Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT, can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found upregulation of LIGHT receptors (LTβR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20 mM glucose) + LIGHT in vitro, and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose-stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by upregulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.
Supplementary Figure 1: persistent hyperglycemia impairs transplanted human islets function in vivo. Panel (A) represents in vivo experiment design showing the double islets transplantation model. First, mice islet grafts (1st graft) were used to achieve normoglycemia in diabetic mice. After two weeks recovery time, minimal human islet grafts (2nd graft) were allowed convalescent period before the 1st grafts removal and dividing animals into two groups based on their non-fasting B-glu measured days post 1st graft removal (B). B-glu level during oral glucose tolerance test (OGTT) on day 15 post 1st graft removal with corresponding AUC represents the differences between groups (C). Assessment of fasting plasma hC-peptide 17 days after 1st graft removal (D). Analysis of mouse pro-inflammatory cytokines IL-6 (E), MCP-1 (F), GRO-α (G) and IP-10 (H) in mice plasma samples harvested at the termination of the study. Difference in non-fasting B-glu was analyzed with two-way ANOVA. Difference in OGTT, hC-peptide and the level of pro-inflammatory cytokines between mice in group NG vs. HG was analyzed with Mann Whitney U-test and presented as mean ± SEM. n=9 mice in group NG (b-glu < 11mM), n=15 mice in group HG (b-glu > 20mM). * p>0.05, ** p>0.01, *** p>0.001, **** p>0.0001 vs. group NG. B-glu, Blood Glucose; hC-peptide, human C-peptide; HG, hyperglycemic; NG, normoglycemic; n, number of mice in each experimental group.
Supplementary Figure 2: IL-22 receptor is expressed in human islets and up-regulated in islets treated with HG+LIGHT. Panel (A) represents immunofluorescent labelling for IL-22R1 and insulin together with DAPI-nuclear staining in dispersed human islets treated with either HG (20mM) and LIGHT (400 ng/ml) and the combination of thereof for 48 hours. Magnification 10x, scale bar 100 µm. Score of IL22-R1-positive cells to the total number of DAPI-stained cells (B). Data is analyzed by one-way ANOVA with Bonferroni corrections and presented as mean ± SEM, n=3. ** p<0.01 vs. untr islets. n, number of independent donors. Untr, untreated; HG, high glucose, IL22-R1: IL-22 receptor subunit 1.