Increase in serum and salivary neutrophil gelatinase-associated lipocalin levels with increased periodontal inflammation

Abstract Objective: This study aimed to determine serum and salivary levels of neutrophil gelatinase-associated lipocalin (NGAL) and evaluate NGAL correlation with key anti-interleukin 10 (IL-10) and pro-inflammatory (IL-1β) cytokines in different severities of periodontal diseases. We also calculated the systemic inflammation using the periodontal inflamed surface area (PISA) to evaluate its correlation with NGAL in the study groups. Methodology: Eighty systemically healthy and non-smoking individuals were separated into four groups of 20: clinically healthy (Group 1), gingivitis (Group 2), stage I generalized periodontitis (Group 3, Grade A), and stage III generalized periodontitis (Group 4, Grade A). Sociodemographic characteristics and periodontal parameters were recorded, and PISA was calculated. The serum and salivary levels of interleukin (IL)-1β, IL-10, and NGAL were determined using the enzyme-linked immunosorbent assay (ELISA). Results: We observed a significant increase in serum and salivary NGAL levels from healthy to periodontitis groups (p=0.000). Group 2 presented significantly higher serum and salivary IL-10 levels and salivary IL-1β levels than Group 3 (p=0.000). Serum and salivary parameters (IL-1β, IL-10, and NGAL levels) were strongly positively correlated to periodontal parameters and PISA values (p=0.000). Groups 2 and 3 showed overlapping PISA values. Conclusion: The overlapping PISA values found in Groups 2 and 3 suggest that gingivitis might progress to a systemic inflammatory burden somewhat comparable to stage I periodontitis. This finding is supported by the higher serum and salivary cytokines/mediators levels in the gingivitis group than in stage I periodontitis group. Serum and salivary NGAL levels increased proportionally to disease severity and PISA. NGAL seems to play a role in the pathogenesis of periodontal disease, within the limitation of our study.


Introduction
Periodontitis is a chronic inflammatory disease In periodontal disease, host inflammatory cells are mobilized on inflammation site and inflammatory cytokines are released from fibroblasts, macrophages, connective tissue, and junctional epithelial cells. 7 One of the most important cytokines in the periodontitisrelated immune response and tissue destruction is interleukin (IL)-1β, which is produced by macrophages, monocytes, lymphocytes, epithelial cells, fibroblasts, and osteoblasts, 8 has a potent pro-inflammatory function, 8 and is reported to be present in increased levels in the serum, saliva, and gingival crevicular fluid (GCF) of periodontitis patients when compared to controls. 9-11 Studies reported a decrease in serum and GCF IL-1β levels after non-surgical periodontal treatment. 11,12 To control immune-inflammatory response and prevent tissue damage, regulatory T cells produce cytokines, such as IL-10, to inhibit the synthesis of pro-inflammatory cytokines and suppress immune-inflammatory-driven alveolar bone resorption. 13 Neutrophil gelatinase-associated lipocalin (NGAL) belongs to the lipocalin family and has the ability to bind iron, fatty acids, prostaglandins, steroids, and matrix metalloproteinases. 14 A study reported NGAL to play an important role in mediating innate immunity response to bacterial infections 15 by sequestrating iron-loaded siderophores. It is also a neutrophils chemoattractant, promoting their maturation, adhesion and extravasation, and their phagocyte capacity, besides activating regulatory T cells. 14 Due to the elevated circulating NGAL levels in the early stages of inflammation, it is considered a risk indicator for nephropathy, kidney dysfunction, and liver homeostasis. 16 IL-1β was found to upregulate NGAL in human epithelial cells 16 and to possibly comprise a determinant for circulating NGAL in systemic inflammations. 17 A study found IL-10 overexpression by primary macrophages to enhance their pro-resolution activity in complex inflammationassociated pathologies. 18 Likewise, blocking of NGAL production in macrophages reduced IL-10 protective effects in a kidney ischemia/reperfusion injury model, substantiating NGAL-associated pro-proliferative and anti-inflammatory properties. 18 Although NGAL role in cases of kidney dysfunction was widely investigated, studies on its function in the pathogenesis of periodontal disease and its association with serum and salivary levels and key cytokines in periodontitis are still scarce. The literature reported NGAL presence in GCF and saliva 18,19 and suggested the neutrophils extravasated is main source of this protein in these biological fluids and in gingiva. 19 NGAL has been reported to be a salivary biomarker in patient monitoring and disease activity control, 20 and urinary NGAL has been indicated for clinicians to screen periodontitis. 21 Considering the lack of studies in the existing literature on periodontitis regarding the role of NGAL and its association with cytokines/ mediators in the pathogenesis of periodontal diseases, it is logical to evaluate NGAL association with IL-1β and IL-10 and determining its function in the pathogenesis of periodontitis.
This study aimed to evaluate serum and salivary NGAL levels in different types of periodontal diseases and different levels of disease severity. We also sought to investigate whether NGAL levels in local (saliva) and systemic environments (serum) proportionally increase with increased severity, bleeding, and size of the ulcerated periodontal inflamed surface area (PISA).  Gingivitis group is characterized by no probing attachment loss (AL), and radiographic bone loss and BOP score ≥ 10% and ≤ 30% (gingivitis in an intact periodontium). 23 Stage I periodontitis is characterized by 1 to 2 mm AL at greatest loss site, radiographic bone loss in coronal third (<15%), and no tooth loss due to periodontitis. 22 Stage III periodontitis is characterized by ≥ 5 mm AL at greatest loss site, radiographic bone loss extending to mid-third or apical third of root, and ≤ 4 teeth loss due to periodontitis. 22 Periodontitis extent was determined as generalized (≤ 30% of teeth Sociodemographic and dental characteristics Sociodemographic (age, gender, education level, monthly income, height, and weight) and dental characteristics (toothbrushing frequency, frequency of dental visits, use of interdental hygiene equipment, such dental floss, waterpick, toothpick) were recorded. Participants were requested to refrain from eating, drinking, chewing gum, or breath mints, as well as performing oral hygiene procedures for at least one hour prior to saliva collection. After collection, samples were placed on ice and aliquoted before being storaged at -80°C.

Serum samples
Fasting blood samples from the antecubital vein were collected using blood collection tubes with anticoagulant (EDTA, Greiner AG, Kremsmünster, Austria) and centrifuged at 3000 × g for 10 minutes.
Before analysis, serum samples were portioned and stored at −80°C.
After all the periodontal measurements were recorded and salivary and serum samples were taken, periodontal therapy was administered according to the individual needs.
Serum samples were diluted 1:10 and saliva 1:50 before analysis, assuming the specific standard curve with eight different concentrations for each parameter.

Statistical analysis
Data were analyzed to verify whether they

Results
Eighty patients (35 females and 45 male) aged between 25 and 63 years participated in the study. Table 1 shows the study population sociodemographic parameters and characteristics. We found no statistically significant differences between Groups 3 and 4 (p=0.966) and Groups 1 and 2 (p=0.142) regarding age. Mean age was significantly lower in  comparisons among groups. Salivary IL-1β levels were significantly higher in Group 2 than in Groups 3 and 1 (p=0.045, and p=0.000), but significantly lower than Group 4 (p=0.000). However, salivary IL-10 levels showed no statistically significant differences between Groups 2 and 3 (p=0.923), but were significantly lower in Group 1 when compared to Group 4 (p=0.000).
Salivary NGAL levels increased significantly alongside disease progression from periodontally health to stage III periodontitis; all groups were significantly different from each other (p=0.000).
Serum IL-1β levels progressively increased from group to group, from periodontal health to periodontitis, and were significantly different among groups (p=0.000). Although serum IL-10 levels increased in the transition from periodontally health to periodontal compromised groups (p=0.000), Groups 2 and 3 presented no significant differences (p=0.450).

Groups 2 and 3 had significantly lower IL-10 levels than
Group 4 and higher levels than Group 1 (p=0.000).
Serum NGAL levels increased significantly alongside disease progression, and all groups were significantly different from each other (p=0.000). All clinical periodontal parameters were significantly and strongly correlated with other clinical periodontal, serum, and salivary parameters. All serum and salivary parameters were significantly and strongly correlated with all other serum and salivary parameters (p=0.000, data not shown, added as supplementary material).

Discussion
This cross-sectional study aimed to investigate whether increased periodontitis severity, bleeding, and ulcerated periodontal inflamed surface area (PISA) increase NGAL levels in local (saliva) and systemic environments (serum). We also investigated whether gingivitis causes a systemic inflammatory burden as high as periodontitis. Our results verified and answered these study questions.

Sociodemographic parameters and characteristics
regarding education level, monthly income, dental visit frequency, and oral hygiene habits were not significantly different among groups. The significant age difference between groups (lower age in Group 1) could be deemed as a limitation. However, to the best of our knowledge, there are no studies in the literature reporting that age could affect serum and salivary IL-1β, IL-10, and NGAL levels. In turn, the available knowledge on the effects of ageing in immunity 31 led us to understand that age differences between groups cause no impact on the investigated parameters, considering the lack of individuals older than 65 years in our study population.
We found significantly higher body mass index (BMI) values in stage III periodontitis group than in healthy group, although similar to other groups.
Our study groups presented mean BMI below obesity  To the best of our knowledge, this is the first study to investigate serum and salivary NGAL levels considering different periodontal diseases and severity. We found all periodontal parameters and PISA values to proportionally increase with increased disease severity and NGAL levels.
Periodontal disease progresses if the balance between pro-and anti-inflammatory cytokines in periodontal tissues is disturbed in favor of proinflammatory cytokines. 7 We investigated IL-1β, a key pro-inflammatory cytokine in tissue damage. Salminen, et al. 39 (2014) showed that salivary IL-1β levels were critical for periodontal disease progression. In our study, serum IL-1β levels progressively increased from healthy (Group 1) to stage III periodontitis (Group 4) and salivary IL-1β levels were higher in gingivitis group than in stage I periodontitis group. This finding confirms that IL-1β is an important cytokine in the transition from gingivitis to periodontitis, corroborating results reported in the literature.
We also investigated another important antiinflammatory cytokine, the IL-10. Information on IL-10 levels reported in the literature are still conflicting: whereas Andrukhov,et al. 40 (2011)  in line with our study hypothesis. This may suggest that periodontal disease may progress more rapid in individuals with low anti-inflammatory capacity, and that the immune system of individuals with gingivitis is as active those with in stage I periodontitis.
A study reported circulating NGAL to be regulated by IL-1β in systemic inflammatory conditions. 16 IL-1β was reported to be the major inductive cytokine of NGAL by NF-kB activation in an experimental acuteon-chronic liver failure model. 16 The positive strong correlations between serum and salivary NGAL and serum and salivary IL-1β found in our study support these studies. Increased serum and salivary IL-1β levels induced the increase in serum and salivary NGAL levels from healthy to severe periodontal disease.
By investigating the association between NGAL and IL-10, a study found that the inhibition of NGAL decreased IL-10 expression by macrophages and the pro-proliferative and anti-inflammatory NGAL role in a renal ischemia-reperfusion model. 18 The role of NGAL in periodontal disease pathogenesis, as regulated by IL-1β and IL-10, could be conjectured within the limitations of this study, as supported by the increased serum levels progressing from health to disease. In our study, serum and salivary NGAL levels were statistically different among the study groups by progressively increasing with increased severity and PISA. Serum and salivary NGAL showed a significant and strong positive correlation with periodontal Increase in serum and salivary neutrophil gelatinase-associated lipocalin levels with increased periodontal inflammation J Appl Oral Sci. 2020;28:e20200276 9/10 clinical parameters and PISA, suggesting that NGAL plays an increasing role in cases of periodontitis with exacerbated inflammation and increased ulcerated bleeding surface area.
Our study poses several limitations. First, its crosssectional design does not enable an analysis of NGAL in the pathogenesis of periodontal disease, which requires future randomized case-control studies investigating the cut-off values of serum and salivary NGAL levels in periodontally healthy and compromised patients, and employing interventional approaches (comparing NGAL levels before and after non-surgical periodontal therapy). Secondly, the presence of obese patients in periodontally compromised groups (although not significant among groups) might have affected salivary and serum NGAL levels. As fat distribution (determined by waist/hip ratio or waist circumference) was reported to be associated with periodontitis rather than higher BMI, 42 the lack of such measurements could pose a limitation to observe clear associations between the investigated parameters.

Conclusion
Despite the limitations of our study, NGAL seems to play a role in the pathogenesis of periodontal disease, as its increasing level is associated with the presence and severity of the disease. This finding is supported by the significant and strong correlations between NGAL and clinical parameters, as well as serum and salivary IL-1β and IL-10 levels.