Critical regulation of early Th17 cell differentiation by interleukin-1 signaling

Abstract

T helper (Th) 17 cells have been recently discovered in both mouse and human. Here we show that interleukin-1 (IL-1) signaling on T cells is critically required for the early programming of Th17 cell lineage and Th17 cell-mediated autoimmunity. IL-1 receptor1 expression in T cells, which was induced by IL-6, was necessary for the induction of experimental autoimmune encephalomyelitis and for early Th17 cell differentiation in vivo. Moreover, IL-1 signaling in T cells was required in dendritic cell-mediated Th17 cell differentiation from naive or regulatory precursors and IL-1 synergized with IL-6 and IL-23 to regulate Th17 cell differentiation and maintain cytokine expression in effector Th17 cells. Importantly, IL-1 regulated the expression of the transcription factors IRF4 and RORgammat during Th17 cell differentiation; overexpression of these two factors resulted in IL-1-independent Th17 cell polarization. Our data thus indicate a critical role of IL-1 in Th17 cell differentiation and this pathway may serve as a unique target for Th17 cell-mediated immunopathology.

Figure 1. IL-1R1 expression on Th17 is critical for induction of EAE

a CD25⁻CD44^loCD62^hiCD4⁺ T cells (Naïve CD4+ T) were FACS-sorted and polarized under Th1, Th2 or Th17 condition for 4 days. b, Naïve CD4+ T cells were stimulated with anti-CD3/CD28 Ab in the presence of indicated cytokine for 24 hours before mRNA expression of IL-1R1 was analyzed. c, FACS-sorted naïve CD4 T cells from WT mice or STAT3 KO, RORα KO, or RORα/γ double KO mice were cultured under Th17 condition for 4 days. d, Naive OT-II CD4⁺ T cells were activated with Ova peptide-pulsed splenic APCs under the neutral (anti-IL-4 and anti-IFN-γ) and coinfected with two bicistronic retroviruses expressing RORα-GFP or GFP vector and RORγt-hCD2 or hCD2. GFP⁺hCD2⁺ cells were FACS sorted. In a, c, and d, cells were restimulated with plate-bound anti-CD3 Ab for four hours before mRNA expression was analyzed by real-time PCR. Data were normalized with expression amounts of Actb. e and f, CD4 T cells were sorted from WT or IL-1R1 KO mice and i.v. transferred into Rag1^−/− mice. The recipient mice were induced EAE and disease incidence (e) and score (f) were measured daily and means ± SEM of all mice in each group were shown (f). *, p<0.05; **, p<0.01 in comparison with WT recipients.

Figure 2. IL-1 signaling in T cells is required for Th17 development in EAE model

Mixed bone marrow chimeric mice were generated and induced EAE. Mononuclear cells in central nervous system (CNS) and spleen were stained with anti-CD45.2 to distinguish WT and IL-1R1 KO compartments and analyzed for CD4+ and CD11b+ cell presence (a), cytokine production profiles in CD4 T cells after PMA/Ionomycin (b and c) or MOG stimulation (b, only for spleen), or Foxp3+ cell in CD4 T cells (d and e). The mice were divided into two groups based on the disease severity (score, 0.5-2 for early phase; 2.5-4.0 for peak phase). *, p<0.05; **, p<0.01 in comparison with WT compartments. Data shown represent two independent experiments with consistent results.

Figure 3. IL-1 signal is required for early differentiation of Th17 cells

a, WT and IL-1R1 KO mice were subcutaneously immunized with KLH in CFA. Three or seven days later, lymphoid cells from draining lymph nodes were restimulated with KLH overnight and IL-17- or IFN-γ-expressing cells were measured by intracellular staining. b, FACS-sorted naïve OT-II T cells from WT or IL-1R1-deficient OT-II mice were intravenously transferred into congenic (CD45.1) mice. The recipient mice were immunized with OVA_323-339 peptide in CFA. Three days later, lymphoid cells from draining lymph nodes were restimulated with PMA and Ionomycin for 5 hours and IL-17- or IFN-γ-expressing cells were measured by intracellular staining. Data shown are on gated CD45.1⁺ CD4⁺ T cells. c and d, FACS-sorted naïve CD4 T cells from WT or IL-1R1−/− mice were cocultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab (0.1μg/ml) and LPS (100 ng/ml) plus TGF-β (1 ng/ml). IL-17- or IFN-γ-expressing cells were measured by intracellular staining (c). At day 4, mRNA expression was assessed by real-time RT-PCR after restimulation of T cells by anti-CD3 for 4 hr (d). Data shown represent two independent experiments and normalized with expression amounts of Actb.

Figure 4. Conversion of Foxp3+ T cells into Th17 cells requires IL-1 signal

a, Mononuclear cells in EAE-induced mixed bone marrow chimeric mice as described in Figure 2 were analyzed for IL-17 and Foxp3 expression. Data shown are on gated CD4+ Foxp3+ cells. Mean values are shown as horizontal bars. **, p<0.01 in comparison with WT compartments. Data shown represent two independent experiments with consistent results. b and c, FACS-sorted CD4+GFP+ cells from naïve Foxp3-GFP reporter mice were cultured with DC and in the presence of soluble anti-CD3 Ab, LPS and TGF-β plus anti-IL-1R1 Ab or rat IgG as a control. IL-17- or GFP-expressing cells were measured by intracellular staining (b). At day 4, mRNA expression was assessed by real-time RT-PCR after restimulation of T cells by anti-CD3 for 4 hr (c). mRNA expression in fresh FACS-sorted CD4+GFP+ cells, stimulated with anti-CD3 Ab for 4 hr, was used as a control. Data shown represent two independent experiments and normalized with expression amounts of Actb.

Figure 5. IL-1 signal expands and maintains Th17 program in the absence of TCR stimulation

FACS-sorted naïve CD4+ T cells from IL-17F-RFP reporter mice were stimulated under Th17 condition (anti-IL-4, anti-IFNγ, IL-6, TGF-β, IL-23). At day 4, RFP+ cells were sorted and the expression of IL-17, IL-17F, IFNγ, Foxp3 or T-bet was analyzed by intracellular staining (a). b-d, The sorted RFP+ cells were labeled with CFSE and cultured with medium alone, IL-23, IL-1 alone, IL-23 plus IL-1, or IL-6 plus TGF-β for additional 3 days. b, IL-17-, IL-17F-, IL-4-, IFNγ-expressing cells were analyzed by intracellular staining after PMA and Ionomycin stimulation for 4 hr. c, Cytokines in the supernatant of 3 day additional culture were measured by ELISA. d, Cells were further restimulated with plate bound anti-CD3 Ab overnight and cytokines in the supernatant were measured by ELISA. ‘Th17’ is the supernatant of the sorted RFP+ cells after anti-CD3 Ab overnight stimuation. Data shown represent four independent experiments.

Figure 6. IL-1 signal regulates Th17 cytokine production in the absence of exogenous TGF-β

FACS-sorted naïve CD4+ T cells from WT or IL-1R1 KO mice were stimulated with anti-CD3/CD28 Abs and anti-IL-4/anti-IFNγ Abs in the presence of indicated cytokines for 4 days. a, IL-17-producing cells were analyzed by intracellular staining. Values are the percentage of IL-17+ cells and mean fluorescence intensity (MFI) of the IL-17 staining of the gated cells. b, Foxp3- and T-bet-expressing cells were analyzed by intracellular staining. c, IL-17- and IFNγ-expressing cells were analyzed by intracellular staining. d, At day 4, cells stimulated under the condition described in c were harvested and restimulated with plated bound anti-CD3 Ab overnight and cytokines in the supernatant were measured by ELSIA. Data shown represent at least two independent experiments.

Figure 7. IL-1R1 signal regulates upregulation of RORγt and IRF4 expression

a, FACS-sorted naïve CD4+ T cells from WT mice were stimulated with anti-CD3/CD28 Abs and anti-IL-4/anti-IFNγAbs in the presence of indicated cytokines for 4 days. mRNA expression was assessed by real-time RT-PCR. b, FACS-sorted naïve CD4 T cells from WT or IL-1R1−/− mice were co-cultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab and LPS plus TGF-β. At days 2 and 4, mRNA expression was assessed by real-time RT-PCR. Data shown represent two independent experiments and normalized with expression amounts of Actb. c and d, FACS-sorted naïve CD4+ T cells from WT or IL-1R1−/− mice were co-cultured with BM-derived DC (WT) in the presence of soluble anti-CD3 Ab and LPS plus TGF-β and infected with bicistronic retrovirus expressing GFP vector (RV-KM), RORγt-GFP or IRF4-GFP. Four days after infection, IL-17- or IFNγ-expressing cells were assessed by intracellular staining. Data shown are gated on GFP+ or GFP− cells and represent two independent experiments. In d, cells were coinfected with two bicistronic retroviruses expressing IRF4-GFP or GFP vector and RORγt-hCD2 or hCD2. GFP+ and hCD2+ cells were sorted and assessed by intracellular staining.