Poster CC17 - Genetic engineering of O-glycan structures to enhance neutrophil capture during acute inflammation

Introduction: Neutrophils, as frontline defenders in the immune system, play an essential role in initiating responses against pathogens and regulating inflammation. Efficient recruitment of neutrophils to inflammation sites involves complex interactions between cell surface glycoproteins and endothelial cells. O-glycans on neutrophil surfaces are pivotal in these interactions, particularly during high-demand situations where precise control is crucial to prevent chronic inflammation. This study targets two specific α(2,3)sialyltransferases, ST3GAL1 and ST3GAL2, which are instrumental in synthesizing sialyl Lewis-X (sLeX), a critical ligand for selectin-mediated cell adhesion[1]. Using a Cas9-expressing neutrophil model (HL-60 cas9), we aim to knock out these genes to investigate their roles in modifying neutrophil adhesiveness by altering O-glycan expression. The hypothesis is that silencing ST3GAL1 and ST3GAL2 will enhance neutrophil recruitment by increasing sLeX levels, making neutrophils more 'sticky' to the endothelium [2].This approach promises to deepen our understanding of the glycosylation pathways critical for immune response modulation and holds potential therapeutic implications for effectively managing inflammation .

Materials and

Methods: In this study, we utilized our previously established HL-60 cas9 cell line to explore the impact of ST3GAL1 and ST3GAL2 knockouts on neutrophil recruitment. Utilizing CRISPR/Cas9 technology, we used specific synthetic single-guide RNAs (sgRNAs) from Synthego to target and disrupt these glycosyltransferase genes. Each sgRNA included a 17-21 nucleotide sequence specific to the target gene, coupled with a 40 nucleotide Cas9 handle hairpin and a 40-nucleotide sequence from Streptococcus pyogenes to enhance the stability of the synthetic RNA. Electroporation was performed using the Neon Transfection Kit (ThermoFisher), which applied optimized parameters (1600V, 10ms, 3 pulses) to ensure the efficient delivery of sgRNAs into the cells. Following this, cells were maintained in IMDM Medium supplemented with 10% FBS, 1% amino acids, and 1% GlutaMAX to support cell growth and recovery. After transfection, we employed flow cytometry, utilizing a variety of fluorescently labeled antibodies and lectins to analyze cell surface changes. Notably, there was a significant increase in PNA lectin binding to the transfected cells, which indicates a decrease in sialylation, likely resulting from the successful knockout of the target genes. Then, we sorted out FACSAria (BD Biosciences) higher than 80% of the highest-expressing PNA FITC-stained knockout cells. Genomic DNA was extracted from the transfected HL-60 cells, and the DNA region encompassing the CRISPR target site was amplified via PCR. The resulting PCR products were then analyzed using the SURVEYOR nuclease assay. This dual approach allowed us to verify the mutation spectrum and the specificity of gene knockouts.

Results, Conclusions, and Discussions: Our CRISPR/Cas9-mediated knockout of ST3GAL1 and ST3GAL2 in HL-60 cells led to significant glycosylation changes, especially in the cell surface structures of sialyl Lewis-X (sLeX) and Lewis X (LeX). To assess these modifications, we employed flow cytometry, utilizing a range of fluorescently labeled antibodies and lectins specifically designed to detect variations in these glycan structures. The antibodies used included HECA (anti-CLA) and CSLEX-1 (anti–CD15s/Sialyl Lewis-X), which target sLeX epitopes, and HI (anti-CD15/Lewis-X), which target LeX structures[1]. Additionally, Peanut Agglutinin (PNA) lectin, which binds to unsialylated Galβ1,3GalNAc structures, was utilized to explore changes in glycosylation that may not involve sialic acid. Flow cytometry and FACS analysis of our CRISPR/Cas9-edited HL-60 cells demonstrated significant increase in PNA lectinbinding demonstrating increased unsialylated glycan structures on the surface of the knockout cells. (Figure1)
Additionally, the analysis revealed an increase in sialyl Lewis-X (sLeX) expression, enhancing the cells' binding affinity to selectins, which is crucial for neutrophil recruitment to inflammation sites. In contrast, alterations in Lewis X (LeX) expression suggested a shift in glycan processing due to the knockout of ST3GAL1 and ST3GAL2, altering glycosylation patterns on the cell surface (Figure 2). This shift increases sLeX levels on the cell surface and changes the overall glycan composition, making the neutrophils more likely to adhere to endothelial cells through interactions with selectins which bind to specific carbohydrate structures like sLeX, play a critical role in the initial steps of neutrophil recruitment to sites of inflammation.
In conclusion, our findings underscore the crucial role of ST3GAL1 and ST3GAL2 in modifying glycosylation patterns that influence cell adhesion, providing insights into potential therapeutic strategies for modulating immune system responses. Moving forward, our studies will extend to assessing the binding capabilities of the mutant HL-60 cells to selectin-coated gels under shear flow conditions. The next phase of our research will utilize microfluidics to closely examine the effects of perturbing ST3Gal-1 or ST3Gal-2 on selectin-mediated adhesion. These advanced studies will enhance our understanding of the dynamic interactions between modified cells and the vascular endothelium, leading to deeper insights into the mechanics of cell trafficking in inflammation.

Acknowledgements (Optional):

References:
1. Mondal, N., et al., ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes. Blood, The Journal of the American Society of Hematology, 2015. 125(4): p. 687-696.
2. Liu, G., et al., Systems-level modeling of cellular glycosylation reaction networks: O-linked glycan formation on natural selectin ligands. Bioinformatics, 2008. 24(23): p. 2740-2747.

This work was supported by NIH NIGMS 143357 to ABJ