Differential expression of interleukin-17A and -17F is coupled to T cell receptor signaling via inducible T cell kinase - PubMed (original) (raw)

Differential expression of interleukin-17A and -17F is coupled to T cell receptor signaling via inducible T cell kinase

Julio Gomez-Rodriguez et al. Immunity. 2009.

Abstract

T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.

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Figures

Figure 1

Figure 1. Reduced IL-17A production from _Itk_−/− and _Itk_−/−_Rlk_−/− CD4+ T cells

a. Naïve (CD62LhiCD44lo) CD4+ T cells (200,000) were stimulated with 1 ug/ml anti-CD3 and 3 ug/ml CD28 in the presence of WT T-depleted splenocytes under either Th1 (40 ng/ml IL-12 and 10 ug/ml anti-IL-4) or Th17-inducing conditions (20 ng/ml IL-6, 5 ng/ml TGF-β1, 10ug/ml anti-IL-4, 10ug/ml anti-IL-12 and 10ug/ml anti-IFN-γ) for 3.5 days, then restimulated with PMA and Ionomycin for 4 hours and cytokine production analyzed by intracellular staining. Cell yields at end of culture are indicated in parentheses below each flow plot. Data is representative of 4 experiments for _Itk_−/−_Rlk_−/− cells and over 10 for _Itk_−/−cells. Right Panel: Percentages of IL-17A CD4+ T cell producers from individual experiments after differentiation for 2–3 days shown as (experiments in IMDM media indicated in red). Statistics were calculated using paired Student’s T-test. b. Naïve (CD62LhiCD44loCD25−) CD4+ T cells were stimulated as above, but restimulated with anti-CD3 plus anti-CD28 prior to intracellular cytokine staining. Scatter plots of IL-17A CD4+ T cell producers from 4 experiments. c. Naïve CD4+ T cells were labeled with CFSE then differentiated and stained as above. For overlay, see Supplemental Figure 1. d. Naïve CD4+ T cells were stimulated for two days under Th0 conditions (anti-CD3, anti-CD28, anti-IL4, anti-IFN-γ and anti-IL12), then infected with a control (MIGR) or MIGR-Itk expressing retroviral vector for 24 hours prior to exposure to Th17-inducing cytokines. 2 days later, cells were restimulated with PMA and Ionomycin and stained for expression of IL-17A and IFN-γ.

Figure 2

Figure 2. Itk is required for efficient transcription of IL-17A, but not Il-17F

a. Naïve CD4+ T cells were differentiated under Th17 conditions and analyzed for mRNA levels of IL-17A, IL-21, IL-22, and IL-17F by qRT-PCR after 3.5 days (84 h) of differentiation (mean of 5 experiments +/− SEM). Similar results were seen at 48 h. b. Intracellular staining for IL-17F of cells differentiated for 2 days under Th17 conditions. c. Scatter plots of total IL-17F CD4+ T cells producers (single IL-17F plus double IL-17F/IL17-A producers) from the subset of experiments in Fig. 1a where IL-17F was measured. Experiments in IMDM are indicated in red. d. IL-17A and IL-17F secreted by WT and _Itk_−/− CD4+ T cells differentiated for 48h, as determined by ELISA (IL-17A: WT 3940 +/− 374 vs _Itk_−/− 256 +/− 73 pg/ml. IL-17F: WT 99700 +/− 5400 vs _Itk_−/− 89700 +/−5390 pg/ml). Statistics were calculated using paired Student’s T-test.

Figure 3

Figure 3. _Itk_−/− mice show impaired production of IL-17A in vivo

a. Lung sections (PAS stained) from naïve and challenged WT and _Itk_−/− mice. b. Expression IL-17A and IL-17F mRNA in lungs from immunized and OVA-challenged WT and _Itk_−/− mice. Data was expressed as 2-ΔΔCT relative to a treated-WT mouse. Thy1 was used for normalization of T cell numbers. Means +/− SEM from 12 mice from four independent experiments are shown.

Figure 4

Figure 4. IL-17A production in _Itk_−/− CD4+ T cells is reduced in response to multiple cytokines, yet normal expression of RORγT and RORα

Naïve CD4+ T cells were differentiated in the presence of the indicated cytokines (plus anti-IL-4, anti-IL12 and anti-IFN-γ) and cytokine production was evaluated by intracellular staining with IL-17A/IFN-γ (a) and IL17-A/Foxp3 (b). Data are representative of over 3 experiments. c. T cells were stimulated with IL-6 plus TGF-β1 for the indicated times, lysed and immunoblotted with anti-phospho-STAT3. Lower panels: total STAT3 levels. Two examples show the range of STAT-3 phosphorylation across 6 experiments. d. Naïve CD4+ T cells were differentiated under Th17 conditions for 84 h and examined for RORγt and _RORα_mRNA by q-RT-PCR (mean of 5 experiments +/− SEM). Similar results were seen at 48 h. e. IL-17A production is poorly rescued in _Itk_−/− T cells by expression of constitutively active STAT3. CD4+ T cells were stimulated and infected with either a control retroviral vector (MIGR) expressing GFP or one expressing constitutively active STAT3-IRES-GFP. GFP+ cells were examined for cytokine production after stimulation under Th17 conditions. Results are from the same experiment as Fig. 1d for comparative purposes, but are representative of 2–3 independent retroviral transduction experiments.

Figure 5

Figure 5. IL-17A production is affected by TCR and NFAT activation

a. WT naïve CD4+ T cells were differentiated in the presence of varying amounts of anti-CD3 (plus 3 ug/ml anti-CD28) and stained for IL-17A and IL-17F. b. WT and _Itk_−/− naïve CD4+ T cells were differentiated with anti-CD3 plus anti-CD28, or anti-CD3 plus Ionomycin and stained for cytokine production. c. WT naïve CD4+ T cells were differentiated with anti-CD3 plus anti-CD28, in the absence or presence of increasing amounts of FK506. (a–c) represent 3 or more experiments, using varying concentrations of antibodies and inhibitors.

Figure 6

Figure 6. IL-17A expression is linked to NFAT activation

a. Conserved NFAT and ROR binding sites across species as predicted using the Mulan software at NCBI DCODE. b. Sequence of the IL-17A promoter conserved NFAT binding site between −3085 to −3077 bp. c. Luciferase assays were performed using Jurkat E6 cells electroporated with IL17Ap luciferase reporter construct (−3500 to +1 from mouse IL-17A promoter subcloned in pGL3-basic) or with IL17Ap-ΔNFAT (potential NFAT-binding site −3085 to −3077 deleted) in the presence or absence of caNFATc1-pMIGR plasmid. After 24h, luciferase activity was quantified and normalized to Renilla luciferase. Results are expressed as fold increase in luciferase activity relative to pGL3-basic plasmid. Means +/− SEM of triplicate samples from four experiments are shown. d. Chromatin immunoprecipitation (CHIP) using anti-NFATc1 antibody and amplifying the region around −3085 bp from the annotated first exon. Data is representative of duplicate experiments, and was normalized to input value and expressed as fold enrichment relative to normal mouse sera.

Figure 7

Figure 7. The IL-17 locus has an open chromatin conformation: caNFATc1 rescues IL-17A defect in _Itk_−/− cells

a. Schematic representation of conserved non coding (CNS) within IL-17A/IL-17F locus (Akimzhanov et al., 2007). b. CHIP using anti-acetylated histone H3 antibody and amplifying regions just upstream of the transcriptional start sites of IL-17A, IL-17F, and CNS 2, 3 and 7 in the IL-17 locus. Data in (b) was normalized to input value and expressed as fold enrichment relative to normal rabbit sera. Data are presented as the mean +/− SEM of 4 independent experiments. c. Improved production of IL-17A after retroviral transduction with an activated NFATc1 construct. Data is from one of three independent experiments.

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