PhD Scientific Days 2025

Molecular Medicine V.

Phagocytosis, 2D migration and spreading activity of neutrophil-like cells derived from conditionally immortalized HoxB8-transduced myeloid progenitors

Name of the presenter

Görög Daniella

Institute/workplace of the presenter

Department of Physiology, Semmelweis University, Budapest, Hungary

Authors

Daniella Görög¹, Áron Pánczél¹, Attila Mócsai¹

1: Department of Physiology, Semmelweis University, Budapest, Hungary

Text of the abstract

Background: In conditionally immortalized HoxB8 progenitors, the estrogen-dependent HoxB8 transcription factor maintains the cells in a myeloid progenitor stage. Upon estrogen withdrawal, HoxB8 progenitors differentiate into neutrophil-like cells (HoxB8 NLCs), offering a suitable framework for genetic modification and functional analysis of the neutrophil lineage. These progenitors were subjected to targeted CRISPR/Cas9 gene editing to disrupt the Cybb, Fcer1g, Itgb2 and Syk genes (CRKO cells).

Aims: We aimed to investigate the phagocytic, 2D migratory and spreading activities of wild-type HoxB8 NLCs compared to progenitor cells and bone marrow neutrophils. We also sought to investigate the functionality of genetically modified HoxB8 NLCs (CRKO cells) lacking Cybb, Fcer1g, Itgb2 and Syk.

Methods: The phagocytosis of GFP-expressing Staphylococcus aureus was followed by flow cytometry. Spreading was tested by stimulating the cells with TNFα or Pam3CSK4 on a fibrinogen surface, or by plating the cells on immobilized Ig immune. Cell shape changes assessed by phase contrast microscopy. 2D chemotactic migration of HoxB8 NLCs were tested in fibrinogen-coated IBIDI µ-slide Chemotaxis chambers towards an fMLP or WKYMVm gradient.

Results: Wild-type HoxB8 NLCs but not HoxB8 progenitors were able to phagocytose S. aureus bacteria. Itgb2 CRKO cells showed partially reduced phagocytic activity. Disrupting Cybb, Fcer1g or Syk did not substantially affect phagocytosis. HoxB8 NLCs were also able to spread over the fibrinogen surfaces upon TNFα or Pam3CSK4 stimulation, or over an immobilized IgG immune complex surface. The Itgb2 and Syk CRKO mutations abrogated all spreading responses, whereas the Fcer1g CRKO cells were only defective on immune complex. We have also been able to test the 2D chemotactic migration of HoxB8 NLCs and found that the Syk CRKO cells show a slightly elevated chemotactic response.
Conclusion: Hoxb8 NLCs derived from conditionally immortalized and genetically modified HoxB8 progenitors provide a robust platform to investigate the mechanisms of neutrophil-mediated immune responses.

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).