Dr. rer. nat. Alexandra Ehrens (Bonn / DE), Celia Nieto Perez (Bonn / DE), Andrea Krez (Bonn / DE), Frederic Risch (Bonn / DE), Benjamin Lenz (Bonn / DE), Nina Offermann (Bonn / DE), Marianne Koschel (Bonn / DE), Melania Capasso (Bonn / DE), Achim Hoerauf (Bonn / DE), Prof. Dr. Marc P. Hübner (Bonn / DE)
Abstract text
Introduction
Eosinophils as well as their cytotoxic granules are important for protective immunity against parasitic filarial nematodes, which can be mediated through extracellular DNA trap cell death (ETosis). Thereby, intracellular DNA is explosively released, entrapping and killing pathogens. Neutrophil ETosis (NETosis) can be induced in a NADPH oxidase (NOX) or Ca2+-dependent manner. Moreover, the activation of the inflammasome can lead to NETosis with gasdermin D (GSDMD) being an essential enzyme in forming pores and thus, releasing the DNA traps. However, eosinophil ETosis (EETosis) is understudied and the exact signaling cascade for filaria-induced ETosis is not known yet.
Objectives
The current study investigated the signaling pathway during EETosis in response to microfilariae (MF) using the rodent filaria Litomosoides sigmodontis.
Materials & Methods
To study EETosis in response to MF, eosinophils were generated from the bone marrow of wild-type and different knockout mice (NOX, caspase-1, ASC, NLRP3 and AIM2) and cells were stimulated with MF. EETosis was investigated using fluorescence microscopy as well as DNA quantification methods.
Results
Our results demonstrate that MF induce EETosis in a NOX-dependent manner since eosinophils generated from NOX knockout mice did not release DNA traps in response to MF and PMA. In addition, similar to PMA, histone citrullination occured during a later state of MF-induced EETosis. Moreover, the canonical inflammasome pathway is involved during EETosis. The canonical inflammasome assembles after activation of a sensor molecule, which recruits pro-caspase-1 and the adaptor molecule ASC. As an executing caspase, caspase-1 is able to cleave GSDMD, which forms pores in the cellular membrane. During MF-induced EETosis, active caspase-1 was detected in DNA traps. Inhibition of caspase-1 as well as eosinophils generated from caspase-1 or ASC knockout mice failed to release DNA in response to MF. Using eosinophils from AIM2 and NLRP3 knockout mice, we identified AIM2 and not NLRP3 as the responsible inflammasome sensor, since AIM2 knockout eosinophils, in contrast to NLRP3 knockout eosinophils, were unable to form DNA traps in response to MF. Moreover, we visualized GSDMD during EETosis and observed pore formation in the membrane and the nucleus.
Conclusion
Our results indicate that EETosis in response to microfilariae is dependent on NOX and the AIM2-caspase 1 inflammasome.