NETosis

Immunity, Pathogenesis and Therapeutics

Geeta Rai

• Publisher: Academic Press
• Year: 2019
• Print ISBN: 9780128161470
• eBook ISBN: 9780128163795
• Topic: Medicine
• Subtopic: Pharmacology

NETosis: Immunity, Pathogenesis and Therapeutics takes a focused approach to the clinical aspects of NETosis and drug development, bringing critical findings. Chapters introduce NETosis, consider mechanisms and antimicrobial strategies regulating NETosis, examine NETosis in neonates, explore the role of NETosis in autoimmunity, delve into NETosis and other diseases, and present therapeutic approaches for dysregulated NETosis. Since Brinkamm, et al, discovered an unrecognized neutrophil anti-microbial mechanism responsible for the extracellular killing of invading pathogens in 2004, the novel process in which nuclear chromatin de-condenses and DNA is ejected into the extra cellular environment, trapping and inactivating tissue pathogens has rapidly evolved.- Presents an up-to-date and detailed analysis of NETosis- Brings together critical findings on NETosis as a comparatively novel immune mechanism- Focuses on the clinical aspects of NETosis that lead to drug development- Covers the topic with a cogency and passion that is based on years of scientific research

Chapter 1

Neutrophil extracellular trap formation: an introduction

Abstract

Neutrophils are the most abundant innate immune effector cells of the human immune system that arrive first at the site of infection. As known previously, it can phagocytose the pathogen or release an arsenal of broad antimicrobials stored in its granules which neutralizes the challenge. However, another novel function of neutrophils, which is release of neutrophil extracellular traps (NETs), has been described. In vitro induction with the pharmacological agents, phorbol-12-myristate-13-acetate, interleukin 8, or lipopolysaccharide, causes neutrophils to release its chromatin embedded with granular proteins by a process termed NETosis. Apart from neutrophils, eosinophils, basophils, mast cells, and monocytes are also being reported of releasing extracellular DNA traps. NETosis might take place by the conventional NADPH oxidase and reactive oxygen species–dependent suicidal pathway, wherein cell lysis takes place by splurging out NETs, or it may take place by nonlytic mechanisms where chromatin bound with nuclear proteins is exocytosed in vesicles termed as vital NETosis.

Keywords

Neutrophil extracellular trap; NETosis; suicidal NETosis; vital NETosis; ROS; chromatin decondensation; histone; citrullination; eosinophil

Neutrophils are the immune cells characterized by a lobulated nucleus, earning them the designation of polymorphonuclear cells (PMNs). Attributed by the abundance of granules in their cytoplasm and their staining with neutral dyes, Paul Ehrlich first called these cells as neutrophils during the 19th century. When the immune system is under a challenge from pathogens, neutrophils are the first cells to exit the circulation and relocate to the area under attack. Through their antimicrobial activity, neutrophils kill microbes, communicate the damage status to other immune cells by releasing small signaling molecules, and initiate healing. With an arsenal of potent antimicrobial proteins at their disposal, neutrophils are the professional phagocytes that, together with the production of reactive oxygen species (ROS), kill microbes inside the phagosome. Through “degranulation,” these antimicrobial proteins are also released. Armed with broadly effective antimicrobials that are stored predominately in specialized granules, neutrophils are the most abundant innate immune effector cells of the human immune system. A third antimicrobial mechanism is the release of neutrophil extracellular traps (NETs). First noticed by Takei et al. (1996), as a unique form of cell death, distinct from necrosis or apoptosis neutrophils were found to kill bacteria by forming extracellular structures called NETs, which are composed mainly of DNA, histones, and proteases, such as neutrophil elastase (NE). This process was further studied and termed as NETosis in 2004 (Brinkmann et al., 2004). In NETosis the neutrophils upon induction of innate immune sensors with pathogenic antigens extrude large amounts of chromatin and granule proteins, such as NE and myeloperoxidase (MPO), which trap and kill microorganisms. NETs contain the invading microorganism to prevent the spread of infection and use their highly localized arsenal of antimicrobial peptides to neutralize and kill the microorganism. With its ability to damage host tissues, neutrophil arsenal deploys itself in a tightly regulated manner during the release of NETs (Brinkmann et al., 2004).

Two distinct forms of NETosis

Current process of NET release follows a bimodal approach. According to the first model, NETosis is a cell death pathway of chromatin decondensation, nuclear and cytoplasmic membrane disintegration followed by the expulsion of the chromatin and granular contents into the extracellular space (Brinkmann et al., 2004, 2007; Fuchs et al., 2010). In contrast to apoptotic cells, neutrophils undergoing NETosis do not appear to display phosphatidylserine and other “eat me” signals, which might prevent their silent clearance by phagocytes. NET disassembly takes place primarily by nucleases (Fuchs et al., 2007).

The second model involves ejection of DNA/serine proteases from intact neutrophils, and also mitochondrial DNA release that works by activating inflammation and is apparently not associated to cell death (Yousefi, Mihalache, Kozlowski, Schmid, & Simon, 2009). In addition to these, autophagy may also contribute to NETosis (Remijsen, Berghe, & Wirawan, 2011).

Suicidal NETosis

Conventional suicidal NETosis is triggered by engagement of IgG–Fc receptors, Toll-like receptors, complement, or cytokines on neutrophils (Brinkmann et al., 2004; Garcia Romo et al., 2011; Munks et al., 2010). These receptors once activated initiates the downstream cascade, wherein calcium sequestered inside endoplasmic reticulum is released as calcium ions into the cytoplasm (Fig. 1.1). This elevation of calcium levels in the cytoplasm increases the activity of protein kinase C (PKC) and phosphorylation of gp91phox, heme binding subunit of the superoxide-generating NADPH oxidase (Kaplan et al., 2012). This initiates the assembly of the subunits of NADPH oxidase in the cytosol and bounds to the membrane into functional complexes at cytoplasmic or phagosomal membranes (also called phagocytic oxidase, PHOX) and the successive generation of ROS (Papayannopoulos, Metzler, Hakkim, & Zychlinsky, 2010). ROS causes rupture of granules and nuclear envelope, and eventually fusing of released nuclear, granular, and cytoplasmic contents occurs. NE (a serine protease of broad specificity) and MPO (peroxidase enzymes that produce antimicrobial hypohalous acids), usually stored in azurophilic granules, move to the nucleus. Subsequently, NE breaks down the linker histone H1 and processes the core histones and MPO which augment chromatin decondensation causing the release of extracellular traps (ETs) (Papayannopoulos et al., 2010). NE also degrades actin cytoskeleton, blocking phagocytosis of neutrophils. Another important enzyme, peptidyl arginine deiminase type 4 (PAD4), causes deamination of histones and their proteolytic cleavage initiated before nuclear breakdown additionally contributes to chromatin decondensation (Pingxin et al., 2010; Saskia, John, Sanja, & Kerra, 2011). The breakdown of the plasma membrane releases NETs and NET-entrapping pathogens, resulting in cell death with loss of viable cell functions like migration and phagocytosis also referred to as beneficial suicide (Papayannopoulos et al., 2010).

Figure 1.1 Suicidal NETosis.
Inducer/stimuli bind to endogenous and exogenous receptors. Raf/MEK/ERK kinases pathway activation leading to increase in cytosolic calcium leads to phosphorylation of gp91phox protein. This activates the NADPH oxidase complex and subsequently produces reactive oxygen species (ROS). Elastase and myeloperoxidase then translocate to the nucleus stimulated by ROS and other yet unknown factors. The chromatin decondenses that result in the loss of lobular shape of the nucleus. Loss of nuclear envelope and granular membrane takes place and decondensed chromatin is associated with cytoplasmic components. Plasma membrane is lost and DNA is released as extracellular traps.

Vital NETosis

As opposed to the conventional form of NETosis, Clark et al. (2007) in a straight forward experiment reported that release of NEs from neutrophils continued without penetration of intracellular DNA staining dye SYTOX Green, emphasizing the fact that neutrophils remain structurally intact. This was evidence for a first alternative pathway of NETosis and the authors coined the term vital NETosis to refer it (Fig. 1.2). This was further supported by electron microscopy images of Staphylococcus aureus–induced NETs which are formed by blebbing of the nuclear envelope and vesicular exportation in vitro and in vivo (Bianchi et al., 2009). Similar catapult, like the release of mitochondrial DNA without lytic cell rupture, has been first reported by Yousefi and colleagues in eosinophils, but subsequently in neutrophils (Yousefi, Gold, & Andina, 2008; Yousefi et al., 2009). Contrary to this, a new report described the formation of eosinophil extracellular trap (EET) resulting in cytolytic granular release (Ueki et al., 2013). Thus, granulocytes, in general, may have both a lytic and a nonlytic pathway of NETosis.

Figure 1.2 Vital NETosis.
Stimuli are recognized by receptors on cell surface. Lobular and multinucleated shape of the nucleus is lost. External and internal nuclear membranes are lost and budding of vesicles takes place. Pearl strings form of DNA filaments are formed inside vesicles in the cytoplasm, dense cytoplasmic granules approach towards intact plasma membrane. DNA is released as extracellular traps through a small area in the cell surface; some cytoplasmic granules also fuse with plasma membrane that is released into extracellular space to associate with DNA.

Differences between suicidal and vital NETosis

The main differences between suicidal NETosis and vital NETosis depend on the nature of inciting stimuli and NET release programming mechanism and requires hours of stimulation for extracellular trap formation. For example, suicidal NETosis has mostly been established in the context of phorbol-12-myristate-13-acetate (PMA)–induced chemical stimulation and requires hours. In contrast, vital NETosis has been exhibited to be activated by microbial-specific molecular patterns called as pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs) recognized by host pattern recognition receptors. One such PAM...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Foreword
• Preface
• Chapter 1. Neutrophil extracellular trap formation: an introduction
• Chapter 2. NETosis: mechanisms and antimicrobial strategies
• Chapter 3. Factors regulating NETosis
• Chapter 4. NETosis in neonates
• Chapter 5. NETosis in Autoimmunity
• Chapter 6. NETosis in other diseases and therapeutic approaches
• Nomenclature list
• Index