What are Neutrophil Extracellular Traps (NETs)?

Neutrophil Extracellular Traps (NETs)

Neutrophil extracellular traps (NETs) are microbicidal structures composed of nuclear proteins, chromatin, histones, and granular antimicrobial proteins that are released extracellularly by neutrophils and eosinophils in response to various stimuli to entrap and eliminate pathogens. 1

Structure and Composition of NETs

NETs consist of:

• Extracellular DNA fibers (chromatin backbone)
• Histones (H1, H2A, H2B, H3, H4)
• Granular antimicrobial proteins including:
  • Myeloperoxidase (MPO)
  • Neutrophil elastase (NE)
  • Cathepsin G
  • Lactoferrin
  • Gelatinase
  • Proteinase 3 1, 2

Formation Mechanisms (NETosis)

NETs can be formed through two primary mechanisms:

1. Vital NETosis (occurs in living neutrophils):

   • Rapid process (30-60 minutes)
   • Triggered by physiological stimuli like GM-CSF, complement fragment C5a, IL-8, and lipopolysaccharide
   • Neutrophils remain viable after NET release
   • No cytosolic proteins detected in NETs
   • NET-releasing cells do not take up exclusion dyes 1

2. Suicidal NETosis (associated with cell death):

   • Triggered by non-physiological stimuli like phorbol-12-myristate-13-acetate (PMA)
   • Characterized by:
     • Massive cytoplasmic vacuolization
     • Rapid chromatin decondensation
     • Breakdown of nuclear and granular membranes
     • Cell death follows NET release 1

Molecular Mechanisms of NETosis

The process of NETosis involves several key steps:

• NADPH oxidase activation - generates reactive oxygen species (ROS) critical for NET formation
• Histone citrullination - mediated by peptidylarginine deiminase 4 (PAD4), which converts positively charged arginine side chains to uncharged citrulline side chains, facilitating chromatin decondensation
• Autophagy components - participate in the process of NET formation
• Nuclear and granular membrane breakdown - allows mixing of nuclear and cytoplasmic contents 1

NETosis is distinct from other cell death pathways as it is:

• Insensitive to caspase inhibitors (unlike apoptosis)
• Insensitive to necrostatin-1 (unlike necroptosis)
• Dependent on NADPH oxidase-mediated superoxide generation
• Dependent on components of the autophagic machinery 1

Physiological Functions of NETs

The primary functions of NETs include:

1. Pathogen entrapment and immobilization - physical containment of microorganisms
2. Pathogen killing - through antimicrobial proteins and histones
3. Limiting pathogen spread - creating a physical barrier 3, 4

Pathological Roles of NETs

Despite their protective role, excessive or dysregulated NET formation contributes to various pathologies:

• Infectious diseases:

  • Bacterial sepsis
  • Viral infections (including COVID-19)
  • Malaria
  • Dengue fever 4

• Inflammatory conditions:

  • Inflammatory bowel disease
  • Autoimmune disorders 3, 5

• Vascular pathologies:

  • Thrombosis - NETs provide a scaffold for platelet adhesion and activation
  • Atherosclerosis - NETs contribute to vascular inflammation
  • Ischemia-reperfusion injury 1, 5

• Cancer - particularly colorectal cancer where NETs may influence tumor progression 3

Detection and Quantification of NETs

NETs can be detected and quantified through various methods:

• Immunofluorescence microscopy - using antibodies against:

  • Extracellular DNA
  • Histone-DNA complexes
  • Granule components (MPO, elastase)

• Biochemical assays:

  • Nuclease activity measurement
  • Quantification of free extracellular DNA
  • Detection of specific NET markers in body fluids 6

Therapeutic Targeting of NETs

Given their role in various pathologies, NETs represent an emerging therapeutic target:

• Direct NET inhibitors - targeting NET formation pathways
• PAD4 inhibitors - preventing histone citrullination
• DNase - degrading the DNA backbone of NETs
• Repurposed FDA-approved drugs with NET-inhibiting properties 4

Clinical Relevance

Understanding NETs is crucial for:

1. Infectious disease management - balancing beneficial antimicrobial effects with detrimental inflammatory consequences
2. Inflammatory disease treatment - targeting excessive NET formation
3. Cardiovascular disease - addressing NET-mediated thrombosis and vascular inflammation 1, 5

NETs represent a double-edged sword in immune function - while critical for pathogen defense, their dysregulation contributes to tissue damage and inflammation across multiple organ systems, making them an important target for therapeutic intervention.