**4. Antimicrobial mechanisms of neutrophils**

Neutrophils are equipped with different antimicrobial mechanisms, which help them to fight a broad spectrum of bacteria, fungi, and protozoa. These mechanisms include phagocytosis, degranulation, and neutrophil extracellular traps (NETs) (**Figure 1**).

#### **4.1. Phagocytosis**

Phagocytosis is a receptor‐mediated process during which a particle is internalized by the cell into a vacuole called the phagosome. Neutrophils recognize pathogens through pattern‐rec‐ ognition receptors (PAMPs), or opsonins (antibody molecules or complement components). Opsonized pathogens are efficiently phagocytosed when they bind with antibody receptors (Fc receptors) or complement receptors on the neutrophil (**Figure 3**). After engulfment, the nascent phagosome matures by fusing with lysosomes. This brings antimicrobial molecules into the phagosomal lumen. The vesicle is now called phagolysosome. Concurrently, reactive oxygen species (ROS) production starts by the assembly of the NADPH oxidase on the phago‐ somal membrane, and the pH inside the phagosome drops to 4.5–5. Also, potassium ions (K<sup>+</sup> ) are pumped into the phagolysosome; this K<sup>+</sup> influx mediates the release of serine proteases. In addition, hydrogen peroxide (H2 O2 ) is also converted into hypochloric acid (HOCl) in a reac‐ tion catalyzed by myeloperoxidase (MPO) [25]. Granules content and ROS create an environ‐ ment toxic to the pathogen. Unfortunately, not all pathogens are killed inside the phagosome. Moreover, some have advanced strategies to survive inside neutrophils. These strategies include interfering with engulfment, modulating phagosome maturation, and creating a more hospitable intraphagosomal environment.

**Figure 3.** Phagocytosis and NETs. (A) Neutrophils recognize opsonized pathogens through Fc Receptors (FcγRIIa) or complement receptors (Mac‐1) on their membrane. The pathogen is internalized into a nascent phagosome, which matures by fusing with lysosomes forming a phagolysosome. (B) Neutrophil extracellular traps (NETs) are formed when neutrophils release decondensed chromatin decorated with antimicrobial molecules, into the extracellular space. ROS, reactive oxygen species.

#### **4.2. Degranulation**

)

influx mediates the release of serine proteases. In

) is also converted into hypochloric acid (HOCl) in a reac‐

**4. Antimicrobial mechanisms of neutrophils**

are pumped into the phagolysosome; this K<sup>+</sup>

hospitable intraphagosomal environment.

addition, hydrogen peroxide (H2

reactive oxygen species.

**4.1. Phagocytosis**

72 Role of Neutrophils in Disease Pathogenesis

degranulation, and neutrophil extracellular traps (NETs) (**Figure 1**).

O2

Neutrophils are equipped with different antimicrobial mechanisms, which help them to fight a broad spectrum of bacteria, fungi, and protozoa. These mechanisms include phagocytosis,

Phagocytosis is a receptor‐mediated process during which a particle is internalized by the cell into a vacuole called the phagosome. Neutrophils recognize pathogens through pattern‐rec‐ ognition receptors (PAMPs), or opsonins (antibody molecules or complement components). Opsonized pathogens are efficiently phagocytosed when they bind with antibody receptors (Fc receptors) or complement receptors on the neutrophil (**Figure 3**). After engulfment, the nascent phagosome matures by fusing with lysosomes. This brings antimicrobial molecules into the phagosomal lumen. The vesicle is now called phagolysosome. Concurrently, reactive oxygen species (ROS) production starts by the assembly of the NADPH oxidase on the phago‐ somal membrane, and the pH inside the phagosome drops to 4.5–5. Also, potassium ions (K<sup>+</sup>

tion catalyzed by myeloperoxidase (MPO) [25]. Granules content and ROS create an environ‐ ment toxic to the pathogen. Unfortunately, not all pathogens are killed inside the phagosome. Moreover, some have advanced strategies to survive inside neutrophils. These strategies include interfering with engulfment, modulating phagosome maturation, and creating a more

**Figure 3.** Phagocytosis and NETs. (A) Neutrophils recognize opsonized pathogens through Fc Receptors (FcγRIIa) or complement receptors (Mac‐1) on their membrane. The pathogen is internalized into a nascent phagosome, which matures by fusing with lysosomes forming a phagolysosome. (B) Neutrophil extracellular traps (NETs) are formed when neutrophils release decondensed chromatin decorated with antimicrobial molecules, into the extracellular space. ROS, In the bone marrow, as precursor cells mature into neutrophils, they synthesize proteins that are sorted into different granules [26]. Granules formation begins in early promyelocytes and continues throughout the various stages of myeloid cell development. The granules are arbitrarily subdivided into three different classes based on their resident cargo molecules: azurophilic, specific, and gelatinase granules (**Figure 1A**, **Table 1**). Neutrophils also form secretory vesicles until the last step of their differentiation (**Figure 1A**, **Table 1**). Granule het‐ erogeneity is explained by regulated expression of the granule protein genes. This regulation is mediated by the combination of myeloid transcription factors that express at specific stages of neutrophil development. Vesicle availability and exocytosis depends on mobilizable intra‐ cellular compartments of the neutrophil. Mature neutrophils are released into the circulation and, in response to infection, they leave the circulation and migrate toward the inflamma‐ tory site. Exocytosis of granules and secretory vesicles plays a crucial role in most neutrophil functions from early activation to the destruction of phagocytosed microorganisms. Secretory vesicles have the highest propensity for extracellular release, followed by gelatinase granules, specific granules, and azurophil granules [27, 28]. For example, neutrophil stimulation with phorbol myristate acetate (PMA) induces complete release of gelatinase granules, restrained release of specific granules, and minimal exocytosis of azurophil granules. In a different way, neutrophil stimulation with fMLF induces release of mostly secretory vesicles without


*Note:* CR, complement receptor; FPR, formyl peptide receptor; NGAL, neutrophil gelatinase‐associated lipocalin; NRAMP1, natural‐resistance‐associated macrophage protein 1.

**Table 1.** Cytoplasmic granules of neutrophils [24–26, 28].

significant release of granules. The hierarchical mobilization of neutrophil granules and secre‐ tory vesicles depends on intracellular Ca2+ level. Gradual elevations in intracellular Ca2+ are induced by ligation of L‐selectin, CD11b/CD18, and the fMLP receptors [26].

#### **4.3. Neutrophil extracellular traps (NETs)**

Neutrophil stimulation can also undergo a mechanism called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. NETosis leads to the release of decondensed chromatin into the extracellular space. The chromatin forms a trap for patho‐ gens that looks like a net, which is why they are called neutrophil extracellular traps (NETs). NETs also contain histones, cytoplasmic proteins, and antimicrobial granular molecules. NETs formation mechanisms are still unknown, nevertheless, NADPH oxidase activation, reactive oxygen species (ROS) production, myeloperoxidase (MPO), and neutrophil elastase (NE) release (**Figure 3**) are required [25].
