Physiologic Doses of Bilirubin Contribute to Tolerance of Islet Transplants by Suppressing the Innate Immune Response

Abstract

Bilirubin has been recognized as a powerful cytoprotectant when used at physiologic doses and was recently shown to have immunomodulatory effects in islet allograft transplantation, conveying donor-specific tolerance in a murine model. We hypothesized that bilirubin, an antioxidant, acts to suppress the innate immune response to islet allografts through two mechanisms: 1) by suppressing graft release of damage-associated molecular patterns (DAMPs) and inflammatory cytokines, and 2) by producing a tolerogenic phenotype in antigen-presenting cells. Bilirubin was administered intraperitoneally before pancreatic procurement or was added to culture media after islet isolation in AJ mice. Islets were exposed to transplant-associated nutrient deprivation and hypoxia. Bilirubin significantly decreased islet cell death after isolation and hypoxic stress. Bilirubin supplementation of islet media also decreased the release of DAMPs (HMGB1), inflammatory cytokines (IL-1β and IL-6), and chemokines (MCP-1). Cytoprotection was mediated by the antioxidant effects of bilirubin. Treatment of macrophages with bilirubin induced a regulatory phenotype, with increased expression of PD-L1. Coculture of these macrophages with splenocytes led to expansion of Foxp3+ Tregs. In conclusion, exogenous bilirubin supplementation showed cytoprotective and antioxidant effects in a relevant model of islet isolation and hypoxic stress. Suppression of DAMP release, alterations in cytokine profiles, and tolerogenic effects on macrophages suggest that the use of this natural antioxidant may provide a method of preconditioning to improve outcomes after allograft transplantation.

Figure 1.

Bilirubin decreases islet cell death in models of transplant-associated stress. AJ mouse islets (100–150 per treatment group) were isolated and maintained in tissue culture (37°C). Dual staining with Hoechst and propidium iodide (PI) was performed, and cell death was estimated based on % PI⁺ staining. Graphs represent pooled data from three independent experiments in each model. (A) Islets were maintained in culture for 48 h after pancreas digestion and gradient isolation to illustrate the effects of isolation stress. Media were unchanged to cause nutrient deprivation. Progressive cell death occurred, peaking at 48 h after recovery. Bilirubin (10 mg/kg) administered intraperitoneally to the islet donor 1 h before islet harvest (Bilirubin IP) caused significant decreases in cell death. (B) Hypoxic stress was induced by subjecting the islets to 1% O₂ for 3 h. Viability was assessed after 24 h of reoxygenation. Hypoxic stress caused marked cell death in the control group. Cell death was significantly decreased by bilirubin IP treatment of the donor prior to islet recovery, but cell death was most effectively prevented by the addition of bilirubin (20 μM/L) to the cell culture media (Bilirubin Media) 1 h before hypoxia. (C) Islets were divided into three size groups based on the measured area of each islet. Percent cell death was greater in large islets exposed to hypoxia, and there was a significant interaction detected between bilirubin treatment and islet size, with bilirubin offering the greatest protection against necrosis of large islets (p = 0.03). A representative image of control islets (inset) shows the large area of central necrosis (arrow) caused by hypoxic injury, while smaller islets were preserved (arrowhead).

Figure 2.

Bilirubin decreases oxidative stress in islets subjected to hypoxic conditions. AJ mice were treated with either vehicle control (Control) or 10 mg/kg of bilirubin intraperitoneally (Bilirubin IP) 1 h before islet harvest. Hypoxic stress conditions were imposed by incubation of mouse islets (250-300 per treatment group) for 3 h at 37°C and 1% O₂. Islets were then reoxygenated for 24 h in a standard 37°C, 20% O₂ environment. Oxidative stress was determined by measuring 8-iso-prostaglandin F_2α levels in the conditioned islet culture media using an enzymatic immunoassay kit. Results of three independent experiments were normalized to values from the unstressed control islets and were expressed as fold change from control. Exposure to hypoxia caused a marked increase in oxidative stress. Donor pretreatment with bilirubin significantly decreased oxidative stress in islets subjected to hypoxia and reoxygenation, maintaining levels of lipid peroxidation similar to unstressed control islets.

Figure 3.

Bilirubin decreases release of HMGB1 and HSP70 at early time points after hypoxic stress. Hypoxic stress conditions were imposed as described in Figure 2, and protein levels of high-mobility group box protein B1 (HMGB1) (A) and heat shock protein 70 (HSP70) (B) were measured in islet culture media after 24 h of recovery. Results were normalized to values from the unstressed control islets and were expressed as fold change from control. Hypoxic stress caused 1.8- and 2.8-fold increases in HMGB1 HSP70 levels in control islets, respectively. Addition of bilirubin (20 μM/L) to islet culture media 1 h before hypoxic stress caused a significant reduction in HMGBI release from treated islets (p = 0.004). While HSP70 release was decreased from a mean of 2.8-fold in the control hypoxia group to 1.6-fold in the bilirubin media group, results were variable and this effect failed to reach statistical significance over four repetitions of the experiment (p = 0.054).

Figure 4.

Bilirubin decreases release of inflammatory cytokines and chemokines from stressed islets. Hypoxic stress conditions were imposed as described in Figure 2, and concentrations of the inflammatory cytokines (IL-1β and IL-6) and the chemokine macrophage chemoattractant protein 1 (MCP-1) were measured in islet culture media after 24 h of recovery. Results were normalized to values from the unstressed control islets and were expressed as fold change from control. Hypoxic stress caused increases in IL-6 (A), IL-1β (B), and MCP-1 (C) in the islet-conditioned media. Addition of bilirubin (20 μM/L) to islet culture media 1 h before hypoxic stress significantly reduced the release of IL-1β (p<0.001), IL-6 (p<0.001), and MCP-1 (p = 0.004) after hypoxia and reoxygenation.

Figure 5.

Bilirubin increases the expression of PD-L1 on macrophages and increased the capacity of Treg induction. C57BL/6 macrophages were seeded in a 12-well tissue culture plate and cultured in the presence of media or 20 μM bilirubin. Additional groups were incubated with 1 μg/ml LPS during the culture period. (A) After 48 h of culture, cells were washed twice and stained for programmed death ligand 1 (PD-L1) for analysis by fluorescence-activated cell sorting (FACS). (B) The ability of these cells (after a 4 day coculture) to induce forkhead box p3 positive (Foxp3⁺) cells from naive Foxp3-enhanced green fluorescent green (EGFP) reporter immune cells was also determined by FACS. Experiments were performed three times to confirm results.

Figure 6.

Schematic illustration of the mechanisms for the tolerogenic effects of bilirubin on islet transplantation. Bilirubin improved islet viability and significantly decreased release of the damage-associated molecular pattern (DAMP) molecule HMGB1, as well as suppressed the release of proinflammatory cytokines IL-1β, IL-2, and the macrophage chemoattractant protein MCP-1. These effects would decrease toll-like receptor 4 (TLR4) stimulation and suppress the innate immune response in both passenger and host APCs. Furthermore, bilirubin was shown to induce expression of the regulatory marker PD-L1 and the formation of Tregs after coculture with naive immune cells. The combined cytoprotective and immunomodulatory effects of bilirubin may lead to the presentation of a smaller dose of antigen by tolerogenic antigen-presenting cells, inducing a state of Treg-mediated tolerance.