OC 19.5 - Epithelial Neuropilin-1 is regulated by the gut microbiota promoting vascularization of small intestinal villus structures

Background: Neuropilin-1 (NRP1) is a multifunctional receptor involved in vascular biology. Indeed, NRP1-knockout mice are embryonically lethal due to defective blood vessel development. In the endothelium, NRP1 acts as co-receptor for vascular endothelial growth factor (VEGF) receptor-2 and affects lacteal maturation through class-3 semaphorin signaling, whose member SEMA3A suppresses VEGF-mediated angiogenesis. Intriguingly, NRP1 expressed in colon adenocarcinoma epithelial cells promotes tumor angiogenesis. In the small intestine (SI), the symbiotic microbiota stimulates the vascularization of villus structures.

Aims: Since our preliminary results identified epithelial NRP1 to be regulated by gut microbiota, to unravel the underlying molecular mechanisms. To investigate the possible effects of epithelial (and not endothelial) NRP1 on the SI vascular phenotype at steady-state conditions.

Methods: The role of microbiota on SI epithelial NRP1 expression was investigated using germ-free, conventionally-raised and toll-like receptor-2 (Tlr2)-knockout mice, along with cell cultures. Tissue-specific effects of SI epithelial NRP1 were unraveled generating a conditional knockout mouse model (Nrp1ΔIEC). Blood and lymphatic vessels in the distal SI of Nrp1ΔIEC mice were visualized by immunofluorescence staining. mRNA and protein levels of NRP1, PECAM-1, SEMA3A and VEGF-A were quantified either by qPCR, Western Blot, flow cytometry, immunofluorescence, or ELISA assays. Statistical analyses were performed by independent samples Student’s t test.

Results: The gut microbiota efficiently downregulated NRP1 through TLR2-induced lysosomal degradation. Compared to WT littermates, Nrp1ΔIEC mice presented decreased Pecam1 mRNA levels along with reduced density of blood capillaries. However, villus and lacteals length and ratio were unchanged. The reduced vessels density can be explained by an elevated SEMA3A expression in the SI epithelium of Nrp1 ΔIEC mice. In contrast, VEGF-A levels were unaffected.

Conclusion(s): Our results reveal that TLR2-stimulation by the microbiota evokes NRP1 lysosomal degradation. Interestingly, NRP1 deficiency in the SI epithelium yielded an intestinal phenotype with reduced villus blood capillaries and an increase of the anti-angiogenic NRP1 ligand SEMA3A.