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Interleukin-1β produced in response to islet autoantigen presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for the timing of tolerizing immunotherapy.

Bertin-Maghit S, Pang D, O'Sullivan B, Best S, Duggan E, Paul S, Thomas H, Kay TW, Harrison LC, Steptoe R, Thomas R - Diabetes (2010)

Bottom Line: IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred.IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells.RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Australia.

ABSTRACT

Objective: The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelB(lo) DCs in the prediabetic period.

Research design and methods: We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro.

Results: Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.

Conclusions: IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies.

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In the IL-1–rich insulitic environment, tolerizing DCs reduce suppressor function at the expense of IL-17. Four-week-old, 12-week-old NOD mice, and 12-week-old NOD.IL1R−/− mice were injected subcutaneously with RelBlo DCs or saline. A: The percentage of CD4+ cells expressing FoxP3 in spleen and the mean fluorescence intensity (MFI) of FoxP3 expressed by CD4+ T-cells were enumerated. B: Four weeks later, CD4+CD25+ T-cells were isolated from spleen and various numbers were added to 1 × 105 CD11c+ splenic DCs and 1 × 105 CD4+CD25− T-cells purified from naïve 6-week NOD mice and stimulated with 0.5 μg/ml anti-CD3. Proliferation was assessed by incorporation of [3H] thymidine. Data represent the mean of triplicate wells ± SE. A total of 6–9 mice were pooled in each of two separate experiments. ***P < 0.0001 (two-way ANOVA). C and D: Six-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs. As DCs were administered, some groups of wild-type mice were administered anti–IL-17 mAb intraperitoneally on alternate days for 10 days or CD4+CD25+ Treg purified from either wild-type or NOD.IL-1R1−/− mice intravenously once. After 4 weeks, Teff and Treg were purified from each group and incubated with DCs in the presence of anti-CD3. IL-17 levels were measured in supernatants by ELISA (C) and cells restimulated with PMA in the presence of brefeldin A were stained for CD4, CD45.2, FoxP3, and IL-17 (D). *P < 0.05; **P < 0.001; ***P < 0.0001 (one-way ANOVA) (C and D).
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Figure 7: In the IL-1–rich insulitic environment, tolerizing DCs reduce suppressor function at the expense of IL-17. Four-week-old, 12-week-old NOD mice, and 12-week-old NOD.IL1R−/− mice were injected subcutaneously with RelBlo DCs or saline. A: The percentage of CD4+ cells expressing FoxP3 in spleen and the mean fluorescence intensity (MFI) of FoxP3 expressed by CD4+ T-cells were enumerated. B: Four weeks later, CD4+CD25+ T-cells were isolated from spleen and various numbers were added to 1 × 105 CD11c+ splenic DCs and 1 × 105 CD4+CD25− T-cells purified from naïve 6-week NOD mice and stimulated with 0.5 μg/ml anti-CD3. Proliferation was assessed by incorporation of [3H] thymidine. Data represent the mean of triplicate wells ± SE. A total of 6–9 mice were pooled in each of two separate experiments. ***P < 0.0001 (two-way ANOVA). C and D: Six-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs. As DCs were administered, some groups of wild-type mice were administered anti–IL-17 mAb intraperitoneally on alternate days for 10 days or CD4+CD25+ Treg purified from either wild-type or NOD.IL-1R1−/− mice intravenously once. After 4 weeks, Teff and Treg were purified from each group and incubated with DCs in the presence of anti-CD3. IL-17 levels were measured in supernatants by ELISA (C) and cells restimulated with PMA in the presence of brefeldin A were stained for CD4, CD45.2, FoxP3, and IL-17 (D). *P < 0.05; **P < 0.001; ***P < 0.0001 (one-way ANOVA) (C and D).

Mentions: Given the IL-1–dependent dysregulation of Treg, coincident with the failure of RelBlo DCs to prevent type 1 diabetes, we analyzed the relationship between RelBlo DC administration and Treg number and function. As NOD mice age, Treg lose expression of FoxP3 (48). The frequency of FoxP3+ Treg in spleen and intensity of FoxP3 expression 4 weeks later were not affected by RelBlo DC administration to 4- or 12-week mice (Fig. 7A). Treg cells purified from untreated 8-week-old NOD mice suppressed Teff purified from naïve 6-week-old NOD mice, but this was significantly reduced by administration of RelBlo DC 4 weeks previously (Fig. 7B). Suppressive capacity of Treg cells purified from 16-week-old NOD mice was reduced relative to the activity of Treg cells purified from 8-week-old mice and significantly reduced by administration of RelBlo DCs 4 weeks previously (Fig. 7B). In contrast, suppressive activity of Treg cells isolated from 16-week-old NOD.IL-1R1−/− recipients of RelBlo DCs administered 4 weeks earlier and untreated NOD.IL-1R1−/− mice was equivalent (Fig. 7B). The data demonstrate that RelBlo DCs exacerbate the age-dependent decline in Treg cell function. Similarly, when 6-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs, IL-17 secretion by splenic Teff and Treg was promoted by RelBlo DCs in an IL-1–dependent manner 4 weeks later (Fig. 7C). IL-1–dependent IL-17 secretion by anti-CD3–stimulated pancreatic draining lymph node cells from these mice was greater after treatment with RelBlo DCs (Fig. 7C). Consistent with this IL-1 dependence, promotion of IL-17 production by RelBlo DCs was not altered by treatment of wild-type recipients with anti–IL-17 mAb (Fig. 7D). Consistent with the inability of IL-1R1−/− Treg to suppress IL-17 production by wild-type cells in vitro (Fig. 6), adoptive transfer of wild-type or NOD.IL-1R1−/− Treg purified from 4-week-old donors to 6-week-old recipients did not impact IL-17 secretion in the presence of RelBlo DCs (Fig. 7D). By staining, IL-17 was almost all produced by host FoxP3lo Treg (data not shown). The data demonstrate that RelBlo DCs exacerbate the age-dependent decline in Treg cell function at the expense of conversion to Th17 in NOD mice including in pancreatic draining lymph node, in an IL-1–dependent manner.


Interleukin-1β produced in response to islet autoantigen presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for the timing of tolerizing immunotherapy.

Bertin-Maghit S, Pang D, O'Sullivan B, Best S, Duggan E, Paul S, Thomas H, Kay TW, Harrison LC, Steptoe R, Thomas R - Diabetes (2010)

In the IL-1–rich insulitic environment, tolerizing DCs reduce suppressor function at the expense of IL-17. Four-week-old, 12-week-old NOD mice, and 12-week-old NOD.IL1R−/− mice were injected subcutaneously with RelBlo DCs or saline. A: The percentage of CD4+ cells expressing FoxP3 in spleen and the mean fluorescence intensity (MFI) of FoxP3 expressed by CD4+ T-cells were enumerated. B: Four weeks later, CD4+CD25+ T-cells were isolated from spleen and various numbers were added to 1 × 105 CD11c+ splenic DCs and 1 × 105 CD4+CD25− T-cells purified from naïve 6-week NOD mice and stimulated with 0.5 μg/ml anti-CD3. Proliferation was assessed by incorporation of [3H] thymidine. Data represent the mean of triplicate wells ± SE. A total of 6–9 mice were pooled in each of two separate experiments. ***P < 0.0001 (two-way ANOVA). C and D: Six-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs. As DCs were administered, some groups of wild-type mice were administered anti–IL-17 mAb intraperitoneally on alternate days for 10 days or CD4+CD25+ Treg purified from either wild-type or NOD.IL-1R1−/− mice intravenously once. After 4 weeks, Teff and Treg were purified from each group and incubated with DCs in the presence of anti-CD3. IL-17 levels were measured in supernatants by ELISA (C) and cells restimulated with PMA in the presence of brefeldin A were stained for CD4, CD45.2, FoxP3, and IL-17 (D). *P < 0.05; **P < 0.001; ***P < 0.0001 (one-way ANOVA) (C and D).
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Figure 7: In the IL-1–rich insulitic environment, tolerizing DCs reduce suppressor function at the expense of IL-17. Four-week-old, 12-week-old NOD mice, and 12-week-old NOD.IL1R−/− mice were injected subcutaneously with RelBlo DCs or saline. A: The percentage of CD4+ cells expressing FoxP3 in spleen and the mean fluorescence intensity (MFI) of FoxP3 expressed by CD4+ T-cells were enumerated. B: Four weeks later, CD4+CD25+ T-cells were isolated from spleen and various numbers were added to 1 × 105 CD11c+ splenic DCs and 1 × 105 CD4+CD25− T-cells purified from naïve 6-week NOD mice and stimulated with 0.5 μg/ml anti-CD3. Proliferation was assessed by incorporation of [3H] thymidine. Data represent the mean of triplicate wells ± SE. A total of 6–9 mice were pooled in each of two separate experiments. ***P < 0.0001 (two-way ANOVA). C and D: Six-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs. As DCs were administered, some groups of wild-type mice were administered anti–IL-17 mAb intraperitoneally on alternate days for 10 days or CD4+CD25+ Treg purified from either wild-type or NOD.IL-1R1−/− mice intravenously once. After 4 weeks, Teff and Treg were purified from each group and incubated with DCs in the presence of anti-CD3. IL-17 levels were measured in supernatants by ELISA (C) and cells restimulated with PMA in the presence of brefeldin A were stained for CD4, CD45.2, FoxP3, and IL-17 (D). *P < 0.05; **P < 0.001; ***P < 0.0001 (one-way ANOVA) (C and D).
Mentions: Given the IL-1–dependent dysregulation of Treg, coincident with the failure of RelBlo DCs to prevent type 1 diabetes, we analyzed the relationship between RelBlo DC administration and Treg number and function. As NOD mice age, Treg lose expression of FoxP3 (48). The frequency of FoxP3+ Treg in spleen and intensity of FoxP3 expression 4 weeks later were not affected by RelBlo DC administration to 4- or 12-week mice (Fig. 7A). Treg cells purified from untreated 8-week-old NOD mice suppressed Teff purified from naïve 6-week-old NOD mice, but this was significantly reduced by administration of RelBlo DC 4 weeks previously (Fig. 7B). Suppressive capacity of Treg cells purified from 16-week-old NOD mice was reduced relative to the activity of Treg cells purified from 8-week-old mice and significantly reduced by administration of RelBlo DCs 4 weeks previously (Fig. 7B). In contrast, suppressive activity of Treg cells isolated from 16-week-old NOD.IL-1R1−/− recipients of RelBlo DCs administered 4 weeks earlier and untreated NOD.IL-1R1−/− mice was equivalent (Fig. 7B). The data demonstrate that RelBlo DCs exacerbate the age-dependent decline in Treg cell function. Similarly, when 6-week wild-type or NOD.IL-1R1−/− mice were untreated or injected with RelBlo DCs, IL-17 secretion by splenic Teff and Treg was promoted by RelBlo DCs in an IL-1–dependent manner 4 weeks later (Fig. 7C). IL-1–dependent IL-17 secretion by anti-CD3–stimulated pancreatic draining lymph node cells from these mice was greater after treatment with RelBlo DCs (Fig. 7C). Consistent with this IL-1 dependence, promotion of IL-17 production by RelBlo DCs was not altered by treatment of wild-type recipients with anti–IL-17 mAb (Fig. 7D). Consistent with the inability of IL-1R1−/− Treg to suppress IL-17 production by wild-type cells in vitro (Fig. 6), adoptive transfer of wild-type or NOD.IL-1R1−/− Treg purified from 4-week-old donors to 6-week-old recipients did not impact IL-17 secretion in the presence of RelBlo DCs (Fig. 7D). By staining, IL-17 was almost all produced by host FoxP3lo Treg (data not shown). The data demonstrate that RelBlo DCs exacerbate the age-dependent decline in Treg cell function at the expense of conversion to Th17 in NOD mice including in pancreatic draining lymph node, in an IL-1–dependent manner.

Bottom Line: IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred.IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells.RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Australia.

ABSTRACT

Objective: The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelB(lo) DCs in the prediabetic period.

Research design and methods: We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro.

Results: Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.

Conclusions: IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies.

Show MeSH
Related in: MedlinePlus