Pro-Angiogenic Activity of TLRs and NLRs: A Novel Link Between Gut Microbiota and Intestinal Angiogenesis

Abstract

In intestinal inflammation the gut microbiota induces an innate immune response by activating epithelial and immune cells that initiate or maintain inflammation. We investigated whether the microbiota also can activate local microvascular cells and induce angiogenesis. Human intestinal microvascular endothelial cells (HIMEC) and human intestinal fibroblasts (HIF) were exposed to bacterial ligands specific for Toll-like receptor (TLR)2/6 and 4, and NOD1 and NOD2, and cell proliferation, migration, transmigration, tube formation, and production of pro-angiogenic factors were measured. The ability of the ligands to induce ex vivo vessel sprouting in an aortic ring assay and in vivo angiogenesis using a collagen gel assay also were assessed. Bacterial ligands induced proliferation, migration, transmigration, tube formation of HIMEC, vessel sprouting, and in vivo angiogenesis; they also stimulated production of angiogenic factors from HIMEC and HIF, and HIF-derived angiogenic factors promoted HIMEC proliferation. To various degrees, all ligands induced angiogenic responses, but these were ligand- and cell type-dependent. Responses were mediated through receptor interacting protein-2 (RIP2)- and tumor necrosis factor receptor-associated factor 6 (TRAF6)-dependent signaling, involved the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways and the up-regulation of vascular endothelial growth factor receptor 2 (VEGF-R2) and focal adhesion kinase (FAK). Knockdown of RIP2 and TRAF6 by RNA interference and neutralization of interleukin-8, basic fibroblast growth factor, and vascular endothelial growth factor inhibited TLR-/NOD-like receptor-induced HIMEC angiogenesis. The gut microbiota can selectively activate mucosal endothelial and mesenchymal cells to promote specific angiogenic responses in a TLR- and NOD-like receptor-dependent fashion. This innate immunity-mediated response may expand the mucosal microvascular network, foster immune cell recruitment, and contribute to chronic intestinal inflammation.