IL-33 Prevents MLD-STZ Induction of Diabetes and Attenuate Insulitis in Prediabetic NOD Mice

Abstract

Type 1 diabetes is an autoimmune disease caused by the immune-mediated destruction of pancreatic β-cells. Prevention of type 1 diabetes requires early intervention in the autoimmune process against beta-cells of the pancreatic islets of Langerhans, which is believed to result from disordered immunoregulation. CD4⁺Foxp3⁺ regulatory T cells (Tregs) participate as one of the most important cell types in limiting the autoimmune process. The aim of this study was to investigate the effect of exogenous IL-33 in multiple low dose streptozotocin (MLD-STZ) induced diabetes and to delineate its role in the induction of protective Tregs in an autoimmune attack. C57BL/6 mice were treated i. p. with five doses of 40 mg/kg STZ and 0.4 μg rIL-33 four times, starting from day 0, 6, or 12 every second day from the day of disease induction. 16 weeks old NOD mice were treated with 6 injections of 0.4 μg/mouse IL-33 (every second day). Glycemia and glycosuria were measured and histological parameters in pancreatic islets were evaluated at the end of experiments. Cellular make up of the pancreatic lymph nodes and islets were evaluated by flow cytometry. IL-33 given simultaneously with the application of STZ completely prevented the development of hyperglycemia, glycosuria and profoundly attenuated mononuclear cell infiltration. IL-33 treatment was accompanied by higher number of IL-13 and IL-5 producing CD4⁺ T cells and increased presence of ST2⁺Foxp3⁺ regulatory T cells in pancreatic lymph nodes and islets. Elimination of Tregs abrogated protective effect of IL-33. We provide evidence that exogenous IL-33 completely prevents the development of T cell mediated inflammation in pancreatic islets and consecutive development of diabetes in C57BL/6 mice by facilitating the induction Treg cells. To extend this finding for possible relevance in spontaneous diabetes, we showed that IL-33 attenuate insulitis in prediabetic NOD mice.

Keywords: C57BL/6 mice; IL-33; NOD mice; diabetes; streptozotocin.

Figure 1

Concomitant treatment with recombinant IL-33 completely abrogates induction of diabetes by MLD-STZ. Effect of four injections of 0.4 μg/mouse IL-33 on glycemia (A), GTT test (B), and glycosuria (C,D). Histology of the islets showed highly significant (p < 0.001) decrease of mononuclear cells influx in IL-33 treated group compared to control group (E,F). An analysis of lymphocytic infiltrates in Langerhans's pancreatic islands was performed by light microscope using a magnifying lens of 40 X.

Figure 2

IL-33 attenuates inflammatory cells and increases IL-5 and IL-13 producing CD4⁺ cells. The percentage and total number of CD4⁺ (A,B), CD8⁺ (C,D), CD4⁺IFN-γ⁺ (E,F), CD8⁺IFN-γ⁺ (G,H), CD4⁺IL-17⁺ (I,J), CD4⁺IL-5⁺ (K,L), CD4⁺IL-13⁺ (M,N) were examined. The animals were treated with 0.4 μg/injection IL-33 together with MLD-STZ (i.p. 40 mg/kg for 5 consecutive days) or of an equimolar dose of PBS or 0.4 μg/injection IL-33 together with citrate buffer. Cells were obtained from pancreatic lymph nodes on day 28 after diabetes induction. Data from two individual experiments with at least 8 mice per group are shown as mean ± SEM; by paired t-test when compared with values obtained with phosphate-buffered saline.

Figure 3

Exogenous IL-33 significantly increases the percentage and total number of regulatory T cells (Foxp3⁺ST2⁺IL-10⁺) in the pancreatic lymph node. The percentage and total number of CD4⁺Foxp3⁺ (A,B), CD4⁺Foxp3⁺ST2⁺ (C,D), CD4⁺Foxp3⁺ST2⁺IL-10⁺ (E,F), CD4⁺Foxp3⁺ST2⁺IL-13⁺ (G,H), CD4⁺Foxp3⁺ST2⁺IL-5⁺ (I,J), CD11c⁺ (K,L), CD11c⁺IL-2⁺ (M,N) were examined. The animals were treated with 0.4 μg/injection IL-33 together with MLD-STZ (i.p. 40 mg/kg for 5 consecutive days) or of an equimolar dose of PBS or with 0.4 μg/injection IL-33 together with citrate buffer. Cells were obtained from pancreatic lymph nodes on day 28 after diabetes induction. Data from two individual experiments with at least 8 mice per group are shown as mean ± SEM; by paired t-test when compared with values obtained with phosphate-buffered saline.

Figure 4

IL-33 attenuates influx of inflammatory cells and promotes regulatory cells infiltration in the pancreatic islets. The percentage and total number of CD4⁺ (A,B), CD4⁺CCR6⁺ (C,D), CD4⁺CXCR3⁺ (E,F), CD4⁺IL-10⁺ (G,H), CD8⁺ (I,J), CD8⁺CXCR3⁺ (K,L), CD4⁺Foxp3⁺ (M,N), CD4⁺Foxp3⁺ST2⁺ (O,P), CD4⁺Foxp3⁺ST2⁺IL-10⁺ (Q,R), CD11b⁺CD11c⁺ (S,T) were examined. IL-33 treatment significant decreases effector CD4⁺CXCR3⁺ and CD8⁺CXCR3⁺ cells and increases percentage of regulatory cells, myeloid dendritic cells. The animals were treated with 0.4 μg/injection IL-33 together with MLD-STZ (i.p. 40 mg/kg for 5 consecutive days) or of an equimolar dose of PBS or with 0.4 μg/injection IL-33 together with citrate buffer. Cells were obtained from pancreatic islets on day 28 after diabetes induction. Data from two individual experiments with at least 8 mice per group are shown as mean ± SEM; by paired t-test when compared with values obtained with phosphate-buffered saline.

Figure 5

Low dose of cyclophosphamide (CY) affects regulatory cells and attenuates protective effect of IL-33. In order to confirm our hypothesis that the effect of Il-33 administration leads to activation and an increase in the number of regulatory T cells, we added a group that, in addition to MLD STZ and IL-33, also received a low dose of CY (2 × 100 mg/kg) that eliminates regulatory T cells (21). Effect of four injections of 0.4 μg/mouse IL-33, MLD STZ and CY on glycemia (A). The number of CD4⁺ (B), CD4⁺Foxp3⁺ (C), CD4⁺Foxp3⁺ST2⁺ (D) in pancreatic lymph nodes and percentage of CD4⁺Foxp3⁺ (E), CD4⁺Foxp3⁺ST2⁺ (F) in pancreatic islets. The animals were treated with MLD-STZ (i.p. 40 mg/kg for 5 consecutive days) with PBS or 0.4 μg/injection of IL-33 or CY or both. Cells were obtained from pancreatic islets and pancreatic lymph nodes on day 28 after diabetes induction. Data from one experiment with at least 8 mice per group are shown as mean ± SEM; by paired t-test.

Figure 6

IL-33 given 6 and 12 days after diabetes induction partially attenuates clinical signs and influx of inflammatory cells in the islets. Effect of four injections of 0.4 μg/mouse IL-33 on glycemia (A,B), GTT test (C,D) and glycosuria (E,F) Histology of the islet showed significantly (p < 0.001) decreased influx of mononuclear cells in IL-33 treated group in comparison with control group (G,H). An analysis of lymphocytic infiltrates in Langerhans's pancreatic islands was performed by light microscope using a magnifying lens of 40 X.

Figure 7

IL-33 decrease insulitis and change composition of mononuclear islet infiltration in prediabetic NOD mice. 16 weeks old animals that were all normoglycemic, free of glycosuria and with normal GTT test were treated with 6 injections of 0,4 μg/mouse IL-33 (every second day) and sacrificed for histological analysis at 18th week of age (A). The percentage and number of CD4⁺ (B,C), CD8⁺ (D,E), CD4⁺IL-17⁺ (F,G), CD3⁺CD4⁺IL-5⁺ (H,I), CD3⁺CD4⁺IL-13⁺ (J,K), CD4⁺Foxp3⁺ (L,M) was examined by flow cytometric analysis. IL-33 treatment significant increase percentage of CD3⁺CD4⁺IL-5⁺ (H,I), CD3⁺CD4⁺IL-13⁺ (J,K) and regulatory CD4⁺Foxp3⁺ (L,M) cells. Data from one experiment with 10 mice per group are shown as mean ± SEM; by paired t-test when compared with values obtained with phosphate-buffered saline.