Alleviation of instant blood-mediated inflammatory reaction in autologous conditions through treatment of human islets with NF-κB inhibitors

Abstract

Background: The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) κB, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-κB inhibitors affected IBMIR using an in vitro human autologous blood islet model.

Methods: Human islets were pretreated with or without NF-κB inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry.

Results: Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-κB inhibitor-treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P<0.001) and CAY10512 (P<0.001) groups.

Conclusions: A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-κB inhibitor could alleviate IBMIR, thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes.

FIGURE 1

Viability of islets after culture in autologous blood. Islet viability was determined by Hoechst 33342–propidium iodide staining. The viability was significantly decreased at 6 hours compared with 1 hour after the culture in the control group (**P < 0.01) but not in either NFκB inhibitor–treated group. When comparing viability at 6 hours, significant differences were found between the control and the withaferin A (WA) and CAY10512 (CAY) groups (**P < 0.01). Data express average ± SE based on 6 independent experiments. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test.

FIGURE 2

Autologous islet damage and the impact of NFκB inhibitors. The plasma levels of C-peptide (A), proinsulin (B), and thrombin-antithrombin (TAT) (C) in an autologous islet tube model are shown. Solid line (black circle), solid line (open circle), and dotted lines show the control, withaferin A (WA), and CAY10512 (CAY) groups, respectively. Symbols indicate the P value compared to baseline within each group of control*, WA^‡ or CAY^# and the significance levels: *P<0.05 and **<0.01. Also, the symbols indicate the P value when compared between the control and WA^§ or CAY^† groups. Data express average ± SE based on 3 independent experiments. Repeated-measurement two-way ANOVA with Dunnett’s multiple comparison test was performed for statistical evaluation.

FIGURE 3

Inhibition of proinflammatory cytokine secretion by NFκB inhibitors. The plasma levels of TNF-α (A), MCP-1 (B), IP-10 (C), IL-8 (D), and IL-6 (E) are shown. Solid line (black circle), solid line (open circle), and dotted lines show control, withaferin A (WA), and CAY10512 (CAY) groups, respectively. Symbols indicate the P value compared to baseline within each group of control* or WA^‡ and the significance levels: *P<0.05 and **<0.01. Data express average ± SE based on 3 independent experiments. Repeated-measurement two-way ANOVA with Dunnett’s multiple comparison test was performed for statistical evaluation.

FIGURE 4

Neutrophil infiltration in islet clots. Neutrophils were stained by Naphthol AS-D chloroacetate esterase reactions (purple), counterstaining with hematoxylin (blue) and insulin (brown). Staining was performed on islet clots obtained after 3 hours of exposure to autologous blood. Representative views of H&E staining in control (A), insulin staining (B), neutrophil staining(C) are shown. Neutrophil staining is also shown in Control (D), Withaferin A treated (E) and CAY10512 (CAY) treated (F) groups. Scale bar indicates 50 μm. The number of neutrophils within an islet was counted (G). Data express average ± SE based on 20 counts from each group. ** P < 0.01 and ***P < 0.0001 evaluated with ANOVA followed by Tukey’s test.

FIGURE 5

Tissue factor (TF) expression in islets after exposure to autologous blood. The expression of TF was evaluated with fluorescence stain (red) where nuclei and insulin were stained as blue and green. The autologous islet-blood samples were taken at 3 hours after the culture initiation. White bar indicates 50 μm. Representative sections are shown. WA indicates withaferin A; CAY, CAY10512.