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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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Related in: MedlinePlus

IL-17 is produced by Teff and reprogrammed Tregs in an IL-1β–dependent manner during insulitis. IL-17 was assayed at 5 (A) or 10–14 weeks (B) in supernatants from the T-cell proliferation assay by ELISA from NOD.Lt cells with or without anti–IL-1 or anti–IL-17 mAb (10 μg/ml) or from NOD.IL-1R1−/− cells. C: CD4+CD25− Teff and CD4+CD25+ Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. After 72 h, IL-17 was measured in supernatants. ***P < 0.0001 (one-way ANOVA). D: Cells from the same experiment were restimulated with PMA in the presence of brefeldin A and stained for CD4, CD45.2, FoxP3, and IL-17. Cells are gated on CD4 and the relevant congenic marker to analyze Teff and Treg FoxP3 and IL-17 expression individually.
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Figure 6: IL-17 is produced by Teff and reprogrammed Tregs in an IL-1β–dependent manner during insulitis. IL-17 was assayed at 5 (A) or 10–14 weeks (B) in supernatants from the T-cell proliferation assay by ELISA from NOD.Lt cells with or without anti–IL-1 or anti–IL-17 mAb (10 μg/ml) or from NOD.IL-1R1−/− cells. C: CD4+CD25− Teff and CD4+CD25+ Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. After 72 h, IL-17 was measured in supernatants. ***P < 0.0001 (one-way ANOVA). D: Cells from the same experiment were restimulated with PMA in the presence of brefeldin A and stained for CD4, CD45.2, FoxP3, and IL-17. Cells are gated on CD4 and the relevant congenic marker to analyze Teff and Treg FoxP3 and IL-17 expression individually.

Mentions: We therefore tested the relationship between NOD mouse age, Treg, Teff, IL-1β, and IL-17 production by adding Treg from NOD or NOD IL-1R1−/− mice to syngeneic Teff stimulated by DCs and anti-CD3 (Fig. 6). IL-17 increased approximately threefold with the addition of Treg cells to Teff from 5-week NOD.Lt mice (Fig. 6A). IL-17 secretion was dependent on IL-1β signaling in vitro and in vivo, as anti–IL-1β mAb blocked the secretion of IL-17 in vitro, and NOD.IL1R1−/− T-cells did not secrete IL-17. By 10 weeks of age, both Teff and Treg secreted IL-17 and this was no longer dependent on IL-1β in vitro (Fig. 6B). However, because T-cells isolated from 10-week NOD.IL1R1−/− mice did not secrete IL-17 ex vivo, the data suggest that IL-1β (and concomitant IL-6) produced in response to antigen presentation promotes the development of Th17 from a young age in NOD.Lt mice through FoxP3+ Treg cell conversion to Th17 (47). To assess this in vitro, Teff and Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. Purified CD45.2 CD25− Teff included 4% CD4+FoxP3+ cells (of which <1% were CD25+), and purified CD45.1 CD25+ Treg comprised 86% FoxP3hi and 14% FoxP3lo cells. After 72 h, IL-17 was measured in supernatants, and PMA-restimulated cells were stained for CD4, CD45.2, FoxP3, and IL-17. Teff and Treg secreted IL-17 in an IL-1–dependent manner. When Teff were mixed with Treg, IL-17 was secreted if either Teff or Treg but not both lacked IL-1R1 (Fig. 6C). The proportion of CD45.1+ FoxP3hi cells fell to 30% in culture (Fig. 6D, i). Approximately 0.6% of the input CD45.2+ Teff and 1.8% of input CD45.1+ Treg expressed IL-17 (Fig. 6D, ii). Of the IL-17+ Treg, the majority were FoxP3lo. When all cells were NOD.IL-1R1−/−, 0.3% of Teff and 0.1% of Treg cells expressed IL-17 in culture (data not shown). However, if NOD.IL-1R1−/− Teff were mixed with wild-type Treg, 4% of input wild-type FoxP3lo and FoxP3hi Treg expressed IL-17 (Fig. 6D, iii), and if NOD.IL-1R1−/− Treg were mixed with wild-type Teff, 0.9% of Teff expressed IL-17 (Fig. 6D, iv). Thus, NOD.IL-1R1−/− Treg were unable to prevent low level of IL-17 production by wild-type Teff. Moreover, FoxP3 expression is unstable after activation in the presence of effector cells, and conversion of FoxP3hi and FoxP3lo Th17 is IL-1 dependent.


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)

IL-17 is produced by Teff and reprogrammed Tregs in an IL-1β–dependent manner during insulitis. IL-17 was assayed at 5 (A) or 10–14 weeks (B) in supernatants from the T-cell proliferation assay by ELISA from NOD.Lt cells with or without anti–IL-1 or anti–IL-17 mAb (10 μg/ml) or from NOD.IL-1R1−/− cells. C: CD4+CD25− Teff and CD4+CD25+ Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. After 72 h, IL-17 was measured in supernatants. ***P < 0.0001 (one-way ANOVA). D: Cells from the same experiment were restimulated with PMA in the presence of brefeldin A and stained for CD4, CD45.2, FoxP3, and IL-17. Cells are gated on CD4 and the relevant congenic marker to analyze Teff and Treg FoxP3 and IL-17 expression individually.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 6: IL-17 is produced by Teff and reprogrammed Tregs in an IL-1β–dependent manner during insulitis. IL-17 was assayed at 5 (A) or 10–14 weeks (B) in supernatants from the T-cell proliferation assay by ELISA from NOD.Lt cells with or without anti–IL-1 or anti–IL-17 mAb (10 μg/ml) or from NOD.IL-1R1−/− cells. C: CD4+CD25− Teff and CD4+CD25+ Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. After 72 h, IL-17 was measured in supernatants. ***P < 0.0001 (one-way ANOVA). D: Cells from the same experiment were restimulated with PMA in the presence of brefeldin A and stained for CD4, CD45.2, FoxP3, and IL-17. Cells are gated on CD4 and the relevant congenic marker to analyze Teff and Treg FoxP3 and IL-17 expression individually.
Mentions: We therefore tested the relationship between NOD mouse age, Treg, Teff, IL-1β, and IL-17 production by adding Treg from NOD or NOD IL-1R1−/− mice to syngeneic Teff stimulated by DCs and anti-CD3 (Fig. 6). IL-17 increased approximately threefold with the addition of Treg cells to Teff from 5-week NOD.Lt mice (Fig. 6A). IL-17 secretion was dependent on IL-1β signaling in vitro and in vivo, as anti–IL-1β mAb blocked the secretion of IL-17 in vitro, and NOD.IL1R1−/− T-cells did not secrete IL-17. By 10 weeks of age, both Teff and Treg secreted IL-17 and this was no longer dependent on IL-1β in vitro (Fig. 6B). However, because T-cells isolated from 10-week NOD.IL1R1−/− mice did not secrete IL-17 ex vivo, the data suggest that IL-1β (and concomitant IL-6) produced in response to antigen presentation promotes the development of Th17 from a young age in NOD.Lt mice through FoxP3+ Treg cell conversion to Th17 (47). To assess this in vitro, Teff and Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1−/− mice were stimulated with DCs and anti-CD3. Purified CD45.2 CD25− Teff included 4% CD4+FoxP3+ cells (of which <1% were CD25+), and purified CD45.1 CD25+ Treg comprised 86% FoxP3hi and 14% FoxP3lo cells. After 72 h, IL-17 was measured in supernatants, and PMA-restimulated cells were stained for CD4, CD45.2, FoxP3, and IL-17. Teff and Treg secreted IL-17 in an IL-1–dependent manner. When Teff were mixed with Treg, IL-17 was secreted if either Teff or Treg but not both lacked IL-1R1 (Fig. 6C). The proportion of CD45.1+ FoxP3hi cells fell to 30% in culture (Fig. 6D, i). Approximately 0.6% of the input CD45.2+ Teff and 1.8% of input CD45.1+ Treg expressed IL-17 (Fig. 6D, ii). Of the IL-17+ Treg, the majority were FoxP3lo. When all cells were NOD.IL-1R1−/−, 0.3% of Teff and 0.1% of Treg cells expressed IL-17 in culture (data not shown). However, if NOD.IL-1R1−/− Teff were mixed with wild-type Treg, 4% of input wild-type FoxP3lo and FoxP3hi Treg expressed IL-17 (Fig. 6D, iii), and if NOD.IL-1R1−/− Treg were mixed with wild-type Teff, 0.9% of Teff expressed IL-17 (Fig. 6D, iv). Thus, NOD.IL-1R1−/− Treg were unable to prevent low level of IL-17 production by wild-type Teff. Moreover, FoxP3 expression is unstable after activation in the presence of effector cells, and conversion of FoxP3hi and FoxP3lo Th17 is IL-1 dependent.

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