PhD Scientific Days 2024

Molecular Medicine I.

Respiratory Burst and In Vitro Migration of Neutrophil-like Cells Differentiated from CRISPR/Cas9 Modified Conditionally Immortalized Myeloid Progenitors

Text of the abstract

Introduction: Neutrophils are terminally differentiated cells, making them difficult to be manipulated by genetic approaches. This has been overcome by HoxB8 progenitors, from which HoxB8 neutrophil like cells (HoxB8 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 proteins, including the Syk tyrosine kinase (Syk CRKO), the CD18 integrin (CD18 CRKO), the Fc receptor γ-chain (FcRγ CRKO), and the gp91phox subunit of NADPH oxidase 2 (Nox2 CRKO).
Aims: The aim of our experiments was to test the in vitro respiratory burst response and the in vitro migration capacity of the various CRKO HoxB8 NLCs.
Methods: HoxB8 progenitors were maintained in the presence of estrogen. 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 the nonphysiological PMA stimulus. Reactive oxygen species (ROS) production was measured by a cytochrome c reduction assay. In vitro migration of HoxB8 NLCs was investigated using a Transwell assay with fMLP as chemoattractant. The number of transmigrated cells were measured by an acid phosphatase assay.
Results: Wild-type HoxB8 NLCs were able to mount a well-measurable ROS production. Syk CRKO HoxB8 NLCs failed to produce ROS upon immune complex or integrin-mediated stimulation but responded normally to PMA stimulation. FcRγ CRKO HoxB8 NLCs did not respond to immune complex stimulation but produced ROS upon integrin-mediated and PMA stimulation. The CD18 CRKO HoxB8 NLCs failed to produce ROS upon integrin-mediated stimulation and upon immune complex but responded mostly normally to PMA stimulation. The Nox2 CRKO HoxB8 NLCs were not able to produce ROS. All types of HoxB8 NLC except CD18 CRKO were able to migrate to fMLP.
Conclusions: Our results indicate that HoxB8 NLCs can produce superoxide upon various stimulation and able to migrate toward chemoattractant. 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) and the HUN-REN Hungarian Research Network (0207007).