Hai-Chon Lee

Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFκB

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is important for the initiation of allergic airway inflammation through a dendritic cell-mediated T helper 2 response. To identify the factors that control TSLP expression, we examined the ability of inflammatory mediators to regulate TSLP production in human airway epithelial cells. We found that both IL-1β and TNF-α were capable of inducing rapid TSLP production in primary human bronchial airway epithelial cells. We further characterized the human TSLP gene promoter, using two human epithelial cell lines, 16HBEo− and A549, and showed that IL-1β- and TNF-α-mediated human TSLP promoter activation in these cells was mediated by an upstream NFκB site. Mutation of this NFκB site abolished activation, as did overexpression of a dominant-negative version of IκB kinase (IKK)β (a kinase acting on IκB, the inhibitor of NFκB). Interestingly, human TSLP mRNA levels were also increased after exposure to Toll-like receptor (TLR) 2, TLR8, and TLR9 ligands, further supporting an important role for NFκB in TSLP gene regulation. Similarly, analysis of the mouse TSLP gene promoter revealed the presence of a similarly situated NFκB site that was also critical for IL-1β-inducible expression of mouse TSLP. Taken together, these results demonstrate that the inflammatory mediators IL-1β and TNF-α regulate human TSLP gene expression in an NFκB-dependent manner.

The IL-7 Receptor Controls the Accessibility of the TCRγ Locus by Stat5 and Histone Acetylation

The IL-7 receptor (IL-7R) plays critical roles in expansion and V(D)J recombination during lymphocyte development. Here we demonstrate that cytokine stimulation rapidly recruits Stat5 and transcriptional coactivators to the Jgamma germline promoter and induces histone acetylation, germline transcription, and accessibility in Ba/F3 cells. We also show that histone acetylation of the TCRgamma locus is significantly reduced in IL-7R-deficient thymocytes and that the introduction of active Stat5 restores the histone acetylation and accessibility of the locus. Furthermore, treatment with histone deacetylase inhibitor recovers the histone acetylation and accessibility in IL-7R-deficient thymocytes. Therefore, these results suggest that Stat5 may recruit the transcriptional coactivators to the Jgamma germline promoter and control the accessibility of the TCRgamma locus by histone acetylation.

TSLP enhances the function of helper type 2 cells

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic inflammation in both humans and mice. TSLP has been shown to promote a Th2‐type response through upregulation of OX40L on dendritic cells, and through direct induction of IL‐4 production in naïve CD4+ T cells. However, its direct effect on effector Th cells has not been extensively investigated. In this study, we show that the level of TSLP receptor (TSLPR) expression on mouse effector Th2 cells is higher than on Th1 and Th17 cells, and that TSLP induced proliferation of effector Th2, but not Th1 nor Th17 cells. TSLP also induced the phosphorylation of signal transducer and activator of transcription (Stat) 5, and expression of the anti‐apoptotic factor Bcl‐2 in Th2 cells. Finally, TSLP‐mediated proliferation on Th2 cells was enhanced by TCR stimulation, through IL‐4‐mediated induction of TSLPR expression. Taken together, these results indicate that TSLP is involved in exacerbation of mouse Th2‐mediated allergic inflammation in a Th2 environment through direct stimulation of Th2 effector cells.

Cutting Edge: Inhibition of NF-κB-Mediated TSLP Expression by Retinoid X Receptor

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has important roles in the initiation of allergic airway inflammation and the activation of dendritic cells. We have shown that the human TSLP gene is regulated in a NF-κB-dependent manner; however the factors that negatively regulate TSLP expression are not known. In this study we demonstrate that 9-cis-retinoic acid (9-cis-RA) is a negative regulator of TSLP expression in airway epithelial cells. This inhibition is manifested as a block in the IL-1β-mediated recruitment of NF-κB to the human TSLP promoter. 9-cis-RA-mediated inhibition is not restricted to TSLP gene expression but rather reflects a general inhibition of NF-κB activation, as other NF-κB-regulated-genes were also inhibited in a similar manner by 9-cis-RA treatment. Taken as a whole, these data demonstrate that inhibition of IL-1β-dependent genes by active retinoid X receptors involves antagonism of NF-κB signaling.

Transcriptional Regulation of the Mouse IL-7 Receptor α Promoter by Glucocorticoid Receptor

Expression of the IL-7R α-chain (IL-7Rα) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7Rα gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7Rα mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7Rα promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of ∼270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7Rα promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7Rα promoter. Thus, this study identifies the IL-7Rα CNS-1 region as a GC-responsive element.

Differential Roles of Cytokine Receptors in the Development of Epidermal γδ T Cells

IL-7 and IL-15 play important roles in γδ T cell development. These receptors transmit proliferation and/or survival signals in γδ T cells. In addition, the IL-7R promotes recombination and transcription in the TCR γ locus. To clarify the role of the cytokine receptors in the development of epidermal γδ T cells, we introduced a Vγ3/Vδ1 TCR transgene, derived from Thy-1+ dendritic epidermal T cells (DETC), into IL-7Rα-deficient mice, and we found that they partly rescued γδ T cells in the adult thymus but not in the spleen. Introduction of an additional Bcl-2 transgene had a minimal effect on γδ T cells in the adult thymus of these mice. In contrast to the adult thymus, the introduction of the Vγ3/Vδ1 TCR transgene into IL-7Rα−/− mice completely restored Vγ3+ T cells in the fetal thymus and DETC in the adult skin. On the contrary, the same Vγ3/Vδ1 TCR transgene failed to rescue DETC in the skin of IL-2Rβ-deficient mice, even with the additional Bcl-2 transgene. These results suggest that the IL-2/IL-15R, rather than the IL-7R, plays an essential role in proliferation and survival of DETC in the fetal thymus and the skin. In contrast, the IL-7R is probably essential in the induction of V-J recombination of TCRγ genes. Thus, this study proves that IL-7R and IL-2/IL-15R serve differential functions in epidermal γδ T cell development.

Induction of Germline Transcription in the Human TCRγ Locus by STAT5

TCR and Ig genes are assembled by V(D)J recombination during lymphocyte development. The enhancer and the germline promoter control the accessibility of each locus for the common recombinase activity. In the mouse TCRγ locus, STAT5 proteins activated by the IL-7R interact with consensus motifs in 5′ regions of Jγ segments and induce germline transcription. To evaluate the role of STAT5 in controlling the accessibility of the TCRγ locus, we characterized the germline transcription of human TCRγ genes and compared it with mouse. We first demonstrated that Jγ-Cγ germline transcripts are induced in a cytokine-dependent human erythroleukemia cell line. STAT consensus motifs are present in 5′ regions of Jγ1.1 and Jγ2.1 gene segments, and activated STAT5 binds to these motifs. By using a reporter assay, we showed that the Jγ1.1 germline promoter is transactivated by STAT5 and that mutations in any of the two STAT motifs abrogate this activity. Thus, this study demonstrates that STAT5 induces germline transcription in the TCRγ locus of both mouse and human and suggests the possibility that this mechanism may play an essential role in controlling the TCRγ locus accessibility. In addition, STAT motifs are conserved among 5′ Jγ germline promoters, 3′ enhancers, and a locus control region-like element, HsA, in both mouse and human TCRγ loci, indicating the possibility that IL-7R/STAT5 signaling probably controls the locus-wide accessibility through these elements.

Accessibility control of TCR Vγ region by STAT5

The signal of the IL-7R and signal transducers and activators of transcription (STAT) 5 plays an essential role in gammadelta T-cell development by inducing V-J recombination in the TCRgamma locus. Previously, we have shown that STAT5 binds to the Jgamma promoters and controls chromatin accessibility by histone acetylation. However, little is known on control mechanism of Vgamma region by the IL-7R. To elucidate the regulation by STAT5, we first analyzed the chromatin status of Vgamma region in primary thymocytes. The levels of histone H3 acetylation are high at Vgamma5, HsA element and Vgamma2 in Rag2(-/-) thymocytes but low in IL-7R alpha-chain (IL-7Ralpha)-deficient early thymocytes, suggesting that IL-7R signaling controls the accessibility of the Vgamma region. In addition, high levels of histone H3 acetylation and germ line transcription were induced at Vgamma5 and HsA by cytokine and STAT5 in cytokine-dependent Ba/F3 and other hematopoietic cell lines. Importantly, the chromatin accessibility of Vgamma5 gene is increased by cytokine signal. Furthermore, STAT5 was not recruited to a non-canonical STAT-binding motif in the endogenous chromatin of the Vgamma5 promoter by cytokine stimulation, while STAT5 binds to a consensus motif in the HsA element. In accordance with this result, STAT5 does not directly activate the Vgamma5 promoter by reporter assay. These results suggested that while STAT5 directly binds to HsA element and induces its histone acetylation, STAT5 indirectly activates the Vgamma5 promoter. Thus, this study implies a potential role of STAT5 in accessibility control of Vgamma region, especially at Vgamma5 and HsA.

Seeding of dendritic epidermal T cells in the neonatal skin is reduced in 129 strain of mice.

Precursors for Thy-1(+) dendritic epidermal T cells (DETC) develop as Vgamma3(+) T cells in the fetal thymus and become distributed in the adult skin. DETC are variably distributed from site to site and from strain to strain. To elucidate the basis of strain variation, we first compared the density of DETC in the ear epidermis among different mouse strains. In the ear epidermis, we detected the highest level of DETC in C57BL/6 mice, intermediate levels in C3H and CBA/J mice, and the lowest levels in other strains including BALB/c and 129 mice. Although BALB/c and 129+Ter/Sv mice showed higher levels of DETC in the abdomen than in the ear, the levels were significantly lower than C57BL/6 mice. Furthermore, in neonatal abdominal epidermis we detected considerably lower numbers of DETC in BALB/c and 129+Ter/Sv mice than in C57BL/6 mice. In contrast, Vgamma3(+) DETC precursors in the fetal thymus are rather increased in 129+Ter/Sv mice. These results suggest that fewer DETC precursors are seeded in the neonatal skin of BALB/c and 129+Ter/Sv mice and that their expansion in the skin during neonatal to adult stages does not reach the levels in C57BL/6 mice.