The predominant immune response to allergens in atopic asthma is mediated by T cells of the helper subtype 2 (Th2). In the past few years, we and others have demonstrated a central role of GATA-3 in Th2 differentiation. Similarly, the transcription factor T-bet was recently shown to be an important regulator of IFN-y production in Th1 cells. Despite these advances, the myriad of molecular events that converge to induce and maintain a stable Th2 phenotype is relatively less well understood. We hypothesize that: 1) a complex signaling event triggered via TCR activation, cytokine (IL-4)/chemokine (MCP-1) signaling and costimulation (CD28) contributes to GATA-3 gene expression and the development and commitment of Th2 cells; 2) GATA-3 plays a crucial role in the establishment of a stable Th2 phenotype; and 3) that GATA-3/T-bet balance plays a determinative role in the Th1/Th2 dichotomy. To address these hypotheses we will:
Aim I. Characterize the signaling events that induce GATA-3 gene expression and the Th2 phenotype in response to stimulation by antigen, cytokine and chemokine. a) The role of NF-kB, which lies downstream of the TCR/CD28/PKC0 axis and is required for Th2 responses in vivo, in the induction of GATA-3 expression will be investigated. The role of b) CD28 and p38 MAPK and 3) MCP-1 and its receptor CCR-2, both implicated in Th2 responses, in GATA-3 expression will be investigated.
Aim II. Investigate the role of GATA-3 in the maintenance of a stable Th2 phenotype. Mice expressing a dominant-negative mutant of GATA-3 (KRR), previously shown by us to drastically limit Th2 responses in vivo, will be used in both in vitro and in vivo experiments. a) DNase I HS assays, chromatin immunoprecipitation assays will be carried out to address the influence of GATA-3 on promoters, enhancers and the conserved region between IL-4/IL-13. Effects on cytokine production will be determined. b) In in vivo studies, KRR expression will be induced by dox treatment after priming, and recall responses will be investigated either by antigenic restimulation of splenic CD4+ cells in vitro or by antigen (ovalbumin; Ova) challenge by inhalation in vivo. Effects on cytokine production, airway inflammation, mucus and IgE production and airway hyperresponsiveness (AHR) will be determined.
Aim III. Investigate the role of the GATA-3/T-bet balance in the Th1/Th2 dichotomy. a) The basal and induced expression of GATA-3 and T-bet in alpha-beta and y8 T cells in mouse strains with different Th1/Th2 bias (C57BL/6 and BALB/c) will be determined and correlated with the development of airway inflammation and AHR using the antigen (Ova) model of airway inflammation. b) Tg mice expressing GATA-3 or T-bet will be tested in Th1-type (DTH) and Th2-type (Ova) models to determine the influence of GATA-3 and T-bet on Th1/Th2 responses in vivo.
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