The Potential Role of Reactive Oxygen Species Produced by Low-Density Neutrophils in Periodontitis

Abstract Objective  Neutrophils own an arsenal of dischargeable chemicals that enable them to handle bacterial challenges, manipulating innate immune response and actual participation in acquired immunity. The reactive oxygen species (ROS) are one of the most important chemicals that neutrophils discharge to eradicate pathogens. Despite their beneficial role, the ROS were strongly correlated to periodontal tissue destruction. Lowdensity neutrophils (LDN) have been recognized for producing enhanced quantities of ROS. However, the potential role of ROS produced by LDN in periodontitis is unknown. The aim of the study was to investigate the impact of ROS produced by LDN in periodontal diseases. Materials and Methods  Venous blood and periodontal parameters were obtained from 100 systemically healthy subjects divided into 40 participants with healthy periodontium in the control group and 60 with unstable periodontitis in the study group. Flow cytometry was used to measure the production of ROS by LDN in both groups. Statistical Analysis  The data were analyzed for normal distribution using the Shapiro-Wilk test at p  < 0.05, Spearman's correlations, and Mann-Whitney U test. Statistical analysis was performed in SPSS v25. Results  No difference between the groups had been obtained in ROS production by LDN. However, a significant positive correlation existed between ROS and clinical attachment loss in periodontitis. Conclusion  LDN exhibits the same ROS generation capacity in the control and periodontitis groups.


Introduction
Periodontitis is a multifactorial progressive condition characterized by continuous destruction of periodontal tissue under the burden of a dysbiotic microflora, host immune response, environmental factors, and subject genetic suscep-tibly. 1,24][5][6][7] Despite their beneficial role in eradicating pathogens, inflammation is a double-edged sword; it is considered a crucial source of periodontal tissue destruction, involving leukocytes, complement, and reactive oxygen species (ROS). 8ROS seem to be short-lived, extremely reactive oxygen-reduced radicals, including superoxide , hydrogen peroxide (H 2 O 2 ), the hydroxyl radical (OH), and singlet oxygen. 9At the cellular level, ROS is crucial for eukaryotic cell physiologic functions such as signaling transmission, differentiation, apoptotic cell death, 8,9 and pathogen oxidative death. 10 clinical study revealed that serum reactive oxygen metabolites were positively correlated with immunoglobulin G antibodies to keystone periodontal pathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia.11,12 Furthermore, the level of ROS markedly regulates many biological events of most cellular components in the periodontium.For instance, a reduced ROS level decreases bone loss by downregulating the osteoclast differentiation marker genes; conversely, it enhances the proliferation and differentiation of human periodontal ligament fibroblasts (hPDLFs) and osteoblasts.13 Low-density neutrophils (LDN) are a unique attentionseeking neutrophil phenotype strongly associated with various immunological diseases, endocrine disorders, systemic diseases, and infections. 10 Bad on previous studies on healthy subjects, the LDN exhibits enhanced ROS-generating capacity compared to the normal-density neutrophil.10 To the best of our knowledge, the potential impact of ROS produced by LDN in the periodontitis has not been studied yet.However, this study tries to illustrate this role in the pathogenesis of periodontitis using a multicolor flow cytometry.

Study Design
This observational research was conducted from August 2022 to March 2023 at the College of Dentistry, University of Baghdad.

Ethical Approval
All procedures used in this study adhered to the guidelines outlined in the Helsinki Declaration of 1964 and its subsequent revisions, specifically concerning research involving human subjects.The protocol for this study has been authorized by the Ethics Committee of the College of Dentistry at the University of Baghdad.The reference number for this approval is 450, the project number is 450622, and the approval date is January 19, 2021.Following the provision of comprehensive information elucidating the nature and objectives of the research, every participant was requested to affix their signature on an informed consent document.Following the participants' agreement, a clinical examination was conducted, and then blood samples were collected from each individual.

Inclusion Criteria
One hundred systemically healthy participants were allocated into two groups: • The control group consisted of 40 subjects with healthy periodontium defined as bleeding on probing (BOP) less than 10% and periodontal pocket depth (PPD) 3 mm.Additionally, there was no evidence of attachment loss during periodontal probing. 14• The periodontitis group consisted of 60 subjects with unstable periodontitis, defined as BOP greater than 10%, with clinical attachment loss (CAL) at the interproximal area at !2 nonadjacent teeth or two teeth with buccal or oral CAL of 3 mm with pocketing greater than 3 mm. 15

Exclusion Criteria
Patients with dental implants, systemic or oral autoimmune illness, infections, inflammatory diseases, drug intake (antibiotics, nonsteroidal anti-inflammatory drugs) within 3 months, endocrine disorders, systemic problems, pregnancy, and smoking habits were excluded.The wisdom teeth were excluded from periodontal examinations.

Subjects Eligibility
Patients with periodontitis referred for periodontal therapy were initially screened (n ¼ 113) to evaluate their eligibility for recruitment.After applying the inclusion/exclusion criteria, 53 patients were excluded for different reasons, and 60 patients were included in the final analysis.Later, the patients with healthy periodontium were included (n ¼ 40) as controls (►Fig. 1).

Blood Collection
After clinical examination and subject allocation, 5 mL of intravenous blood was collected and placed in a 10-mm ethylenediaminetetraacetic acid (EDTA) tube.The collected blood was preserved at 4°C protected from light for 30 minutes.LDN purification must done within a period not exceeding 6 hours to purify pure, healthy, and activated LDN. 10

Purification of LDN
The LDN were purified by mixing 5 mL of anticoagulated blood with 2 mL of 6% dextran T500 by inversion and allowing it to rest for 45 minutes to precipitate red blood cells.Next, the layer of leukocyte-rich plasma was placed carefully on top of 5-mL Lympho-Paque at 1.077 g/mL density.The sample was centrifuged at 520 g for 20 minutes at 4°C using a Thermo Scientific Megafuge 8R Small Benchtop Centrifuge.A layer of mononuclear cells (MNC), including monocytes, lymphocytes, and LDNs, is located at the plasma-lympho-paque interface (►Fig. 2).LDNcontaining MNC was collected, diluted 1:2 with phosphate buffer saline (PBS), and stored in ice until use.
European Journal of Dentistry © 2024.The Author(s).
Reactive Oxygen Species in Periodontitis Mousa et al.

Descriptive Data
In the control group, the descriptive data show the minimum, maximum, mean, and standard deviation of different variables.
The descriptive data in the periodontitis group showed the minimum, maximum, mean, and standard deviation of different variables (as shown in ►Table 1).
Intergroup comparisons showed a significantly higher BOP and PI in the periodontitis group than in the control group (p < 0.01) (as shown in ►Table 2).
However, no significant difference was observed in ROS production between the two groups (as shown in ►Table 3).

Correlations in the Control Group
The observed data shows a significant positive correlation between BOP% and PI in the control group.However, no  Reactive Oxygen Species in Periodontitis Mousa et al.
correlation was observed between ROS and the other variables in the control group (►Table 4).

Correlations in the Periodontitis Group.
The collected data show a significant positive correlation among all periodontal parameters; however, ROS was significantly and positively correlated with CAL in the periodontitis group (p ¼ 0.275; 2-tailed ¼ 0.034) (►Table 5).

Discussion
Neutrophils are the predominant leukocytes in circulation and are considered the main cellular arm of innate immunity, which responds to inflammation and infection. 17In inflamed tissues, neutrophils perform several antimicrobial tasks, 18 such as degranulation, 19 ROS generation, 20 phagocytosis, and neutrophil extracellular trap (NET) formation. 21In addition to innate immunological activities, neutrophils modulate adaptive immune responses. 22In fact, neutrophils do not appear to be a homogeneous population that often behaves similarly; however, multiple subpopulations of neutrophils have been proposed in diverse health and disease situations. 23DN is a unique neutrophil phenotype that exists in large numbers in various pathological disorders like inflammations, infections, cancer, and immunosuppression. 24Based on a previous study, LDN has also been detected in the peripheral blood of systemically healthy subjects and exhibits an enhanced capacity for ROS compared to the normal high-density neutrophil (HDN). 10nterestingly, the outcomes of our study revealed no significant difference in ROS produced via LDN between  Reactive Oxygen Species in Periodontitis Mousa et al.Reactive Oxygen Species in Periodontitis Mousa et al.
the study groups, which may indicate that LDN is an excellent ROS producer regardless of the periodontal status.Despite this, LDN exhibits more ROS production capacity in the periodontitis group at 89% compared to the healthy control group at 85%.However, this may be due to the systemic dissemination of periodontal pathogens and their products via the ulcerated pocket epithelium. 25,26dditionally, a significant positive correlation was observed between ROS and CAL in the periodontitis group.Based on this outcome in healthy subjects, ROS is beneficial in pathogen eradication and differentiation of cells. 11On the other hand, in periodontitis patients, increasing ROS coincided with increasing destruction of the periodontal tissue (CAL).In conclusion, enhanced ROS benefits the healthy periodontium; in contrast, it is harmful in periodontitis.
However, the schizophrenic behavior of ROS in the current study could be related to the homeostatic imbalance between ROS and antioxidant defense systems, 27,28 as the periodontitis patients showed lower plasma and serum total antioxidant concentrations (TAOC) levels than healthy controls. 28,29he TAOC is the main body defense system for neutralizing ROS.Furthermore, decreased TAOC and elevated ROS might be risk factors for periodontitis or might be induced by periodontal inflammation. 30However, to estimate the precise effect of TAOC, the analysis of site-specific LDNs purified from saliva, gingival crevicular fluid (GCF), and the junctional epithelium is mandatory.Indeed, in the murine model, the LDN can be characterized by the expression of CD11b and Ly6G þ markers. 313][34] Consequently, the density gradient centrifugation of peripheral blood is the sole method for the purification of LDN.The above-mentioned considerations present a major challenge to accurately evaluate the actual impact of TAOC in neutralizing LDN ROS.

Conclusion
Irrespective of the periodontal status, LDN demonstrates an increased capability for generating ROS.The enhanced ROS benefits the healthy periodontium; conversely, it is harmful in periodontitis.
ROS production was measured by detecting fluoresce changes in LDNs loaded with dihydrorhodamine 123 (DHR-123).Cells (1 Â 10 6 ) were suspended in 100 mL of 15 mM DHR-123 in PBS and incubated for 15 minutes at 37°C in the dark.Cells were washed with 1 mL PBS, resus-pended in 100 mL of PBS containing 20 nM of Probol-12myristate-13-acetate (PMA), and incubated at 37°C in the dark for 50 minutes.Next, cells were washed in cold PBS and resuspended in 1% paraformaldehyde in PBS.Finally, cells were kept in a cold and dark place until analyzed by flow cytometry.The cells were gated by a dot-plot analysis, and 10,000 cells were acquired per sample.The cells were analyzed in BD FACSCanto II multicolor flow cytometry with the BD FACSDiva Software v9.0 (►Fig.3).

Table 1
Descriptive data in the control group Abbreviations: BOP, bleeding on probing; PI, plaque index; ROS, reactive oxygen species.European Journal of Dentistry © 2024.The Author(s).

Table 2
Descriptive data in the periodontitis group

Table 4
The correlations in the control group a Refers to the significant correlation coefficient.Ã significant at P-value <0.05.

Table 5
The correlations in the periodontitis group European Journal of Dentistry © 2024.The Author(s).
Abbreviationsa Refers to the significant correlation coefficient.b Refers to the Sig.(2-tailed).