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Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice.

Emamaullee JA, Davis J, Merani S, Toso C, Elliott JF, Thiesen A, Shapiro AM - Diabetes (2009)

Bottom Line: Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates.GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity.Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta,Canada. juliete@ualberta.ca

ABSTRACT

Objective: The T helper 17 (Th17) population, a subset of CD4-positive T-cells that secrete interleukin (IL)-17, has been implicated in autoimmune diseases, including multiple sclerosis and lupus. Therapeutic agents that target the Th17 effector molecule IL-17 or directly inhibit the Th17 population (IL-25) have shown promise in animal models of autoimmunity. The role of Th17 cells in type 1 diabetes has been less clear. The effect of neutralizing anti-IL-17 and recombinant IL-25 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, was investigated in this study.

Research design and methods and results: Although treatment with either anti-IL-17 or IL-25 had no effect on diabetes development in young (<5 weeks) NOD mice, either intervention prevented diabetes when treatment was started at 10 weeks of age (P < 0.001). Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates. Both treatments also decreased GAD65 autoantibody levels. Analysis of pancreatic lymph nodes revealed that both treatments increased the frequency of regulatory T-cells. Further investigation demonstrated that IL-25 therapy was superior to anti-IL-17 during mature diabetes because it promoted a period of remission from new-onset diabetes in 90% of treated animals. Similarly, IL-25 delayed recurrent autoimmunity after syngeneic islet transplantation, whereas anti-IL-17 was of no benefit. GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity.

Conclusions: These studies suggest that Th17 cells are involved in the pathogenesis of autoimmune diabetes. Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

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Related in: MedlinePlus

IL-25 treatment reduces the frequency of autoreactive Th2 cells and Th17 cells and leads to the formation of a CD4-positive splenocyte population that can prevent type 1 diabetes development in an adoptive transfer model. At 1 month after the completion of treatment, splenocytes were harvested from either anti–IL-17–treated, IL-25–treated, or control animals (n = 3–4 animals per treatment group; all normoglycemic) that had been previously treated beginning at 10 weeks of age. Purified splenocytes were analyzed using cytokine ELISpot assays or adoptively transferred into NOD-RAG−/− recipients. A: No difference in the frequency of GAD65-responsive, IFN-γ–secreting splenocytes was observed in either treatment group versus controls. B: IL-25 treatment resulted in a reduction in the number of GAD65-responsive, IL-4–secreting splenocytes compared with both anti–IL-17–treated animals (*P < 0.02 for IL-25 vs. anti–IL-17 and control by ANOVA), suggesting that IL-25 treatment can reduce the frequency of autoreactive Th2 cells. C: Whereas IL-25 treatment was associated with a reduction in the number of GAD65-responsive, IL-17–secreting splenocytes (# P < 0.05 vs. control by ANOVA), anti–IL-17 treatment significantly increased the frequency of this population (*P < 0.001 by ANOVA vs. control and IL-25). D: CD4-positive lymphocytes were further purified from these splenocyte preparations using magnetic beads. Naïve NOD-RAG−/− males received either 1 × 107 splenocytes harvested from spontaneously diabetic NOD mice (diabetic splenocytes) combined with 2 × 106 CD4-positive splenocytes isolated from mice previously treated with anti–IL-17, 1 × 107 diabetic splenocytes combined with 2 × 106 CD4-positive splenocytes from mice previously treated with IL-25, or 1 × 107 diabetic splenocytes with no CD4 supplementation (control). Animals were monitored three times per week thereafter for diabetes onset. Supplementation with CD4-positive cells harvested from mice previously treated with IL-25 resulted in a significant dominant protective effect, preventing diabetes development in 75% of the animals in this group (P < 0.005 vs. anti–IL-17 and control by log-rank). Supplementation with CD4-positive cells from animals previously treated with anti–IL-17 had no effect, with 100% of the animals becoming diabetic within 60 days post-transfer, which was comparable to the diabetes incidence observed in the control group.
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Figure 6: IL-25 treatment reduces the frequency of autoreactive Th2 cells and Th17 cells and leads to the formation of a CD4-positive splenocyte population that can prevent type 1 diabetes development in an adoptive transfer model. At 1 month after the completion of treatment, splenocytes were harvested from either anti–IL-17–treated, IL-25–treated, or control animals (n = 3–4 animals per treatment group; all normoglycemic) that had been previously treated beginning at 10 weeks of age. Purified splenocytes were analyzed using cytokine ELISpot assays or adoptively transferred into NOD-RAG−/− recipients. A: No difference in the frequency of GAD65-responsive, IFN-γ–secreting splenocytes was observed in either treatment group versus controls. B: IL-25 treatment resulted in a reduction in the number of GAD65-responsive, IL-4–secreting splenocytes compared with both anti–IL-17–treated animals (*P < 0.02 for IL-25 vs. anti–IL-17 and control by ANOVA), suggesting that IL-25 treatment can reduce the frequency of autoreactive Th2 cells. C: Whereas IL-25 treatment was associated with a reduction in the number of GAD65-responsive, IL-17–secreting splenocytes (# P < 0.05 vs. control by ANOVA), anti–IL-17 treatment significantly increased the frequency of this population (*P < 0.001 by ANOVA vs. control and IL-25). D: CD4-positive lymphocytes were further purified from these splenocyte preparations using magnetic beads. Naïve NOD-RAG−/− males received either 1 × 107 splenocytes harvested from spontaneously diabetic NOD mice (diabetic splenocytes) combined with 2 × 106 CD4-positive splenocytes isolated from mice previously treated with anti–IL-17, 1 × 107 diabetic splenocytes combined with 2 × 106 CD4-positive splenocytes from mice previously treated with IL-25, or 1 × 107 diabetic splenocytes with no CD4 supplementation (control). Animals were monitored three times per week thereafter for diabetes onset. Supplementation with CD4-positive cells harvested from mice previously treated with IL-25 resulted in a significant dominant protective effect, preventing diabetes development in 75% of the animals in this group (P < 0.005 vs. anti–IL-17 and control by log-rank). Supplementation with CD4-positive cells from animals previously treated with anti–IL-17 had no effect, with 100% of the animals becoming diabetic within 60 days post-transfer, which was comparable to the diabetes incidence observed in the control group.

Mentions: Although both anti–IL-17 and IL-25 therapies were able to reduce the incidence of type 1 diabetes during the effector phase leading into type 1 diabetes, only IL-25 therapy was able to control diabetes once the disease was established. To further investigate the different mechanisms by which these two therapies function, splenocytes from normoglycemic treated animals in the prevention studies (Fig. 1) were examined ex vivo for autoreactive T-cell populations using GAD65-stimulated ELISpot assays at 1 month after the completion of treatment. Although no difference in IFN-γ–secreting GAD65-responsive splenocytes was observed compared with controls (Fig. 6A), a significant reduction in IL-4–secreting GAD65-responsive splenocytes was observed in IL-25–treated animals compared with both anti–IL-17–treated and control animals (P < 0.02) (Fig. 6B). Paradoxically, anti–IL-17 treatment resulted in an increased frequency of IL-17–secreting GAD65-reponsive splenocytes, whereas the opposite occurred after IL-25 treatment, where a significant reduction in this autoreactive Th17 population was observed (P < 0.001 for anti–IL-17 vs. control and IL-25, and P < 0.05 by ANOVA for IL-25 vs. control) (Fig. 6C).


Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice.

Emamaullee JA, Davis J, Merani S, Toso C, Elliott JF, Thiesen A, Shapiro AM - Diabetes (2009)

IL-25 treatment reduces the frequency of autoreactive Th2 cells and Th17 cells and leads to the formation of a CD4-positive splenocyte population that can prevent type 1 diabetes development in an adoptive transfer model. At 1 month after the completion of treatment, splenocytes were harvested from either anti–IL-17–treated, IL-25–treated, or control animals (n = 3–4 animals per treatment group; all normoglycemic) that had been previously treated beginning at 10 weeks of age. Purified splenocytes were analyzed using cytokine ELISpot assays or adoptively transferred into NOD-RAG−/− recipients. A: No difference in the frequency of GAD65-responsive, IFN-γ–secreting splenocytes was observed in either treatment group versus controls. B: IL-25 treatment resulted in a reduction in the number of GAD65-responsive, IL-4–secreting splenocytes compared with both anti–IL-17–treated animals (*P < 0.02 for IL-25 vs. anti–IL-17 and control by ANOVA), suggesting that IL-25 treatment can reduce the frequency of autoreactive Th2 cells. C: Whereas IL-25 treatment was associated with a reduction in the number of GAD65-responsive, IL-17–secreting splenocytes (# P < 0.05 vs. control by ANOVA), anti–IL-17 treatment significantly increased the frequency of this population (*P < 0.001 by ANOVA vs. control and IL-25). D: CD4-positive lymphocytes were further purified from these splenocyte preparations using magnetic beads. Naïve NOD-RAG−/− males received either 1 × 107 splenocytes harvested from spontaneously diabetic NOD mice (diabetic splenocytes) combined with 2 × 106 CD4-positive splenocytes isolated from mice previously treated with anti–IL-17, 1 × 107 diabetic splenocytes combined with 2 × 106 CD4-positive splenocytes from mice previously treated with IL-25, or 1 × 107 diabetic splenocytes with no CD4 supplementation (control). Animals were monitored three times per week thereafter for diabetes onset. Supplementation with CD4-positive cells harvested from mice previously treated with IL-25 resulted in a significant dominant protective effect, preventing diabetes development in 75% of the animals in this group (P < 0.005 vs. anti–IL-17 and control by log-rank). Supplementation with CD4-positive cells from animals previously treated with anti–IL-17 had no effect, with 100% of the animals becoming diabetic within 60 days post-transfer, which was comparable to the diabetes incidence observed in the control group.
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Related In: Results  -  Collection

Show All Figures
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Figure 6: IL-25 treatment reduces the frequency of autoreactive Th2 cells and Th17 cells and leads to the formation of a CD4-positive splenocyte population that can prevent type 1 diabetes development in an adoptive transfer model. At 1 month after the completion of treatment, splenocytes were harvested from either anti–IL-17–treated, IL-25–treated, or control animals (n = 3–4 animals per treatment group; all normoglycemic) that had been previously treated beginning at 10 weeks of age. Purified splenocytes were analyzed using cytokine ELISpot assays or adoptively transferred into NOD-RAG−/− recipients. A: No difference in the frequency of GAD65-responsive, IFN-γ–secreting splenocytes was observed in either treatment group versus controls. B: IL-25 treatment resulted in a reduction in the number of GAD65-responsive, IL-4–secreting splenocytes compared with both anti–IL-17–treated animals (*P < 0.02 for IL-25 vs. anti–IL-17 and control by ANOVA), suggesting that IL-25 treatment can reduce the frequency of autoreactive Th2 cells. C: Whereas IL-25 treatment was associated with a reduction in the number of GAD65-responsive, IL-17–secreting splenocytes (# P < 0.05 vs. control by ANOVA), anti–IL-17 treatment significantly increased the frequency of this population (*P < 0.001 by ANOVA vs. control and IL-25). D: CD4-positive lymphocytes were further purified from these splenocyte preparations using magnetic beads. Naïve NOD-RAG−/− males received either 1 × 107 splenocytes harvested from spontaneously diabetic NOD mice (diabetic splenocytes) combined with 2 × 106 CD4-positive splenocytes isolated from mice previously treated with anti–IL-17, 1 × 107 diabetic splenocytes combined with 2 × 106 CD4-positive splenocytes from mice previously treated with IL-25, or 1 × 107 diabetic splenocytes with no CD4 supplementation (control). Animals were monitored three times per week thereafter for diabetes onset. Supplementation with CD4-positive cells harvested from mice previously treated with IL-25 resulted in a significant dominant protective effect, preventing diabetes development in 75% of the animals in this group (P < 0.005 vs. anti–IL-17 and control by log-rank). Supplementation with CD4-positive cells from animals previously treated with anti–IL-17 had no effect, with 100% of the animals becoming diabetic within 60 days post-transfer, which was comparable to the diabetes incidence observed in the control group.
Mentions: Although both anti–IL-17 and IL-25 therapies were able to reduce the incidence of type 1 diabetes during the effector phase leading into type 1 diabetes, only IL-25 therapy was able to control diabetes once the disease was established. To further investigate the different mechanisms by which these two therapies function, splenocytes from normoglycemic treated animals in the prevention studies (Fig. 1) were examined ex vivo for autoreactive T-cell populations using GAD65-stimulated ELISpot assays at 1 month after the completion of treatment. Although no difference in IFN-γ–secreting GAD65-responsive splenocytes was observed compared with controls (Fig. 6A), a significant reduction in IL-4–secreting GAD65-responsive splenocytes was observed in IL-25–treated animals compared with both anti–IL-17–treated and control animals (P < 0.02) (Fig. 6B). Paradoxically, anti–IL-17 treatment resulted in an increased frequency of IL-17–secreting GAD65-reponsive splenocytes, whereas the opposite occurred after IL-25 treatment, where a significant reduction in this autoreactive Th17 population was observed (P < 0.001 for anti–IL-17 vs. control and IL-25, and P < 0.05 by ANOVA for IL-25 vs. control) (Fig. 6C).

Bottom Line: Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates.GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity.Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Alberta, Edmonton, Alberta,Canada. juliete@ualberta.ca

ABSTRACT

Objective: The T helper 17 (Th17) population, a subset of CD4-positive T-cells that secrete interleukin (IL)-17, has been implicated in autoimmune diseases, including multiple sclerosis and lupus. Therapeutic agents that target the Th17 effector molecule IL-17 or directly inhibit the Th17 population (IL-25) have shown promise in animal models of autoimmunity. The role of Th17 cells in type 1 diabetes has been less clear. The effect of neutralizing anti-IL-17 and recombinant IL-25 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, was investigated in this study.

Research design and methods and results: Although treatment with either anti-IL-17 or IL-25 had no effect on diabetes development in young (<5 weeks) NOD mice, either intervention prevented diabetes when treatment was started at 10 weeks of age (P < 0.001). Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates. Both treatments also decreased GAD65 autoantibody levels. Analysis of pancreatic lymph nodes revealed that both treatments increased the frequency of regulatory T-cells. Further investigation demonstrated that IL-25 therapy was superior to anti-IL-17 during mature diabetes because it promoted a period of remission from new-onset diabetes in 90% of treated animals. Similarly, IL-25 delayed recurrent autoimmunity after syngeneic islet transplantation, whereas anti-IL-17 was of no benefit. GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity.

Conclusions: These studies suggest that Th17 cells are involved in the pathogenesis of autoimmune diabetes. Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

Show MeSH
Related in: MedlinePlus