Patients with COVID-19: in the dark-NETs of neutrophils

Abstract

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Fig. 1. Potential mediators for the induction of NET-formation in the infected and inflamed tissues.

Viruses (SARS-CoV-2), ROS, calcium oxalate, co-infecting microorganisms, cytokines and chemokines, cationic antimicrobial peptides, nanodiamonds, monosodium urate (MSU), and platelets reportedly induce NET formation. See main text for references. Original illustration from the authors.

Fig. 2. Mechanisms of NET formation.

Pathways that regulate NET formation (see body text for references). Pattern recognizing receptors (PRR) initiate NADPH oxidase activation and a spike of cytosolic calcium activating neutrophil peptidylarginine deiminase 4 (PADI4) causing histone citrullination (yellow circle) and DNA decondensation. Chromatin and/or mitochondrial DNA is expelled and form NETs. Several necrotic cell death pathways may contribute to NETosis. Necroptosis involves RIPK1/RIPK3-mediated activation of MLKL and plasma membrane permeabilization contributing to the release of NETs. Pyroptosis involves canonical or non-canonical inflammasome activation by the caspases-1 or 4, respectively. Caspase-1 and 4 as well as NE cleave GSDMD and generates the N-GSDMD fragment with a pore-forming activity that enables the release of NETs. In addition, autophagic processes contribute to the release of NETs. Original illustration from the authors.

Fig. 3. The role of aggregation and degradation of NETs in vascular occlusions.

Increased numbers of patrolling neutrophils in inflamed tissues form aggregated neutrophil extracellular traps (aggNETs). These are prone to occlude the ducts and glands of the pancreas, gall bladder, and ocular surface. The occlusions precipitate organ pathogeneses like pancreatitis and cholelithiasis. AggNETs also occlude blood vessels in particular the microvasculature of lungs, liver, kidney, heart, and thus cause pathogenesis. Original illustration from the authors.

Fig. 4. NETs induced by SARS-CoV-2.

NET formation of human blood-derived neutrophils after treatment with SARS-CoV-2. Immunofluorescence staining of NETs was done using antibodies against elastase (red) and DNA-histone1-complexes (green), with a counterstain of DNA (blue). Yellow staining indicates colocalization of NETs (histone-DNA fibers) with elastase. The Bars represent 25 µm (left) and 50 µm (middle and right). Original illustration from the authors.

Fig. 5. Occlusion of pulmonary vessels by aggNETs in COVID-19.

Occlusion of small and intermediate-sized pulmonary vessels in COVID-19 published by Leppkes et al 2020 [59]. The former is marked by asterisks and the latter by a white frame. Note, that large fields of the (micro)-vasculature are occluded by NETs identified by extracellular neutrophil elastase (green). The nuclei of the cells were stained with propidium iodide (red). The bar represents 1000 µm. Original illustration from the authors.