The Septin Cytoskeleton is a Novel Regulator of Intestinal Epithelial Barrier Integrity and Mucosal Inflammation

The intestinal epithelium comprising a single layer of polarized intestinal epithelial cells IECs connected by cell-cell junctions represents the bodys largest interface with the external environment. This monolayer is crucial for nutrient uptake and defense against pathogens and its disruption may lead to mucosal inflammation and increased susceptibility to colorectal cancer. Cytoskeletal components are important regulators of cell-cell junction integrity and epithelial polarity. While the functions of actin microtubules and intermediate filaments in these processes are well-documented the contribution of the filament-forming septin family is poorly understood. This highlights an important knowledge gap since dysregulation of septins especially septin 9 SEPT9 in humans has been linked to colon cancer pathogenesis. To address this gap we investigated the role of SEPT9 in regulating intestinal epithelial homeostasis and mucosal inflammation using SEPT9-NeonGreen knockin mice inducible intestinal epithelial specific SEPT9 knockout KO mice SEPT9-knockout human intestinal epithelial cell IEC and SEPT9-interactomics. We observed accumulation of SEPT9 at epithelial apical junctions in intestinal mucosa of SEPT9-NG mice and immunolabeled human IEC cells where it colocalized with tight junction TJ proteins ZO1 claudin-3 and non-muscle myosin IIC NM IIC all of which were found to be part of the SEPT9 interactome in DLD-1 human colonic epithelial cells. Loss of SEPT9 in mouse intestinal epithelial cells in vivo and human IEC in vitro resulted in intestinal epithelial barrier disruption according to dextran-permeability and transepithelial electrical resistance measurements. We isolated ileal and colonic epithelial cells of SEPT9 cKO mice and their controls and performed bulk RNA sequencing analysis of these cells. The analysis revealed transcriptional reprograming of SEPT9 deficient IEC manifested by large numbers of upregulated and downregulated genes. The pathway enrichment analysis revealed upregulation of transcripts related to acute phase and inflammatory response and down-regulation of contractile genes in colonic mucosa of SEPT9 cKO mice