Gap Ryol Lee

Interchromosomal associations between alternatively expressed loci

The T-helper-cell 1 and 2 (TH1 and TH2) pathways, defined by cytokines interferon-γ (IFN-γ) and interleukin-4 (IL-4), respectively, comprise two alternative CD4+ T-cell fates, with functional consequences for the host immune system. These cytokine genes are encoded on different chromosomes. The recently described TH2 locus control region (LCR) coordinately regulates the TH2 cytokine genes by participating in a complex between the LCR and promoters of the cytokine genes Il4, Il5 and Il13. Although they are spread over 120 kilobases, these elements are closely juxtaposed in the nucleus in a poised chromatin conformation. In addition to these intrachromosomal interactions, we now describe interchromosomal interactions between the promoter region of the IFN-γ gene on chromosome 10 and the regulatory regions of the TH2 cytokine locus on chromosome 11. DNase I hypersensitive sites that comprise the TH2 LCR developmentally regulate these interchromosomal interactions. Furthermore, there seems to be a cell-type-specific dynamic interaction between interacting chromatin partners whereby interchromosomal interactions are apparently lost in favour of intrachromosomal ones upon gene activation. Thus, we provide an example of eukaryotic genes located on separate chromosomes associating physically in the nucleus via interactions that may have a function in coordinating gene expression.

Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13–induced tissue responses and apoptosis

Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39−/− mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39−/− animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.

Regulation of the Th2 Cytokine Locus by a Locus Control Region

The Th2 cytokine genes IL4, IL5, and IL13 are clustered and expressed in a cell lineage-specific manner. We investigated the global locus-specific regulation of these genes using BAC transgenic mice containing the murine Th2 cytokine cluster carrying an IL4 promoter-luciferase reporter. IL4 promoter activity in effector CD4 T cells from these transgenic mice was strong, Th2 specific, and copy number dependent, suggesting the presence of an LCR in the locus. The production of IL4 and IL13, but not IL5, by these cells was also copy number dependent. Deletion analysis defined a 25 kb fragment in the RAD50 gene as the region containing the LCR activity. Expression of the IL4 promoter-luciferase reporter was transactivated by GATA-3 irrespective of position in the locus, suggesting the global nature of this regulation. The LCR itself, however, does not respond directly to GATA-3.

Hypersensitive site 7 of the T H 2 locus control region is essential for expressing T H 2 cytokine genes and for long-range intrachromosomal interactions

Several regulatory regions are important for the expression of genes encoding T helper type 2 (TH2) cytokines, including TH2-specific DNase I hypersensitivity sites in the TH2 cytokine locus control region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid TH2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in TH2 cell differentiation, we have generated RHS7-deficient mice. CD4+ T cells and mast cells showed a notable reduction in TH2 cytokine expression in vitro and TH2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the locus control region and promoters of the TH2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of TH2 cytokine gene expression.

γ-Secretase Inhibitor Reduces Allergic Pulmonary Inflammation by Modulating Th1 and Th2 Responses

RATIONALE Gamma-secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. In asthma, a subset of CD4(+) T cells is believed to initiate and perpetuate the disease. OBJECTIVES The aim of this study was to evaluate the therapeutic potential of GSI against allergic asthma. METHODS GSI was administered to an ovalbumin-sensitized mouse via an intranasal route at the time of ovalbumin challenge. MEASUREMENTS AND MAIN RESULTS The administration of GSI inhibits asthma phenotypes, including eosinophilic airway inflammation, goblet cell metaplasia, methacholine-induced airway hyperresponsiveness, and serum IgE production. GSI treatment of bronchoalveolar lavage cells stimulated via TCR or non-TCR pathways led to a decrease in Th2 cytokine production with a concomitant increase in Th1 cytokine secretion. Expression of Hes-1, a target of Notch signaling, was down-regulated in conjunction with a reduction of Notch intracellular domain and GATA-3 levels after GSI treatment of bronchoalveolar lavage cells. GSI treatment resulted in an inhibition of NF-kappaB activation, and combined treatment with GSI and an NF-kappaB inhibitor augmented IFN-gamma production in a synergistic manner. CONCLUSIONS These data suggest that GSI directly regulates Th1 and Th2 responses in allergic pulmonary inflammation through a Notch signaling-dependent pathway and that GSI is of high therapeutic value for treating asthma by inhibiting airway inflammatory responses.

HHQ and PQS, two Pseudomonas aeruginosa quorum‐sensing molecules, down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4‐hydroxy‐2‐alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild‐type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor‐α and interleukin‐6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild‐type did not. The addition of 4‐hydroxy‐2‐heptylquinoline (HHQ) or 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild‐type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor‐κB (NF‐κB) binding to its binding sites and the expression of NF‐κB target genes, and PQS delayed inhibitor of κB degradation, indicating that the effect of HHQ and PQS was mediated through the NF‐κB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

Th2 LCR is essential for regulation of Th2 cytokine genes and for pathogenesis of allergic asthma

Previous studies have shown that Th2 cytokine genes on mouse chromosome 11 are coordinately regulated by the Th2 locus control region (LCR). To examine the in vivo function of Th2 LCR, we generated CD4-specific Th2 LCR-deficient (cLCR KO) mice using Cre-LoxP recombination. The number of CD4 T cells in the cLCR KO mouse was comparable to that in wild-type mice. The expression of Th2 cytokines was dramatically reduced in in vitro-stimulated naïve CD4 T cells. Deletion of the LCR led to a loss of general histone H3 acetylation and histone H3-K4 methylation, and demethylation of DNA in the Th2 cytokine locus. Upon ovalbumin challenge in the mouse model of allergic asthma, cLCR KO mice exhibited marked reduction in the recruitment of eosinophils and lymphocytes in the bronchoalveolar lavage fluid, serum IgE level, lung airway inflammation, mucus production in the airway walls, and airway hyperresponsiveness. These results directly demonstrate that the Th2 LCR is critically important in the regulation of Th2 cytokine genes, in chromatin remodeling of the Th2 cytokine locus, and in the pathogenesis of allergic asthma.

Transcription factor YY1 is essential for regulation of the Th2 cytokine locus and for Th2 cell differentiation

The Th2 locus control region (LCR) has been shown to be important in efficient and coordinated cytokine gene regulation during Th2 cell differentiation. However, the molecular mechanism for this is poorly understood. To study the molecular mechanism of the Th2 LCR, we searched for proteins binding to it. We discovered that transcription factor YY1 bound to the LCR and the entire Th2 cytokine locus in a Th2-specific manner. Retroviral overexpression of YY1 induced Th2 cytokine expression. CD4-specific knockdown of YY1 in mice caused marked reduction in Th2 cytokine expression, repressed chromatin remodeling, decreased intrachromosomal interactions, and resistance in an animal model of asthma. YY1 physically associated with GATA-binding protein-3 (GATA3) and is required for GATA3 binding to the locus. YY1 bound to the regulatory elements in the locus before GATA3 binding. Thus, YY1 cooperates with GATA3 and is required for regulation of the Th2 cytokine locus and Th2 cell differentiation.

Twisting the Th1/Th2 immune response via the retinoid X receptor: Lessons from a genetic approach

The immune system is influenced by environmental factors such as hormones and nutrients. Previous studies have suggested that vitamins A and D influence the process of naive T helper (Th) cell differentiation into Th1 or Th2 cells. Vitamins A and D signal through the retinoid X receptor (RXR), which partners with either the retinoic acid receptor or the vitamin D receptor. Most previous studies into the role of RXR in Th differentiation have been performed in vitro and it was necessary for these to be verified in a physiological environment. However, in vivo study has been hindered since RXRα deficient mice are embryonic lethal. Du et al. in this issue of the European Journal of Immunology, overcome this obstacle using “pinkie” mice that harbor a hypomorphic mutation in the Rxrα gene. The authors report that the mutant mice have an exaggerated Th1 immune response which is attributed to the aberrant antigen‐presenting cell and CD4 T cell function. This study confirms previous studies indicating that RXR signaling plays an important role in Th cell differentiation and also provides a valuable tool with which to study the mechanisms and the in vivo functions of this signaling pathway.

Hypersensitive site 6 of the Th2 locus control region is essential for Th2 cytokine expression

The T helper type 2 (Th2) cytokine genes Il4, Il5, and Il13 are contained within a 140-kb region of mouse chromosome 11 and their expression is controlled by a locus control region (LCR) embedded within this locus. The LCR is composed of a number of DNase I–hypersensitive sites (HSs), which are believed to encompass the regulatory core of the LCR. To determine the function of these sites, mutant mice were generated in which combinations of these HSs had been deleted from the endogenous LCR, and the effect on Th2 cytokine expression was assessed through the use of in vivo and in vitro models. These experiments revealed that, although all of the hypersensitive sites analyzed are important for appropriate LCR function, some sites are more important than others in regulating cytokine expression. Interestingly, each LCR mutation showed contrasting effects on cytokine expression, in some cases with mutants displaying opposing phenotypes between in vitro cultures and in vivo immunizations. These studies indicated that Rad50 hypersensitive site 6 was the singularly most important HS for Th2 cytokine expression, displaying consistent reductions in cytokine levels in all models tested. Furthermore analysis of chromatin modifications revealed that deletion of Rad50 hypersensitive site 6 impacted epigenetic modifications at the promoters of the Il4, Il5, and Il13 genes as well as other regulatory sites within the Th2 locus.