Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury

Abstract

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.

Regulation of inflammation and fibrosis by macrophages in lymphedema

“…These findings are consistent with previous studies demonstrating that macrophages play active roles in the regulation of fibrosis in other organ systems (6,33). In addition to other studies, consistent with known roles of macrophages in the regulation of lymphangiogenesis (7,8,23), we have shown that macrophages in lymphedematous tissues strongly express VEGF-C (33)(34)(35)(36). However, although it is clear that lymphedema promotes macrophage infiltration, the precise role of these cells in the regulation of fibrosis and lymphangiogenesis in response to sustained lymphatic fluid stasis has not been assessed through direct loss-of-function studies.…”

“…However, we have also previously reported that the number of macrophages in the lymphedematous tissues is increased substantially compared with controls (37). In addition, in a recent report using a mouse tail model of lymphedema, we found that animals lacking Toll-like receptors had decreased macrophage accumulation and increased fibrosis/adipose deposition (35). These findings are consistent with previous studies demonstrating that macrophages play active roles in the regulation of fibrosis in other organ systems (6,33).…”

A chronic and latent lymphatic insufficiency follows recovery from acute lymphedema in the rat foreleg

“…Inflammation has been shown to play an important role in determining the amount of swelling in lymphedema (22,45,46) and may be an important stimulus promoting the appearance and perpetuation of the swelling (8). Indeed, the initial appearance of the chronic form of lymphedema in the human can occur rapidly (within a few hours to days) following an acute inflammatory insult (4), although subsequently it may slowly worsen.…”

Diabetic foot ulcers in conjunction with lower limb lymphedema: pathophysiology and treatment procedures

“…54 Studies in a mouse model of post-surgical lymphedema deficient for TLR2, TLR4, and TLR9 demonstrated significantly worse effects post injury compared with those in a wild-type model, as evidenced by increased tissue edema, reduced lymphangiogenesis, increased fibrosis, and increased leucocyte infiltration but reduced monocyte infiltration. 55 The data suggest a role for TLRs in the normal repair of lymphatic injury and resolution of lymphedema. 55 Consequently, although TLRs appear to have a role in normal inflammation and swelling, their role in the diabetic patient with lymphedema may be confused, and the excessive TLR-mediated inflammation associated with diabetes is counterproductive with resultant chronic lymphedema.…”

“…55 The data suggest a role for TLRs in the normal repair of lymphatic injury and resolution of lymphedema. 55 Consequently, although TLRs appear to have a role in normal inflammation and swelling, their role in the diabetic patient with lymphedema may be confused, and the excessive TLR-mediated inflammation associated with diabetes is counterproductive with resultant chronic lymphedema.…”