Neutrophil depletion for early allogeneic islet survival in a methacrylic acid (MAA) copolymer-induced, vascularized subcutaneous space

Abstract

Islet transplantation is a promising treatment for type I diabetes (T1D). Despite the high loss of islets during transplantation, current islet transplant protocols continue to rely on portal vein infusion and intrahepatic engraftment. Because of the risk of portal vein thrombosis and the loss of islets to instant blood mediated inflammatory reaction (IBMIR), other transplantation sites like the subcutaneous space have been pursued for its large transplant volume, accessibility, and amenability for retrieval. To overcome the minimal vasculature of the subcutaneous space, prevascularization approaches or vascularizing biomaterials have been used to subcutaneously deliver islets into diabetic mice to return them to normoglycemia. Previous vascularization methods have relied on a 4 to 6 week prevascularization timeframe. Here we show that a vascularizing MAA-coated silicone tube can generate sufficient vasculature in 2 to 3 weeks to support a therapeutic dose of islets in mice. In order to fully harness the potential of this prevascularized site, we characterize the unique, subcutaneous immune response to allogeneic islets in the first 7 days following transplantation, a critical stage in successful engraftment. We identify neutrophils as a specific cellular target, a previously overlooked cell in the context of subcutaneous allogeneic islet transplantation. By perioperatively depleting neutrophils, we show that neutrophils are a key, innate immune cell target for successful early engraftment of allogeneic islets in a prevascularized subcutaneous site.

Keywords: immunosuppression; islet transplantation; methacrylic acid; neutrophil depletion.; prevascularization.

Figure 1

Neutrophils were the predominant immune cell in the first 24 h post-islet transplantation. Silicone tubes (3 cm long) were dip-coated in a 40% MAA-co isodecyl acrylate (IDA) solution (A), then inserted into the upper dorsum of BALB/c mice. After 14 days, 250 islet equivalents (IEQ) isolated from C57Bl/6J mice were suspended in collagen and injected into the subcutaneous site at the time of silicone tube removal (B,C). The subcutaneous tissue was digested and the immune response to the 250 allogeneic islets was analyzed by flow cytometry 1, 3, and 7 days after transplantation (D–F). (D) There was the greatest CD45+ immune cell recruitment 3 days post transplantation. (E) Live, single, CD45+ immune cells were further gated to reveal a dynamic wave of neutrophil then macrophage and then dendritic cell (DC) recruitment in the week following transplantation. Neutrophils were gated as F480-Ly6G+; macrophages as F480+CD11b+; natural killer cells (NKs) as CD49b+; DCs as F480-CD11c+. (F) Macrophage polarization was further determined by CD206 and MHCII expression. M1(CD206-MHCII+); M2(CD206+MHCII-); DP (CD206+MHCII+); DN(CD206-MHCII-). Data shown as mean ± SEM; n = 3-6; analyzed using two-way ANOVA; *p < 0.05, **p < 0.005, ****p < 0.0001.

Figure 2

Perioperative anti-Ly6G treatment returned diabetic mice to normoglycemia post-transplantation even after neutrophils began to return (A) after 14-21 days of prevascularization, 600 islet equivalents (IEQ) isolated from C57Bl/6J mice were suspended in collagen and injected into the subcutaneous site of diabetic BALB/c mice at the time of silicone tube removal. Mice received daily immunosuppressants, dexamethasone (5 mg/kg, i.p.) and fingolimod (1 mg/kg, i.p.) from 1 day before (D-1) to 7 days after (D7) transplantation, or anti-Ly6G alone (40 μg/mouse, i.p.) from 3 days before (D-3) to 7 days after (D7) transplantation. (B) Blood glucose (BG) levels of diabetic BALB/c mice receiving prednisolone and fingolimod (n = 3) remained high while the BG of mice receiving only anti-Ly6G (n = 3) all dropped to normoglycemic range after transplantation. (C) The proportion of CD45+ Ly6G+ cells in peripheral blood was examined by flow cytometry to monitor the effect of the anti-Ly6G antibody during (D5) and after stopping treatment (D10, D14). The effect of the antibody was compared to a no treatment control. Data shown as mean ± SEM; n = 3–4.

Figure 3

Low dose rapamycin enabled longer term blood glucose control and but did not alter systemic T cells proportions. (A) After 14-21 days of prevascularization, 600 islet equivalents (IEQ) isolated from C57Bl/6J mice were suspended in collagen and injected into the subcutaneous site of diabetic BALB/c mice at the time of silicone tube removal. All mice received daily anti-Ly6G (40 μg/mouse; i.p.) from 3 days before (D-3) to 7 days after (D7) transplantation, and some mice continued to receive an immunosuppressant (rapamycin, 0.2 mg/kg, i.p.) for another 7 days (D8-D14). (B,C) Grafts failed earlier in mice receiving only anti-Ly6G (n = 3) whereas mice that received subsequent rapamycin remained normoglycemic for >30 days with some grafts still surviving past 75 days post-transplantation (n = 4). Mice were considered diabetic when they had BG levels above 20 mM for two consecutive readings. (D) Peripheral blood samples showed little changes in peripheral T cell (CD45+ Ly6G- CD4+; CD45+ Ly6G- CD8+) populations after 7 daily doses of rapamycin (D14). Data shown as mean ± SEM; n = 2-5.