**2. Role of neutrophils in rheumatoid arthritis**

#### **2.1. Neutrophil trafficking from blood to the synovial cavity**

Neutrophil recruitment is an important stage in the inflammatory development process, including autoimmune diseases such as RA. Among the circulating cells, neutrophils are the first ones to reach the synovium and are the most abundant cells in the synovial fluid [24]. In this section, we discuss the cascade of events that culminates in neutrophil entry into inflamed joints. The leukocyte recruitment cascade involves the following commonly recognized steps: capture, rolling, firm adhesion, and finally transendothelial migration.

component in which genetic and environmental risk factors contribute to disease development. Its prevalence in the world population is between 0.3 and 1%, and it affects three times

The pathophysiology of RA is complex and appears to be initiated when the adaptive immune system (cellular or humoral) recognizes self-joint antigens as non-self, which triggers a variety of distinct inflammatory effector mechanisms, including the recruitment of leukocytes [5–8]. RA is characterized by intense inflammatory processes and joint damage that are mediated by the influx of immune system cells to the synovial space such as neutrophils, macrophages, and lymphocytes [1, 2]. A critical factor that contributes to tissue damage is the excessive production of inflammatory mediators by resident and/or infiltrated cells. Among the primary mediators involved in joint damage are free radicals, enzymes that degrade the matrix, and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and

IL-1β, as well as chemokines such as CXCL-8, lipid mediators, such as leukotriene B<sup>4</sup>

[9, 10], and endothelin (ET) [11, 12]. Inflamed synovial tissue is invasive and called pannus, which can be formed by synovial cell proliferation, angiogenesis, and the accumulation of

Neutrophils are crucial cells that have significant roles in diverse inflammatory diseases, including acute, chronic, autoimmune, infectious, and non-infectious conditions [14]. The most wellknown effector function of neutrophils is their role in innate immunity. However, recent studies have identified neutrophils as active cells during adaptive immunity, facilitating the recruitment and activation of antigen-presenting cells or directly interacting with T cells. Neutrophils are the most abundant leukocytes in inflamed joints, and the importance of these cells in the initiation and progression of human RA as well as in murine models has been demonstrated [15–18]. Therefore, neutrophils play an essential role in joint inflammation, and the modulation of neutrophil functions is considered a potential target for pharmacological intervention in arthritis [19–21].

The pharmacologic treatment options for arthritis are diverse. The current treatments are mostly symptomatic and include non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs), and biologic therapies. High costs and an increased risk of malignancies limit the use of these agents, in addition to the potential side effects that all therapies possess. Plant-derived products, such as polyphenols, sesquiterpenes, flavonoids, and tetranortriterpenoids, which are herbal metabolites with anti-inflammatory activity, may provide new therapeutic agents and cost-effective treatments [22, 23]. This chapter focuses on the role of neutrophils in the pathogenesis of arthritis and the action of

Neutrophil recruitment is an important stage in the inflammatory development process, including autoimmune diseases such as RA. Among the circulating cells, neutrophils are the first ones to reach the synovium and are the most abundant cells in the synovial fluid [24]. In this section,

substances from natural products as putative antirheumatic therapies.

**2. Role of neutrophils in rheumatoid arthritis**

**2.1. Neutrophil trafficking from blood to the synovial cavity**

 (LTB<sup>4</sup> )

more women than men [3, 4].

90 Role of Neutrophils in Disease Pathogenesis

macrophages, lymphocytes, and neutrophils [13].

Neutrophil release from the bone marrow to the circulating blood occurs immediately after the first signal of inflammation, serving to increase the number of neutrophils available for recruitment into the tissue in response to inflammation [25]. The mobilization of neutrophils from the bone marrow is orchestrated by the hematopoietic cytokine granulocyte colony-stimulating factor (G-CSF). G-CSF mobilizes neutrophils indirectly by shifting the balance between CXCR4 and CXCR2 ligands [26]. In response to the release of inflammatory mediators such as TNF-α and IL-17, the adjacent vascular endothelium becomes activated. Cell surface proteins of the selectin family termed E- and P-selectin and their ligands (L-selectin) mediate this initial neutrophil capture. Neutrophil rolling through the endothelium facilitates their contact with chemotactic factors that promotes neutrophil activation [27]. Chemokines (CXCR-1 or 2 ligands, such as IL-8), the C5a fragment of the complement system, and leukotriene B4 (LTB<sup>4</sup> ) are responsible for neutrophil mobilization to the synovial fluid [28–30].

Firm adhesion is mediated by interactions between β<sup>2</sup> integrins (LFA-1, CD11a/CD18, and MAC-1, CD11b/CD18) and their ligand (ICAM-1). Integrins are usually in an inactive state on neutrophil and become activated after the triggering of G protein-coupled receptors such as chemokine receptors [31]. The binding of integrins to their ligands activates signaling pathways in neutrophils stabilizing adhesion and initiating cell motility [32, 33]. This signaling also regulates actin polymerization, which controls the direction of neutrophil movement [34, 35]. The final stage in the adhesion cascade is the ultimate migration of the neutrophil from the vasculature into the inflamed tissue. Passage through the endothelial cell layer occurs both paracellularly (between endothelial cells) and by a transcellular route (over the endothelial cell). Paracellular migration of neutrophils is mediated by binding to endothelial proteins that target neutrophils to intercellular junctions and facilitate their passage through them. To reach the inflamed joint, neutrophils must pass over the basal membrane, which occurs through the degradation of extracellular matrix molecules by proteases stored inside the cells, such as matrix metalloproteinases (MMPs) and serine proteases [14].

In inflammatory foci, neutrophils find immune complexes on the synovium that bind to Fcγ receptors on the neutrophil membrane, triggering their degranulation and reactive oxygen species (ROS) production [36]. In RA pathology, oxidative stress is a result of inadequate ROS release by neutrophils [37]. Oxygen radicals cause DNA damage and oxidation of lipids, proteins, and lipoproteins and may be involved in immunoglobulin mutations that lead to rheumatoid factor (RF) formation [38, 39]. Moreover, proteins from neutrophil degranulation are found at high concentrations in the RA synovial fluid and could be responsible for cartilage and tissue damage, activation of cytokines and soluble receptors, inhibition of chondrocyte proliferation and activation of synoviocytes proliferation and invasion [40–43]. In addition, activated neutrophils also generate chemoattractants (such as IL-8 and LTB<sup>4</sup> ) that promote further neutrophil recruitment and amplify the inflammatory response (see **Figure 1**).

**Figure 1.** Overview of the role of neutrophils in arthritis. Neutrophils leave blood vessels after chemotactic signals from inflamed tissues that promote the firm adhesion of neutrophils to endothelial cells mediated by adhesion molecules, which induce neutrophil activation and actin filament formation followed by transendothelial migration toward the inflammatory foci. Immune complexes and proinflammatory molecules activate neutrophils, which then produce ROS and release enzymes responsible for cartilage destruction. Activated neutrophils communicate with other cells of the immune system through the secretion of cytokines and chemokines and by antigen presentation in conjunction with MHC class II. Neutrophils can undergo a special form of cell death called NETosis. This results in the release of a complex of nuclear and granule molecules called NETs contributing to tissue damage. Activated neutrophils also generate chemoattractants (such as IL-8 and LTB<sup>4</sup> ), forming a positive-feedback loop that promotes further neutrophil recruitment and amplifies the acute inflammatory response. Finally, effective neutrophil apoptosis is required for the resolution of inflammation. However, delayed neutrophil apoptosis occurs in the inflamed joint, which results in persistent inflammation and tissue damage due to the continued release of ROS, granule enzymes, and cytokines.

#### **2.2. Neutrophil action in rheumatoid arthritis**

Neutrophils are key cells in articular inflammation that are abundant in the synovial fluid and pannus of patients with active RA [44], a typical knee joint may have 2 × 10<sup>9</sup> cells, of which 90% are neutrophils [24]. These cells are mobilized to synovial tissue by chemoattractant mediators, such as CXCL1, CXCL2, endothelin (ET)-1, and leukotriene B<sup>4</sup> , a process in which resident macrophages play a central role [11, 45, 46].

For many years, the major contribution of neutrophils to the pathology of RA was thought to be their cytotoxic potential, since neutrophils participate in the pathogenesis of arthritis by promoting the inflammatory process and cartilage degradation, as well as bone resorption. However, neutrophils are now recognized to have an active role in orchestrating the progression of inflammation through regulating the functions of other immune cells [47, 48], and current research has shown that these cells are involved in RA onset [49, 50].

In the synovial cavity, activated neutrophils exhibit an increased expression of plasma membrane receptors such as major histocompatibility complex (MHC) class II molecules and present antigens to T lymphocytes, an immune function that they share with macrophages and dendritic cells (DCs) [51]. In addition, the interaction of neutrophils with other cells induces the secretion of MMP-8 and MMP-9, and a repertoire of cytokines (IL-1β, IL-12, IL-18, IL-23, and TNF-α) and chemokines (CCL-2, CCL-4, CCL-5, and CXCL-8), including TNF ligand superfamily member (RANKL) [52, 53] and TNFSF13B (also known as BLyS or BAFF) [54], which are implicated in the activation of osteoclasts and B lymphocytes, respectively, regulate the function of other immune cells [48, 55–57].

Neutrophils from patients with RA are functionally very different from those isolated from healthy individuals. RA blood neutrophils are already primed for ROS production [58] and striking differences in gene and protein expression exist between peripheral blood neutrophils from patients with RA and their healthy counterparts [18], including higher levels of membrane-expressed TNF and myeloblastin (also known as PR-3 or cANCA antigen) in RA [59].

In RA patients, neutrophils can be activated by immune complexes, such as RF or anti-citrullinated protein antibodies (ACPAs), both within the synovial fluid and deposited on the articular cartilage surface [60]. These complexes engage Fcγ receptors and thereby trigger neutrophil activation, which release ROS and RNS [61, 62], collagenases, gelatinases, neutrophil myeloperoxidase (MPO), elastase, and cathepsin G into the synovial fluid and joints [14, 55, 56, 63] due to frustrated phagocytosis [60].

#### *2.2.1. Pain in rheumatoid arthritis and neutrophils*

**2.2. Neutrophil action in rheumatoid arthritis**

generate chemoattractants (such as IL-8 and LTB<sup>4</sup>

92 Role of Neutrophils in Disease Pathogenesis

which resident macrophages play a central role [11, 45, 46].

Neutrophils are key cells in articular inflammation that are abundant in the synovial fluid

recruitment and amplifies the acute inflammatory response. Finally, effective neutrophil apoptosis is required for the resolution of inflammation. However, delayed neutrophil apoptosis occurs in the inflamed joint, which results in persistent inflammation and tissue damage due to the continued release of ROS, granule enzymes, and cytokines.

**Figure 1.** Overview of the role of neutrophils in arthritis. Neutrophils leave blood vessels after chemotactic signals from inflamed tissues that promote the firm adhesion of neutrophils to endothelial cells mediated by adhesion molecules, which induce neutrophil activation and actin filament formation followed by transendothelial migration toward the inflammatory foci. Immune complexes and proinflammatory molecules activate neutrophils, which then produce ROS and release enzymes responsible for cartilage destruction. Activated neutrophils communicate with other cells of the immune system through the secretion of cytokines and chemokines and by antigen presentation in conjunction with MHC class II. Neutrophils can undergo a special form of cell death called NETosis. This results in the release of a complex of nuclear and granule molecules called NETs contributing to tissue damage. Activated neutrophils also

), forming a positive-feedback loop that promotes further neutrophil

which 90% are neutrophils [24]. These cells are mobilized to synovial tissue by chemoattrac-

For many years, the major contribution of neutrophils to the pathology of RA was thought to be their cytotoxic potential, since neutrophils participate in the pathogenesis of arthritis by promoting the inflammatory process and cartilage degradation, as well as bone resorption. However, neutrophils are now recognized to have an active role in orchestrating the progression of inflammation through regulating the functions of other immune cells [47, 48], and

In the synovial cavity, activated neutrophils exhibit an increased expression of plasma membrane receptors such as major histocompatibility complex (MHC) class II molecules and present antigens to T lymphocytes, an immune function that they share with macrophages and dendritic cells (DCs) [51]. In addition, the interaction of neutrophils with other cells induces the secretion of MMP-8 and MMP-9, and a repertoire of cytokines (IL-1β, IL-12, IL-18, IL-23,

cells, of

, a process in

and pannus of patients with active RA [44], a typical knee joint may have 2 × 10<sup>9</sup>

tant mediators, such as CXCL1, CXCL2, endothelin (ET)-1, and leukotriene B<sup>4</sup>

current research has shown that these cells are involved in RA onset [49, 50].

One of the most prevalent symptoms of RA is the increase in sensitivity to joint pain (hyperalgesia), which causes movement limitations. Despite its clinical relevance, strategies for the treatment of arthralgia remain limited. In animal models, hyperalgesia (inflammatory pain) is defined as hypernociception (a decreased nociceptive threshold) [64]. It is broadly accepted that articular hypernociception results mainly from the direct and indirect effects of inflammatory mediators on the sensitization (increased excitability) of primary nociceptive fibers that innervate the inflamed joints [65–67]. Prostaglandins and sympathetic amines are the key mediators of this process. Furthermore, other mediators, such as the cytokines TNF-α, IL-1β, IL-6, and IL-17 play a crucial role in the pathogenesis of arthritis, increasing the recruitment of neutrophils into the joint and driving the enhanced production of chemokines and degradative enzymes [68–70]. In addition, endothelin-1 (ET-1), acting directly or indirectly, also sensitizes primary nociceptive neurons [71–74].

During the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, by mediating the release of hyperalgesic molecules (such as MPO, MMPs, hypochlorite, superoxide anion, and PGE<sup>2</sup> ) capable of activating nociceptive neurons and causing pain [17, 75–78].

Indeed, decreased inflammation and joint destruction have been directly correlated with reduced neutrophil influx into the joints, as observed in mouse models by means of antibody blockade or the gene deletion of chemoattractant receptors such as CXCR1, CXCR2, and BLT1 (LTB<sup>4</sup> receptor) [15, 79]. Therefore, the blockade of neutrophil migration could be a target in the development of new analgesic drugs [77].

#### *2.2.2. Citrullinated autoantigens and NETs in rheumatoid arthritis*

Citrullination is the natural posttranslational conversion of arginine to citrulline mediated by peptidyl arginine deiminases (PADs), enzymes present in macrophages, dendritic cells, and neutrophils. Experimental evidence indicates that citrullination is involved in the breakdown of immune tolerance and may generate neoantigens (neoAgs) that become additional targets during epitope spreading [80]. Citrullinated residues stimulate the production of anti-citrullinated protein antibodies (ACPAs) in predisposed individuals. It has been observed that ACPAs can be present for several years before any clinical signs of arthritis appear [81–83]. A substantial increase in the number and titer of many antibodies against posttranslationally modified proteins is also seen shortly before the onset of arthritis. Citrullinated Ags have increased immunogenicity and arthritogenicity, and their presence in arthritic joints correlates with disease severity [80, 84–86].

Osteoclasts are dependent on citrullinating enzymes for their normal maturation and display citrullinated antigens on their cell surface in a non-inflamed state. In humans, the binding of ACPAs to osteoclasts in the bone compartment induces IL-8 secretion. In turn, IL-8 sensitizes and/or activates sensory neurons by binding to CXC chemokine receptor (CXCR) 1 and CXCR2 on peripheral nociceptors [87–90], producing IL 8 dependent joint pain that is associated with ACPA-mediated bone loss.

IL-8 release contributes to the chemoattraction of neutrophils [49], which play critical roles in initiating and maintaining joint-inflammatory processes that have been described in experimental arthritis [36, 91]. However, the exact roles that neutrophils play in the posttranslational modification of proteins and disease initiation and progression in RA remain unclear. Recent evidence suggests that, among the various mechanisms by which neutrophils cause tissue damage and promote autoimmunity, aberrant formation of neutrophil extracellular traps (NETs) could play important roles in the pathogenesis of RA [50].

NETs are released during a process of cellular death named NETosis. NETosis occurs with neutrophils upon contact with bacteria, fungi [92], or under several inflammatory stimuli. This process is associated with changes in the morphology of the cells, which eventually lead to cell death with extrusion of NETs [93, 94]. This process requires calcium mobilization, reactive oxygen species (ROS) produced by NADPH oxidase, neutrophil chromatin decondensation mediated by neutrophil elastase (NE) and myeloperoxidase (MPO), and chromatin modification via the citrullination of histones by peptidyl arginine deiminase 4 (PAD4) [95–99]. NETs are a network of extracellular fibers, which contain nuclear compounds as DNA and histones and that are covered with antimicrobial enzymes and granular components, such as MPO, NE, cathepsin G, and other microbicidal peptides [93, 94]. In the extracellular environment, NET fibers entrap microorganisms, and their enzymes and granular substances reach locally high concentrations and are thus able to cleave virulence factors and kill microorganisms [95, 100, 101].

Although NETs play a key role in the defense against pathogens, they may cause undesirable effects to the host, which has increased the interest in the role of neutrophils and NETs in autoimmunity. Augmented NET formation was first described in preeclampsia and ANCA-associated vasculitis and followed by the description in a series of autoimmune conditions, including psoriasis, systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome (APS), and RA [50, 100, 102–105]. Neutrophil extracellular traps are an obvious source of nuclear material. Among these are a range of cytoplasmic and extracellular citrullinated antigens, well-established targets of the ACPAs found in RA [50, 100]. The protein contents of NETs not only serve as targets for autoantibody and immune complex formation but also induce further NETosis, resulting in a harmful positive-feedback loop. These factors form an inflammatory microenvironment that may trigger a strong autoimmune response in individuals with the corresponding susceptibility [106, 107]. Pro-inflammatory cytokines, such as TNF-α and IL-17, as well as autoantibodies stimulate the formation of NETs and affect their protein composition [50]. Additionally, NETs have been shown to stimulate autoimmunity via the production of interferons and activation of the complement cascade. Interferons activate both the innate and adaptive immune systems, inducing a Th1 immune response and stimulating B cells toward the generation of autoantibodies [108]. The deposition of NETs observed in various inflammatory pathologies is associated with the circulating cell-free DNA (cfDNA) levels in biological fluids, such as plasma and serum, from patients [100, 101, 109]. Therefore, circulatory cfDNA could eventually be utilized as a marker of NETs in these pathologies, while the determination of the DNA levels might facilitate the monitoring of disease activity and assessment of the effectiveness of a selected therapeutic strategy.

Neutrophils have been traditionally viewed as short-lived cells that die at sites of inflammation; however, some evidence suggests that they can prolong their life span upon specific stimuli and transmigrate away from inflammatory loci [48, 110, 111]. Conditions within the synovial joint, such as hypoxia [112] and the presence of antiapoptotic cytokines (including TNF, granulocytemacrophage colony-stimulating factor (GM CSF), and IL 8) [113, 114], can increase neutrophil survival for up to several days [115, 116], which contributes to enhanced tissue damage.

As described above, neutrophils play an essential role on innate and adaptive immunity in RA physiopathology, contributing to tissue lesions in RA, and therefore represent a promising pharmacological target in RA. Pharmacological strategies that inhibit or reduce neutrophil mobilization or activation could be successful in RA treatment.
