Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation

Abstract

Neutrophils are the most abundant type of white blood cells. As part of their immune defense mechanisms they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well understood.Here, we study how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide gels with varied elasticity (Young's modulus E) within the physiologically relevant range from 1 kPa to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen).Neutrophils were cultured on these substrates and stimulated with chemical inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). Additionally, we show that LPS-induced NETosis correlates with the cell contact area indicating that adhesion signaling and NETosis pathways are connected. On the other hand, PMA-induced NETosis did not require adhesion signaling.In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

Identification of a Novel HIF-1α-αMβ2 Integrin-NET Axis in Fibrotic Interstitial Lung Disease

“…Moreover, several studies have shown that α M β 2 blockade reduced NET release ( 39 , 40 , 69 , 70 ), which indirectly supports our work. Our findings that α M β 2 interaction with ligand may regulate NET formation builds on earlier findings, which showed that PMA stimulation led to high levels (>80%) of chromatin decondensation (a prelude to NET formation) and was not affected by substrate ( 41 ). In contrast, LPS stimulation led to lower basal levels of decondensation (~20%) and levels increased with matrix stiffening and increased cell spreading on β 2 and β 1 integrin ligands.…”

“…Having found an effect on NET release, we next examined integrin activation and neutrophil adhesion, which are also implicated in NET induction ( 39 – 41 ). We measured neutrophil adhesion to primary human endothelial cells in the absence or presence of PMA (a general integrin activator) or LPS (to mimic infectious stimuli), stimuli that induce NETs via distinct pathways ( 42 ).…”

“…Our analysis of NET formation ultimately focuses on the end stages culminating in NET release into cell supernatants. Further work could explore the effects of hypoxia upon the preceding stages such as cell spreading and chromatin decondensation/nuclear swelling ( 41 , 87 ), to better understand how hypoxia affects the entire NET formation process.…”

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation

“…This manuscript has been released as Pre-Print at BioRxiv (56). We thank Andreas Janshoff and Claudia Steinem for fruitful discussions and support.…”

Reply to Liu: The disassembly of the actin cytoskeleton is an early event during NETosis

“… Liu’s flow cytometry analysis requires cells in suspension, while ours and other studies report actin disassembly during NETosis of adherent cells ( 1 , 3 , 5 ). Neutrophils in situ adhere via integrins to extracellular matrix, other cells, or clots, and integrin engagement to an immobile ligand contributes to NETosis ( 6 , 7 ). While PMA induces integrin activation, the absence of immobile ligands in suspension would fail to initiate integrin-mediated activation of actin regulatory pathways ( 8 ) during NETosis.…”