A71-14 Impaired Sphingolipid Synthesis Reduces Rhinovirus Entry in Airway Epithelial Cells

Rationale

Asthma-associated genetic variations regulate ORMDL3, influencing sphingolipid metabolism. Variants at the 17q21 locus are also associated with wheezing illness caused by human rhinovirus (HRV) in early life and show a significant interaction effect on asthma risk. Although the 17q21 locus can affect sphingolipid de novo synthesis through increased expression of ORMDL3, the effect of reduced sphingolipids on airway epithelial cells remains unclear. We investigate the impact of impaired sphingolipid synthesis on HRV infection of the airway epithelial cells.

Methods

To assess the effect of impaired sphingolipid synthesis in airway epithelial cells, we generated serine palmitoyltransferase (SPT)-deficient BEAS-2B cells by genetically modifying SPTLC1 using CRISPR-Cas9. Cell topology and stiffness of the SPT-deficient BEAS-2B cells were evaluated by atomic force microscopy (AFM). To pharmacologically decrease SPT activity, the airway epithelial cells were treated with the SPT inhibitor myriocin, infected with HRV16, and viral entry was quantified by real-time PCR. Cell surface expression of ICAM-1 (the receptor for HRV16) and GM1 (a lipid raft marker) was analyzed by flow cytometry to assess potential changes in receptor availability and lipid raft-associated entry mechanisms. Gene expression was analyzed by RNA sequencing (RNA-seq).

Results

Genetic knockdown of SPTLC1 resulted in increased cellular stiffness (p<0.05) and reduced HRV entry (p<0.001) without affecting ICAM-1 cell surface expression or virus binding. HRV entry remained ICAM-1 dependent but was associated with reduced lipid raft clustering. Transcriptomic analysis revealed that SPT deficiency altered endocytosis and actin cytoskeleton pathways, linking altered membrane trafficking and cytoskeletal dynamics to increased stiffness and constrained viral internalization. Pharmacologic SPT inhibition with myriocin similarly reduced HRV entry across multiple airway epithelial cells (p<0.01 in BEAS-2B, p<0.05 in immortalized human airway epithelial cell line BCi-NS1.1, and p<0.05 in primary human nasal epithelial cells differentiated at the air-liquid interface). RNA-seq analysis revealed altered endocytic and cytoskeletal gene programs in the myriocin-treated nasal cells while largely preserving antiviral immune signaling.

Conclusions

Impaired sphingolipid synthesis by decreased SPT activity in airway epithelial cells suppresses rhinovirus entry into airway epithelial cells, potentially through sphingolipid-dependent regulation of endocytosis and actin cytoskeletal dynamics. These findings identify sphingolipid biosynthesis as a key regulator of epithelial biomechanics and membrane trafficking that affects HRV entry, highlighting SPT as a potential therapeutic target to limit HRV infection.

This abstract is funded by: R01 AI171390-01A1