Probing the Roots of Inflammation-Induced Lymphatic Dysfunction: A Model to Investigate the Role of Mesenteric Lymphatics in the Development of Crohn’s Disease

Abstract

BACKGROUND: Crohn’s disease (CD) is a chronic inflammatory bowel disease characterized by significant mesenteric lymphatic alterations downstream of the inflamed gut but whether the lymphatic changes occur first and contribute to the upstream enteritis is unknown. Here we investigated the spatial and temporal development of the mesenteric lymphatic alterations (including lymphadenopathy, lymphangiogenesis, lymphangiectasia, collecting lymphatic vessel (CLV) immune cell recruitment, lymph leakage, and CLV-associated (CA) tertiary lymphoid organ (TLO) development) in the TNFΔARE/+ mouse, a transgenic model which spontaneously develops a TNFα-driven, microbiota-dependent CD-like ileitis. Due to the microbiota-driven phenotype, we reassessed these changes in germ-free mice to determine whether the lymphatic alterations are microbiota-dependent. METHODS: Eight-, 12-, 20-, and 28-week-old TNFΔARE/+ and wildtype (WT) littermate mice were assessed for ileal inflammation via myeloperoxidase activity (MPO) and mesenteric lymphatic alterations by confocal immunofluorescence imaging. These measurements were repeated in 28-week germ-free (GF) TNFΔARE/+ mice and their WT littermate controls. Immunofluorescence imaging was also performed on mesenteric CLVs, of 12- and 28-week-old WT and TNFΔARE/+ mice, to assess for changes to junctional proteins while vessel permeability was assessed in 12-week WT and TNFΔARE/+ CLVs by measuring the rate of leakage of fluorescent dextrans out of the vessel at 5 cmH2O luminal pressure. To investigate the effect of shear stress on the susceptibility of junctional protein loss human dermal lymphatic endothelial cells (HDLECs) were exposed to shear stress for 48 hours and stimulated with or without 100 ng/mL TNFα for the final 24 hours and assessed for gene expression changes to junctional proteins. RESULTS: Eight-week-old TNFΔARE/+ mice present with lymphadenopathy but not significant ileitis while the CLVs have no abnormal CD45+ immune cell recruitment nor changes in LYVE1+macrophages. By 12 weeks, the ileal MPO peaks and the mesenteric CLVs are dilated with CD45+immune cell clustering around them, however, without junctional protein loss nor lymph leakage. At 20 weeks lymphangiogenesis is evident and CA-TLOs are found where immune cell clusters were previously located and these clusters recruit LYVE1+ macrophages while they are reduced around CLVs. By 28 weeks, the CLVs further dilate, lose junctional protein expression and organization, and CLV-associated LYVE1+ macrophages were depleted. Twenty-eight-week GF TNFΔARE/+ mice do not show gross ileal inflammation but have increased ileal TNFα. Critically, lymphadenopathy, CLV dilation, and lymphangiogenesis are present within the mesentery, however, immune cells remain associated with the CLVs and CA-TLOs are absent. CLV stimulation with TNFα identified a loss of CD31 at the post-valve region and induced an increase in vessel leakage. Shear stress did not alter CD31 and VECAD expression in HDLECs, and TNFα decreased CD31 specifically but not differentially in shear stress conditions. CONCLUSION: The TNFΔARE/+ mice develop mesenteric lymphadenopathy before terminal ileal inflammation. CLV dilation occurs alongside ileitis while ILV lymphangiogenesis develops when the inflammation spills into the mesentery. CLV dilation and ILV lymphangiogenesis develop independently of ileitis in the TNFΔARE/+ mice but can be worsened by it. Changes to CLV-associated immune cells and CA-TLOs are dependent on the presence of a microbiome. CLV leakage occurs due to chronic inflammation and loss of junctional protein expression. While TNFα decreases CD31 expression and increases leakage, shear stress does not sensitize HDLECs to this loss.