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Abstract

Lymphatic capillaries collect interstitial fluid and dendritic cells from the periphery and deliver them to the lymph nodes for immune surveillance and tolerance maintenance. Upon injury and inflammation, lymphatic drainage can increase rapidly, and thus the lymphatic capillaries experience a broad range of fluid stresses and need to respond rapidly to such changes. Our understanding of their functional biology, adaptive ability and response to pathological changes in the biophysical environment is still very limited. This thesis focuses on how the lymphatic capillary endothelium senses and responds to changes of its biophysical environment with respect to its function, including fluid and cell transport, in the context of physiological and pathophysiological conditions. Using in vitro and in vivo models, we demonstrate that lymphatic endothelium is exquisitely sensitive to transmural flow, modulating both fluid and cell transport functions even in the absence of inflammatory cytokines. Flow increased lymphatic permeability and dendritic cell (DC) transmigration, which were coincident with changes in gene and protein expression of factor known to be involved in these, including chemoattractant chemokines, adhesion molecules and junctional proteins. These data provide the first evidence that lymphatic endothelium can regulate its transport functions in response to flow cues. They also show for the first time that transmural flow can, in the absence of inflammatory cues like TNF-a, drive expression of DC adhesion molecules like ICAM-1 and chemokines like CCL21 by lymphatic endothelium. Based on these findings we suggest that lymphatic flow is an important mediator of lymphatic function, particularly with respect to DC recruitment and trafficking to the lymph node. Furthermore, the response of the lymphatic endothelium to flow may be specific to the type of low, as lymphatic endothelial cells (LECs) might react differently to luminal shear flow, at physiological and pathophysiological levels, compared to transmural flow. To address this we used laminar shear stress chambers to expose only the apical surface of the lymphatic endothelial cells (LECs) to shear stress and demonstrated that lymphatic endothelium is indeed sensitive to luminal flow, fine-tuning its expression of adhesion molecules to modulate DC adhesion according to the shear stress. Specifically, at extremely low shear, adhesion was enhanced, unlike at higher shear, adhesion was downregulated. This is consistent with the notion that DC adhesion to LECs is only needed for entry but not transport in conducting vessels, since only the absorbing capillaries have low shear stresses. The interactions between DCs and LECs were strongly mediated by CCL21 and its receptor CCR7. These findings suggest that luminal shear stress acts as an active mechanosignal in LECs to potentially facilitate the traffic of immune cells inside of the lymphatic vessel to reach the lymph node and trigger an immune response. Additionally, we demonstrated that lymphatic vessels respond differentially to immunogenic and tolerogenic inflammatory stimulus. We used in vitro models to evaluate lymphatic uptake and lymphatic participation in dendritic cell transmigration in the presence of various inflammatory stimuli. We found that lymphatic endothelium exposed to TNF-a or complement activation were more conductive to dendritic cell transmigration, while LPS and TGF-β had opposite effect, affecting DCs to decrease DC transmigration. We further presented some insights in similarities in tumor and dendritic cells intravasation into inflamed lymphatic capillaries. These findings imply that lymphatic activation and participation in immune cells transport is not a universal phenomenon, but is strongly dependent on the nature of the inflammation. In conclusion, this work highlights the critical role of biophysical environment in lymphatic endothelial function, and how exquisitely sensitive the lymphatic capillaries are to their extracellular environment and how they use multiple cues to sense inflammation and danger, modulating their transport functions accordingly to regulate the delivery of antigens and immune cells to the lymph nodes.

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