influence on GCF cytokine levels (including leptin) rather than the increased systemic inflammatory burden due to obesity.
It is well recognized that tobacco smoking has been shown to be deleterious for periodontal health [43,44]. Studies on GCF assay have also shown cytokine concentrations to be low in habitual tobacco smokers owing to its immunosuppressant state as compared to nonsmokers [45]. Worthy of note, however, is that the subjects included in the studies fulfilling our inclusion criteria were nonsmokers. This again suggests that the intensity of periodontal inflammation alone may mainly be responsible for the increased GCF cytokine concentrations in subjects with periodontitis with and without obesity [46]. Weight management in obese subjects has shown a reduction in systemic inflammatory burden as expressed by lower levels of serum cytokines [47,48]. It is also reported that weight control could reduce the amounts of MMP-8, MMP-9, and IL-1 in GCF of obese subjects with healthy periodontium [49]. The effect of periodontal therapy on obesity has been reported in recent study [50]; however, the effect of weight control on GCF cytokine profile in obese patients with CP still needs to be assessed. Moreover, the effect of periodontal treatment including recent adjunctive therapies (such as laser and photodynamic therapy) on the levels of proinflammatory needs to be explored [51,52].
The review of included studies suggests that considerable heterogeneity existed in the studies reviewed (methodology, cytokines assessed, systemic health of subjects, and cytokine collection sites). Therefore, in light of the systematic review and assessment of available data, it remains arguable whether patients having chronic periodontitis with obesity have elevated proinflammatory GCF cytokine levels compared to nonobese individuals.