360 CAD, a Nucleotide Synthesis Enzyme, Inhibits NOD2 Anti-Bacterial Function in Human Intestinal Epithelial Cells

Abstract

intense lymphangiogenesis is initially an appropriate and beneficial adaptation that ultimately becomes dysregulated due to the chronicity of inflammation, or is an abnormal response from the onset of IBD remains to be established. Methods: We addressed these issues In Vivo by systemic inhibition of the receptor VEGFR-3 or delivery of the lymphangiogenic factor VEGF-C in the dextran sulfate sodium (DSS) model of colitis by a blocking antibody or adenovirus transfer, respectively. Whole mount of proximal and distal colons were stained during acute and chronic colitis with antibodies against LYVE-1 and CD31 to measure area density and dimension of lymphatic vessels. Lymphatic drainagewas assessed by intramucosal injection of Evans blue dye. Moreover, In Vitro tubulogenesis was used to assess human intestinal lymphatic endothelial cells (HILEC) organization into capillary tubules using a Matrigel system. Results: Despite enhanced lymphangiogenesis, lymph flow was significantly reduced in DSS-induced colitis (p<0,001), particularly in the chronic stage. Systemic inhibition of VEGFR-3 blocked lymphangiogenesis, reducing both area density (from 35 ±0,8 to 14 ±0,3 vessels/mm2, p<0,01) and lymphatic vessel dimension (from 81 ±1,4 μm to 39 ±0,9 μm, p<0,01), while increasing inflammatory edema formation and inhibiting disease resolution, as assessed by weight loss, colitis activity index and histological score. In contrast, although lymphatic drainage was decreased in the chronic phase of colitis, it was enhanced by systemic delivery of the lymphangiogenic factor VEGF-C, which in turn significantly improved colitis both clinically and histologically. HILEC isolated from CD biopsies showed reduced capacity to undergo tubulogenesis In Vitro (30 ± 1,8 number of NL tubes vs 11 ± 0,5 number of CD tubes) and downregulation of VEGFR3 expression compared to healthy control tissues. Treatment with recombinant VEGFC induces tubulogenesis of CD HILEC while anti-VEGFR3 antibody inhibits tube formation of control HILEC, proving that VEGFR3 pathway is required for HILEC tubulogenesis and that reduced lymphatic function observed In Vivo is VEGFR3-dependent. Conclusion: Our findings demonstrate that stimulation of functional lymphangiogenesis via VEGFR-3 accelerates disease resolution and inhibits chronic inflammation. Correction of defective lymphatic function may therefore offer an entirely novel therapeutic strategy for IBD.