Tuesday, August 25, 2015

Neutrophil throwing a NET


Across the mammalian class the most common defense cell are the neutrophils. In humans these cells constitute about 40% of the circulating WBC in blood. Their function is to seek and destroy dead cells, pathogens and debris. They form a kind of first line defense against the incoming pathogens, long before any other specialized cells come into the battlefield. The neutrophils predominantly ingest the pathogens and subsequently degrade the pathogens inside their phagosome. Interestingly, certain pathogens have made use of this phenomenon. For example, Salmonella can sit inside neutrophils and avoid activation of phagosome thereby using the neutrophil as a free ride and a safe house.

Pathogens are not something that are so easy to catch up with. Funny to describe it seems but at the scale of cells, pathogens can be slippery to catch and sometimes they may simply disperse and run. If you haven't seen the famous video of a neutrophil chase you must see it once. It was known for a longtime that neutrophils have a limited life span. Once the neutrophil has digested enough or has encountered pathogen, it will undergo self degradation. In physiological context, this happens at a very small fraction that we don't notice. But when scaled up the neutrophil liquefaction is so high that it forms a thick matter or what is known commonly as the pus. It was believed that the pus traps agents such as bacteria thus making it harder for the organism to run around.

Till recently about a decade ago, it was believed that neutrophil functions at two fronts. 1st, they engulf bacteria and debris and kill the invaders intracellularly. 2nd, upon stimulation by inflammatory mediators they can degranulate thereby releasing molecules such as defensins which then acts as antimicrobial at an extracellular level. It is now clear that there is a third mechanism. It appears that neutrophils can throw up internal chemical contents with its DNA to form a netted structure to catch up the pathogens. In other words kind of throwing a net to catch the contents. In contrast to what was earlier believed this was not a passing event but a thoroughly regulated process. For lack of a better explanatory word, it was called as Neutrophil Extracellular traps (NETs) and the process came to be known as Netosis. Precisely, NETs are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens.

Careful analysis of the contents of NET showed that they contained molecules from primary, secondary and tertiary granules of neutrophils. Interestingly, these NETs don't contain cytoplasmic materials such as actin and tubulin. This is the first indication that the NET is not just the liquefacted neutrophil product rather a well programmed and directed effort to capture bacteria. Interestingly, more recently, it appears that other WBC such as eosinophils can also possess similar ability to form extracellular traps. Hence, the more recent nomenclature is "ETosis" to reflect extracellular traps.

Fig 1:Mechanism of NET release. Source
It is not clear on how exactly, the process starts. However, it appears that bacterial components and inflammatory mediators can kick start the process. For example, within 10 min of stimulation with S aureus, NETs are produced. The key molecule in the process is PAD4 (Peptidylarginine deiminase 4), a marker of Netosis. In brief, PAD4 causes citrullination of histones leading to chromatin opening. Other set of molecules dissolve nuclear membrane and opened chromatin forms a net-like structure. This is ejected from the cell (Like casting the net). The bacteria trapped is now immobilized making it easy for subsequent attack. Interestingly, content molecules of NET have antimicrobial molecules such as defensins and proteases. Further, the histone itself has been shown to have toxic property in very low concentrations. All this time the the neutrophil ghost cell is still there. (This is what makes it different from the classical liquefactive process). But most of the part of NET is interlinked DNA which forms the NET. Now you see the importance of DNAase produced by S aureus, and its importance in viruelnce. The DNAase allows the bacteria to escape from the DNA trap. Many other bacteria are also known to make DNAase which functions similarly.

As already explained Netosis is a highly regulated process. Presence of DNA floating for a longtime around is not such a good thing. Once the pathogens are trapped, macrophages come into the picture and clean up. It is being theorized that improper cleanup of the process can lead to autoimmune conditions such as SLE where the antibodies are developed against DNA and (or) nuclear antigens. Formation of NET has been implicated in several infections and also in the process of disease.

NETs probably have other roles that is yet to be deciphered. For example, In a recently published research, it was shown that monocytes with NETs prepared from cholesterol crystal-stimulated neutrophils induced a modest increase in IL-1β, whereas pretreatment of monocytes with NETs followed by stimulation with cholesterol crystals induced very good IL-1β secretion, suggesting that NETs provide the priming signal for cholesterol crystal-induced maturation of IL-1β in macrophages.

Isn't that now an interesting story.

Brinkmann, V. (2004). Neutrophil Extracellular Traps Kill Bacteria Science, 303 (5663), 1532-1535 DOI:10.1126/science.1092385

Guimarães-Costa AB, Nascimento MT, Wardini AB, Pinto-da-Silva LH, & Saraiva EM (2012). ETosis: A Microbicidal Mechanism beyond Cell Death. Journal of parasitology research, 2012 PMID:22536481

Branzk N, & Papayannopoulos V (2013). Molecular mechanisms regulating NETosis in infection and disease. Seminars in immunopathology, 35 (4), 513-30 PMID: 23732507

Donna L. Bratton, & Peter M. Henson (2012). Neutrophil Clearance: when the party’s over, cleanup begins Trends Immunol., 32(8), 350-357 : PMCID: PMC3151332