J Periodont Res. 2020;55:267–276. wileyonlinelibrary.com/journal/jre © 2019 John Wiley & Sons A/S.
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267 Published by John Wiley & Sons Ltd1 | INTRODUCTION
The immune-inflammatory response in periodontitis, both innate and acquired immunity, is complex and various mediators produced by
bacteria and host contribute to modulation of the inflammatory process and tissue destruction.1 Beside these, the nervous system, an element of human immune defense system, has been known as a critical regu-lator of inflammation in periodontal diseases through cell recruitment.2 Received: 15 May 2019
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Revised: 19 September 2019|
Accepted: 27 October 2019DOI: 10.1111/jre.12712
O R I G I N A L A R T I C L E
Netrin-1 and its receptor Unc5b as markers of periodontal
disease
Sadiye Gunpinar
1| Bilge Meraci
1| Niyazi Dundar
2 1Department of Periodontology, Facultyof Dentistry, Bolu Abant Izzet Baysal University, Bolu, Turkey
2Faculty of Dentistry, Research Center, Selcuk University, Konya, Turkey
Correspondence
Sadiye Gunpinar, Periodontology Department, Faculty of Dentistry, Bolu Abant Izzet Baysal University, Bolu, Turkey. Email: coskunersadiye@gmail.com
Funding information
Bolu Abant Izzet Baysal University, Coordination Unit of Scientific Research Projects, Grant/Award Number: 2018.06.05.1269
Abstract
Objectives: The aim of this present study was to evaluate the levels of Netrin-1 and
Unc5b in periodontal health and disease.
Background: Netrin-1, acting via its receptor UNC5b, regulates the inflammatory
re-sponse and takes apart in bone destructive diseases.
Methods: Samples of gingival crevicular fluid (GCF), whole saliva, and serum were
taken from systemically healthy, nonsmoking 20 periodontitis , 20 gingivitis patients, and 20 periodontally healthy subjects at baseline and 4 weeks after nonsurgical periodontal treatment (NSPT). Whole-mouth and site-specific clinical periodontal parameters were recorded. Netrin-1 and Unc5b levels were measured by enzyme-linked immunosorbent assay. Data were analyzed by nonparametric tests.
Results: Total amount of Netrin-1 in GCF was significantly higher in
periodonti-tis than the others, and the levels were significantly reduced after NSPT. Salivary and serum concentrations of Netrin-1 were significantly different among the study groups (P = .000), and NSPT significantly increased the concentration levels of both salivary and serum Netrin-1 (P < .05). Healthy subjects had significantly lower GCF (P = .001) and conversely, higher salivary and serum levels of Unc5b than the other groups (P = .002). The GCF levels of Unc5b were significantly reduced (P < .01), and conversely, serum concentrations were significantly increased after NSPT (P < .05). GCF Netrin-1 and Unc5b total amounts were positively correlated with clinical pa-rameters (P < .01 and P < .05) whereas salivary Netrin-1 and Unc5b concentrations were negatively correlated with clinical parameters (P < .01 and P < .05).
Conclusions: The results of this study indicate that Netrin-1 and its receptor Unc5b
may have essential roles in periodontal inflammation and those can be assumed as useful therapeutic agent to control inflammation and periodontal breakdown.
K E Y W O R D S
cellular immunology, gingival crevice fluid, inflammation, macrophages, nonsurgical periodontal therapy, periodontal disease
Netrin-1 is a laminin-related protein and identified as a neuroim-mune guidance cue. During development of the nervous system,3 this molecule has been shown to play essential role via directing axons to its targets through interaction with its one of the main re-ceptor uncoordinated (UNC) 5 family members: UNC5A, UNC5B, UNC5C, and UNC5D.4 Recent studies have demonstrated that the expression of Netrin-1 is not limited with the nervous system. This molecule functions in both acute and chronic inflammatory diseases via controlling angiogenesis,5,6 regulation of immune cell recruit-ment,7 macrophage reprogramming,8 and regulation of cyclooxy-genase-2 pathways.9
Netrin-1, acting via its receptor UNC5b, is shown to be a po-tent inhibitor of monocyte and lymphocyte migration in vitro and in vivo.7 This molecule suppressed TNF-α-induced vascular adhesion molecules and hampered the adhesion of monocytes to endothe-lial cells. Moreover, Netrin-1 prevented NF-κB activation and re-vealed an anti-inflammatory function.10 It has been reported that Netrin-1 regulates the inflammatory response through suppression of COX-2–mediated PGE2 production9 and HIF-1alpha-dependent induction of Netrin-1 has been demonstrated to attenuate hypox-ia-elicited inflammation at mucosal surfaces.11 One mechanism of the anti-inflammatory effect of Netrin-1 is induction of M2 macro-phage polarization through PPAR pathways and therefore promotes inflammation resolution.8
Tadagavadi, et al12 reported that administration of recombinant Netrin-1 before or after renal ischemia-reperfusion reduced renal tubular cell apoptosis, monocyte and neutrophil infiltration, serum and renal cytokine, and chemokine production thus reduced kidney injury. Conversely, neutralization of UNC5B receptor reduced this Netrin-1–mediated renal protection.12
In addition to the anti-inflammatory properties, Netrin-1/ Unc5b interaction takes apart in bone destructive diseases13,14 impacting through osteoclast fusion. Blockade of Netrin-1/Unc5b axis has been shown to prevent bone destruction.14 On the other hand, increased expression of Netrin-1 and its receptor Unc5b in obese adipose tissue15 and in atherosclerotic plaques is responsible for chronic inflammation via promoting macrophage retention.16
In the shade of these informations, we hypothesized that Netrin-1 and its receptor Unc5b may be involved in the inflammatory mechanism of periodontal disease and may reveal anti-inflamma-tory effects and promote inflammation resolution (see in Figure 1). Therefore, we measured the gingival crevicular fluid (GCF), saliva, and serum levels of Netrin-1 and Unc5b in periodontally healthy subjects, gingivitis, and periodontitis patients.
2 | MATERIALS AND METHODS
2.1 | Study population and clinical examination
One hundred and seventy four systemically healthy individuals were examined in the Department of Periodontology, Faculty of Dentistry, Bolu Abant Izzet Baysal University from January 2018 to
February 2019. Twelve did not want to participate in the study and 89 did not meet the inclusion criteria. Thus, 73 patients were en-rolled in this clinical and biochemical trial. On the other hand, nine patients did not come to the first-month visit and four patients did not follow the oral hygiene instructions. As a result, 60 subjects completed the study. The methodology of the study was approved by the Ethics Committee of the School of Medicine, Bolu Abant Izzet Baysal University (decision number: 2017/87) and registered in Clinical Trials Registry (NCT03919006). Before the study, all par-ticipants were verbally informed about the protocol and written in-formed consent was received.
A total of 60 individuals, comprising 20 patients with periodonti-tis (P group; aged 23-42 years), 20 patients with gingiviperiodonti-tis (G group; aged 22-37 years), and 20 periodontally healthy controls (H group; aged 26-38 years) were involved in the study. The patients were diagnosed according to the new classification scheme.17 Stage and extent of periodontitis were also determined.18
The clinical periodontal criteria for the diagnosis of the groups were as follows: the periodontitis group had interdental clinical at-tachment loss (CAL) ≥5 mm at site of greatest loss and periodontal probing depth (PD) ≥6 mm in one or more sites of the two nonadja-cent teeth in at least two quadrants of the mouth. Accordingly, stage III generalized periodontitis patients were included in the study. Patients were diagnosed as gingivitis with bleeding on probing ≥50%, PD ≤ 3 mm, and no radiographic bone loss or CAL. Periodontally healthy individuals had no recorded history of periodontal problems, with PD ≤ 3 mm, no radiographic bone loss, good oral hygiene, no gingival inflammation.
Inclusion criteria were as follows: (a) aged > 18 years, (b) having at least 20 natural teeth (excluding third molar), (c) nonsmokers with no history of smoking, and (d) not having any diagnosed medical illness or drug intake that could affect the periodontal condition. The pa-tients who had (a) taken antibiotics, nonsteroidal anti-inflammatory, or any other drugs within the past 3 months, (b) received nonsurgical or surgical periodontal treatment, (c) a restorative and endodontic therapy requirement, and (d) a removable partial denture and/or having orthodontic therapy were excluded from the study. Current pregnancy or lactation and having serum CRP > 3 mg/L were also the exclusion criteria.
2.1.1 | Periodontal measurements and treatment
All individuals were examined at baseline and four weeks after nonsurgical periodontal treatment including, whole mouth prob-ing depth (PD), CAL, the presence of bleedprob-ing on probprob-ing (BOP), papillary bleeding index (PBI),19 gingival index (GI),20 and plaque index (PI)21 except the third molars. PD and CAL were measured at six sites per tooth using a manual periodontal probe (Unc15, Hu-Friedy).
The nonsurgical periodontal treatment for periodontitis group cluded supra- and subgingival scaling, root planing, and oral hygiene in-structions. Subgingival scaling and root planing was performed under
local anesthesia (Ultracaine DS Forte; Sanofi, Aventis) in two sessions within the 48-72 hours. Gingivitis group had supra- and subgingival scaling, polishing, and oral hygiene instructions. The treatment of gin-givitis and periodontitis patients were performed by a periodontist (BM) using hand (Gracey curettes, Hu-Friedy) and ultrasonic (Cavitron select, Dentsply) instruments. All measurements were performed by the same calibrated examiner (SG). Intraexaminer calibrations were performed in 10 (five gingivitis and five periodontitis) patients who were not included in this study. PD and CAL were recorded on six surfaces, and GI was recorded on four surfaces of each tooth, in two separate sessions, 48 hours apart. The intraclass correlation coeffi-cients ranged between .831 and .923 for PD and between .912 and .937 for CAL. The Cohen's kappa (κ) value of GI was .834.
2.2 | Collection of biological samples
Saliva, GCF, and serum samples were collected in the morn-ing between 8.30 am and 10.30 am, 48-72 hours after the initial
periodontal recording. The samplings from periodontitis and gin-givitis patients were repeated four weeks after nonsurgical peri-odontal treatment. First of all, unstimulated saliva samples were collected. Accordingly, all subjects were asked to avoid eating, drinking, brushing, and flossing at least 30 minutes before the saliva sample collection. Each subject was instructed to rinse the mouth completely with water for 1 minutes, and then expectorate whole saliva into a sterile polypropylene tube for 5 minutes. A vol-ume of 1.5 to 2 mL saliva samples was vortexed for 3 minutes and centrifuged at 10 000 g for 10 minutes at 4°C. Supernatants were aliquoted on ice and frozen at −40°C first and then − 80°C until the analysis.
Following the saliva collection, GCF samples were obtained from two nonadjacent interproximal sites in one single-rooted and one multi-rooted teeth. GCF was sampled from two sites with GI < 1, PD ≤ 3 mm, and without BOP in the healthy group; two sites with GI ≥ 2, PD ≤ 3 mm, and positive BOP (visible signs of inflammation) in the gingivitis group; and two deepest pocket (≥5 mm) with GI ≥ 2 and positive BOP in periodontitis group.
F I G U R E 1 Possible mechanisms of Netrin-1/Unc5b pathway in periodontal disease. (Unc5b: uncoordinated-5 receptor B; BMP: bone
morphogenetic protein; NF-κB: nuclear factor kappa B; COX-2: cyclooxygenase-2; PG: prostaglandin; TNF-α: tumor necrosis factor-alpha; MCP: monocyte chemoattractant protein; IL: interleukin)
The selected sampling areas were isolated with cotton rolls, and supragingival plaque was removed from the sampling area. The area was then slightly dried to avoid contamination with saliva. Standardized paper strips (PerioPaper, Proflow) were gently inserted 1 mm into the gingival sulcus/pocket and leaved there for 30 sec-onds. If the strips were contaminated with blood, then they were discarded. The absorbed GCF volume was measured with a precal-ibrated electronic device (Periotron 8010, Oraflow), and two strips obtained from each individual were then placed into one Eppendorf tube and stored at −80°C for laboratory analysis. The periotron readings were converted to an actual volume (microliter, µL) by ref-erence to the standard curve.
Serum samples were taken following saliva and GCF sampling before the periodontal treatment. Six milliliters of venous blood was obtained by a standard venipuncture method, and the serum was separated from blood by centrifugation at 1500 g for 20 min-utes. Enumerated serum samples were then stored at −80°C until analysis.
2.3 | Detection of Netrin-1 and Unc5b levels
in biofluids
The Netrin-1 (Human NTN1(Netrin-1) ELISA Kit, FineTest) and Unc5b levels (Human UNC5B( Netrin Receptor UNC5B) ELISA Kit, FineTest) in GCF, saliva, and serum were measured by ELISA kits in line with the guidelines of the manufacturer. The assay procedures of both Netrin-1 and Unc5b ELISA kits were the same. First, GCF samples were diluted by 500-µL phosphate-buffered saline (PBS). One hundred μL of standard blank or sample were added to each well and covered with the adhesive strip. The plate was incubated for 90 minutes at 37°C. Then, 100-μL Biotin-detection antibody working solution was added to each well appropriately. After incu-bation at 37°C for 60 minutes, it was aspirated and then washed 3 times with wash buffer. One hundred μL SABC working solution was added to each well properly, incubated for 30 minutes at 37°C and aspirated, then washed 5 times. Afterward, 90 μL of TMB sub-strate was added into each well and incubated at 37°C in dark within 15-30 minutes. Then, 50 μL of stop solution was added to each well and the plate was tapped gently to ensure thorough mixing. The ab-sorbance of the reacting solution in wells was read at 450 nm in a mi-croplate reader (ELx800, BioTek) immediately after adding the stop solution. The concentrations of Netrin-1 and Unc5b were calculated according to the standard absorbance curve. The detection range of Netrin-1 and Unc5b kits were 31.25-2000 pg/mL with a sensitiv-ity < 18.75 pg/mL and 0.313-20 ng/mL with a sensitivsensitiv-ity < 0.188 ng/ mL, respectively. Total amount of Netrin-1 (pg/30 seconds) and Unc5b (ng/30 seconds) levels in GCF was determined by multiply-ing the absorbance values with diluent volume (0.5 mL) and then di-vided by 2 (two paper strips). The GCF concentrations (pg/µL) were calculated by multiplying the GCF absorbance values by 0.5 mL and dividing by the GCF volume. The findings in saliva and serum were expressed as concentrations.
2.4 | Statistical analysis
The required number of patients for each group was determined by power analysis (G*Power version 3.1.9.2, Heinrich Heine University). According to the calculation, minimum 20 subjects were necessary to determine the difference between groups at least 0.42 f-type effect size level, under one-way ANOVA method, with a power of 80% and at
α = .05 significance level.
All data were entered in Microsoft Excel (MS office version 2010) and transferred to the statistical software sheet (SPSS v.20.0, IBM). The normal distribution of the data was evaluated by Shapiro-Wilk's normality test. Comparisons of clinical and biochemical parameters between the study groups were performed using the Kruskal-Wallis with Mann-Whitney U test with Bonferroni correction method. The intragroup comparisons (at baseline and first month) were per-formed using Wilcoxon test for paired samples. Associations among levels of the GCF, saliva, and serum biomarkers and clinical parame-ters were also examined using the Spearman rank correlation anal-ysis. A P < .05 value was considered to be statistically significant.
3 | RESULTS
3.1 | Clinical findings
Table 1 summarizes the demographic characteristics and clinical per-iodontal parameters of the three groups. Subjects in the periodonti-tis group were significantly older than subjects in both gingiviperiodonti-tis and healthy groups (P = .000) whereas there was no statistical difference among gingivitis and healthy groups (P > .05). No significant differ-ence was observed in gender among the groups (P > .05), but peri-odontitis patients had less teeth than the others (P < .01).
3.2 | Laboratory findings
Netrin-1 and Unc5b levels in biofluids are shown in Table 2.
3.2.1 | GCF, saliva, and serum Netrin-1 levels
Total amount of Netrin-1 in GCF was significantly higher in the periodontitis group than the other groups and also gingivitis group had significantly higher GCF Netrin-1 levels than healthy group (P = .000). Nonsurgical periodontal treatment significantly reduced the total amount of Netrin-1 in both periodontitis (P = .000) and gingivitis group (P = .012), but the first month Netrin-1 levels did not differ between periodontitis and gingivitis groups (P > .05). The GCF concentration of Netrin-1 was significantly higher in the peri-odontitis group than that of healthy subjects (P = .000) and gingivitis patients (P = .023), but there was no statistically difference between gingivitis and healthy controls (P > .05). The concentration of GCF Netrin-1 was statistically reduced after periodontal treatment in
periodontitis group (P < .05), but the concentration at the first month was similar between periodontitis and gingivitis groups (P > .05).
Salivary and serum concentrations of Netrin-1 were significantly different among the study groups (P = .000). Nonsurgical periodon-tal treatment significantly increased the concentration levels of both salivary and serum Netrin-1 (P < .05), but the levels did not differ between periodontitis and gingivitis at the first month (P > .05).
3.2.2 | GCF, saliva, and serum Unc5b levels
Healthy subjects had significantly lower GCF Unc5b total levels than the other groups (P = .001), and the levels were similar between gingi-vitis and periodontitis subjects (P > .05). Nonsurgical periodontal treat-ment significantly reduced the total Unc5b levels (P < .01), but those levels did not differ between gingivitis and periodontitis (P > .05).
TA B L E 1 The demographic and clinical periodontal parameters of the study population
Periodontitis Gingivitis Healthy P
Age (years) 44.80 ± 8.36a,b 35.55 ± 9.21 32.80 ± 10.31 0.000A
Gender (Female/male) 12/8 13/7 15/5 0.592B
Number of teeth 23 (16-28)a,b 26 (17-28) 26 (20-28) 0.006C
Whole mouth periodontal parameters
PI T0 1.61 (0.80-2.44)a,b 1.29 (0.87-1.60) a 0.12 (0.02-0.51) 0.000C T1 0.20 (0.06-0.56)c 0.34 (0.05-1.24)c - 0.185D GI T0 1.78 (1.55-1.93) a 1.74 (1.39-2.03) a 0.33 (0.03-0.68) 0.000C T1 0.43 (0.13-0.72)c 0.42 (0.24-0.64)c - 0.379D PBI T0 2.09 ± 0.53 a 1.97 ± 0.68 a 0.18 ± 0.15 0.000A T1 0.32 ± 0.18c 0.37 ± 0.15c - 0.411E BOP (%) T0 80.73 ± 7.61a,b 69.37 ± 14.08 a 7.19 ± 2.84 0.000A T1 16.84 ± 6.79c 17.94 ± 5.55c - 0.578E PD (mm) T0 4.76 (2.37-6.88)a,b 2.04 (1.67-2.68) 1.95 (1.68-2.16) 0.000C T1 2.07 (1.52-3.84)c 1.99 (1.63-2.53)c - 0.213D CAL (mm) T0 4.89 (4.03-6.57)a,b 0.00 (0.00-0.00) 0.00 (0.00-0.00) 0.000C T1 3.45 (1.61-4.80)c 0.00 (0.00-0.00)c - 0.000D
Sampling site periodontal parameters
PI T0 1.75 (1.00-3.00)a 1.50 (1.00-3.00)a 0.00 (0.00-0.50) 0.000C T1 0.00 (0.00-1.50)c 0.00 (0.00-2.00)c - 0.731D GI T0 2.00 (1.00-2.50) a 2.00 (2.00-2.50)a 0.00 (0.00-0.00) 0.000C T1 0.00 (0.00-1.00)c 0.00 (0.00-1.00)c - 1.000D PBI T0 2.50 (1.50-4.00) a 3.00 (1.00-4.00)a 0.00 (0.00-0.00) 0.000C T1 0.00 (0.00-1.00)c 0.00 (0.00-0.00)c - 0.038D BOP (%) T0 100.00 (50.00-100.00) a 100.00 (100.00-100.00) a 0.00 (0.00-0.00) 0.000C T1 0.00 (0.00-50.00)c 0.00 (0.00-0.00)c - 0.075D PD (mm) T0 5.50 (4.50-6.50)a,b 3.00 (1.50-3.00) a 2.00 (1.50-2.50) 0.000C T1 2.50 (2.00-4.00)c 2.50 (1.00-3.00)c - 0.009D (Continues)
TA B L E 2 GCF, saliva, and serum levels of Netrin-1 and Unc5b of the study groups
Periodontitis Gingivitis Healthy P*
Netrin-1 GCF total amount (pg/30 s) T0 72.40 (6-248.40)a,b 13.95 (0.70-153.80)a 2.95 (2.60-57.90) .000 T1 4.05 (2.60-88.20)c 2.90 (0.40-57.70)c - .267 GCF concentration (pg/µL) T0 131.30 (9.50-213.20)a,b 20.20 (1.80-241.90) 17.20 (4.50-214.40) .001 T1 14.60 (4.00-173.80)c 15.60 (1.70-198.60) - .827 Saliva (pg/mL) T0 66.02 (11.10-196.14)a,b 96.48 (28.70-103.10)a 172.46 (25.93-407.41) .000 T1 110.82 (34.26-239.84)c 128.24 (40.74-258.77)c - .079 Serum (pg/mL) T0 1166.91 (652.11-1670.93)a,b 1474.78 (553.52-2612.41)a 1921.39 (595.78-3350.81) .000 T1 1725.59 (1229.97-2115.97)c 1812.05 (578.87-2323.82)c - .330 Unc5b GCF total amount (pg/30 s) T0 115 (10-607)a 116 (10-315)a 22 (1-400) .001 T1 22 (10-202)c 25 (10-112)c - .774 GCF concentration (pg/µl) T0 200 (10-1030)a 300 (10-821)a 10 (10-140) .018 T1 10 (10-80)c 10 (10-130) - .382 Saliva (pg/mL) T0 700 (100-1305)a 800 (100-4800)a 1900 (100-6800) .002 T1 701 (10-8600) 900 (210-2820) - .371 Serum (pg/mL) T0 1200 (200-1890)a 1800 (200-1970)a 7100 (900-9300) .012 T1 3300 (300-15700)c 6000 (600-14300)c - .256
Bold values indicate statistical significance.
Abbreviations: GCF, gingival crevicular fluid; T0, baseline; T1, first month.
aSignificant difference compared with the healthy group (P < .05).
bSignificant difference compared with the gingivitis group (P < .05).
cIntragroup comparison showed significant difference compared with T0 according to Wilcoxon signed-rank test (P < .05).
*Kruskal-Wallis, Mann-Whitney U test with Bonferroni correction.
Periodontitis Gingivitis Healthy P
CAL (mm) T0 6.00 (5.00-7.50)a,b 0.00 (0.00-0.00) 0.00 (0.00-0.00) 0.000C T1 4.50 (3.00-6.50)c 0.00 (0.00-0.00)c - 0.000D GCF volume (µL) T0 0.54 (0.31-0.68)a,b 0.38 (0.24-0.67) a 0.19 (0.06-0.60) 0.000C T1 0.27 (0.21-0.68)c 0.24 (0.08-0.77)c - 0.119D
Values (except for gender) are given as mean ± standard deviation or median (minimum-maximum).
(A) One-way ANOVA and Tukey tests, (B) Chi-squared test, (C) Kruskal-Wallis, Mann-Whitney U test with Bonferroni correction, (D) Mann-Whitney U test with Bonferroni correction, (E) Independent samples t test.
Abbreviations: PI, plaque index; GI, gingival index; PBI, papillary bleeding index; BOP, bleeding on probing; PD, probing depth; CAL, clinical attachment loss; GCF, gingival crevicular fluid; T0, baseline; T1, first month.
aSignificant difference compared with the healthy group (P < .05).
bSignificant difference compared with the gingivitis group (P < .05).
cIntragroup comparison showed significant difference compared with T0 according to paired samples t test (whole mouth PBI and BOP) or Wilcoxon
signed rank test (P < .05). TA B L E 1 (Continued)
The concentration of Unc5b in GCF was significantly lower in healthy than gingivitis and periodontitis groups (P = .018). On the other hand, salivary and serum concentrations of Unc5b were higher in the healthy group than the other groups (P = .002). There was no statistically difference between gingivitis and periodontitis groups in GCF, salivary, and serum levels of Unc5b (P > .05). The GCF levels of Unc5b were significantly reduced and conversely, serum concen-trations were significantly increased after nonsurgical periodontal treatment in both gingivitis and periodontitis groups (P < .05). But nonsurgical periodontal treatment did not alter the salivary concen-trations of Unc5b level (P > .05).
3.3 | Correlations
GCF Netrin-1 and Unc5b total amounts were positively correlated with all site-specific clinical parameters (P < .01 and P < .05) whereas there was no correlation between GCF Netrin-1 and Unc5b total amounts (P > .05) (Table 3).
Salivary/serum Netrin-1 and salivary Unc5b concentrations were negatively correlated with all full mouth clinical parameters (P < .01 and P < .05). On the other hand, salivary Netrin-1 was positively cor-related with salivary Unc5b (r = .532, P = .000) and serum Netrin-1 (r = .271, P = .036) concentrations (Table 4).
GCF Netrin-1 concentration was negatively correlated with salivary Unc5b (r = −.360, P = .007) and serum Netrin-1 concen-trations (r = −.394, P = .003). There were negative correlations be-tween serum Unc5b and full mouth PI, GI, PBI, and BOP (P < .01 and
P < .05).
4 | DISCUSSION
In this study, the levels of Netrin-1 and Unc5b in healthy subjects and those with gingivitis and periodontitis patients and also the alteration of those levels after nonsurgical periodontal treatment were compared. To the best of our knowledge, this study is the first that demonstrates the GCF, saliva, and serum levels of Netrin-1 and Unc5b in periodontal health and disease.
In the current study, the GCF levels of Netrin-1 and Unc5b were presented as both total amounts and concentrations. The GCF con-centrations of these cytokines were directly affected by the volume of GCF and as is known the healthy sites have low GCF volume than the periodontally disease sites.22 Therefore, in general, the GCF con-centrations of cytokines will increase in healthy sites and this will cause a misleading evaluation about the role of studied cytokines in periodontal disease. In the present study, although the GCF vol-umes are low in healthy sites, the results of concentrations and total amounts of Netrin-1 and Unc5b are in consistent with each other and the levels were significantly lower in healthy group than the other groups. This means that in the present study, the increase in the levels of studied biomarkers in GCF was more than the increase in the levels of GCF volume so that the results of both concentration and total amount supported with each other.
Studies in human and animal models have revealed that Netrin-1 can modulate monocyte/macrophage migration and takes part in limiting the transmigration of these cells into the tissues in inflam-matory conditions via its receptor Unc5b7,10,15,23 thus controlling the inflammation. This interaction is important to hamper exces-sive inflammatory reactions. Studies have revealed that mono-cytes/macrophages are the key components of the host response and the accumulation of those cells into the periodontal tissues cause the prolonged chronic inflammation.24 In addition to the reg-ulation of immune cell migration, Netrin-1 was shown to regulate the inflammatory response of macrophages through suppression of COX-2–mediated PGE2 production9 and also promote macro-phage reprogramming to induce inflammation resolution.8,25,26 Furthermore, Netrin-1 binding with its receptor Unc5b takes part in osteoclast biology14 and thus suggested as a therapeutic target for inflammatory bone destructive diseases.13,27 In this study, Netrin-1 and its receptor Unc5b were detected in all samples. To the best of our knowledge, there is no published paper in the literature, which investigates the relationship between those biomarkers and peri-odontitis. Therefore, we compared the results of the present study with the outcomes of the other inflammatory conditions which may have common pathogenesis with periodontal disease.
In the literature, it has been reported that Netrin-1 and its re-ceptor Unc5b levels were increased in the local inflammatory area. Ramkhelawon, et al15 studied the role of Netrin-1/Unc5b axis in TA B L E 3 Correlation coefficients for total amounts of Netrin-1 and Unc5b in GCF with site-specific clinical parameters
PI GI PBI BOP PD CAL
GCF Netrin-1 total amount (pg/30 s)
GCF Unc5b total amount (pg/30 s)
GCF Netrin-1 total amount (pg/30 s)
.480** .488** .536** .540** .623** .545** 1.000 .219 GCF Unc5b total amount
(pg/30 s)
.334** .416** .349** .459** .323* .308* .219 1.000 Bold values indicate statistical significance.
Abbreviations: PI, plaque index; GI, gingival index; PBI, papillary bleeding index; BOP, bleeding on probing; PD, probing depth; CAL, clinical attachment loss; GCF, gingival crevicular fluid.
*Correlation is significant at the .05 level (2-tailed) by Spearman rank correlation test. **Correlation is significant at the .01 level (2-tailed) by Spearman rank correlation test.
obesity. The researchers reported that Netrin-1 and its recep-tor Unc5b are highly expressed in obese adipose tissue of humans and mice. They showed that the high expression of those markers in obese adipose tissue was associated with high expression of the macrophage marker and the proinflammatory cytokine genes. They identified Netrin-1 as a macrophage retention signal in adipose tis-sue during obesity, which promotes chronic inflammation and insulin resistance. In an in vivo acute pulmonary inflammation model, the expression of Netrin-1 by vascular endothelium was down-regulated during inflammation concurrent with the migration of neutrophils to the tissue.7 In a study conducted by Lin, et al10 revealed that Netrin-1 blocked the attachment of monocytes to endothelial cells via suppres-sion of TNF-α-induced vascular adhesuppres-sion molecules and suppressuppres-sion of TNF-α-induced NF-κB activation and production of MCP-1, IL-1β, and IL-6. In another study, the role of Netrin-1 and its receptor was investigated in atherosclerosis and it has been shown that Netrin-1 was secreted in human and mouse atheroma and inhibited CCL2- and CCL19-directed macrophage migration acting via its receptor UNC5b.28 In parallel, Ramkhelawon, et al16 explained the mechanism of the macrophage retention and survival in atherosclerotic plaques by hypoxia and HIF-1α. Therefore, the researchers concluded that Netrin-1 promotes atherosclerosis by retaining macrophages in the artery wall. An acute experimental colitis model in mice showed en-hanced both protein and gene expression of Netrin-1 and exogenous Netrin-1 attenuated colonic inflammation. The underlying mecha-nism is down-regulation of neutrophil tissue infiltration rather than attenuating intestinal epithelial cell apoptosis.23 Maruyama, et al13 determined high levels of Netrin-1 in the synovial fluid of rheumatoid arthritis patients and Netrin-1 treatment attenuated bone erosion. In line with, blockade of Netrin-1/Unc5b prevented bone destruction and reduced the severity of arthritis.27 Rosenberger, et al11 reported that Netrin-1 was strongly induced by hypoxia and pretreatment with exogenous Netrin-1 abolished the hypoxia-dependent increases in plasma cytokine concentrations and PMN numbers into the colon, lungs, and kidneys, thus ameliorate mucosal inflammation.
In our study, the GCF levels of Netrin-1 and Unc5b increased in periodontal disease sites and also the levels were positively cor-related with the inflammatory clinical parameters. Furthermore, the local levels of those markers decreased after resolution of the in-flammation. Conversely, saliva concentrations, which also reflects local environment of the periodontal condition, decreased in line with the periodontal inflammation, and the levels were negatively correlated with the whole mouth clinical parameters. Furthermore, the concentrations were increased with the resolution of the in-flammation. The increased levels of those markers in GCF may be explained by inhibition of macrophage migration and thus retention of tissue macrophages so that promoting chronic inflammation. But why the saliva concentrations of Netrin-1 and Unc5b in periodontitis patients were lower than healthy subjects? We hypothesize that the periodontitis patients might be inactive rather than the quiescent state so if the source of those markers are GCF, the concentrations of saliva levels will increase with the gingival fluid flow. On the other hand, based on these results it can be concluded that the sources
T A B LE 4 C or re la tio ns o f s al iv ar y a nd s er um N et rin -1 a nd U nc 5b c on ce nt ra tio ns w ith f ul l-m ou th c lin ic al p er io do nt al p ar am et er s, a nd w ith e ac h o th er PI GI PB I BO P PD C A L G C F N etr in -1 (pg /µL ) G C F U nc 5b ( pg /µ L) Sal iv ar y N etr in -1 (p g/m L) Sa liv ar y U nc 5b (p g/m L) Se ru m N etr in -1 (p g/m L) Se ru m U nc 5b (p g/m L) Sa liv ar y N et rin -1 (p g/m L) −. 37 2* * −. 36 6* * −. 333 ** −. 40 6* * −. 39 6* * −.3 54 ** −. 02 1 −. 23 9 1. 000 .5 32 ** .2 71* .2 14 Sa liv ar y U nc 5b (p g/m L) −.4 73* * −. 39 2* * −. 39 5* * −. 43 8* * −. 40 7* * −. 32 9* −. 36 0* * −.1 00 .5 32 ** 1. 000 .17 8 .0 61 Se ru m N et rin -1 (p g/m L) −.4 83* * −. 39 5* * −. 39 1* * −. 40 0* * −. 31 5* −. 47 0* * −. 394* * −. 21 9 .2 71* .17 8 1. 000 .2 20 Se ru m U nc 5b (p g/m L) −. 26 9* −. 37 3* * −. 36 7* * −. 35 2* * −. 21 9 −. 24 4 −. 02 7 −. 13 5 .2 14 .0 61 .2 20 1. 000 PI , p la qu e i nd ex ; G I, g in gi va l i nd ex ; P B I, p ap ill ar y b le ed in g i nd ex ; B O P, b le ed in g o n p ro bi ng ; P D , p ro bi ng d ep th ; C A L, c lin ic al a tt ac hm en t l os s; G C F, g in gi va l c re vi cu la r f lu id . B ol d valu es in di ca te s ta tis tic al s ign ifi ca nc e. *C or re la tio n i s s ig ni fic an t a t t he . 05 l ev el ( 2-ta ile d) b y S pe ar m an r an k c or re la tio n t es t. ** C or re la tio n i s s ig ni fic an t a t t he . 01 l ev el ( 2-ta ile d) b y S pe ar m an r an k c or re la tio n t es t.
of Netrin-1 and Unc5b in saliva may be serum rather than the GCF or this may be a compensation mechanism of the host to keep the severity of inflammation and periodontal breakdown under control.
Circulating levels of Netrin-1 and Unc5b in chronic inflammatory diseases have been determined in several studies. In an acute kid-ney injury, the increased urinary Netrin-1 levels concurrent with the down-regulation of its plasma levels were reported.29 In renal isch-emia-reperfusion injury, serum Netrin-1 levels were decreased and administration of recombinant Netrin-1 reduced kidney injury and monocyte and neutrophil infiltration. Furthermore, neutralization of UNC5B receptor-induced monocyte and neutrophil infiltration thus increased kidney injury.12 In line with these results, obese patients15 and patients with multiple sclerosis30 showed significantly lower cir-culating Netrin-1 levels than those of controls. Liu, et al31 reported that plasma Netrin-1 levels were lower in newly diagnosed type 2 diabetes mellitus patients than those in healthy controls and also the levels were negatively correlated with blood glucose levels and insulin resistance. Conversely, increased serum Netrin-1 concentra-tions in patients with type 2 diabetes have been reported in recent studies.32,33 Our results demonstrated decreased serum Netrin-1 and Unc5b levels in periodontitis patients and also the levels were negatively correlated with the whole mouth inflammatory clinical pa-rameters. In addition, we determined that the serum concentrations increased after nonsurgical periodontal treatment in both gingivitis and periodontitis groups. Therefore, considering these findings, it can be suggested that the decreased serum levels of Netrin-1 and Unc5b in periodontal disease may be responsible for the egression of monocytes through vascular endothelium and Netrin-1/Unc5b inter-action may be essential to resolve the inflammation in periodontitis.
The analysis of Netrin-1 and its receptor in all biologic fluids re-lated to periodontal inflammation and also the alteration after peri-odontal treatment are strengths of our study to clarify the causal relationship between those biomarkers and periodontal status. An important limitation of the present study might be the lack of vari-ous inflammatory cytokines analysis to understand the mechanism of Netrin-1 and Unc5b in periodontal disease. Determining gene expression of Netrin-1/Unc5b in gingival tissue samples could be better to understand the early term alterations of those markers in periodontal inflammation. Moreover, split-mouth design would be preferred to overcome patient-related differences especially differ-ent inter-individual monocyte responses and cytokines expression, but parallel design is preferred to be able to evaluate the levels of saliva and serum. The last limitation of this study could be short-term reevaluation period, but only the alteration of those markers was aimed to analyze after inflammation resolution rather than analyzing the effect of periodontal treatment.
5 | CONCLUSIONS
The present study showed increased GCF total amounts and de-creased saliva and serum concentration of Netrin-1 and Unc5b in
periodontitis. In line with, the GCF levels were decreased and the saliva and serum levels were increased concurrent with the inflam-mation resolution. Furthermore, the GCF levels were positively and the saliva and serum levels were negatively correlated with clinical measurements. All these findings indicate that Netrin-1 and its re-ceptor Unc5b may have essential roles in periodontal inflammation and those can be assumed as useful therapeutic agent to control inflammation and periodontal breakdown. Future studies are neces-sary to clarify the pathway that is regulated by Netrin-1/Unc5b axis and elucidate its potential for diagnosis and treatment of periodon-tal disease. Development of Netrin-1/Unc5b based therapies will be able to treat the inflammatory periodontal condition.
ACKNOWLEDGEMENTS
This study was supported by Bolu Abant Izzet Baysal University, Coordination Unit of Scientific Research Projects (Project number 2018.06.05.1269).
CONFLIC TS OF INTEREST
The authors declare that they have no conflict of interest.
ORCID
Sadiye Gunpinar https://orcid.org/0000-0001-6100-322X
Bilge Meraci https://orcid.org/0000-0001-9848-0927
Niyazi Dundar https://orcid.org/0000-0002-6558-3775
REFERENCES
1. Giannobile WV. Host-response therapeutics for periodontal dis-eases. J Periodontol. 2008;79(8 Suppl):1592-1600.
2. Tracey KJ. The inflammatory reflex. Nature. 2002;420(6917):853-859. 3. Tessier-Lavigne M, Goodman CS. The molecular biology of axon
guidance. Science. 1996;274(5290):1123-1133.
4. Barallobre MJ, Pascual M, Del Rio JA, Soriano E. The Netrin family of guidance factors: emphasis on Netrin-1 signalling. Brain Res Brain Res Rev. 2005;49(1):22-47.
5. Lu X, Le Noble F, Yuan L, et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature. 2004;432(7014):179-186.
6. Larrivee B, Freitas C, Trombe M, et al. Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis. Genes Dev. 2007;21(19):2433-2447.
7. Ly NP, Komatsuzaki K, Fraser IP, et al. Netrin-1 inhibits leu-kocyte migration in vitro and in vivo. Proc Natl Acad Sci U S A. 2005;102(41):14729-14734.
8. Li Y, Wan S, Liu G, et al. Netrin-1 promotes inflammation resolution to achieve endothelialization of small-diameter tissue engineering blood vessels by improving endothelial progenitor cells function in situ. Adv Sci (Weinh). 2017;4(12):1700278.
9. Ranganathan PV, Jayakumar C, Mohamed R, Dong Z, Ramesh G. Netrin-1 regulates the inflammatory response of neutrophils and macrophages, and suppresses ischemic acute kidney in-jury by inhibiting COX-2-mediated PGE2 production. Kidney Int. 2013;83(6):1087-1098.
10. Lin Z, Jin J, Bai W, Li J, Shan X. Netrin-1 prevents the attachment of monocytes to endothelial cells via an anti-inflammatory effect. Mol Immunol. 2018;103:166-172.
11. Rosenberger P, Schwab JM, Mirakaj V, et al. Hypoxia-inducible fac-tor-dependent induction of Netrin-1 dampens inflammation caused by hypoxia. Nat Immunol. 2009;10(2):195-202.
12. Tadagavadi RK, Wang W, Ramesh G. Netrin-1 regulates Th1/Th2/ Th17 cytokine production and inflammation through UNC5B re-ceptor and protects kidney against ischemia-reperfusion injury. J Immunol. 2010;185(6):3750-3758.
13. Maruyama K, Kawasaki T, Hamaguchi M, et al. Bone-protective Functions of Netrin 1 Protein. J Biol Chem. 2016;291(46): 23854-23868.
14. Mediero A, Ramkhelawon B, Wilder T, et al. Netrin-1 is highly ex-pressed and required in inflammatory infiltrates in wear particle-in-duced osteolysis. Ann Rheum Dis. 2016;75(9):1706-1713.
15. Ramkhelawon B, Hennessy EJ, Menager M, et al. Netrin-1 promotes adipose tissue macrophage retention and insulin resistance in obe-sity. Nat Med. 2014;20(4):377-384.
16. Ramkhelawon B, Yang Y, van Gils JM, et al. Hypoxia induces Netrin-1 and Unc5b in atherosclerotic plaques: mechanism for macrophage retention and survival. Arterioscler Thromb Vasc Biol. 2013;33(6):1180-1188.
17. Caton JG, Armitage G, Berglundh T, et al. A new classification scheme for periodontal and peri-implant diseases and conditions – introduction and key changes from the 1999 classification. J Periodontol. 2018;89(Suppl 1):S1-S8.
18. Papapanou PN, Sanz M, Buduneli N, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Periodontol. 2018;89(Suppl 1):S173-S182.
19. Saxer UP, Muhlemann HR. Motivation and education. SSO Schweiz Monatsschr Zahnheilkd. 1975;85(9):905-919.
20. The LH. Gingival Index, the Plaque Index and the Retention Index. Systems. J Periodontol. 1967;38(6):610–616.
21. Silness J, Loe H. Periodontal Disease in Pregnancy. Ii. Correlation between Oral Hygiene and Periodontal Condtion. Acta Odontol Scand. 1964;22:121-135.
22. Curtis MA, Griffiths GS, Price SJ, Coulthurst SK, Johnson NW. The total protein concentration of gingival crevicular fluid. Variation with sampling time and gingival inflammation. J Clin Periodontol. 1988;15(10):628-632.
23. Aherne CM, Collins CB, Masterson JC, et al. Neuronal guidance molecule Netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis. Gut. 2012;61(5):695-705.
24. Yu T, Zhao L, Huang X, et al. Enhanced activity of the macrophage M1/M2 phenotypes and phenotypic switch to M1 in periodontal infection. J Periodontol. 2016;87(9):1092-1102.
25. Ranganathan PV, Jayakumar C, Ramesh G. Netrin-1-treated macro-phages protect the kidney against ischemia-reperfusion injury and suppress inflammation by inducing M2 polarization. Am J Physiol Renal Physiol. 2013;304(7):F948-957.
26. Mao XG, Xing H, Mao AH, et al. Netrin-1 attenuates cardiac isch-emia reperfusion injury and generates alternatively activated mac-rophages. Inflammation. 2014;37(2):573-580.
27. Mediero A, Wilder T, Ramkhelawon B, Moore KJ, Cronstein BN. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis. FASEB J. 2016;30(11):3835-3844. 28. van Gils JM, Derby MC, Fernandes LR, et al. The neuroimmune
guid-ance cue Netrin-1 promotes atherosclerosis by inhibiting the emigra-tion of macrophages from plaques. Nat Immunol. 2012;13(2):136-143. 29. Layne K, Ferro A, Passacquale G. Netrin-1 as a novel therapeutic target in cardiovascular disease: to activate or inhibit? Cardiovasc Res. 2015;107(4):410-419.
30. Mulero P, Cordova C, Hernandez M, et al. Netrin-1 and multiple sclerosis: a new biomarker for neuroinflammation? Eur J Neurol. 2017;24(9):1108-1115.
31. Liu CX, Ke XJ, Wang Y, et al. The level of Netrin-1 is decreased in newly diagnosed type 2 diabetes mellitus patients. BMC Endocr Disord. 2016;16:33.
32. Yim J, Kim G, Lee BW, et al. Relationship between circulating Netrin-1 concentration, impaired fasting glucose, and newly diag-nosed type 2 diabetes. Front Endocrinol. 2018;9:691.
33. Ay E, Marakoglu K, Kizmaz M, Unlu A. Evaluation of Netrin-1 levels and albuminuria in patients with diabetes. J Clin Lab Anal. 2016;30(6):972-977.
How to cite this article: Gunpinar S, Meraci B, Dundar N.
Netrin-1 and its receptor Unc5b as markers of periodontal disease. J Periodont Res. 2020;55:267–276. https ://doi. org/10.1111/jre.12712
