Neutrophil N1 and N2 Subsets and Their Possible Association with Periodontitis: A Scoping Review

Periodontitis is a chronic non-communicable disease caused by dysbiotic changes that affect the subgingival microbiota. During periodontitis, neutrophils play a central role in the initial recognition of bacteria, and their number increases with the appearance of the first signs of periodontal inflammation. Recent evidence has led to the proposition that neutrophils can also functionally polarize, determining selective activity patterns related to different diseases. Two well-defined neutrophil phenotypes have been described, the pro-inflammatory N1 subset and the suppressor N2 subset. To date, it has not been established whether these different neutrophil subtypes play a role in the pathogenesis of periodontitis. Thus, this scoping review aimed to determine whether there was evidence to suggest that the neutrophils present in periodontal tissues can be associated with certain phenotypes. The research question, population, concept, and context sought to identify original articles, in humans, that detected the presence of neutrophils in the periodontal tissues of people affected by periodontitis. Based on the search strategy, we found 3658 studies. After removing the papers with abstracts not related to the outcome measures and eligibility criteria, 16 articles were included for qualitative analysis. Several studies identified the presence of different neutrophil subsets, specifically, the naive, pro- and para-inflammatory, hyper-reactive and hyper-active, and high- and low-responder phenotypes. The existing evidence demonstrates the presence of pro-inflammatory, hyper-reactive and high-responder neutrophils in periodontal tissues affected with periodontitis. There is no evidence demonstrating the presence of the N1 or N2 phenotypes in periodontal tissues during periodontitis. However, the existence of pro-inflammatory phenotypes, which increase NETosis and degranulation, and increase the production of pro-inflammatory cytokines, could be suggestive of the N1 phenotypes.


Introduction
Neutrophils are polymorphonuclear cells derived from bone marrow granulocyte precursors [1]. Neutrophils have a relatively short lifespan in the peripheral blood circulation, approximately 4 to 5 h. However, a longer lifespan is evidenced when they infiltrate the connective tissues, where they recognize microbiota-or pathogen-associated molecular patterns (MAMPs or PAMPs). Neutrophils are between 60 to 70% of the total polymorphonuclear cells [2]. Neutrophils are the first cell population to be recruited at inflamed sites, and can migrate by chemotaxis from the peripheral blood circulation to the tissues. In the connective tissues, they fulfill their main role: phagocytosis [3,4]. In recent years, in tumor-related pathologies, two subtypes of neutrophils have been defined, with phenotypic and functional differences: neutrophil type 1, or anti-tumorigenic N1, and N2, or pro-tumorigenic neutrophils [5,6]. Additionally, these same phenotypes have been Figure 1. PRISMA flow diagram. Summary of the article search. From a universe of 3658 articles 147 passed the first review of abstract and title. Subsequently, systematic reviews with or withou meta-analysis and narrative reviews were eliminated until 101 articles remained, which were re viewed in full-text format. Finally, 14 articles were selected to prepare this systematic review. (Cre ated with http://biorender.com, accessed on March 2020). and lowresponder neutrophils in Figure 1. PRISMA flow diagram. Summary of the article search. From a universe of 3658 articles, 147 passed the first review of abstract and title. Subsequently, systematic reviews with or without meta-analysis and narrative reviews were eliminated until 101 articles remained, which were reviewed in full-text format. Finally, 14 articles were selected to prepare this systematic review. (Created with http://biorender.com, accessed on March 2020).  [18] To identify the presence of oral neutrophil hyperactivity among refractory periodontitis patients, and to determine if the hyperactivity is related to a history of periodontal disease severity. Characterization of high-responder neutrophils in patients with periodontitis, and low-responder neutrophils in healthy subjects.
Refractory Disease Unit, Dental Research Institute, University of Toronto, Canada. 2 Borenstein et al., 2018 [21] To determine the morphological diversity between different groups of patients, as well as the response induced in naïve neutrophils after incubation with bacteria. Oral neutrophils had lower number of granules per cytoplasm area compared to blood neutrophils. Bacteria-stimulated oral neutrophils were more granular, had more phagosomes than the blood neutrophils. Neutrophils that migrated into the connective tissue have a lighter cytoplasmic density, fewer granules and higher euchromatin fraction. Gingival tissue neutrophils had increased euchromatin/heterochromatin ratio.

Toronto General Hospital 2 Nephrology Center and
University of Toronto's Graduate Periodontology Clinic, Toronto, Canada. 3 Dutzan et al., 2016 [20] To characterize the human immunological cell network patrolling the oral barrier in health with a particular focus on the gingival area. To demonstrate the presence of para-inflammatory neutrophils in the healthy oral cavity and pro-inflammatory neutrophils in patients with periodontitis. Oral neutrophils from patients with periodontitis are in a pro-inflammatory activation state when compared with healthy oral neutrophils. Neutrophils from patients affected with periodontitis were characterized by elevated degranulation, phagocytosis, ROS production, and NETosis.
Toronto General Hospital's Nephrology Center and the University of Toronto's Graduate Periodontology Clinic 5 Johnstone et al., 2007 [24] To compare the generation of oxygen radicals in peripheral neutrophils from patients with aggressive, chronic, and periodontally healthy after stimulation with phorbol myristate acetate (PMA). Additionally, to examine the phagocytotic ability of the neutrophils.  [19] To present a rapid method for oral neutrophil isolation and to characterize and compare the neutrophil gene expression profile in the blood and oral compartment of healthy individuals.  [16] To confirm the reported FcgR hyper-reactivity of peripheral neutrophils in chronic periodontitis using more relevant physiological conditions, and to determine whether the ROS could be detected. To assess the longitudinal variation in the expression of the adhesion and activation markers of neutrophils, to determine the neutrophil maturation stage based on CD surface markers, and to evaluate the suppressive neutrophil phenotypes. Blood sample PD and BOP correlated with neutrophil subsets. In particular, neutrophils over-expressed CD11b, CD16b, and CD66b, and under-expressed CD62L.
The Royal Dental Hospital of Melbourne and Melbourne Dental Clinic, The University of Melbourne. 9 Papantonopoulos et al., 2019 [22] To investigate in datasets of immunologic parameters from early onset and late-onset periodontitis patients (EOP and LOP), the existence of hidden random fluctuations (anomalies or noise), which may be the source for increased frequencies and longer periods of exacerbation, resulting in rapid progression in EOP. In early-onset periodontitis, there was an increase in IL-1, IL-4, and IFN-γ compared with late-onset periodontitis. CD8, CD20, CD4/CD8 ratio and IL-2 were higher in late-onset periodontitis. Additionally, chemotaxis, phagocytosis, and adhesion of neutrophils were higher in late-onset periodontitis, than early-onset.

Okayama University
Dental Hospital 10 Rudin et al., 2020 [29] To determine whether the CD177+ and CD177− neutrophil subsets differ in their propensity to migrate to both aseptic-and microbe-triggered inflamed human tissues.
Not specified Buffy coats CD177+ neutrophil subtype was recruited to inflammatory exudate in periodontitis. Increased levels of CD177+ neutrophils in blood of periodontitis patients were detected, as compared to healthy controls. To test the hypothesis that PDK1 regulates chemotaxis in neutrophils and is responsible for the abnormal neutrophil chemotaxis in LAP.

Author/Year
To determine the population of neutrophils in the periodontal tissues of patients with or without periodontal disease To isolate neutrophils from peripheral blood samples and evaluate phenotypes.
To determine the neutrophils phenotype or cytokine analysis in patients with inflammatory diseases 1 Aboodi et al., 2011 [18] neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Phenotypic Characterization of Infiltrating Neutrophils in Perio
During periodontitis, there is an increase in neutrophils in the compared to the absence of periodontitis [20,26]. Neutrophils are cha CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64 CD177+, and elastase+ [20,23,26]. In addition, they exhibit inc phagocytosis, degranulation, production of cytokines, MPO and RO 29]. In fact, in periodontitis-affected tissues, NET degradation is lo subjects [26]. Additionally, 2.4% of oral neutrophils produce the rece nuclear factor-κB ligand (RANKL) [30]. Additionally, in patie periodontitis, neutrophil ROS production is correlated with increase (PI), bleeding on probing (BOP), pocket deep (PD), and clinical atta 2 Borenstein et al., 2018 [21] The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Phenotypic Characterization of Infil
During periodontitis, there is an inc compared to the absence of periodontitis [ CD11b+, CD15+, CD16+, CD18+, CD55 CD177+, and elastase+ [20,23,26]. In a phagocytosis, degranulation, production 29]. In fact, in periodontitis-affected tissu subjects [26]. Additionally, 2.4% of oral n nuclear factor-κB ligand (RANKL) [30 periodontitis, neutrophil ROS production (PI), bleeding on probing (BOP), pocket 4 Fine et al., 2016 [27] PPD: probing of pocket depth, CAL: clinical attachment loss, BOP: bleeding on probing, OP: oral 97 plaque, PI: plaque index, GI: Gingival index, CD: cluster of differentiation, NET: neutrophil 98 extracellular trap. 99 The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research.

105
In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory 117 periodontitis, neutrophil ROS production is correlated with increased periodontal index The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

105
In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory 117 periodontitis, neutrophil ROS production is correlated with increased periodontal index The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.
The selected articles correspond to st neutrophils in periodontal tissues based o findings described. In addition, Table 2 sh the results that answer our research quest

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.
The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.
The selected articles correspond to st neutrophils in periodontal tissues based o findings described. In addition, Table 2 sh the results that answer our research quest

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Phenotypic Characterization of Infil
During periodontitis, there is an inc compared to the absence of periodontitis [ CD11b+, CD15+, CD16+, CD18+, CD55 CD177+, and elastase+ [20,23,26]. In a phagocytosis, degranulation, production 29]. In fact, in periodontitis-affected tissu subjects [26]. Additionally, 2.4% of oral n nuclear factor-κB ligand (RANKL) [30 periodontitis, neutrophil ROS production (PI), bleeding on probing (BOP), pocket 7 Matthews PPD: probing of pocket depth, CAL: clinical attachment loss, BOP: bleeding on probing, OP: oral 97 plaque, PI: plaque index, GI: Gingival index, CD: cluster of differentiation, NET: neutrophil 98 extracellular trap. 99 The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103 In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Phenotypic Characterization of Infiltrating Neutrophils in Perio
During periodontitis, there is an increase in neutrophils in the compared to the absence of periodontitis [20,26]. Neutrophils are cha CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64 CD177+, and elastase+ [20,23,26]. In addition, they exhibit inc phagocytosis, degranulation, production of cytokines, MPO and RO 29]. In fact, in periodontitis-affected tissues, NET degradation is lo subjects [26]. Additionally, 2.4% of oral neutrophils produce the rece nuclear factor-κB ligand (RANKL) [30]. Additionally, in patie periodontitis, neutrophil ROS production is correlated with increase (PI), bleeding on probing (BOP), pocket deep (PD), and clinical atta 8 Medara The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103 In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Phenotypic Characterization of Infil
During periodontitis, there is an inc compared to the absence of periodontitis [ CD11b+, CD15+, CD16+, CD18+, CD55 CD177+, and elastase+ [20,23,26]. In a phagocytosis, degranulation, production 29]. In fact, in periodontitis-affected tissu subjects [26]. Additionally, 2.4% of oral n nuclear factor-κB ligand (RANKL) [30 periodontitis, neutrophil ROS production (PI), bleeding on probing (BOP), pocket 10 Rudin et al., 2020 [29]  The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103

105
In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Phenotypic Characterization of Infiltrating Neutrophils in Perio
During periodontitis, there is an increase in neutrophils in the compared to the absence of periodontitis [20,26]. Neutrophils are cha CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64 CD177+, and elastase+ [20,23,26]. In addition, they exhibit inc phagocytosis, degranulation, production of cytokines, MPO and RO 29]. In fact, in periodontitis-affected tissues, NET degradation is lo subjects [26]. Additionally, 2.4% of oral neutrophils produce the rece nuclear factor-κB ligand (RANKL) [30]. Additionally, in patie periodontitis, neutrophil ROS production is correlated with increase (PI), bleeding on probing (BOP), pocket deep (PD), and clinical atta The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103

105
In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory The selected articles correspond to studies in humans characterizing the presence of 100 neutrophils in periodontal tissues based on their functions. Table 1 summarizes the main 101 findings described. In addition, Table 2 shows the main findings for each study, indicating 102 the results that answer our research question and gaps in the research. 103

105
In Table 2, the relevance of each selected source to the objectives of this scoping 106 review is identified. 107  During periodontitis, there is an increase in neutrophils in the periodontal tissues 110 compared to the absence of periodontitis [20,26]. Neutrophils are characterized by CD10+, 111 CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64+, CD66+, CD138+, 112 CD177+, and elastase+ [20,23,26]. In addition, they exhibit increased chemotaxis, 113 phagocytosis, degranulation, production of cytokines, MPO and ROS, and NETosis [27-114 29]. In fact, in periodontitis-affected tissues, NET degradation is lower than in healthy 115 subjects [26]. Additionally, 2.4% of oral neutrophils produce the receptor activator of the 116 nuclear factor-κB ligand (RANKL) [30]. Additionally, in patients with refractory The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Phenotypic Characterization of Infiltrating Neutrophils in Perio
During periodontitis, there is an increase in neutrophils in the compared to the absence of periodontitis [20,26]. Neutrophils are cha CD11b+, CD15+, CD16+, CD18+, CD55+, CD62L+, CD63+, CD64 CD177+, and elastase+ [20,23,26]. In addition, they exhibit inc phagocytosis, degranulation, production of cytokines, MPO and RO 29]. In fact, in periodontitis-affected tissues, NET degradation is lo subjects [26]. Additionally, 2.4% of oral neutrophils produce the rece nuclear factor-κB ligand (RANKL) [30]. Additionally, in patie periodontitis, neutrophil ROS production is correlated with increase (PI), bleeding on probing (BOP), pocket deep (PD), and clinical atta 13 Wright The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Phenotypic Characterization of Infil
During periodontitis, there is an inc compared to the absence of periodontitis [ CD11b+, CD15+, CD16+, CD18+, CD55 CD177+, and elastase+ [20,23,26]. In a phagocytosis, degranulation, production 29]. In fact, in periodontitis-affected tissu subjects [26]. Additionally, 2.4% of oral n nuclear factor-κB ligand (RANKL) [30 periodontitis, neutrophil ROS production (PI), bleeding on probing (BOP), pocket 14 Yagi The selected articles correspond to studies in humans character neutrophils in periodontal tissues based on their functions. Table 1 s findings described. In addition, Table 2 shows the main findings for e the results that answer our research question and gaps in the researc

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the object review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each se review is identified.

Inclusion of Sources of Evidence
In Table 2, the relevance of each selected source to the objectives of this scoping review is identified.
When analyzing the neutrophil phenotypes described, there is no clear consensus. Some studies mention hyper-reactive or hyperactive phenotypes [16], naïve, pro-inflammatory, and para-inflammatory subsets [21,27]. These phenotypes can be described based on their capability to produce ROS [16,18], their euchromatin/heterochromatin ratio, and their higher or lesser phagocytic activity [21,27]. Interestingly, when patients are treated and a follow-up of 3, 6, or 12 months is carried out, it is observed that the CD16b + CD66 + CD11b + CD62L − population decreases, and has a positive correlation with the BOP index [23]. Curiously, CD177 + cells predominate in gingival crevicular fluid (GFC) during periodontitis, while the CD177population predominates under healthy conditions. Curiously, CD177 + cells have a higher apoptotic activity than CD177 − cells [29].

Para-and Pro-Inflammatory Neutrophils in Periodontitis
Periodontal tissues can be affected by different causes that induce inflammation. Periodontitis has been recognized as the main chronic inflammatory disease, but it is not the only one. There may be a form of periodontitis resulting from trauma, or there may be an adaptation phenomenon [32]. Thus, when periodontitis occurs due to dysbiosis of the microbiota, we speak of pro-inflammatory phenomena, and when periodontitis is due to an adaptive process, we speak of para-inflammatory phenomena [33,34]. This is noteworthy to mention, because the cause of the inflammation defines the type of inflammatory response, and the pro-and para-inflammatory phenotypes have been described in periodontal tissues [21]. In this context, a para-inflammatory and pro-inflammatory phenotype characterized by low granulation, light cytoplasm, and a large amount of euchromatin was detected [21]. Additionally, in healthy periodontal tissues, two different populations of oral neutrophils have been observed-para-inflammatory 1 and para-inflammatory 2-and both of them have a similar profile to naïve blood neutrophils and a lower state of activation [21].

Degranulation and NETosis
Considering degranulation and NETosis as an important part of neutrophil function, it is curious that only one study has evaluated NETosis [16]. The N1 phenotypes produce NET to induce the pro-inflammatory response, and aggNET enhances the N1 function. During periodontitis, the infiltration of neutrophils with high capability of triggering NETosis subsets is essential for determining the greater or lesser recruitment of phagocytes, which influences the increase or decrease in inflammatory signs. Additionally, it is not unusual to find a positive correlation between the presence of NET-producer neutrophils with the PI, and BOP index, or even CAL loss.

Review Question
Is there evidence characterizing the existence or the possible role of the N1 and N2 neutrophil phenotypes in periodontal tissues affected by periodontitis?

Participants, Concept, and Context
We include studies conducted in humans, in which the population is clearly identified. In particular, studies were selected where samples of adults between 20 and 80 years of age were used, regardless of the distribution by sex, socioeconomic level or ethnicity (participants). Additionally, the studies described both the periodontal diagnosis and the clinical criteria used to diagnose (concept). We selected the studies that analyzed or characterized the presence of neutrophils in the periodontal tissues of healthy people or those affected by periodontitis (concept), and the origin of each participant (context).

Source of Evidence
The present scoping review was carried out following the indications of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The protocol related to the data search, selection, extraction, and analysis was discussed and established by all of the authors in a dynamic drive and without further modifications. There were no language, year, or publication status restrictions for inclusion, and when eligible studies in languages other than English, Spanish, French or Portuguese were detected, a qualified translator was consulted. The selected articles were included in a drive for full text analysis, and the articles not selected were included in a table indicating the reasons for their non-inclusion or exclusion.
We included primary research studies, randomized or non-randomized clinical trials, and case-control and cohort studies. We excluded systematic reviews with or without meta-analysis from the analysis, as they are not a primary source of information. However, we left open the option to include any source of evidence directly associated with the research question and that met the eligibility criteria.

Search Strategy
An electronic search was performed in the Latindex, SciELO, Cochrane Library (CEN-TRAL) and Medline via Pubmed databases, from 30 January 2021 to 30 June 2022. Two reviewers (DS-E and JD-Z) performed the electronic database survey independently and in duplicate. The search strategy was performed using Mesh terms "neutrophil", "bacteria", "host-pathogens interactions", "periodontitis", "subsets", and "phenotypes" in the Medline database and adapted for the other databases. The complete search strategy is provided in Supplementary file S1.

Source of Evidence Screening and Selection
Evidence screening and selection were performed independently and in duplicate, by assessing titles and abstracts to determine their inclusion. Furthermore, the selected full-texts were analyzed to determine whether they met the inclusion criteria. In case of disagreement, the article selection was discussed until a consensus was reached on whether the article could be included or excluded. If excluded, the motifs were recorded in the PRISMA flow chart (Figure 1). In cases where consensus was not reached, a third reviewer made the decision (SM-R).

Data Extraction
For each paper included, the following information was extracted: first author and year of publication, study objective, population (number, sex, age), concept (periodontal diagnosis criteria, interventions or phenomena of interest, samples, analysis methods, and results), and context (group definition and origin) ( Table 1).

Outcome Measures
To assess the characterization of the neutrophils in the gingival tissues, the primary outcome was to determine the population of neutrophils in the periodontal tissues of patients with or without periodontal disease. As a secondary outcome, articles that isolated neutrophils from peripheral blood samples and evaluated phenotypes were selected. As a tertiary outcome, studies that included a complete phenotype or cytokine analysis in patients with inflammatory diseases were chosen.

Discussion
Periodontitis is one of the most prevalent pathologies in the oral cavity, which affects the supporting tissues of the teeth: periodontal ligament, root cement and alveolar bone; and its characteristic is the progressive destruction of these tissues with the possibility of a subsequent tooth loss [35,36]. Additionally, it is considered a non-communicable chronic disease, characterized by a high concentration of pro-inflammatory mediators in the affected periodontal tissue, which can spread to the peripheral circulation and induce a chronic low-grade inflammatory phenotype (CLIP) response [36]. The main cause of periodontitis is the dysbiosis of the subgingival microbiota, which begins with an alteration in the amount of bacteria [34,37].
Generally, periodontitis begins with a pro-inflammatory phenomenon, characterized by the presence of bacteria in the gingiva-dental sulcus and their recognition by Langerhans cells pattern recognition receptors (PRR) within the epithelium [38][39][40]. The Langerhans cells secrete IL-8, which will spread into the connective tissue creating a concentration gradient [38][39][40]. Furthermore, neutrophils can recognize the chemoattractant signal through their CD177 and migrate to the thickness of the epithelium [29,39,40]. Once neutrophils reach the site of inflammation and are activated, they facilitate microbial removal through several different processes, including phagocytosis, generation of myeloperoxidase (MPO) and ROS, release of granular content, cytokine production, and NETosis [24,[41][42][43].
Other characteristics that have been observed in neutrophils are the degranulation capacity and death induced by NETosis. Neutrophils produce NET under different conditions, and it is made up of elastase, histones, MPO, and cathepsin-G, among other proteins [43].
In the presence of bacterial invasion, NETosis causes the release of IL-1β in the surrounding neutrophils and increase the production of chemokines such as CXCR2, CXCL8, CXCL1, CXCL3, and CXCL3, which produce a higher infiltrate of neutrophils favoring inflammation by degranulation [51]. Additionally, the neutrophils trigger the aggregation of NET, which initiates the anti-inflammatory process by trapping dead cells, bacteria, or phagocytes, facilitating the macrophages phagocytosis [52]. Interestingly, the NET production depends directly of the C-type lectin receptor Dectin-1, which is a size microorganism sensor [53]. The activation of Dectin-1 activates elastase, and elastase translocates to the nuclei and promotes histone degradation [43]. Once the concentration of neutrophils in the affected site of bacterial invasion exceeds a certain threshold, the NET begins to aggregate, building the aggNET [52]. AggNETs sequester DNA and proteins from neutrophil granules, and degrade pro-inflammatory cytokines through DNA-bound serine proteases. Indeed, as aggNET increases, more control exists over the pro-inflammatory phenomenon [43,52].
Aboodi and Johnstone described the high-and low-responder phenotypes based on ROS production, corresponding to the N1 and N2 subsets, respectively [18,24]. Additionally, the presence of neutrophils with CD15, CD16, CD62L, and CXCL10 markers corresponds to the N1 subset, and neutrophils with higher levels of CD11b and lower levels of CD62L correspond to the N2 subset [20,23,27]. In the presence of pathogenic mechanisms, neutrophils are primed by TLR2, TLR4, CD14, or even NOD1 [68]. Both subsets produce divergent amounts of CXCR4, which allows neutrophils to migrate to lymph nodes and present antigens to T lymphocytes. N1 neutrophils produce lower levels of CXCR4, while N2 neutrophils produce higher levels of CXCR4 than the N1 subset [69]. In this sense, the pro-inflammatory response triggered by N1 neutrophils could occur in situ after priming, and the modulatory response triggered by N2 probably occurs in the lymph-nodes. Nevertheless, plausible mechanisms for resolvign the gap in the knowledge regarding the capability of N2 to modulate the adaptive response in periodontitis is still unresolved.
Although we can identify that there is evidence in animals that neutrophils N1 and N2 have been described outside of a tumor environment, there are no studies that have characterized them during periodontal disease or in periodontal tissues [9]. A first approach for neutrophil subsets was related to their high or low density. High-density neutrophils (HDN) are Ly6G HIGH CD11b HIGH , mature, with segmented nuclei, cytotoxic, with a high capacity of migration, phagocytosis, and oxidative burst, non-suppressive, pro-inflammatory, and antitumorigenic [70][71][72]. Conversely, low-density neutrophils (LDN) are Ly6G HIGH CD11b LOW , mature, with segmented nuclei, non-cytotoxic, with a reduced migration, phagocytosis, and ROS production, suppressive, anti-inflammatory, and pro-tumorigenic. Additionally, HDN were named as belonging to a pro-inflammatory or anti-tumor phenotype, or N1, while LDN were denoted as belonging to an anti-inflammatory or pro-tumor phenotype, or N2 [70,72].
HDN subsets are characterized by Toll-like receptor (TLR)-2, TLR4, TLR5, TLR8, CD11b LOW CD49 HIGH CD177 HIGH expression and IL-12 production [73]. HDN subsets also produce higher amounts of TNF, CXCL10, and ROS, express higher levels of ICAM-1, CCL3, and CD95, and lower levels of CXC4, vascular endothelial growth factor (VEGF), and IL-8 [5,[74][75][76]. Conversely, LDN neutrophils express TLR2, TLR4, TLR7, TLR9, and CD11b HIGH CD49 LOW CD177 LOW , and produce IL-10 [73]. LDN has a long lifespan and produces large amounts of arginase, which in turn inactivates T-cells, produce higher levels of CCL2, CXCR2, CXCR4, VEGF, IL-8, IL-10, and TGF-β1, and produce low levels of ICAM-1 and CCL3 [5,75,77]. LDN neutrophils also express MPO and produce ROS, but at a lower rate than HDN [76]. The greater or lesser extent of ROS production can be explained by its pro-inflammatory or homeostatic role. In fact, Aboodi [18] demonstrated the presence of two responder phenotypes, according to ROS production. HDN cells produce an oxidative burst, which contributes to local degranulation and bacterial killing, while LDN decreases ROS in order to produce genotoxicity in other immune cells [76]. When comparing published data, it is possible to identify the presence of different neutrophil phenotypes; however, whether they are N1 or N2 subsets can only be speculated ( Figure 2). Curiously, in systemic inflammation, a third phenotype has been described, characterized by being CD62 LOW CD11b HIGH CD11c HIGH , and by its strong capacity to inhibit T lymphocyte phenotypes by direct contact through Mac1 integrin and ROS production [53,78]. Additionally, two studies analyzed a population of neutrophils, detecting a third circulating phenotype, which could be the granulocytic myeloid-derived suppressor cells (gMSCD) [16,23,72].
In general terms, there are no human studies demonstrating the presence of the N1 or N2 phenotypes of neutrophils in periodontal tissues. The closest approximations are related to the increase in pro-inflammatory functions in people with periodontitis, which in itself does not imply that there is an N1 phenotype. Thus, the absence of evidence does not allow us to demonstrate that these phenotypes exist in the periodontium, nor does it allow us to identify a possible role. Thus, in vitro and experimental studies are required to demonstrate the role of the two neutrophil phenotypes during periodontitis. described, characterized by being CD62 LOW CD11b HIGH CD11c HIGH , and by its strong capacity to inhibit T lymphocyte phenotypes by direct contact through Mac1 integrin and ROS production [52,77]. Additionally, two studies analyzed a population of neutrophils, detecting a third circulating phenotype, which could be the granulocytic myeloid-derived suppressor cells (gMSCD) [15,22,71].  Neutrophil subsets N1 and N2. N2 neutrophils are differentiated in the presence of IL-10, IL-35, TGF-β1, and G-CSF. N2 cells are characterized by producing ROS, IL-10 and TGF-β1, expressing CD11b, CD16b, CD62L, CD66 and CD177LOW, and having a pro-tumor and antiinflammatory function. On the other hand, N1 neutrophils differentiate in the presence of IFN-γ and TNF-α. They are characterized by producing oxidative burst, IFN-γ, TNF-α, CCL3, CXCL4 and expressing CD16b, CD11b, CD66 and CD177HIGH, and fulfill an anti-tumor and pro-inflammatory role. References: [16,18,23,24,72,76]. (Created with http://biorender.com).

Future Research Directions
The evidence of neutrophil subsets in periodontitis is an unexplored field. Even though several studies have determined different neutrophil roles, the role of the N1 or N2 subsets in periodontal health or disease still cannot be concluded. It is necessary to determine the pathways by which oral bacteria may be able to induce N1 or N2 response in in vitro or experimental models. Additionally, it is necessary to determine the frequency of detection of these neutrophil phenotypes in periodontal health or disease.

Conclusions
The presence of neutrophils increases in tissues affected by periodontitis compared to healthy tissues. These neutrophils have a pro-inflammatory phenotype characterized by increased phagocytosis, degranulation, production of pro-inflammatory cytokines, and NETosis. However, neither study defines detected neutrophils as being either N1 or N2.

Implication of the Findings for Research
This scoping review demonstrates that there is a gap in knowledge. In this context, new research hypotheses can be developed that must be resolved in the future. Additionally, Figure 3 represents a hypothesis regarding the role of both subsets during periodontitis, which must be proven in the future.

Implication of the Findings for Research
This scoping review demonstrates that there is a gap in knowledge. In this context, new research hypotheses can be developed that must be resolved in the future. Additionally, Figure 3 represents a hypothesis regarding the role of both subsets during periodontitis, which must be proven in the future.  . N1 and N2 neutrophils and their role during periodontal health or disease. Neutrophils, by recognizing symbiotic bacteria, have the ability to differentiate into the N2 phenotype. Cytokines produced by N2 cells will polarize macrophages towards M2-anti-inflammatory subsets. In addition, they will migrate to the regional lymph-node and present the antigen to naïve lymphocytes to allow their differentiation into the Th2 and Treg phenotypes, which will naturally maintain tissue homeostasis. Conversely, in the presence of keystone bacteria or pathobionts, neutrophils polarize towards the N1 phenotype, which secretes cytokines that allow the differentiation of pro-inflammatory M1 macrophages and pro-inflammatory dendritic cells. Together, they present antigens to T lymphocytes at the affected periodontal site, differentiating them towards Th1 and Th17 effector phenotypes. Thus, the response initiated by N2 neutrophils is a regional response, and the one triggered by N1 neutrophils is localized. References: [5,34,57,[59][60][61][62][63][64][65][66]69]. (Created with http://biorender.com).

Implication of the Findings for Practice
Understanding the role of neutrophils as the first line of immune response will make it possible to design therapeutic alternatives for the treatment of pro-inflammatory diseases in order to avoid N1 polarization or induce N2 polarization. Specifically, periodontitis is caused by keystone pathogens that trigger a pro-inflammatory response. In this context, determining what type of neutrophil response each bacterium induces would be important for assessing the microbiological or immunological susceptibility of each individual. Data Availability Statement: The study did not report any data.