Regulation of Inflammation and Fibrosis

43 Introduction: Lymphedema, a common complication of cancer treatment, is characterized by 44 inflammation, fibrosis, and adipose deposition. We previously have shown that macrophage 45 infiltration is increased in mouse models of lymphedema. Because macrophages are regulators of 46 lymphangiogenesis and fibrosis, this study aimed to determine the role of these cells in lymphedema 47 using depletion experiments. 48 49 Methods: Matched biopsy specimens of normal and lymphedema tissues were obtained from 50 patients with unilateral upper extremity breast cancer-related lymphedema and macrophage 51 accumulation was assessed using immunohistochemistry. In addition, we used a mouse tail model of 52 lymphedema to quantify macrophage accumulation and analyze outcomes of conditional 53 macrophage depletion. 54 55 Results: Histological analysis of clinical lymphedema biopsies revealed significantly increased 56 macrophage infiltration. Similarly, in the mouse tail model, lymphatic injury increased the number 57 of macrophages and favored M2 differentiation. Chronic macrophage depletion using lethally 58 irradiated wild-type mice reconstituted with CD11b-DTR mouse bone marrow did not decrease 59 swelling, adipose deposition, or overall inflammation. Macrophage depletion after lymphedema had 60 become established significantly increased fibrosis, accumulation of CD4+ cells, and promoted Th2 61 differentiation while decreasing lymphatic transport capacity and VEGF-C expression. 62 63 Conclusion: Our findings suggest that macrophages home to lymphedematous tissues and 64 differentiate into the M2 phenotype. In addition, our findings suggest that macrophages have an 65 anti-fibrotic role in lymphedema and either directly or indirectly regulate CD4 cell accumulation 66 and Th2 differentiation. Finally our findings suggest that lymphedema associated macrophages are a 67 major source of VEGF-C and that impaired macrophage responses after lymphatic injury results in 68 decreased lymphatic function. 69