Investigating the Antigens Driving T-Cell Responses in Lymphedema: An Interrogation of Insulin and Insulin Receptor Signaling

PURPOSE: Secondary lymphedema (LE) is a common complication of cancer treatment and is characterized by progressive fibrosis and inflammation. Using immunosequencing of CD4+ T-cells in clinical lymphedema skin biopsies, we have discovered T-cell clones in lymphedematous tissues that recognize insulin (1). Other studies have shown that diabetes is a significant clinical risk factor for lymphedema development and that insulin sensitizing medications improve lymphedema outcomes in preclinical mouse models (2, 3). Taken together, these studies suggest that insulin and insulin receptor (INS-R) signaling may play a role in the pathophysiology of LE. The purpose of this study was therefore to use mouse models of LE to investigate insulin resistance and insulin signaling in LE tissue. METHODS: Secondary lymphedema was induced in mice by tail lymphatic excision and compared with sham-operated animals (n= 8-10/group). Tail fluid was collected at 2 and 6-weeks following tail lymphatic ablation and insulin concentration was quantified using an insulin ELISA. Insulin tolerance test was performed in 4-hr fasted LE and control mice by serial blood glucose measurements following intraperitoneal insulin administration. Insulin signaling was compared between lymphedema and control mouse tail skin biopsy samples and in the gastrocnemius muscle after tissue harvest and ex-vivo stimulation with insulin (10nM). RESULTS: The concentration of insulin in lymph fluid was significantly increased 6 weeks after surgery compared with fluid collected 2 weeks post-op (p<0.05). Fasting blood glucose levels and insulin resistance following an insulin bolus was modestly, though significantly increased in mice with lymphedema compared to sham controls (p<0.05 for both). Insulin receptor expression was decreased in muscle biopsies and increased in tail skin biopsies collected from mice with lymphedema. The expression of insulin receptor adaptor proteins as well as insulin receptor substrates (IRS) 1 and 2 was markedly decreased in tail skin biopsies collected from mice with lymphedema; in contrast we found no differences in these proteins in muscle biopsies suggesting that lymphedema can alter insulin receptor signaling locally. CONCLUSION: Our study suggests that lymphatic injury results in increased tissue levels of insulin, insulin resistance, and decreased insulin receptor signaling in lymphedematous tissues. Lymphatic injury also has mild effects on systemic glucose tolerance. Our findings are consistent with and provide a molecular mechanism for previous studies demonstrating a protective role for insulin-sensitizing drugs. Future studies are needed to understand the effects of insulin signaling on lymphatic endothelial dysfunction and inflammatory cell activation. REFERENCES: 1. Campbell A-C, Mehrara BJ, Brown S. T Cell Repertoire Diversity in Lymphedema: Investigating the Antigens Driving the T Cell Response. Plastic and Reconstructive Surgery – Global Open. 2022;10(10S):107. 2. Wei MW, Liangliang; Liu, Xin; Deng, Yaping; Yang, Sanhong; Pan, Wenjie; Zhang, Xiaoshan; Xu, Guangchao; Xiao, Shune; Deng Chengliang. Metformin Alleviates Inflammation and Fibrosis in Lymphoedema by Activating AMPK Signalling. Social Science Research Network (SSRN). 2023:35. 3. Chen Z, Ghavimi SAA, Wu M, McNamara J, Barreiro O, Maridas D, et al. PPARγ agonist treatment reduces fibroadipose tissue in secondary lymphedema by exhausting fibroadipogenic PDGFRα+ mesenchymal cells. JCI Insight. 2023;8(24).