Abstract
Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but 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 studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent 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). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.
Highlights
Neutrophilic granulocytes are the most abundant type of circulating white blood cells
To investigate the effect of substrate elasticity/stiffness on NETosis, freshly isolated human neutrophils were seeded on PAA gels coated with either collagen I or fibrinogen which are both known to interact with integrins on neutrophils [30, 37]
Neutrophils on 4 or 128 kPa PAA collagen-I- or fibrinogen-coated gels were incubated with Roswell Park Memorial Institute (RPMI) media only and did not show significant neutrophil extracellular traps (NETs) formation (Supplementary Figures S1, S2)
Summary
Neutrophilic granulocytes are the most abundant type of circulating white blood cells. In a process termed NETosis, they release neutrophil extracellular traps (NETs), web-like structures composed of decondensed chromatin decorated with antimicrobial proteins [1, 2]. During NETosis, the nuclear chromatin swells until both the nuclear envelope and the outer cell membrane rupture. NETosis is considered an important immune defense mechanism as neutrophils can bind and kill bacteria and other pathogens via NETs. Apart from physiological stimuli such as pathogens, chemokines (e.g., CXCL8), activated platelets or urea crystals there are several additional NET-inducers like phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharides (LPS), which induce NETosis in vitro [4]. The question which environmental factors play a role in this process and may influence the course of diseases is highly important
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