Neutrophil Biology and Defense Against Pathogens

NEUTROPHIL BIOLOGY Specific granules Gelatinase granules Secretory vesicles PHAGOCYTOSIS Phagocytosis is a key defense mechanism that neutrophils use against invading pathogens. Phagocytosis proceeds in a series of phases to engulf and destroy the pathogen. 1) Pathogen Recognition > Internalization 2) Phagosome Formation > Maturation PAD gp91phox Rac p40 phox p22 phox p47 phox NADPH 2O2 H2O2 Oxidative Burst Degradation HOCl 2H+ + H+ H+ 2O2 ·- NADP+ Phagosome Azurophilic granules NEUTROPHIL ACTIVATION Neutrophils are a key component of the innate immune system, the first line of defense against invading microorganisms. They are rapidly recruited to sites of inflammation and express several surface and intracellular receptors to recognize and respond to inflammatory stimuli. Lytic NETosis: · Occurs slowly · NADPH activation/ROS-dependent · Nuclear envelope disassembly · Plasma membrane rupture · NETs released & neutrophil dies Vital NETosis: · Occurs rapidly · PAD activation · NETs expelled via vesicles · Neutrophil remains alive as functional anuclear cytoplast NADPH oxidase ↑Ca2+ MPO © 2019 Cayman Chemical, Revised February 2023 NH2 O ROS Proton channel LYTIC VERSUS VITAL NETosis NET PATHOPHYSIOLOGY · Autoimmunity · COVID-19 · Cancer metastasis · Infection · Sepsis · Thrombosis NET COMPONENTS · Actin · Azurocidin · Calprotectin · Cathepsin G · CitH3 · Defensins · DNA/mtDNA · Lactoferrin · Lipocalin · MPO · NE · Proteinase 3 · Vimentin NETosis Numerous pathogens, inflammatory components, and other endogenous and exogenous stimuli can induce the formation of neutrophil extracellular traps (NETs). NETs are composed of a web of DNA, citrullinated histones, and granule enzymes like NE, MPO, and various proteases. During NETosis, NETs are extruded from neutrophils to trap and immobilize pathogens. They also have direct antimicrobial activity and alert other immune cells such as macrophages to the presence of these pathogens but can have deleterious consequences if not properly cleared. Triggers of NET Formation · Activated platelets · Cholesterol & urate crystals · Cytokines (e.g., IL-8, IL-1β, TNF-α) · DAMPs (e.g., HMGB1) · Immune complexes & antibodies · Ionophores (e.g., A23187, nigericin) · PMA · Pathogens · ROS (e.g., H2O2) Intracellular Signaling DEGRANULATION Neutrophils have granules containing an arsenal of antimicrobial proteins, enzymes, and other cytotoxic agents. Upon neutrophil activation, granules are mobilized to the phagosome or plasma membrane where they fuse with the membrane to release their cargo, destroying the invading pathogen. This process is tightly regulated to avoid bystander injury to neighboring cells and tissues. Azurophilic granules contain many potent antimicrobial compounds and lytic enzymes. Azurophilic granules function primarily at the phagosome to kill intracellular pathogens. Upon fusion with the phagosome, azurophilic granules release: · Neutrophil elastase (NE) · Myeloperoxidase (MPO) · Cathepsins · Collagenase (MMP-8) · Lactoferrin · Lipocalin Specific granules expel their contents mainly at the plasma membrane via exocytosis. These granules contain antimicrobial compounds as well as certain proteins that modulate neutrophil functions, including: Gelatinase granules share many properties with specific granules but are distinguishable by the expression of gelatinase, also known as MMP-9, a protease that promotes matrix remodeling. Gelatinase granules contain: · Gelatinase (MMP-9) · Arginase I · Complement receptors · CD11b/CD18 · CD14 Secretory vesicles are the first type of granule to be released upon neutrophil activation. They expel their contents via exocytosis and act mainly as storage compartments for membrane-associated receptors that function early in the neutrophil inflammatory process. They contain: · Defensins · Proteases · Cathelicidin · Cytochrome b558 · Cytochrome b558 · Ficolin I · CD16 · Albumin · Alkaline phosphatase Phagosome Oxidative Burst NLRs NE NE NE Endosome TLR7/9 Endosome Complement receptors TLRs Bacteria ParasitesLipid metabolites Chemokines Complement Platelets Fc receptors Integrins Cytokine receptors GPCRs Cytokines Antibodies INFLAMMATION Neutrophils release an array of soluble mediators that have roles in both the promotion and resolution of inflammation. Chemokines recruit additional immune cells to the site of inflammation, and cytokines tailor the immune response. Neutrophils highly express 5-lipoxygenase (5-LO), which produces a variety of lipid mediators such as 5(S)-HETE, which can have broad-ranging effects on other immune cells. Chemokines Lipid metabolites Cytokines MPO NE Viruses Ca2+ H2O NH4 + PAD H2N + NH2 NH Protein H2N O NH Protein Peptidyl Arginine (+ charge) Peptidyl Citrulline (neutral) Citrullination Targets: · β-Catenin · Cytokeratin · α-Enolase · Fibrinogen · Filaggrin · Heat shock proteins · Histones · VimentinMPO CITRULLINATION p67 phox RLRs