Defense strategies of neutrophils - degranulation and phagocytosis

To attack intruders, neutrophils have to be recruited and guided to the place of infection.

This process has to be tightly regulated since uncontrolled neutrophil activation can lead to massive tissue damage as discussed in paragraph 1.2.5 for NETosis. The complex mechanisms of neutrophil recruitment are reviewed in detail by Kolaczkowska and Kubes [66].

In the classical scenario of diapedesis, neutrophils have to migrate through the intact endothelium into the tissue to reach the site of infection [132]. Therefore, the cells have to show particularly high flexibility and compensate the mechanical forces due to the shear stress of the blood flow as well as pressure of the surrounding cells. This process is tightly regulated and involves characteristic changes of the nuclear morphology and its interplay with the cytoskeleton as recently extensively reviewed [94, 133]. The unique morphology of neutrophils also allows them to reach a remarkable migration speed of 19 +/-6 µm/min in vitro [134].

Neutrophil infiltration from the vasculature into the tissue is mainly induced by mast cells and macrophages as well as pathogens and their products recognized by receptors for pathogen-associated molecular patterns (PAMPs) [135]. To guide neutrophils to the site of infection, the endothelium, in the first step, expresses E- and P-selectin in proximity to the stimulus and neutrophils bind by glycosylated ligands such as P-selectin glycoprotein ligand 1 (PSGL1) [136] enabling them to adjustably tether and roll along the endothelium [137].

Under high shear stress, even thin membrane extensions can be built, so-called ‘slings’, which wrap around a neutrophil and guide the rolling cell on the endothelial lining [138].

Further stimulation of neutrophils by e.g., chemokines attached to the endothelium [139], leads to activation of integrins on the cell’s surface (inside-out-signaling). The main integrins involved are 2-integrins (lymphocyte function-associated antigen 1 (LFA-1), macrophage-1 antigen (Mac-1)) and 1-integrins (very late antigen-4 (VLA-4)). These integrins mediate the cell’s binding to intercellular or vascular cell adhesion molecule-1/2 (ICAM-1/2, VCAM-1) expressed by the endothelium [140]. Subsequently, neutrophils reduce rolling speed, crawl and flatten the cell body until they attach firmly and start to migrate para- and transcellularly through the endothelium [141]. Thereafter, cells are guided to the site of infection by chemotactic gradients (e.g., chemokines, leukotrienes, complement factors or bacterial products) [66, 142]. In addition to this classical way, alternative recruiting pathways of neutrophils into specific tissues such as liver, brain or spleen were reported [14, 66]. For

CHAPTER 1 - Scientific background

Dissertation - Elsa Neubert

distances in complex cell cluster movements. This process, also referred to as neutrophil swarming, can be mediated by leukotriene B4 [144].

Having arrived at the target, neutrophils attack pathogens by different strategies including degranulation, phagocytosis and NETosis (Fig. 3). One of the most important tools to exert these functions is the directed release of different granules. As already alluded to paragraph 1.1.2, neutrophils contain at least four different types of granules (AGs, SGs, GGs, VSs) with unique compositions of dissolved and membrane-bound molecules [145-147]

(Fig. 1 and below). The granules discharge their contents in a precisely defined sequence in response to multiple signals and thresholds (reviewed in [148]). In general, they are released inversely to their generation and with increasing content of antimicrobial substances (SVs GGs SGs AGs). The precise regulation of degranulation is complex (Fig. 3 (1)). The current knowledge including the underlying signaling cascades was only recently summarized by Yin et al. [148].

SVs [149, 150] contain a large fraction of membrane-bound proteins including receptors (e.g., fMLPR, CR1/3, CXCR2), MMP-25, membrane fusion proteins (e.g., VAMP2), integrins (Mac-1) as well as membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components (gp91^phox/NOX2, p22^phox) (Fig. 1; for abbreviations see figure legend). Furthermore, they contain a surprisingly high amount of cytoplasmic proteins [149], most likely originating from an endocytotic formation of these vesicles [146].

Importantly, SVs are already mobilized during rolling [151] by the induced modest calcium increase and simultaneous activation by chemokines [152, 153]. Thereby, integrins [154] and receptors [155] are transported to the cell’s surface, which modulate neutrophil activation as well as firm adhesion to the endothelium.

Specific (lactoferrin positive) and gelatinase (gelatinase positive) granules (SGs, GGs) have similar functions and overlapping contents [45, 50]. They contain moderate amounts of antimicrobials (SG > GGs) and several membrane-bound receptors and integrins (e.g., fMLPR, TNFR1, Mac-1) to guide neutrophils to the site of infection and initiate the antimicrobial defense [148]. Degranulation of SGs and GGs occurs in response to stronger calcium signals (calcium flickers [156]) induced by the increasing chemotactic signal and subsequent G protein-coupled receptor activation (GPCR) [157]. Thereby, the majority of these granules is released onto the cell surface and only a few granules (mainly SGs) into the phagosome during phagocytosis (Fig. 3 (2)) [158].

AGs contain a large number of antimicrobial effectors such as myeloperoxidase (MPO), serine proteases (neutrophil elastase (NE), proteinase 3, cathepsin G (CG) and neutrophil serine protease 4 (NSP4)), bactericidal/permeability-increasing protein, defensins and lysosomal proteins. Hence, the main function of AGs is the effective killing of pathogens. To carry out this function, AGs can be secreted onto the cell surface (membrane-targeting, actin-dependent [159]) or into the phagosome (phagosome-targeting, tubulin-dependent [160]) [161-163]. Their degranulation is triggered by an additive calcium signal induced by combinations of GPCR, phagocytic receptor [164] and complement receptor [165] activation

Figure 3: Defense strategies of neutrophil granulocytes. Neutrophils can attack pathogens by three main mechanisms. 1) The release of antimicrobial substances by degranulation. 2) Phagocytosis of bacteria. 3) Immobilization and attack of bacteria by the release of NETs. Neubert et al., in preparation.

Phagocytosis is one of the most important defense strategies of neutrophils first described by Metchnikoff in 1905 [167] (Fig. 3 (2)). Neutrophils can engulf opsonized pathogens by pseudopod extension in so-called phagosomes after binding of immunoglobulin G (IgG) by phagocytic receptors (e.g., FcyRIIa and FcyRIIIb) or complement factors by complement receptors [168]. Importantly, neutrophil phagocytosis is closely connected to degranulation.

Unlike macrophages, they do not perform the classical endosome-lysosome pathway, but fuse AGs and partly SGs, directly with the phagosome in a remarkably fast manner within seconds [169] (reviewed in detail by Nordenfelt and Tapper [168]). During this process, the degranulation of granules can occur already prior to and during phagosome formation at the plasma membrane [170]. In this way, neutrophils can directly attack pathogens by antimicrobial substances from the first contact on. This antimicrobial response is supported by the generation of reactive oxygen species (ROS) (oxidative burst), one of the key steps in phagocytosis [171]. For ROS generation, the protein complex NADPH oxidase first assembles at granular, plasma and phagosomal membranes triggered by soluble agonists such as phorbol 12-myristate 13-acetate (PMA) and fMLP [172, 173] or in the context of phagocytosis activation [174]. Upon activation, the membrane-bound components (gp91^phox/NOX2, p22^phox), which are mainly localized at the membranes of SGs, GGs and to a lower extent SVs [145, 146], associate with the cytosolic components (p47^phox, p67^phox, p40^phox) as well as with the Ras-related C3 botulinum toxin substrate (Rac) [168] (Fig. 1 and

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Dissertation - Elsa Neubert

(H2O2). The formation of H2O2 is the basis for further ROS production. It can be used for instance by MPO to generate hypochlorous acid (HClO) or in Fenton reactions to create hydroxyl radicals (OH^.) catalyzed by metal ions (e.g., Fe²⁺) [175].

Furthermore, neutrophils are able to ‘choose’ a certain defense strategy [176]. For instance, NETosis is considered to be preferred over phagocytosis with increasing pathogen size as reported for fungi hyphae [177]. During this process, the decision for NETosis depends on multiple factors such as the accessibility of MPO and NE in granules [177], successful activation of phagocytosis via pathogen detection as shown for Dectin-1 [177] or cytoskeletal integrity [176, 177].

Beside the main defense pathways shown in Fig. 3, neutrophils can modulate the immune response by de novo synthesis of specific proteins or generation of microparticles [35].

Microparticles are membrane vesicles filled with heterogeneous content depending on the surrounding conditions and the activating stimulus. They can be released to carry out diverse functions in health and diseases [178]. These functions include contributions to reactions against opsonized particles [179, 180] or induction of pro-inflammatory [181] as well as anti-inflammatory [182, 183] responses.

Over time, activated neutrophils decrease their resistance against anti-apoptotic signaling [184] and, after successfully targeting the pathogen, they undergo apoptosis or, under certain circumstances, autophagy [185]. In this context, they can express ‘find-me’ or ‘eat-me’ signals and are silently cleared by phagocytosis [35, 185, 186]. According to a model of Kennedy and DeLeo, this process can be dysregulated by certain pathogens or in a disease-related context. Importantly, this dysregulation can induce a switch to pro-inflammatory cell death pathways such as pyroptosis, oncosis and NETosis [185, 187] accompanied by subsequent tissue damage and acute as well as chronic inflammation [35].