Functional characterization of an allergy-associated regulatory variant at the human IL13 locus

T helper type--2 (Th2) immunity orchestrates responses against extracellular pathogens under normal conditions and mediates pathogenic responses against innocuous substances when dysregulated, leading to allergic disease. Among the cytokines expressed by Th2 cells, interleukin (IL)--13 has emerged as a critical effector molecule in Th2 responses and common IL13 variants are associated with allergy--related phenotypes in populations of distinct ethnic background. IL13 expression in human T cells is paralleled by extensive IL13 locus remodeling, which results in the appearance of multiple DNase I hypersensitive (HS) sites. Among these, HS4 in the distal IL13 promoter is constitutively present in both naive and polarized Th2 cells, and spans a single nucleotide polymorphism, IL13--1512AC (rs1881457), which is common and strongly associated with total serum IgE levels. This dissertation combines in vitro and ex vivo approaches to characterize the role of HS4 in the regulation of IL13 gene expression and to provide novel insights into the mechanisms that underlie the association between IL13--1512AC and allergic disease.The results showed that HS4 acts as a novel cis--acting element that up--regulates IL13 transcription in activated Th2 cells. The enhancing activity of HS4 mapped within the 3' end of this element and was dependent on binding/recruitment of the transcription factors NF90 and NF45. Moreover, the IL13--1512C risk allele significantly enhanced HS4--dependent IL13 expression by creating a binding site for the transcription factor Oct--1. The increased expression of the --1512C allele was dependent on endogenous levels of Oct--1. Collectively, these results illustrate how a functional variant in an important regulatory element may modulate susceptibility to a common complex disease

Experimental Models of Inflammatory Bowel DiseasesSummary

The understanding of the intestinal inflammation occurring in the inflammatory bowel diseases (IBD) has been immeasurably advanced by the development of the now numerous murine models of intestinal inflammation. The usefulness of this research tool in IBD studies has been enabled by our improved knowledge of mucosal immunity and thus our improved ability to interpret the complex responses of mice with various causes of colitis; in addition, it has been powered by the availability of models in which the mice have specific genetic and/or immunologic defects that can be related to the origin of the inflammation. Finally, and more recently, it has been enhanced by our newly acquired ability to define the intestinal microbiome under various conditions and thus to understand how intestinal microorganisms impact on inflammation. In this brief review of murine models of intestinal inflammation, we focus mainly on the most often used models that are, not incidentally, also the models that have yielded major insights into IBD pathogenesis. Keywords: Cell Transfer Colitis, DSS Colitis, IL10 Deficiency, Murine Colitis Models, NKT Cells, Oxazolone Colitis, TNBS Colitis, TH1 Cells, TH17 Cells, Treg

NF90 and NF45 regulate Interleukin-13 (IL13) gene transcription in human T cells

The Th2 cytokine IL13 is a critical effector of allergic inflammation. We previously showed human IL13 gene expression is paralleled by chromatin remodeling, leading to the formation of multiple DNase I hypersensitive (HS) sites throughout the locus. The distal IL13 promoter harbors two HS sites, HS4 and HS5, detected in both naïve and polarized CD4⁺ T helper cells. We now show HS4, which is located ~1.5 kb upstream of the IL13 ATG, acts as a cis-regulatory element that enhances transcription driven by the IL13 promoter in transiently transfected, activated Jurkat T cells. The enhancing activity mapped to the 3’half of HS4 (HS4-3’), a region which binds Nuclear Factor-90 (NF90) and NF45 as demonstrated by DNA-affinity chromatography and tandem mass spectrometry. The NF90/45 binding motif in HS4-3’ was further mapped and dissected by gel shift analysis. Chromatin immunoprecipitation confirmed recruitment of NF90 and NF45 to the HS4-containing region in the endogenous IL13 locus upon T cell stimulation. Moreover, stable overexpression of NF90 and NF45 increased HS4-mediated enhancement of IL13 transcription by 3-fold and 4-fold, respectively. Collectively, our results identify NF90 and NF45 as important regulators of human IL13 transcription in response to T cell activation.1 page(s

NF45 and NF90 regulate HS4-dependent interleukin-13

Expression of the cytokine interleukin-13 (IL13) is critical for Th2 immune responses and Th2-mediated allergic diseases. Activation of human IL13 expression involves chromatin remodeling and formation of multiple DNase I-hypersensitive sites throughout the locus. Among these, HS4 is detected in the distal IL13 promoter in both naive and polarized CD4+ T cells. We show herein that HS4 acts as a position-independent, orientation-dependent positive regulator of IL13 proximal promoter activity in transiently transfected, activated human CD4+ Jurkat T cells and primary murine Th2 cells. The 3′-half of HS4 (HS4-3′) was responsible for IL13 up-regulation and bound nuclear factor (NF) 90 and NF45, as demonstrated by DNA affinity chromatography coupled with tandem mass spectrometry, chromatin immunoprecipitation, and gel shift analysis. Notably, the CTGTT NF45/NF90-binding motif within HS4-3′ was critical for HS4-dependent up-regulation of IL13 expression. Moreover, transfection of HS4-IL13 reporter vectors into primary, in vitro differentiated Th2 cells from wild-type, NF45+/−, or NF90+/− mice showed that HS4 activity was exquisitely dependent on the levels of endogenous NF45 (and to a lesser degree NF90), because HS4-dependent IL13 expression was virtually abrogated in NF45+/− cells and reduced in NF90+/− cells. Collectively, our results identify NF45 and NF90 as novel regulators of HS4-dependent human IL13 transcription in response to T cell activation.12 page(s