The Woodruff School

SUMMARY

The lymphatic system serves important roles in fluid balance and immune system regulation within the body. Through both passive and active transport of fluid, the lymphatic network transports interstitial fluid back into the circulatory system. When the lymphatic system fails, that excess fluid can no longer be properly transported back into the circulation. This leads to a disease called lymphedema, which manifests as swelling of distal limbs and normally occurs following injury to the lymphatic network. The mechanisms of lymphedema development are not completely understood, but the immune response is known to play an important role in lymphedema pathogenesis. The main goal of this thesis is to investigate both the functional response of the intact lymphatic vasculature and changes in leukocyte populations within draining lymph nodes (dLNs) during lymphedema progression. In the first aim, we used near-infrared (NIR) imaging techniques to quantify changes in lymphatic function in vivo following induction of lymphedema in mice using a novel lymphedema model. We specifically investigated the effect of two potential therapeutic mechanisms, antagonism of leukotriene B4 (LTB4) production and deletion of epsin, on lymphatic function following lymphedema surgery. Further in vivo and ex vivo analysis was performed to investigate potential mechanisms regulating the effect of LTB4 on lymphatic contractile function. In the second aim, we used flow cytometry to investigate changes in leukocyte populations within dLNs during acute lymphedema progression. Our novel lymphedema model leaves a pair of intact collecting lymphatic vessels on one side of the tail while other tail lymphatics are ligated, allowing for analysis of the immune response within dLNs experiencing differences in drainage. Further analysis using a nanoparticle delivery system was used to quantify differences in particle uptake between dLNs as lymphedema progressed. The effect of LTB4 antagonism on the immune response was also elucidated. Overall, this work furthers understanding of the mechanisms driving lymphedema pathogenesis, by combining comprehensive analysis of changes in lymphatic contractile function in vivo and ex vivo with investigation of changes in the immune response within dLNs.

SUBJECT:	Ph.D. Dissertation Defense

BY:	Matthew Cribb

TIME:	Wednesday, December 1, 2021, 1:30 p.m.

PLACE:	Krone Engineered Biosystems Building, CHOA

TITLE:	Investigation of Functional Lymphatic Changes and the Immune Response During Lymphedema Development

COMMITTEE:	Dr. J. Brandon Dixon, Chair (ME) Dr. Susan Thomas (ME) Dr. Krishnendu Roy (BME) Dr. Rudolph Gleason (ME) Dr. Mark Nicolls (Stanford University School of Medicine)

SUMMARY The lymphatic system serves important roles in fluid balance and immune system regulation within the body. Through both passive and active transport of fluid, the lymphatic network transports interstitial fluid back into the circulatory system. When the lymphatic system fails, that excess fluid can no longer be properly transported back into the circulation. This leads to a disease called lymphedema, which manifests as swelling of distal limbs and normally occurs following injury to the lymphatic network. The mechanisms of lymphedema development are not completely understood, but the immune response is known to play an important role in lymphedema pathogenesis. The main goal of this thesis is to investigate both the functional response of the intact lymphatic vasculature and changes in leukocyte populations within draining lymph nodes (dLNs) during lymphedema progression. In the first aim, we used near-infrared (NIR) imaging techniques to quantify changes in lymphatic function in vivo following induction of lymphedema in mice using a novel lymphedema model. We specifically investigated the effect of two potential therapeutic mechanisms, antagonism of leukotriene B4 (LTB4) production and deletion of epsin, on lymphatic function following lymphedema surgery. Further in vivo and ex vivo analysis was performed to investigate potential mechanisms regulating the effect of LTB4 on lymphatic contractile function. In the second aim, we used flow cytometry to investigate changes in leukocyte populations within dLNs during acute lymphedema progression. Our novel lymphedema model leaves a pair of intact collecting lymphatic vessels on one side of the tail while other tail lymphatics are ligated, allowing for analysis of the immune response within dLNs experiencing differences in drainage. Further analysis using a nanoparticle delivery system was used to quantify differences in particle uptake between dLNs as lymphedema progressed. The effect of LTB4 antagonism on the immune response was also elucidated. Overall, this work furthers understanding of the mechanisms driving lymphedema pathogenesis, by combining comprehensive analysis of changes in lymphatic contractile function in vivo and ex vivo with investigation of changes in the immune response within dLNs.