PhD Scientific Days 2025

Poster Session I. - A: Molecular Medicine

Interrogation of Antibacterial and Autoimmune Responses Using Gene-edited Neutrophilic Granulocytes

Name of the presenter

Havasi Márk

Institute/workplace of the presenter

Élettani Intézet, Semmelweis Egyetem

Authors

Márk Havasi¹, Csaba Papp², Attila Gácser², Áron Pánczél¹, Attila Mócsai^1,3

1: Department of Physiology, Semmelweis University, Budapest, Hungary;
2: Institute of Biology, University of Szeged; Szeged, Hungary
3: HUN-REN-SE Inflammation Physiology Research Group, Budapest, Hungary

Text of the abstract

Introduction:
Although the role of several genes was thoroughly characterized in the migration and immune-complex induced activation of neutrophilic granulocytes (neutrophils), this process is considerably hampered by the unfeasibility of direct genetic manipulation of this cell type.
Aim:
We tried to circumvent this obstacle using CRISPR-editing of in vitro maintained, conditionally immortalised neutrophil progenitors (so-called HoxB8 cells), followed by the characterization of the resulting phenotypes in vivo.
Methods:
HoxB8 progenitors were genetically targeted using CRISPR/Cas and transplanted into irradiated recipient mice for in vivo neutrophilic differentiation. Neutrophil host-defence functions were tested in a Staphylococcus-induced peritonitis model and immune complex- induced inflammation was examined using the K/B×N serum transfer arthritis and reverse passive Arthus (RPA) reaction models.
Results:
Using our above approach, we successfully generated circulating neutrophils deficient for the Itgb2, Fcer1g or Cybb genes. Survival in our bacterial infection model was impaired in the absence of Itgb2 and Cybb genes but seemed unaffected by Fcer1g-disruption. On a mechanistic level we found severely reduced infiltration of the infected site by Itgb2-deficient neutrophils and higher intracellular bacterial burden in case of Cybb deficiency, suggesting delayed degradation of phagocytosed microbes. Inflammatory cytokine production was exaggerated in both genotypes with decreased survival. On the other hand, immune complex-induced inflammation both in terms of macroscopic signs and cytokine-production was mitigated in the absence of Itgb2 or Fcer1g but not in that of Cybb in both models (K/B×N and RPA) used.
Conclusion:
Based on our results, CRISPR-modified HoxB8 progenitors represent a rapid and efficient way to examine neutrophil functions at a single protein resolution, allowing the effect of targeted genetic manipulation on neutrophil functions to be examined in a living organism in various disease models, facilitating discovery of novel regulators of innate immune responses potentially differently involved in antibacterial and autoimmune processes and thus of great therapeutic importance.

Funding: EKÖP-2024-266.