**1. Introduction**

Periodontal disease is a major public health problem due to its high prevalence worldwide [1]. Periodontitis is a more advanced inflammatory form of periodontal disease. It is a chronic inflammatory disease that causes tooth loss, by destroying the periodontium. Periodontal destruction may be caused by different factors, including accumulation of dental biofilm, poor oral hygiene, and loss of balance between oral microbiota and immune response. Dysbiosis

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(an alteration of oral microbiota) is thought to be the initial trigger for periodontitis [2]. The accumulation of bacteria biofilm leads to an increase in the inflammatory infiltrate, composed mainly by neutrophils into oral tissues. In this chapter, we will discuss the role of neutrophils in periodontal disease.

#### **2. Neutrophil homeostasis**

Neutrophils are considered to be the first line of defense during infections and inflammation [3]. They are the most abundant leukocytes in blood and can live for much longer than pre‐ viously thought. It is estimated that neutrophils half‐life is days instead of hours [4]. When microorganisms invade the organism, an inflammatory response is induced. Neutrophils are recruited from the circulation into the tissues where they destroy microorganisms by phagocy‐ tosis, by releasing antimicrobial substances, or by NETosis (**Figure 1**). This last mechanism was

**Figure 1.** Neutrophil homeostasis involves production, trafficking, and clearance of these cells. (A) Production of neutrophils takes place at the bone marrow. Neutrophils maturate in the bone marrow accumulating different granules (arrow heads), azurophil, specific, and gelatinase. Finally, they also produce secretory vesicles. Lines show the moment of appearances of granules and vesicles. Neutrophils are released from the bone marrow to the circulation by interfering with the CXCR4‐CXCL12 interaction. (B) Neutrophils mobilization to infection site through a leukocyte adhesion cascade that includes capture, rolling, firm adhesion, and transmigration of neutrophils (thin arrows). Senescent circulating neutrophils increase the expression of CXCR4, and respond to CXCL12 by homing back to the bone marrow. (C) Neutrophils kill bacteria by phagocytosis, degranulation, and NETs formation. Apoptotic neutrophils are cleared by macrophage phagocytosis. The process of "neutrostat" that maintains steady‐state neutrophil levels (molecules with green background and green arrows). In an infected site, macrophages produce IL‐23, which activates IL‐17. IL‐17 induces G‐CSF that promotes neutrophil differentiation and release from the bone marrow (thick arrows). After macrophages phagocyte apoptotic neutrophils, they downregulate the production of IL‐23 and produce IL‐10 and TGF‐β, this events stop the recruitment of neutrophils. CXC, chemokine receptor; IL, interleukin; G‐CSF, granulocyte colony‐stimulating factor.

recently discovered and consists on the formation of neutrophil extracellular traps (NETs) [5]. Activated neutrophils produce a variety of chemokines and cytokines, directing the inflam‐ matory and the immune responses [6]. Unfortunately, if there is not a proper clearance of neutrophils after an infection, the proteases released from neutrophils into the surrounding tissue can cause damage to the host [7]. Bacteria biofilm deposited on teeth induces a constant recruitment of neutrophils (>95%) to the gingival sulcus (**Figure 2**) [8, 9]. Therefore, neutrophil homeostasis is important to prevent collateral damage to the host by the potent proinflamma‐ tory and antimicrobial effects of these cells. As neutrophils are the most abundant leukocytes, their excess or absence in the mouth leads to periodontal tissue damage. Moreover, neutro‐ phil distribution and numbers are essential in maintaining oral health. Neutrophil homeostasis involves production, trafficking, and clearance of these cells [10].

#### **2.1. Production**

(an alteration of oral microbiota) is thought to be the initial trigger for periodontitis [2]. The accumulation of bacteria biofilm leads to an increase in the inflammatory infiltrate, composed mainly by neutrophils into oral tissues. In this chapter, we will discuss the role of neutrophils

Neutrophils are considered to be the first line of defense during infections and inflammation [3]. They are the most abundant leukocytes in blood and can live for much longer than pre‐ viously thought. It is estimated that neutrophils half‐life is days instead of hours [4]. When microorganisms invade the organism, an inflammatory response is induced. Neutrophils are recruited from the circulation into the tissues where they destroy microorganisms by phagocy‐ tosis, by releasing antimicrobial substances, or by NETosis (**Figure 1**). This last mechanism was

**Figure 1.** Neutrophil homeostasis involves production, trafficking, and clearance of these cells. (A) Production of neutrophils takes place at the bone marrow. Neutrophils maturate in the bone marrow accumulating different granules (arrow heads), azurophil, specific, and gelatinase. Finally, they also produce secretory vesicles. Lines show the moment of appearances of granules and vesicles. Neutrophils are released from the bone marrow to the circulation by interfering with the CXCR4‐CXCL12 interaction. (B) Neutrophils mobilization to infection site through a leukocyte adhesion cascade that includes capture, rolling, firm adhesion, and transmigration of neutrophils (thin arrows). Senescent circulating neutrophils increase the expression of CXCR4, and respond to CXCL12 by homing back to the bone marrow. (C) Neutrophils kill bacteria by phagocytosis, degranulation, and NETs formation. Apoptotic neutrophils are cleared by macrophage phagocytosis. The process of "neutrostat" that maintains steady‐state neutrophil levels (molecules with green background and green arrows). In an infected site, macrophages produce IL‐23, which activates IL‐17. IL‐17 induces G‐CSF that promotes neutrophil differentiation and release from the bone marrow (thick arrows). After macrophages phagocyte apoptotic neutrophils, they downregulate the production of IL‐23 and produce IL‐10 and TGF‐β, this events stop the recruitment of neutrophils. CXC, chemokine receptor; IL, interleukin; G‐CSF, granulocyte

in periodontal disease.

colony‐stimulating factor.

**2. Neutrophil homeostasis**

68 Role of Neutrophils in Disease Pathogenesis

Thousands of neutrophils are daily produced in the bone marrow and released into the cir‐ culation [11]. Three pools of neutrophil population reside in the bone marrow: the stem cell pool, the mitotic pool, and the postmitotic pool (**Figure 1A**). The first pool consists of undif‐ ferentiated pluripotent hematopoietic stem cells (HSCs), the second pool consists of committed granulocytic progenitor cells that proliferate and differentiate. Finally, the third pool consists of fully differentiated neutrophils, which form the bone marrow reserve, available for release [12]. HSCs differentiate into myeloblasts, a developmental cell type committed to becoming granulocytes (**Figure 1A**). Granulocyte colony‐stimulating factor (G‐CSF) regulates both, pro‐ duction or granulopoiesis, and neutrophil release from the bone marrow. G‐CSF regulates granulopoiesis by inducing proliferation of granulocytic precursors in the bone marrow [10]. A large postmitotic pool is retained in the bone marrow by the interaction of CXC chemokine receptor 4 (CXCR4) on neutrophils with chemokine CXCL12 (stromal‐derived factor‐1/SDF‐1)

**Figure 2.** Neutrophil infiltrate and inflammation state. (A) In health conditions few neutrophils are recruited to the gingival sulcus to maintain symbiotic microbial community and the gum is not inflamed. (B) During gingivitis more neutrophils are recruited to the gingival sulcus and the gum in moderate inflamed. The junctional epithelium is starting to detach from the tooth. (C) During periodontitis a mayor neutrophil infiltrate is recruited to the periodontal pocket and the gum in severely inflamed. Inflammatory response activates osteoclasts, which in turn reabsorb bone.

produced by bone marrow stromal cells (**Figure 1A**). G‐CSF regulates mature neutrophil release from the bone marrow by interfering with the CXCR4‐CXCL12 interaction [12]. In addi‐ tion, interleukin‐17 (IL‐17) endorses granulopoiesis and neutrophil release by upregulation of G‐CSF (**Figure 1**) [10]. IL‐17 builds on an interesting positive loop of neutrophil recruitment. For example, in chronic inflammation sites, neutrophils produce IL‐17 and can also attract IL‐17‐producing CD4<sup>+</sup> T lymphocytes (Th17 cells) [13]. Neutrophils also release CCL20 and CCL2 chemokines, which are ligands for CCR6 and CCR2 chemokine receptors, respectively, on Th17 cells. This interaction maintains Th17 cells at inflammation sites. Therefore, Th17 cells secrete more IL‐17 and more neutrophils are recruited [14].

#### **2.2. Trafficking**

Circulating neutrophils can be quickly mobilized to infection or inflammation sites through a systematically controlled process known as the leukocyte adhesion cascade, which achieves neutrophil transmigration (**Figure 1B**) [15]. The process initiates when endothelial cells get activated and upregulated the expression of adhesion receptors such as E‐ and P‐selec‐ tins. Neutrophils recognize these selectins and begin rolling on endothelial cells. This roll‐ ing depends on transient interactions of selectins with glycoprotein ligands on neutrophils. Next, neutrophils get activated by chemokines, which induce a high affinity state in integrins, another group of adhesion receptors. Interaction of both selectins and integrins with their cor‐ responding ligands leads to slow neutrophils rolling followed by a firm adhesion that brings neutrophils to a full stop. Finally, neutrophils crawl on the endothelium and transmigrate into infection or inflammation sites. This last process is regulated mainly by β2 integrins. Integrins are heterodimeric receptors formed by a unique α (CD11) and a common β (CD18) subunit that interact with adhesion ligands such as intercellular adhesion molecule‐1 (ICAM‐1) and ICAM‐2 on endothelial cells (**Figure 1B**). This leukocyte adhesion cascade is positive regu‐ lated by tissue‐derived cytokines and by tissue‐derived chemokines. Cytokines control the expression of endothelial adhesion molecules and chemokines induce integrins to change conformation into a high affinity state [16]. Once neutrophils move into tissues, they follow chemoattractant gradients to reach infection or inflammation sites. Some chemoattractants for neutrophils are activated by complement components, such as the anaphylatoxin C5a, and bacterial components, such as formyl‐methionyl‐leucyl‐phenylalanine (fMLF). Recently, it has been discovered that the leukocyte adhesion cascade is also negatively regulated by endog‐ enous inhibitors such as Del‐1 (developmental endothelial locus‐1), pentraxin 3, and growth‐ differentiation factor 15 [17].

#### **2.3. Clearance**

Neutrophils are mostly cleared in tissues (**Figure 1C**) and possibly also in the bone mar‐ row (**Figure 1A**). In tissues, once neutrophils have completed their antimicrobial duty, they undergo apoptosis. Resident phagocytes, for instance, macrophages and dendritic cells, clear neutrophils locally. Phagocytosis of apoptotic neutrophils reprograms mac‐ rophages to initiate an anti‐inflammatory response, characterized by the synthesis of tumor growth factor (TGF)‐β and IL‐10, and by a reduction in IL‐23 synthesis (**Figure 1**) [18]. IL‐23 cytokine induces IL‐17 synthesis; thus, the reduced IL‐17 levels lead to less G‐CSF production and in consequence, less neutrophil production. This process is a control loop that has been described as a "neutrostat" (neutrophil rheostat), and main‐ tains steady‐state neutrophil levels (**Figure 1**) [14]. Senescent circulating neutrophils are recruited for clearance in the bone marrow. These neutrophils increase the expression of CXCR4, and respond to CXCL12 by homing back to the bone marrow (**Figure 1**) [19]. Apoptosis and proper removal of apoptotic cells are key aspects of inflammation resolu‐ tion. Neutrophils death is influenced by environmental conditions including hypoxia and presence of inflammatory mediators, such as granulocyte/monocyte colony‐stimulating factor (GM‐CSF) and lipopolysaccharides (LPS). Neutrophil clearance depends on signals that apoptotic neutrophils express on their surface. These signals allow macrophages to recognize and ingest the neutrophils (**Figure 1C**) [20]. Failure to clear these apoptotic cells results in secondary necrosis and release of products that generate proinflammatory sig‐ nals. Neutrophils express molecules that regulate their survival. Some of these molecules are survivin, cyclin‐dependent kinases and proliferating cell nuclear antigen (PCNA). Survivin is expressed more highly in immature neutrophils than in mature ones, but its expression can be reestablished in mature cells by inflammatory signals, for instance, GM‐ CSF or G‐CSF [21]. Similarly, cyclin‐dependent kinases function as prosurvival factors. Their inhibition induces caspase‐dependent apoptosis. PCNA in neutrophils associates with procaspases in the cytosol and is thought to prevent their activation. During apopto‐ sis, PCNA is targeted for proteosomal degradation, which correlates with an increase in caspase‐3 and caspase‐8 activities [11].
