Neutrophil Biology and NET Formation Quiz

Immune Cell Function in Innate Immunity

• Objectives include defining neutrophil diapedesis steps and interactions, mechanisms for bacteria killing by neutrophils and macrophages, describing how immune cells cause host pathology, and differentiating between M1 and M2 macrophage functions.

The Leukocytes: Meet the O'Phils

• The Gran Team displays diagrams of basophils, eosinophils, and neutrophils.
• A cartoon depiction of a neutrophil consuming bacteria is shown.
• The text expresses the need to feed 'Phil' (neutrophil).

Primary, Secondary, and Tertiary Granules

• Neutrophil granules contain proteins with different synthesis timing during differentiation.
• Granules release according to hierarchy; secretory granules exocytose most readily, while azurophil granules undergo partial exocytosis.
• Specific proteins are listed within each granule type, including myeloperoxidase, neutrophil elastase, cathepsin G, proteinase 3, azurocidin, lactoferrin, cathelicidin, lysozyme, collagenase, leukolysin, cytochrome b558, NGAL, and defensins.

Granule Exocytosis

• Neutrophil granule exocytosis is triggered by chemokines, cytokines, and chemoattractants.
• Specific and azurophil granules fuse with phagocytic vacuole membranes to release their contents.
• Secretory granules fuse with the plasma membrane for extracellular release.
• A hierarchy exists in granule mobilization, with secretory granules first, followed by gelatinase, specific, and lastly azurophil granules.

Major Secretory Granule Proteins

• CR1 (aka CD35, C3bR, C4bR) is a receptor for particles opsonized with C3b.
• CR3 (aka CD11b, Mac1) is a receptor for fibrinogen, ICAM1, and C3b.
• FPR is a receptor for N-formylated peptides (G protein coupled receptor).
• CD14 is a receptor for LPS.
• CD16 is a low-affinity receptor for IgG.

Important Concepts

• Neutrophils act as the first line of defense against invading pathogens.
• Neutrophils have a short life span and are continually entering and leaving the circulation.
• Neutrophil function depends on granule proteins' mobilization in an ordered fashion.
• The most rapidly mobilized proteins are crucial for chemotaxis, neutrophil adhesion, and bacterial opsonization.
• Slowly mobilized proteins promote bacteria killing.

Road Rash and Innate Immunity

• The diagram shows the activation of dendritic cells, macrophages, and NK cells in response to microbes.
• TNF, IL-1, and chemokines are released to facilitate neutrophil response in blood vessels.

Neutrophil Rolling

• Leukocytes roll along the endothelium via selectins and integrins.
• LFA-1 (low-affinity state) is involved in rolling.
• Selectin ligands interact with selectins.
• Integrin ligands, like ICAM2, allow for stable adhesion through integrins (high-affinity state).
• Chemokines, and the extracellular matrix components like fibrin/fibronectin, drive migration through endothelium.

Key Points

• Interstitial macrophages encounter bacteria and release cytokines, primarily TNF and IL-1.
• Activated endothelial cells produce P-selectin, ICAMs, and chemokines, like IL8.
• Neutrophils roll on P-selectins, engage IL-8, activate LFA-1, engage ICAM-2, and adhere stably.
• Neutrophils extravasate through interactions of LFA-1, MAC-1, and PECAM-1 with endothelium's ICAM-1 and PECAM.
• After extravasation, neutrophils adhere to ECM proteins like fibronectin/fibrinogen, phagocytose bacteria, and release chemokines, like IL-8.

Activation of NADPH Oxidase by Chemokines

• Chemokines activate NADPH oxidase through G-protein-coupled receptors.
• NADPH oxidase is crucial for generating reactive oxygen species.

The NADPH Oxidase/Myeloperodixase System

• Azurophil granules contain MPO, which, in combination with activated NADPH and halide, forms toxic agents.
• Toxic agents include superoxide anion, hydrogen peroxide, hydroxyl radicals, and hypochlorous acid (HOCI).

Disorders of Oxygen-Dependent Antimicrobial Systems

• Myeloperoxidase deficiency is relatively common (1:2000 individuals).
• Typically, these patients do not have a higher risk of infection.
• While phagocytosis is normal, candidacidal activity may be moderately impaired.
• The disorder is characterized by delayed respiratory burst deactivation and enhanced H2O2 production; H2O2 is itself bactericidal, potentially compensating for reduced toxic substance production.
• Granulomatous diseases do not stem from this deficiency, and metabolizing chemoattractants/inflammation mediators is unaffected.

Chronic Granulomatous Disease

• Chronic granulomatous disease (CGD) is an inherited X-linked or autosomal recessive disorder affecting 1 to 500,000 patients.
• Sufferers typically experience recurrent infections, producing granulomas in response to inflammation.
• A characteristic bacterium causing infections is S. aureus.
• The disorder stems from a failure in cytochrome b558 generation due to a defect in gp91phox (60% of patients) or p47phox (30%) components of NADPH oxidase, impeding superoxide anion generation.

Azurophile Granule Contents

• Acid mucopolysaccharides are negatively charged polysaccharides which absorb azure stain.
• Granule proteins are cationic and bind tightly to acid mucopolysaccharides.
• Microbicidal enzymes include myeloperoxidase and lysozyme.
• Neutral proteases include serprocidins (which also contain elastase, cathepsin G, and proteinase 3), gelatinase, and collagenase.

Antimicrobial Serprocidins

• Cathepsin G stimulates the acute phase response, acting as an antimicrobial protease against Gram-positive bacteria.
• Elastase, an antimicrobial protease, plays a role in killing Gram-negative bacteria by degrading outer membrane proteins.

Wegeners Granulomatosis

• ANCA (anti-neutrophil cytoplasmic antibodies) induce necrotizing vasculitis by activating neutrophil and monocyte respiratory burst and degranulation.
• Endothelial injury is a consequence.
• The disease necessitates an initiating inflammatory event, leading to antibody formation against shielded granule proteins like proteinase 3 and MPO.

Diagnosing and treating ANCA-associated vasculitis

• ANCA-associated vasculitides (AAV) are rare, systemic necrotizing small-vessel vasculitides, 80-90% consisting of microscopic polyangiitis and granulomatosis with polyangiitis.
• Patients with suspected AAV, manifest with features like chronic rhinosinusitis, cavitated lung nodules, palpable purpura, and acute kidney injury, undergo ANCA testing for diagnosis and further investigations.
• Estimated average Granulomatosis with polyangiitis incidence is 2-13 per million population.
• Risk factors include exposure to silica dust, farming, and chronic Staphylococcus aureus carriage.

Dual Function of BPI

• Bactericidal Permeability Increasing Factor (BPI) is a cationic protein (50-55 kDa) with selective cytotoxicity towards Gram-negative bacteria, found within azurophil granules and the surface of PMNs.
• BPI exhibits high affinity for lipopolysaccharide (LPS), disrupting the bacterial membrane permeability barrier and causing cell death.
• BPI synergizes with defensins and phospholipase A2 to exhibit microbicidal activity in inflammatory exudates.
• BPI has a role in moderating inflammation by interacting with LPS.

Summary of Neutrophil Function

• Granules play roles in eradicating pathogens and guiding neutrophil migration to infection sites.
• Bactericidal proteins (defensins, phospholipase, BPI, protease) act within phagolysosomes, but are also released into inflammatory exudates at biologically active concentrations.
• Unregulated neutrophil degranulation has adverse effects.
• NADPH oxidase plays a prominent role in promoting neutrophil's antimicrobial activity.
• Oxidative and proteolytic mechanisms are important in pathogen elimination but may cause host tissue damage.

NETs (Neutrophil Extracellular Traps)

• NETosis involves chromatin decondensation and nuclear membrane disintegration, with granules moving toward the nucleus.
• Pathogens get trapped within NETs formed by extruded fibrillar networks of DNA.
• The 15x greater volume of NETs is noteworthy.
• NETs are vital for pathogen trapping, and NET composition includes proteins such as MPO, cathepsin G, BPI, lactoferrin, gelatinase, H1, H2A, H2B, H3, H4, CD63, and annexin I.
• Microbial virulence factors are degraded via NET proteins; NET fibers are bactericidal.

NET Formation

• Reactive oxygen species (ROS) generated by NADPH oxidase stimulate MPO to activate neutrophil elastase and serprocidin, and their transfer into the nucleus.
• Elastase acts proteolytically on histones, disrupting chromatin packaging.
• MPO and elastase independently bind and decondense chromatin.

Macrophages

• Macrophages are derived from blood monocytes, originating from bone marrow stem cells.
• Macrophage forms include microglia (CNS), Kupffer cells (liver), alveolar macrophages, osteoclasts (bone).
• Macrophages differentiate and are activated in tissues. The process of maturation entails progenitor movement from blood to tissue.

Liver Anatomy

• The liver contains liver lobules and portal fields.
• Liver lobules are characterized by hepatocytes, Kupffer cells, and sinusoids.
• Portal fields house the hepatic artery, portal vein, and bile ductule.
• The space of Disse and endothelial cells surround hepatocytes.

Macrophage Function: Liver Kupffer Cells

• Kupffer cells are macrophages residing in the liver sinusoids.
• They are vital for clearing S. aureus from the bloodstream.
• A video (part 1), titled "Catching of MRSA by Kupffer cells", showcases this process.
• Video 2 (part 1) details the ability of Kupffer cells to capture Staphylococcus aureus (in vivo).

Neutrophils Form NETs That Persist in Liver Vasculature and Cause Local Necrosis in MRSA Sepsis

• Neutrophils create NETs within liver vasculature, persisting for extended periods in MRSA sepsis.
• Staining with eDNA, neutrophil elastase, and histone highlights the persistence of NETs in liver sinusoids after 4, 8, and 24 hours.

NETs Cause Damage to the Vasculature

• S. aureus entering the circulation is rapidly cleared by Kupffer cells; failure leads to necrosis.
• Neutrophil recruitment, activation, and NET release contribute to necrosis.
• NET structures adhere to vascular walls via von Willebrand factor.
• Neutrophil elastase remains active on immobilized NET structures, causing necrosis.

Comparison of Macrophages and Neutrophils

• Both cells play roles in phagocytosis, but the timing/duration/methods differ.
• Macrophages operate over several months within tissues, whereas neutrophils act swiftly (over hours).
• Macrophage phagocytosis involves endocytosis, phagosome formation, and eventually phagolysosome fusion; neutrophils, using gelatinase/azurophilic granules, demonstrate a quicker, more aggressive approach.
• Different enzymes and proteins are crucial for various stages and final products (e.g. specific proteins, cytokines).

Macrophage Bactericidal Functions

• Macrophages utilize different mechanisms for pathogen killing, including NADPH oxidase, nitric oxide synthase, lysosomal hydrolases, vacuolar ATPase regulation, and iron/manganese/copper/zinc trafficking.
• The presence of these functionalities suggests extensive molecular processes.

A Reminder: Pro-inflammatory role of macrophages

• Macrophages actively participate in inflammation by activating dendritic cells, recruiting neutrophils, and releasing pro-inflammatory cytokines (TNF, IL-1, chemokines).

F1000Prime Reports 2014, Inflammatory M1 and Anti-inflammatory M2 Macrophages

• M1 macrophages exhibit pro-inflammatory characteristics, producing cytokines (IFN-γ, LPS, TNF) and associated factors like iNOS, RNI, and ROI, aiding in killing intracellular pathogens, tissue destruction, and inflammatory conditions.
• Conversely, M2 macrophages are anti-inflammatory, promoting a wide range of responses (IL-4, IL-13, TGF-β) that are fundamental for immune regulation.

Microbial Biofilms Promotes Differentiation of M2 Macrophage

• Microbial biofilms can influence the activation state of macrophages by altering their differentiation into M1 or M2 subtypes to promote bacterial clearance or persistence, respectively.

Macrophage and Fibroblast Interactions in Biomaterial-Mediated Fibrosis

• Biomaterials, such as hip implants, become coated with extracellular matrix, leading to microbial biofilm formation and M2 macrophage polarization, predisposing to fibrosis.

The catheterized bladder environment induces dysregulation of macrophage polarization exacerbating bacterial UTI

• Mice with catheterized bladders show increased fibrin deposition and M2 macrophage polarization in response to E. coli/E. faecalis compared with naïve controls.

Classically activated macrophages

• Classically activated macrophages (M1s) produce pro-inflammatory cytokines (IL-1, TNF, IL-18), display strong microbicidal activity, and are important for eradicating intracellular pathogens.
• iNOS, an enzyme, plays a key role in oxidative killing and response to intracellular pathogens.
• Activated M1s participate in numerous inflammatory disorders and promote autoimmunity in some cases (e.g. Inflammatory Bowel Disease, Rheumatoid Arthritis).

Alternatively activated macrophages

• Stimulated by various signals (IL-4, IL-10, TGF-β, and helminth infections), alternatively activated macrophages (M2s) produce anti-inflammatory cytokines and have a prominent role in tissue remodeling and wound healing.
• M2 macrophages reduce inflammation by producing IL-10, decoy receptors, and receptor antagonists.
• Arginase, produced by M2 macrophages, depletes the substrate arginine, suppressing nitric oxide synthase (iNOS) activity.

Test your understanding of neutrophil elastase activation, NET formation processes, and their impact during MRSA sepsis. This quiz covers the roles of different receptors and granule exocytosis in neutrophil function. Challenge your knowledge of macrophage types and their functions as well.