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Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling.

Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ - Nature (2010)

Bottom Line: Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β.T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model.These data indicate an alternative mode for T(H)17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. ghoreschik@mail.nih.gov

ABSTRACT
CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-β signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

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IL-23-induced Th17 cells express T-bet but not IL-9 and IL-10a, Naïve CD4+ T cells were polyclonally stimulated in the presence of IL-6, IL-1β and either TGF-β (Th17(β) cells) or IL-23 (Th17(23) cells). Microarray analysis demonstrates the differential expression of genes encoding cytokines and receptors in the two subsets of Th17 cells. Mean values from two independent experiments are shown. b, Th17(β) and Th17(23) cells were polarized, expanded in IL-2, restimulated with anti-CD3/anti-CD28 antibodies (1 μg ml−1), cytokines, expanded and then analyzed for IL-17, IL-9 or IL-10 expression by intracellular staining. c, Transcription factor expression in Th17(β) and Th17(23) cells as assessed by microarray analysis. Mean values of two independent experiments are shown. d–f, Th17(23) but not Th17(β) express T-bet. Naïve CD4+ T cells were activated by IL-6, IL-1β with either IL-23 or TGF-β. IL-17 expression was not altered in T-bet−/− Th17(β) cells compared to T-bet+/+ Th17(β) cells. In contrast, loss of T-bet expression enhanced IL-17 production in Th17(23) cells. A representative experiment is depicted in d and pooled data are shown in e (n=4) and f (n=3, error bars are s.e.m., *P<0.05).
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Figure 3: IL-23-induced Th17 cells express T-bet but not IL-9 and IL-10a, Naïve CD4+ T cells were polyclonally stimulated in the presence of IL-6, IL-1β and either TGF-β (Th17(β) cells) or IL-23 (Th17(23) cells). Microarray analysis demonstrates the differential expression of genes encoding cytokines and receptors in the two subsets of Th17 cells. Mean values from two independent experiments are shown. b, Th17(β) and Th17(23) cells were polarized, expanded in IL-2, restimulated with anti-CD3/anti-CD28 antibodies (1 μg ml−1), cytokines, expanded and then analyzed for IL-17, IL-9 or IL-10 expression by intracellular staining. c, Transcription factor expression in Th17(β) and Th17(23) cells as assessed by microarray analysis. Mean values of two independent experiments are shown. d–f, Th17(23) but not Th17(β) express T-bet. Naïve CD4+ T cells were activated by IL-6, IL-1β with either IL-23 or TGF-β. IL-17 expression was not altered in T-bet−/− Th17(β) cells compared to T-bet+/+ Th17(β) cells. In contrast, loss of T-bet expression enhanced IL-17 production in Th17(23) cells. A representative experiment is depicted in d and pooled data are shown in e (n=4) and f (n=3, error bars are s.e.m., *P<0.05).

Mentions: To compare IL-23/IL-6-induced Th17 [Th17(23)] cells with conventional Th17 cells [Th17(β)], we assessed global gene expression in both cells and found more than 2000 genes differentially expressed (Supplementary Fig. 4a, b). As shown in Fig. 3a, conventional Th17(β) cells expressed higher levels of Il9, Il10 and Ccl20, whereas Th17(23) cells expressed higher levels of Il2, Il33 and Il18r1. These differences were confirmed by quantitative PCR and measurement of protein expression (Fig. 3b, Supplementary Fig. 4 c–e). To assess whether IL-17-producing cells with a phenotype similar to those induced in vitro in the absence of TGF-β are generated in vivo, mice were immunized with ovalbumin and adjuvant. By day 7 the majority of IL-17-producing OT-II CD4+ T cells also expressed IL-18R1 and produced IL-26 (Supplementary Fig. 4f). Collectively, these data support the in vivo relevance of TGF-β-independent generation of Th17 cells.


Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling.

Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ - Nature (2010)

IL-23-induced Th17 cells express T-bet but not IL-9 and IL-10a, Naïve CD4+ T cells were polyclonally stimulated in the presence of IL-6, IL-1β and either TGF-β (Th17(β) cells) or IL-23 (Th17(23) cells). Microarray analysis demonstrates the differential expression of genes encoding cytokines and receptors in the two subsets of Th17 cells. Mean values from two independent experiments are shown. b, Th17(β) and Th17(23) cells were polarized, expanded in IL-2, restimulated with anti-CD3/anti-CD28 antibodies (1 μg ml−1), cytokines, expanded and then analyzed for IL-17, IL-9 or IL-10 expression by intracellular staining. c, Transcription factor expression in Th17(β) and Th17(23) cells as assessed by microarray analysis. Mean values of two independent experiments are shown. d–f, Th17(23) but not Th17(β) express T-bet. Naïve CD4+ T cells were activated by IL-6, IL-1β with either IL-23 or TGF-β. IL-17 expression was not altered in T-bet−/− Th17(β) cells compared to T-bet+/+ Th17(β) cells. In contrast, loss of T-bet expression enhanced IL-17 production in Th17(23) cells. A representative experiment is depicted in d and pooled data are shown in e (n=4) and f (n=3, error bars are s.e.m., *P<0.05).
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Figure 3: IL-23-induced Th17 cells express T-bet but not IL-9 and IL-10a, Naïve CD4+ T cells were polyclonally stimulated in the presence of IL-6, IL-1β and either TGF-β (Th17(β) cells) or IL-23 (Th17(23) cells). Microarray analysis demonstrates the differential expression of genes encoding cytokines and receptors in the two subsets of Th17 cells. Mean values from two independent experiments are shown. b, Th17(β) and Th17(23) cells were polarized, expanded in IL-2, restimulated with anti-CD3/anti-CD28 antibodies (1 μg ml−1), cytokines, expanded and then analyzed for IL-17, IL-9 or IL-10 expression by intracellular staining. c, Transcription factor expression in Th17(β) and Th17(23) cells as assessed by microarray analysis. Mean values of two independent experiments are shown. d–f, Th17(23) but not Th17(β) express T-bet. Naïve CD4+ T cells were activated by IL-6, IL-1β with either IL-23 or TGF-β. IL-17 expression was not altered in T-bet−/− Th17(β) cells compared to T-bet+/+ Th17(β) cells. In contrast, loss of T-bet expression enhanced IL-17 production in Th17(23) cells. A representative experiment is depicted in d and pooled data are shown in e (n=4) and f (n=3, error bars are s.e.m., *P<0.05).
Mentions: To compare IL-23/IL-6-induced Th17 [Th17(23)] cells with conventional Th17 cells [Th17(β)], we assessed global gene expression in both cells and found more than 2000 genes differentially expressed (Supplementary Fig. 4a, b). As shown in Fig. 3a, conventional Th17(β) cells expressed higher levels of Il9, Il10 and Ccl20, whereas Th17(23) cells expressed higher levels of Il2, Il33 and Il18r1. These differences were confirmed by quantitative PCR and measurement of protein expression (Fig. 3b, Supplementary Fig. 4 c–e). To assess whether IL-17-producing cells with a phenotype similar to those induced in vitro in the absence of TGF-β are generated in vivo, mice were immunized with ovalbumin and adjuvant. By day 7 the majority of IL-17-producing OT-II CD4+ T cells also expressed IL-18R1 and produced IL-26 (Supplementary Fig. 4f). Collectively, these data support the in vivo relevance of TGF-β-independent generation of Th17 cells.

Bottom Line: Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β.T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model.These data indicate an alternative mode for T(H)17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. ghoreschik@mail.nih.gov

ABSTRACT
CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-β signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

Show MeSH
Related in: MedlinePlus