Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that >5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4 T-cell inflammation and T-helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4 cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin-4 (IL-4) or IL-13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4 inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.

IntroductionLymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema.MethodsWe used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis.ResultsLymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model.ConclusionsLymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis.

Mechanisms regulating lymphedema pathogenesis remain unknown. Recently, we have shown that lymphatic fluid stasis increases endogenous danger signal expression, and these molecules influence lymphatic repair (Zampbell JC, et al. Am J Physiol Cell Physiol 300: C1107-C1121, 2011). Endogenous danger signals activate Toll-like receptors (TLR) 2, 4, and 9 and induce homeostatic or harmful responses, depending on physiological context. The purpose of this study was to determine the role of TLRs in regulating tissue responses to lymphatic fluid stasis. A surgical model of lymphedema was used in which wild-type or TLR2, 4, or 9 knockout (KO) mice underwent tail lymphatic excision. Six weeks postoperatively, TLR KOs demonstrated markedly increased tail edema compared with wild-type animals (50-200% increase; P < 0.01), and this effect was most pronounced in TLR4 KOs (P < 0.01). TLR deficiency resulted in decreased interstitial and lymphatic transport, abnormal lymphatic architecture, and fewer capillary lymphatics (40-50% decrease; P < 0.001). Lymphedematous tissues of TLR KOs demonstrated increased leukocyte infiltration (P < 0.001 for TLR4 KOs), including higher numbers of infiltrating CD3+ cells (P < 0.05, TLR4 and TLR9 KO), yet decreased infiltrating F4/80+ macrophages (P < 0.05, all groups). Furthermore, analysis of isolated macrophages revealed twofold reductions in VEGF-C (P < 0.01) and LYVE-1 (P < 0.05) mRNA from TLR2-deficient animals. Finally, TLR deficiency was associated with increased collagen type I deposition and increased transforming growth factor-β1 expression (P < 0.01, TLR4 and TLR9 KO), contributing to dermal fibrosis. In conclusion, TLR deficiency worsens tissue responses to lymphatic fluid stasis and is associated with decreased lymphangiogenesis, increased fibrosis, and reduced macrophage infiltration. These findings suggest a role for innate immune responses, including TLR signaling, in lymphatic repair and lymphedema pathogenesis.

Background Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goals of this two-part study were to determine the effect of lymphatic fluid stasis on adipogenesis and inflammation (part 1) and how these changes regulate the temporal and spatial expression of fat differentiation genes (part 2). Methods Adult female mice underwent tail lymphatic ablation and were sacrificed 6 weeks after surgery (n=20). Fat deposition, fibrosis, and inflammation were then analyzed in the regions of the tail exposed to lymphatic fluid stasis as compared with normal lymphatic flow. Results Lymphatic fluid stasis in the tail resulted in significant subcutaneous fat deposition with a 2-fold increase in fat thickness (p<0.01). In addition, lymphatic stasis was associated with subcutaneous fat fibrosis and collagen deposition. Adipogenesis in response to lymphatic fluid stasis was associated with a marked mononuclear cell inflammatory response (5-fold increase in CD45+ cells; p<0.001). In addition, we noted a significant increase in the number of monocytes/macrophages as identified by F4/80 immunohistochemistry (p<0.001). Conclusions The mouse-tail model has pathological findings that are similar to clinical lymphedema including fat deposition, fibrosis, and inflammation. Adipogenesis in response to lymphatic fluid stasis closely resembles this process in obesity. This model therefore provides an excellent means to study the molecular mechanisms that regulate the pathophysiology of lymphedema.

Background Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goal of this study was to determine how lymphatic fluid stasis regulates adipogenic gene activation and fat deposition. Methods Adult female mice underwent tail lymphatic ablation and sacrifice at 1, 3, or 6 weeks post-operatively (n=8/group). Samples were analyzed by immunohistochemistry and western blot. An alternative group of mice underwent axillary dissections or sham incisions and limb tissues were harvested 3 weeks post-operatively (n=8/group). Results Lymphatic fluid stasis resulted in significant subcutaneous fat deposition and fibrosis in lymphedematous tail regions (p<0.001). Western blot analysis demonstrated that proteins regulating adipose differentiation including CCAAT/enhancer binding protein-alpha (CEBP-α) and adiponectin were markedly upregulated in response to lymphatic fluid stasis in the tail and axillary models. Expression of these markers increased in edematous tissues according to the gradient of lymphatic stasis distal to the wound. Immunohistochemical analysis further demonstrated that adiponectin and peroxisome proliferator-activated receptor gamma (PPAR-γ), another critical adipogenic transcription factor, followed similar expression gradients. Finally, adiponectin and PPAR-γ expression localized to a variety of cell types in newly formed subcutaneous fat. Conclusions The mouse-tail model of lymphedema demonstrates pathological findings similar to clinical lymphedema including fat deposition and fibrosis. We show that lymphatic fluid stasis potently upregulates the expression of fat differentiation markers both spatially and temporally. These studies elucidate mechanisms regulating abnormal fat deposition in lymphedema pathogenesis and therefore provide a basis for developing targeted treatments.