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The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes.

Hill T, Krougly O, Nikoopour E, Bellemore S, Lee-Chan E, Fouser LA, Hill DJ, Singh B - Cell Regen (Lond) (2013)

Bottom Line: This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas.Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22.We conclude that IL-22 may contribute to the regeneration of β-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON Canada.

ABSTRACT

Background: In Type 1 diabetes, the insulin-producing β-cells within the pancreatic islets of Langerhans are destroyed. We showed previously that immunotherapy with Bacillus Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) of non-obese diabetic (NOD) mice can prevent disease process and pancreatic β-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas.

Results: We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg3δ (INGAP) mRNA expression in isolated pre-diabetic NOD islets treated with IL-22. We measured IL-22, and IL-22 receptor(R)-α mRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice. Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22.

Conclusions: We conclude that IL-22 may contribute to the regeneration of β-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

No MeSH data available.


Related in: MedlinePlus

Detection of pancreatic islet regeneration after IL-22 treatment. Immunofluorescence localization of nuclear EdU and cytoplasmic insulin within pancreatic islets was done in 5 to 6-week-old NOD mice. Islets were treated with either DMEM media alone (A) or 10 ng/mL of recombinant IL-22 for 48 hrs (B) (red = EdU; green = insulin; blue = DAPI). Examples of dual-labeled cells are shown with arrows. The size bar = 50 μm. (C) The mean percentage ± SEM of β-cells immunopositive for both EdU and insulin relative to insulin alone. Results were taken from 26 control islets and 46 IL-22-treated islets derived from 4 animals for each group. The asterisk (*) denotes a significant difference from the control (P<0.001). As described in the Methods section, in parallel experiments we explored the nuclear expression of Ki/67 as a measure of islet cell proliferation (data not shown). This further confirmed the proliferation of islet cells by IL-22 treatment.
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Fig5: Detection of pancreatic islet regeneration after IL-22 treatment. Immunofluorescence localization of nuclear EdU and cytoplasmic insulin within pancreatic islets was done in 5 to 6-week-old NOD mice. Islets were treated with either DMEM media alone (A) or 10 ng/mL of recombinant IL-22 for 48 hrs (B) (red = EdU; green = insulin; blue = DAPI). Examples of dual-labeled cells are shown with arrows. The size bar = 50 μm. (C) The mean percentage ± SEM of β-cells immunopositive for both EdU and insulin relative to insulin alone. Results were taken from 26 control islets and 46 IL-22-treated islets derived from 4 animals for each group. The asterisk (*) denotes a significant difference from the control (P<0.001). As described in the Methods section, in parallel experiments we explored the nuclear expression of Ki/67 as a measure of islet cell proliferation (data not shown). This further confirmed the proliferation of islet cells by IL-22 treatment.

Mentions: To determine whether IL-22 can promote DNA synthesis in islet β-cells, we performed immunohistochemical analysis on isolated islets from 5 to 6-week-old NOD mice treated with recombinant IL-22 (10 ng/ml) for 48 hours and stained for nuclear EdU and cytoplasmic insulin (Figure 5A and B). The percentage of EdU positive β-cells was found to be significantly higher in islets treated with recombinant IL-22 when compared to control islets treated with media alone (P<0.001) (Figure 5C). To confirm results for DNA synthesis, slides containing IL-22 treated islets were stained for nuclear Ki/67 protein, which is associated with cell proliferation. The islets were stained with mouse anti-Ki/67 followed by incubation with secondary antibody. The positive expression of Ki/67 in the islets validated the induction of islet β-cells by IL-22 (data not shown). The ability of IL-22 to increase the percentage of β-cells undergoing DNA synthesis, as detected by nuclear labeling with EdU, was inversely related to islet size (y = −29logx + 56, r2 = 0.31, p<0.001, n=46). Islet size was estimated from the total number of insulin-immuno-reactive β-cells present per islet within tissue sections. No such relationship existed between islet size and the percent nuclear labeling of β-cells with EdU for control incubations (y = 2.2logx + 4.3, r2 = 0.01, non-significant, n=26). These findings suggest that the mitogenic actions of IL-22 on β-cells were greatest for the smaller islets.Figure 5


The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes.

Hill T, Krougly O, Nikoopour E, Bellemore S, Lee-Chan E, Fouser LA, Hill DJ, Singh B - Cell Regen (Lond) (2013)

Detection of pancreatic islet regeneration after IL-22 treatment. Immunofluorescence localization of nuclear EdU and cytoplasmic insulin within pancreatic islets was done in 5 to 6-week-old NOD mice. Islets were treated with either DMEM media alone (A) or 10 ng/mL of recombinant IL-22 for 48 hrs (B) (red = EdU; green = insulin; blue = DAPI). Examples of dual-labeled cells are shown with arrows. The size bar = 50 μm. (C) The mean percentage ± SEM of β-cells immunopositive for both EdU and insulin relative to insulin alone. Results were taken from 26 control islets and 46 IL-22-treated islets derived from 4 animals for each group. The asterisk (*) denotes a significant difference from the control (P<0.001). As described in the Methods section, in parallel experiments we explored the nuclear expression of Ki/67 as a measure of islet cell proliferation (data not shown). This further confirmed the proliferation of islet cells by IL-22 treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230743&req=5

Fig5: Detection of pancreatic islet regeneration after IL-22 treatment. Immunofluorescence localization of nuclear EdU and cytoplasmic insulin within pancreatic islets was done in 5 to 6-week-old NOD mice. Islets were treated with either DMEM media alone (A) or 10 ng/mL of recombinant IL-22 for 48 hrs (B) (red = EdU; green = insulin; blue = DAPI). Examples of dual-labeled cells are shown with arrows. The size bar = 50 μm. (C) The mean percentage ± SEM of β-cells immunopositive for both EdU and insulin relative to insulin alone. Results were taken from 26 control islets and 46 IL-22-treated islets derived from 4 animals for each group. The asterisk (*) denotes a significant difference from the control (P<0.001). As described in the Methods section, in parallel experiments we explored the nuclear expression of Ki/67 as a measure of islet cell proliferation (data not shown). This further confirmed the proliferation of islet cells by IL-22 treatment.
Mentions: To determine whether IL-22 can promote DNA synthesis in islet β-cells, we performed immunohistochemical analysis on isolated islets from 5 to 6-week-old NOD mice treated with recombinant IL-22 (10 ng/ml) for 48 hours and stained for nuclear EdU and cytoplasmic insulin (Figure 5A and B). The percentage of EdU positive β-cells was found to be significantly higher in islets treated with recombinant IL-22 when compared to control islets treated with media alone (P<0.001) (Figure 5C). To confirm results for DNA synthesis, slides containing IL-22 treated islets were stained for nuclear Ki/67 protein, which is associated with cell proliferation. The islets were stained with mouse anti-Ki/67 followed by incubation with secondary antibody. The positive expression of Ki/67 in the islets validated the induction of islet β-cells by IL-22 (data not shown). The ability of IL-22 to increase the percentage of β-cells undergoing DNA synthesis, as detected by nuclear labeling with EdU, was inversely related to islet size (y = −29logx + 56, r2 = 0.31, p<0.001, n=46). Islet size was estimated from the total number of insulin-immuno-reactive β-cells present per islet within tissue sections. No such relationship existed between islet size and the percent nuclear labeling of β-cells with EdU for control incubations (y = 2.2logx + 4.3, r2 = 0.01, non-significant, n=26). These findings suggest that the mitogenic actions of IL-22 on β-cells were greatest for the smaller islets.Figure 5

Bottom Line: This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas.Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22.We conclude that IL-22 may contribute to the regeneration of β-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON Canada.

ABSTRACT

Background: In Type 1 diabetes, the insulin-producing β-cells within the pancreatic islets of Langerhans are destroyed. We showed previously that immunotherapy with Bacillus Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) of non-obese diabetic (NOD) mice can prevent disease process and pancreatic β-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas.

Results: We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg3δ (INGAP) mRNA expression in isolated pre-diabetic NOD islets treated with IL-22. We measured IL-22, and IL-22 receptor(R)-α mRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice. Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet β-cell DNA synthesis in vitro in the presence of IL-22.

Conclusions: We conclude that IL-22 may contribute to the regeneration of β-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

No MeSH data available.


Related in: MedlinePlus