Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT

in Journal of Molecular Endocrinology
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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.

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  • 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.

 

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    Hyperglycemia upregulates the expression of LTβR and HVEM receptors in transplanted human islets. Representative images showing immunofluorescent double staining for LTβR/insulin (A), HVEM/insulin (B) and TUNEL labeling (C) together with DAPI-nuclear staining in human islets engrafted under normoglycemic (NG) or hyperglycemic (HG) mice kidney capsule. Magnification 40× with scale bars 20 µm. Quantification of the area of LTβR-positive (D) and HVEM-positive cells (E) as well as the area of insulin-positive cells (F) and the ratio of TUNEL-positive cells to DAPI-nuclear staining (G) within transplanted human islet grafts. In all analysis, data is analyzed with Mann Whitney U test and presented as mean ± s.e.m. n = 9 mice in group NG (b-glu <11 mM), n = 15 mice in group HG (b-glu >20 mM), *P < 0.05, **P < 0.01, ***P < 0.001 vs group NG. HG, hyperglycemic; n, number of mice in each group; NG, normoglycemic.

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    IL-22 reverses the effect of HG + LIGHT on expression of LTβR and HVEM receptors. Western blot analysis of LTβR and HVEM in human islets treated with either HG (20 mM) or LIGHT (400 ng/mL) and HG + hrLIGHT with or without IL-22 (50 ng/mL) for 48 h (A). Quantification of LTβR (B) and HVEM (C) band densities normalized to housekeeping gene GAPDH and presented as fold of untr islets. Representative images showing immunofluorescent staining of dispersed human islets treated with HG (20 mM) or LIGHT (400 ng/mL) and combined HG + LIGHT with or without IL-22 (50 ng/mL) for LTβR (D) and HVEM (F) together with insulin and DAPI-nuclear counter-staining. Magnification 10×, scale bar 100 µm. Score of LTβR-positive (E) and HVEM-positive cells (G) to the total number of counted cells. For all analysis, data is analyzed by one-way ANOVA with Bonferroni corrections and presented as mean ± s.e.m., n = 3. *P < 0.05, ***P < 0.001 vs untr islets, #P < 0.05, ###P < 0.001 vs HG + LIGHT-treated islets. HG, high glucose; n, number of independent donors; Untr, untreated.

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    IL-22 recovers the adverse effect of HG + LIGHT on human islets function and mass. Insulin secretion in response to basal (1.67 mM) and stimulated (20 mM) levels of glucose was measured by EIA (A) and calculated as stimulation index (SI) (B) on human islets exposed to either HG (20 mM) or LIGHT (400 ng/mL) and the combination of HG + LIGHT with or without IL-22 (50 ng/mL) for 48 h. Insulin content was measured by EIA in human islets treated with HG or LIGHT and combined HG + LIGHT with or without IL-22 for 48 h and normalized to total protein concentration (C). In A, data is analyzed with Wilcoxon matched paired signed rank test and Mann Whitney U-test. In B and C, data is analyzed by one-way ANOVA with Bonferroni corrections. All data are presented as mean ± s.e.m., n = 7. *P < 0.05 vs untr islets, #P < 0.05 vs HG + LIGHT-treated islets. §P < 0.05 vs stimulated glucose level. HG, high glucose; n, number of independent donors; Untr, untreated.

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    IL-22 might at least partly protect human islets from HG + LIGHT-induced ER stress. Western blot analysis of p-IRE1α, BiP, CHOP and GAPDH in human islets treated with HG (20 mM) or LIGHT (400 ng/mL) and HG + LIGHT with or without IL-22 (50 ng/mL) for 48 h (A). Quantification of p-IRE1α (B), BiP (C) and CHOP (D) band densities normalized to GAPDH and presented as fold of untreated islets. For all analysis data is analyzed with one-way ANOVA with Bonferroni corrections and presented as mean ± s.e.m., n = 3. *P < 0.05 vs untr islets. HG, high glucose; n, number of independent donors; Untr, untreated.

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    IL-22 protects human islets from inflammatory responses induced by HG + LIGHT. Assessment of pro-inflammatory mediators IL-6 (A), IL-8(B), MCP-1 (C), IP-10 (D) and TF (E) measured by multiplex assay in cell-free supernatant collected after human islets were cultured with either HG (20 mM) or LIGHT (400 ng/mL) and combination of HG + LIGHT with or without IL-22 (50 ng/mL) for 48 h. In all analysis, data is presented as a fold changes vs untreated islets with mean ± s.e.m., n = 8 and analyzed by one-way ANOVA with Bonferroni corrections. *P < 0.05, **P < 0.01, ***P < 0.001 vs untr islets, #P < 0.05, ##P < 0.01 vs HG + LIGHT-treated islets. HG, high glucose; n, number of independent donors; TF, tissue factor; Untr, untreated.

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    IL-22 reverses elevated apoptosis induced by HG + LIGHT in human islets. Apoptosis evaluated in cytoplasmic fraction of human islets treated with either HG (20 mM) or LIGHT (400 ng/mL) and the combination of HG + LIGHT with or without IL-22 (50 ng/mL) for 48 h by Cell death ELISAPLUS (A) and Caspase 3/7 activity measurement (B) (n = 7). Fluorescein diacetate (FDA)/propidium iodide (PI) staining followed by islets visualization with fluorescent microscope plus the bright-field images taken from the same islets showing islets integrity (C). Magnification 10×, scale bar 200 µm. Protein expression assessment of Bad (D), Bim (E), Bcl-xl (F) and Mcl-1 (G) by multiplex assay in cytoplasmic and mitochondrial fractions of human islets treated with either HG or LIGHT and combined HG + LIGHT with or without IL-22. Data is presented as fold of untreated islets, mean ± s.e.m. and analyzed by one-way ANOVA with Bonferroni corrections, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001 vs untr islets, #P < 0.05, ##P < 0.01 vs HG + LIGHT-treated islets. HG, high glucose; n, number of independent donors; Untr, untreated.

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