We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like
dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid
chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells
and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA).
Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of
the cortical F-actin at spatially confined areas, the NET extrusion sites, effected
by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding
proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity
of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself
appeared to be proteolyzed by neutrophil elastase possibly to confine its activity
to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited
by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry
showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion
pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma
membrane of the remaining cell body. A similar distribution was observed for myosin
that concentrated together with cortical F-actin along the periphery of the remaining
cell body suggesting force production by acto-myosin interactions supporting NET expulsion
as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin.
Isolated human neutrophils displayed differences in their content of certain cytoskeletal
proteins. After stimulation neutrophils with high gelsolin content preferentially
formed “cloud”-like NETs, whereas those with low or no gelsolin formed long “filamentous”
NETs.
