Regulation of T cell activation by Notch ligand, DLL4, promotes IL-17 production and Rorc activation - PubMed (original) (raw)

Regulation of T cell activation by Notch ligand, DLL4, promotes IL-17 production and Rorc activation

Sumanta Mukherjee et al. J Immunol. 2009.

Abstract

The activation and differentiation of T cells are dependent upon numerous initiating events that are influenced by the immune environment, nature of the Ag, as well as the activation state of APCs. In the present studies we have investigated the role of a specific notch ligand, delta-like 4 (Dll4). In particular, our data have indicated that Dll4 is inducible by pathogen-associated signals through TLR activation on dendritic cells but not early response inflammatory cytokines, IL-1 and IL-18 that also activate cells via MyD88 adapter pathway. Our observations from in vitro cultures confirmed earlier reports demonstrating that Dll4 inhibits Th2 cytokine production. Furthermore, Dll4 influences the generation of IL-17-producing T cells in the presence of additional skewing cytokines, IL-6 and TGF-beta. In the absence of notch signals, IL-17 production was significantly inhibited even under specific skewing conditions. These studies further demonstrate that Dll4 up-regulates Rorc expression in T cells and that both Rorc and Il17 gene promoters are direct transcriptional notch targets that further enhance the differentiation of Th17 cell populations. Thus, facilitation of efficient T cell differentiation may depend upon the activation of T cells via specific notch ligand stimulation.

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Figures

Figure 1

Figure 1. Upregulation of notch ligands Delta like by TLR but not inflammatory cytokines

Bone marrow derived dendritic cells (BMDCs) were stimulated with different Toll like receptor ligands or with individual cytokines for 24 hours. Following stimulation total RNA was harvested, reverse transcribed to cDNA and utilized for Sybr green real time amplification for different notch ligands. The data was normalized to a house keeping gene GAPDH. The data is expressed as the mean ± SE and (*) p<0.05 was considered significant.

Figure 2

Figure 2. TLR, Dll4 mediated IL17 production

DO-11.10 splenocytes were stimulated with either ova or LPS or both together in the presence or absence of Dll4 antibody for 48 hours. Cytokine levels were measured using a luminex system. The experiment was repeated two times in triplicates and the data represents the mean ± SE. * p ≤ 0.02

Figure 3

Figure 3. Modulation of T cell responses by Dll4 under Th17 skewed conditions

A) CD4+T cells were activated under Th17 conditions in the presence or absence of rDll4 (2.5μg/ml) or rJag1 (0.5_μ_g/ml) and the supernatants were analyzed for indicated cytokines. B) CD4+ T cells were differentiated under Th17 conditions in the presence or absence of rDll4 for 5 days, rested for 3 days and then viable cells were re-stimulated with plate bound anti-CD3 and soluble anti-CD28 for 24 hours. Following stimulation the supernatant was analyzed IL17. Each experiment was repeated at least four times with similar results. The data are expressed as the mean± the SEM and (*) p ≤ 0.05 was considered significant. C) MACS purified CD4+ T cells from WT and IL17−/− mice were stimulated under Th17 conditions and after 48 hours the supernatant were analyzed for indicated cytokines using a luminex system. The data represents the mean ± the SEM and * p = 0.004.

Figure 4

Figure 4. Notch activation is necessary for Dll4 enhanced IL17 production in CD4+ T cells

A) CD4+ T cells were MACS purified from the spleen of Balb/c mice and were skewed under Th17 conditions in the presence or absence of Dll4 or GSI or control (DMSO) for 48 hours. The supernatants were analyzed for IL17 production. The data represents the mean ± the SEM of triplicate determinants and are representative of four independent experiments. * p = 0.004 and ** p = 0.002. B) CD4+ T cells were MACS purified from the spleen Balb/c mice and were skewed under Th17 conditions in the presence or absence of Dll4 for 4 hours or 20 hours. The whole cell lysate was analyzed by Western blot for the notch intracellular domain (N-ICD) and β-actin was used as loading control. The experiment was repeated twice with similar results. C) Using the same conditions as above Dll4 enhances not only IL-17 but also the mRNA expression of IL-22 and the Notch pathway target gene hes5. Data represents the mean ± SE from 3 separate experiments.

Figure 5

Figure 5. Differential regulation of transcription factors ror_γ-t_ and g_ata_3 by Dll4

A) CD4+ T cells were stimulated under Th17 conditions in the presence or absence of rDll4 for varying time. Total RNA was extracted, reverse transcribed and cDNA was amplified using specific primers for rorγ-t or gata-3. The data is expressed as the mean± the SEM. * p = 0.031. B) CD4+ T cells were stimulated under Th17 conditions in the presence or absence of IL6 or TGF_β_ or rDll4. The whole cell lysate was the analyzed by western blot for phosphorylation of STAT-3. _β_-actin was used as a loading control.

Figure 6

Figure 6. RBPj-κ (CSL) directly interacts with rorγ-t promoter

Diagram for rorγ-t promoter with CSL binding sites (underlined) 5′ upstream of exon 1 is shown and forward and reverse primers were designed to amplify the indicated regions. MACS purified splenic CD4+ T cells were stimulated under Th17 conditions for 20 hours and fixed for ChIP. Antibodies used for IP are anti-CSL (_α_CSL) and control Ig (cIg). Total input DNA before IP was used for normalization of data. The graphs represent quantitative PCR analysis of the ratio of enriched rorγ-t promoter with CSL binding sites to the input DNA. CSL binding sites were amplified using ror_γ_-t specific promoter primers. Data represents mean ± SE of a representative of 3 independent experiments each performed in triplicate.

Figure 7

Figure 7. RBPj-κ (CSL) directly interacts with IL17 promoter on specific binding sites

CD4+ T cells were activated under Th17 skewing conditions in the presence of rDll4 or in the presence or absence of GSI for 24 hours. The cells were the fixed for ChIP. IP was carried out using anti CSL antibody. The three CSL binding sites on IL17 promoter (p1, p2 and p3) were amplified and analyzed by quantitative RT-PCR. The data represents the normalized percentage amounts relative to the input DNA before IP. The experiment is one representative of three independent experiments. The data is expressed as the mean ± the SE and is representative of four independent experiments.

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