ANCA-dependent neutrophil activation induces a multitude of cellular events including the generation of NETs. NETS are lattice-like structures containing DNA, histone and neutrophil granule proteins including both most common ANCA antigens MPO and PR3. NETs are thought to play a major role in AAV but their exact roles and regulation remain to be described.
We recently put in evidence that NETs generation is regulated by the activation of a programmed form of cell death (necroptosis) via receptor-interacting protein kinase 1 and 3 (RIPK1/3) and the mixed lineage kinase domain-like protein (MLKL). We further showed that NETs generated by ANCA-stimulated neutrophils caused direct damage to endothelial cells in vitro. This effect was prevented by I) pharmacological inhibition of necroptosis, II) enzymatic NETs degradation and III) genetic deletion of RIPK3. In vivo, mice with a genetic deletion of RIPK3 or MLKL were protected from the development of AAV.
We could also demonstrate that NETs provided a scaffold for the activation of alternative complement, which in turn further contributed to the endothelial cell damage in vitro. Altogether, we identified a mechanistic link between ANCA-activated neutrophils, NETs, necroptosis, the alternative complement and endothelial damage. We are currently looking for additional pathways and mechanisms, which might be involved in the regulation and formation of NETs.