Colonic Insult Impairs Lymph Flow, Increases Cellular Content of the Lymph, Alters Local Lymphatic Microenvironment, and Leads to Sustained Inflammation in the Rat Ileum

Abstract

Background Lymphatic dysfunction has been linked to inflammation since the 1930’s. Lymphatic function in the gut and mesentery is grossly underexplored in models of IBD despite the use of lymphatic occlusion in early models of IBD. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS. Methods To evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics. Results Lymph transport fell 24hrs after TNBS administration and began recovering at 72hrs. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased MPO activity. These changes were preceded by increased MHCII+ cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction. Conclusions Alterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.

The pro‐inflammatory cytokineTNF‐α inhibits lymphatic pumping via activation of theNF‐κB‐iNOSsignaling pathway

“…Although the PCR experiments on intact lymphatics do not permit us to determine in which cell type the iNOS upregulation occurred, we observed that both lymphatic endothelial and muscle cells in culture exhibited upregulated iNOS mRNA expression following TNF-α treatment (Rehal, Roizes & von der Weid, unpublished data) and we can reasonably infer that both cell types are sensitive to TNF-α and contribute to lymphatic contractile dysfunction by upregulating iNOS expression and NO production. This proposal is further supported by other studies demonstrating iNOS upregulation in cultured lymphatic endothelial cells isolated from sheep, mouse and rat mesenteric lymphatic vessels upon stimulation with LPS, TNF-α, and other cytokines [17,42,45] and in mesenteric lymphatic vessels from old rats, which display an inflamed phenotype[51] Alternatively, NO could also be generated by immune cells reminiscent of macrophages that have recently been reported to populate the wall of mesenteric lymphatic vessels [12,16,40]. Indeed, some of these cells display NF-κB activation (see arrowheads in Fig.…”

“…However in these conditions, as demonstrated in rodent models of TNBS-induced ileitis [49,77], the ability of the mesenteric lymphatics to pump and thus promote intestinal lymph drainage is compromised. The findings presented here suggest that part of this dysfunction might be due to TNF-α, one of the pro-inflammatory cytokines upregulated in the ileum during TNBS-induced inflammation [16,64]. Indeed, we found that mRNA expressions of TNF-α and the TNF-α receptor TNFR1 were significantly upregulated in lymphatic vessels following TNBS-induced ileitis, suggesting that the inflammation spread to the mesentery and that the lymphatic vessels were indeed inflamed.…”

Anatomy and roles of lymphatics in inflammatory diseases

“…121,122 In rat and guinea pig models of intestinal inflammation, indeed, the contractile capacity within isolated collecting mesenteric lymphatics is compromised, and this effect correlates with lymphatic dysfunction, lymphatic vessel dilation and mucosal inflammation. 88,103,123,124 Furthermore, these studies have shown cyclooxygenase (cyclooxygenase 1/2) or inducible nitric oxide synthase inhibitors improve lymphatic pumping in non-contracting lymphatics in experimental IBD, suggesting prostanoids and nitric oxide released during intestinal inflammation contribute to diminished lymphatic contractile activity.…”

“…Lymph flow increases in acute inflammation, but reductions in lymph transport during chronic inflammation have also been reported. 91,[101][102][103] Lymph flow increases within minutes of acute intestinal inflammation and under acute edemagenic stress (i.e. infusion of isotonic saline).…”

“…Conversely, lymph flow increased during recovery in acute skin and intestinal inflammation models, but not in chronic skin inflammation models. [101][102][103] The immune system can differentially regulate lymph flow through inflammatory mediators modulating both drainage and lymphatic pumping. 99 Although negative inflammatory effects on lymphatic flow can potentially prevent the spread of pathogens and/or inflammatory mediators to the main circulation, decreased lymph flow can intensify antigen presentation activating the immune system towards non-pathogenic stimuli, resulting in persistent infection and edema.…”