PhD Scientific Days 2025

Molecular Medicine IV.

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

Name of the presenter

Jex Emiliána

Institute/workplace of the presenter

Semmelweis University, Department of Physiology

Authors

Emiliána Jex¹, Áron Pánczél¹, Attila Mócsai¹

1: Semmelweis University, Department of Physiology

Text of the abstract

Introduction: 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 target genes, including Syk (Syk CRKO), Itgb2 (Itgb2 CRKO), Fcer1g (Fcer1g CRKO), and Cybb (Cybb CRKO).
Aims: The aim of our experiments was to test the respiratory burst response, the degranulation and the migration capacity of the various CRKO HoxB8 NLCs.
Methods: HoxB8 NLCs were generated by estrogen withdrawal along with G-CSF treatment for 4 days. We used two different methods to measure respiratory burst. In one of the methods, HoxB8 NLCs 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, W-peptide or by the non-physiological PMA stimulus. Reactive oxygen species (ROS) production and degranulation was measured by flow cytometry. In vitro migration of HoxB8 NLCs was investigated using a Transwell assay.
Results: Wild-type HoxB8 NLCs but not HoxB8 progenitors were able to produce ROS under all tested stimulation conditions. Upon immune complex stimulation, all CRKO NLCs failed to produce superoxide. Only Fcer1g CRKO NLCs produced ROS upon integrin-mediated stimulation. Wild-type, Syk CRKO, Itgb2 CRKO, Fcer1g CRKO HoxB8 NLCs were able to produce ROS upon fMLP, W-peptide and PMA stimulation, only Cybb CRKO HoxB8 NLCs failed to produce ROS. All CRKO genotypes showed a normal degranulation response. Itgb2 CRKO NLCs failed to migrate to fMLP.
Conclusions: Our results indicate that HoxB8 NLCs can produce superoxide upon various stimulation and are able to degranulate and migrate toward chemoattractants. 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).