The great obstruction The mechanism of neutrophil extracellular trap (NET) formation and their role in occlusive disorders

Field: Immunology and Microbiology (Life Sciences)

A healthy human body encounters several unicellular and multicellular organisms daily. Combatting invading organisms relies heavily on our innate immune system, the first line of defense against these invading entities. One of the first responders from the innate immune system are the polymorphonuclear (PMN) leukocytes or neutrophils. They can be considered as the armed soldiers of our innate immune system with various strategies such as degranulation, phagocytosis, and neutrophil extracellular trap (NET) formation up their sleeves to eliminate pathogens and limit and invasion. The generation of neutrophils occurs in the bone marrow, and in circumstances of infection, the production of neutrophils can be elevated 10- fold up to 1012 cells per day. Interestingly, about 55-60% of the bone marrow is committed to producing neutrophils. 50-70% and 10-25% of all circulating leukocytes in humans and mice are neutrophils, respectively. In the circulation, mature neutrophils harbor a segmented nucleus and are about ~10-15 μm in diameter, possessing a cytoplasm with secretory vesicles and granules. The rapid localization of neutrophils to the site of infection is essential for a successful defense. Traditionally, neutrophils were only thought of as fighting against pathogenic organisms. However, they also have immune regulatory functions and have been associated with numerous pathologies. Any impairment in the effector functions of neutrophils, specifically the formation of NETs, can lead to adverse consequences. NETs are web-like structures unleashed by neutrophils to immobilize and neutralize pathogens, but an accelerated and/or dysfunctional NETs formation can lead to occlusive disorders. Recent findings have demonstrated that the functions of NETs extend beyond the roles of defense and protection. In addition, these reports have also widened our purpose to investigate the mechanisms and the pathologic formation of NETs. With the same context in mind, through this dissertation I seek to achieve and report : (I) The elucidation of the molecular mechanism that modulates the unlocking of condensed chromatin during the formation of NETs. We report the influx of calcium as necessary into the cytoplasm to activate the PAD-4 enzyme which triggers the cysteine protease calpain-1 to cleave the outer nuclear membrane protein nesprin-1 and weakens the nuclear membrane to promote NET release. (II) To present a collective report that discusses current literature focussing on the role of NETs in various occlusive disorders where we summarize the ‘side job’ of the extracellular chromatin during NETs formation and its role in disease pathologies specifically those of occlusive diseases such as coronavirus disease 2019 (COVID-19) and Meibomean gland dysfunction (MGD). (III) To categorize heterogeneous pulmonary vessel occlusions and a novel detection technique in patients with COVID-19; here we present the utilization of native endogenous fluorescence or NEF from hemoglobin-alpha releasing fluorescence signal from occluded vessels and three types of occlusive morphologies whose occurrence is influenced by age and/or sex. (IV) A step-by-step protocol to demonstrate to induce allergic eye disease (AED) caused by NETs occluding ocular Meibomian glands (MG) in established murine models and novel approaches to obtain conjunctiva and cornea from AED mice.