Table 1.

The main properties and biofunctions of ET-associated proteins in neutrophils and MCs

Producing cell	ET-associated proteins	Main properties and biofunctions of biomolecules and mediators attached to DNA strands	Ref
Neutrophil	Neutrophil elastase (NE)	• A serine protease expressed in primary granules	[95]
		• In humans, NE translocates from azurophilic granules to the nucleus upon formation of NET where it cleaves histones and contributes to chromatin decondensation by partially degrading specific histones	[96]
		• Neutrophils of NE−/− mice produce NETs when stimulated by PMA	[97]
		• Maintains its catalytic ability after being localized to DNA	[98]
		• It is suggested that NE blocking would largely abrogate the protease activity associated with NETs	[99]
	Myeloperoxidase	• Synergies with NE in decondensation of chromatin during NETosis	[96]
		• A granule component of neutrophil that possesses antiviral activity	[100]
	Cathepsin G	• Cleaves the pro-IL-1α precursor and produces more IL-1α through which it activates endothelial cells	[101]
		• Plays a role in platelet activation, platelet aggregation, and dense granule secretion	[102, 103]
	Leukocyte proteinase 3	• Has similar substrates, structural and functional characteristics with NE	[104]
		• it is a neutral protease identified as the principal antigen of antineutrophil cytoplasm autoantibodies (c-ANCA)	[104]
		• Like other NET-associated proteases (NE and cathepsin G), leukocyte proteinase 3 is activated by dipeptidyl peptidase I (DPPI) in mature neutrophils	[105]
	Lactoferrin	• Deprives the bacteria of iron by capturing iron	[106]
		• Polysialic acid modulates the Binding of external lactoferrin in NETs	[106]
		• Binds DNA through interactions of positively charged residues located in the N-terminal with negatively charged DNA	[107]
		• Similar to elastase, lactoferrin is present in the cytoplasm of unstimulated neutrophils but is localized to the cell membrane after 2 h PMA- stimulation	[107]
		• Lactoferrin has been reported to inhibit the release of NET	[106]
	Gelatinase	• Matrix metalloproteinases (MMPs) are zinc-dependent proteases that degrade extracellular matrix and mediate the tissue remodeling	[108]
		• MMP-9 cleaves laminin, chondroitin sulfate, collagen IV, and collagen V	[109]
		• MMP-9 activates the endothelial MMP-2 and drives endothelial dysfunction	[110]
	Lysozyme	• NETs carry lysozyme upon exposure to several microorganisms including Pseudomonas aeruginosa	[111]
	Calprotectin	• Structurally is a heterodimer and acts as an effective antifungal component in NETs	[112]
	Cathelicidins	• LL-37 is the only human cathelicidin which is an amphipathic and cationic peptide and has been reported to act as chemotactic AMP. It has immunomodulatory properties	[113]
		• May lose its antimicrobial properties when it binds to DNA	[114]
		• LL-37 induces the formation of NETs in ex vivo experiments	[115]
		• LL-37 has been reported in structure of NETs when neutrophils are exposed to microbes including bacteria and parasites	[116, 117]
	Defensins	• Human β-defensin 1 (hBD-1) is produced by epithelial surfaces and acts mainly against gram-negative bacteria	[118]
		• Mature hBD-1 under influence of thioredoxin is modified and produces redhBD-1 by elimination of disulfide bonds	[119]
		• NET formation induces the production of hBD-2 by keratinocytes in psoriasis	[120]
Mast cell	Histones	• Produced and released as the component of MCETs when MCs are exposed to intra/extracellular pathogens such as L. monocytogenes, Streptococcus pyogenes, and Leishmania	[12, 59, 63]
		• Histones have been reported to have antimicrobial properties, i.e., H3 and H4 histones cause membrane damage accompanied with blebbing and pore formation, while H2B disrupts the integrity of the cell	[121]
	Tryptase	• The most abundant protease found in the MC secretory granules, that is associated with the pathologies including allergy, inflammation, and tissue remodeling	[122]
		• Tryptase acts as a ligand for protease activated receptor-2 (PAR-2); the cleavage of PAR-2 is the activation mechanism through which tryptase activates PAR-2	[123, 124]
		• Tryptase β has been reported to effectively detoxify various venoms	[125]
		• Since MCs are the only producers of tryptase and that tryptase is a component of MCETs, immunofluorescence microscopy to identify tryptase and DAPI staining together form the routine protocol to visualize MCETs.	[8]
	LL-37	• LL-37 is formed from an 18-kDa precursor protein (hCAP-18)	[126]
		• Other immune cells rather than MCs produce LL-37 including monocytes, neutrophils, MCs, NK cells, and B and T cells.	[126]
		• LL-37 possesses antimicrobial activity, induces the release of nucleic acids by MCs however, it has been reported not to play a role in formation of MCETs.	[61]
		• Its effectiveness against bacteria is due to its pore-forming activity	[62]