PhD Scientific Days 2026

Poster Session 1.A - Molecular Medicine

Respiratory burst and degranulation of neutrophil-like cells differentiated from CRISPR/Cas9 modified conditionally immortalized myeloid progenitors

Name of the presenter

Jex, Emiliána

Institute/workplace of the presenter

Department of Physiology, Semmelweis University

Authors

Emiliána Jex¹, Áron Pánczél¹, Attila Mócsai¹
1: Department of Physiology, Semmelweis University

Text of the abstract

Neutrophils are difficult to be manipulated by genetic approaches. This has been overcome by conditional immortalization of mouse myeloid progenitors (HoxB8 progenitors), from which HoxB8 neutrophil like cells (NLCs) can be differentiated. We have set up a system for the CRISPR/Cas9-based genetic manipulation of HoxB8 progenitors and were able to delete several isoforms of a protein family, including the PKC-α (Prkca CRKO), the PKC-β (Prkcb CRKO), the PKC-δ (Prkcd CRKO), and the PKC-ε (Prkce CRKO).
The aim of our experiments was to investigate the respiratory burst and the degranulation of the various CRKO HoxB8 NLCs.
HoxB8 NLCs were generated by estrogen withdrawal along with G-CSF treatment for 4 days. For the respiratory burst experiments, the cells were stimulated by immobilized IgG immune complexes, proinflammatory agonists on an integrin ligand surface, or by PMA stimulus. Superoxide production was measured by a cytochrome c reduction assay. For the other respiratory burst assay and degranulation experiments, the cells were stimulated by fMLP, or by PMA stimulus. Reactive oxygen species (ROS) production and degranulation was measured by flow cytometry.
Wild-type HoxB8 NLCs were able to produce superoxide upon various stimulation. Upon these stimulation, Prkcb CRKO and Prkcd CRKO NLCs showed reduced superoxide production. Wild-type, Prkca CRKO, Prkce CRKO NLCs were able to produce ROS upon PMA stimulation, but in case of Prkcb CRKO and Prkcd CRKO HoxB8 NLCs the ROS production was decreased. The wild-type, Prkca CRKO, and Prkce CRKO NLCs exhibited a normal degranulation response, whereas the degranulation reaction was reduced in Prkcb CRKO and Prkcd CRKO NLCs.
Our results indicate that PKC-β and PKC-δ may play a role in the respiratory burst and degranulation response of HoxB8 NLCs. Therefore, our CRISPR-based targeting strategy is suitable for deleting functionally relevant proteins from NLCs.
Funding: Hungarian National Research, Development and Innovation Office (KKP-129954, K-146160 and TKP2021-EGA-24), the HUN-REN Hungarian Research Network (0207007) and the Hungarian Academy of Sciences (LP2024-16/2024).