IntroductionObesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system.MethodsAdult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8–10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up.ResultsHigh fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages.ConclusionsObesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.

Lymphedema, a common complication of cancer treatment, is characterized by inflammation, fibrosis, and adipose deposition. We have previously shown that macrophage infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema using depletion experiments. Matched biopsy specimens of normal and lymphedema tissues were obtained from patients with unilateral upper extremity breast cancer-related lymphedema, and macrophage accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of lymphedema to quantify macrophage accumulation and analyze outcomes of conditional macrophage depletion. Histological analysis of clinical lymphedema biopsies revealed significantly increased macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally irradiated wild-type mice reconstituted with CD11b-diphtheria toxin receptor mouse bone marrow did not decrease swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had become established significantly increased fibrosis and accumulation of CD4(+) cells and promoted Th2 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. Our findings suggest that macrophages home to lymphedematous tissues and differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an antifibrotic role in lymphedema and either directly or indirectly regulate CD4(+) cell accumulation and Th2 differentiation. Finally, our findings suggest that lymphedema-associated macrophages are a major source of VEGF-C and that impaired macrophage responses after lymphatic injury result in decreased lymphatic function.

Background Although lymph node transplantation has been shown to improve lymphatic function the mechanisms regulating lymphatic vessel reconnection and functional status of lymph nodes remains poorly understood. Methods We developed and used LacZ lymphatic reporter mice to examine the lineage of lymphatic vessels infiltrating transferred lymph nodes. In addition, we analyzed lymphatic function, expression of vascular endothelial growth factor (VEGF-C), maintenance of T and B cell zone, and anatomic localization of lymphatics and high endothelial venules (HEVs). Results Reporter mice were specific and highly sensitive in identifying lymphatic vessels. Lymph node transfer was associated with rapid return of lymphatic function and clearance of Tc99 secondary to a massive infiltration of recipient mouse lymphatics and putative connections to donor lymphatics. T and B cell populations in the lymph node were maintained. These changes correlated with marked increases in the expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Newly formed lymphatic channels in transferred lymph nodes were in close anatomic proximity to HEVs. Conclusions Transferred lymph nodes have rapid infiltration of functional host lymphatic vessels and maintain T and B cell populations. This process correlates with increased endogenous expression of VEGF-C in the perinodal fat and infiltrating lymphatics. Anatomic proximity of newly formed lymphatics and HEVs supports the hypothesis that lymph node transfer can improve lymphedema by exchanges with the systemic circulation.

Although obesity is a major clinical risk factor for lymphedema, the mechanisms that regulate this effect remain unknown. Recent reports have demonstrated that obesity is associated with acquired lymphatic dysfunction. The purpose of this study was to determine how obesity-induced lymphatic dysfunction modulates the pathological effects of lymphatic injury in a mouse model. We used a diet-induced model of obesity in adult male C57BL/6J mice in which experimental animals were fed a high-fat diet and control animals were fed a normal chow diet for 8-10 wk. We then surgically ablated the superficial and deep lymphatics of the midportion of the tail. Six weeks postoperatively, we analyzed changes in lymphatic function, adipose deposition, inflammation, and fibrosis. We also compared responses to acute inflammatory stimuli in obese and lean mice. Compared with lean control mice, obese mice had baseline decreased lymphatic function. Lymphedema in obese mice further impaired lymphatic function and resulted in increased subcutaneous adipose deposition, increased CD45(+) and CD4(+) cell inflammation (P < 0.01), and increased fibrosis, but caused no change in the number of lymphatic vessels. Interestingly, obese mice had a significantly increased acute inflammatory reaction to croton oil application. In conclusion, obese mice have impaired lymphatic function at baseline that is amplified by lymphatic injury. This effect is associated with increased chronic inflammation, fibrosis, and adipose deposition. These findings suggest that obese patients are at higher risk for lymphedema due to impaired baseline lymphatic clearance and an increased propensity for inflammation in response to injury.

Although recent studies have shown that obesity decreases lymphatic function, the cellular mechanisms regulating this response remain unknown. In the current study, we show that obesity results in perilymphatic accumulation of inflammatory cells and that local inhibition of this response with topical tacrolimus, an inhibitor of T cell differentiation, increases lymphatic vessel density, decreases perilymphatic iNOS expression, increases lymphatic vessel pumping frequency, and restores lymphatic clearance of interstitial fluid to normal levels. Although treatment of obese mice with 1400W, a selective inhibitor of iNOS, also improved lymphatic collecting vessel contractile function, it did not completely reverse lymphatic defects. Mice deficient in CD4+ cells fed a high fat diet also gained weight relative to controls but were protected from lymphatic dysfunction. Taken together, our findings suggest that obesity-mediated lymphatic dysfunction is regulated by perilymphatic accumulation of inflammatory cells and that T cell inflammatory responses are necessary to initiate this effect.

Background/Objectives High-fat diet (HFD)-induced obesity has significant negative effects on lymphatic function, but it remains unclear whether this is a direct effect of HFD or secondary to adipose tissue deposition. Methods We compared the effects of HFD on obesity-prone and obesity-resistant mice and analyzed lymphatic function in vivo and in vitro. Results Only obesity-prone mice had impaired lymphatic function, increased perilymphatic inflammation, and accumulation of lipid droplets surrounding their lymphatic endothelial cells (LECs). LECs isolated from obesity-prone mice, in contrast to obesity-resistant animals, had decreased expression of VEGFR-3 and Prox1. Exposure of LECs to a long-chain free fatty acid increased cellular apoptosis and decreased VEGFR-3 expression, while inhibition of intracellular inhibitors of VEGFR-3 signaling pathways increased cellular viability. Conclusions Collectively, our studies suggest that HFD-induced obesity decreases lymphatic function by increasing perilymphatic inflammation and altering LEC gene expression. Reversal of diminished VEGFR-3 signaling may rescue this phenotype and improve lymphatic function.

Lymphedema (LE) is a morbid disease characterized by chronic limb swelling and adipose deposition. Although it is clear that lymphatic injury is necessary for this pathology, the mechanisms that underlie lymphedema remain unknown. IL-6 is a known regulator of adipose homeostasis in obesity and has been shown to be increased in primary and secondary models of lymphedema. Therefore, the purpose of this study was to determine the role of IL-6 in adipose deposition in lymphedema. The expression of IL-6 was analyzed in clinical tissue specimens and serum from patients with or without LE, as well as in two mouse models of lymphatic injury. In addition, we analyzed IL-6 expression/adipose deposition in mice deficient in CD4+ cells (CD4KO) or IL-6 expression (IL-6KO) or mice treated with a small molecule inhibitor of IL-6 or CD4 depleting antibodies to determine how IL-6 expression is regulated and the effect of changes in IL-6 expression on adipose deposition after lymphatic injury. Patients with LE and mice treated with lymphatic excision of the tail had significantly elevated tissue and serum expression of IL-6 and its downstream mediator. The expression of IL-6 was associated with adipose deposition and CD4+ inflammation and was markedly decreased in CD4KO mice. Loss of IL-6 function resulted in significantly increased adipose deposition after tail lymphatic injury. Our findings suggest that IL-6 is increased as a result of adipose deposition and CD4+ cell inflammation in lymphedema. In addition, our study suggests that IL-6 expression in lymphedema acts to limit adipose accumulation.

T cell-mediated responses have been implicated in the development of fibrosis, impaired lymphangiogenesis, and lymphatic dysfunction in secondary lymphedema. Here we show that CD4+ T cells are necessary for lymphedema pathogenesis by utilizing adoptive transfer techniques in CD4 knockout mice that have undergone tail skin and lymphatic excision or popliteal lymph node dissection. We also demonstrate that T cell activation following lymphatic injury occurs in regional skin-draining lymph nodes after interaction with antigen-presenting cells such as dendritic cells. CD4+ T cell activation is associated with differentiation into a mixed T helper type 1 and 2 phenotype, as well as upregulation of adhesion molecules and chemokines that promote migration to the skin. Most importantly, we find that blocking T cell release from lymph nodes using a sphingosine-1-phosphate receptor modulator prevents lymphedema, suggesting that this approach may have clinical utility.