Increased expression of IRF4 and ETS1 in CD4+ cells from patients with intermittent allergic rhinitis.

BACKGROUND: The transcription factor (TF) IRF4 is involved in the regulation of Th1, Th2, Th9, and Th17 cells, and animal studies have indicated an important role in allergy. However, IRF4 and its target genes have not been examined in human allergy. METHODS: IRF4 and its target genes were examined in allergen-challenged CD4(+) cells from patients with IAR, using combined gene expression microarrays and chromatin immunoprecipitation chips (ChIP-chips), computational target prediction, and RNAi knockdowns. RESULTS: IRF4 increased in allergen-challenged CD4(+) cells from patients with IAR, and functional studies supported its role in Th2 cell activation. IRF4 ChIP-chip showed that IRF4 regulated a large number of genes relevant to Th cell differentiation. However, neither Th1 nor Th2 cytokines were the direct targets of IRF4. To examine whether IRF4 induced Th2 cytokines via one or more downstream TFs, we combined gene expression microarrays, ChIP-chips, and computational target prediction and found a putative intermediary TF, namely ETS1 in allergen-challenged CD4(+) cells from allergic patients. ETS1 increased significantly in allergen-challenged CD4(+) cells from patients compared to controls. Gene expression microarrays before and after ETS1 RNAi knockdown showed that ETS1 induced Th2 cytokines as well as disease-related pathways. CONCLUSIONS: Increased expression of IRF4 in allergen-challenged CD4(+) cells from patients with intermittent allergic rhinitis leads to activation of a complex transcriptional program, including Th2 cytokines.

HIV-1 Tat represses transcription from the mannose receptor promoter.

The mannose receptor is expressed on mature macrophages and immature dendritic cells, and functions to mediate phagocytosis of pathogens and capture of Ags for delivery to MHC class II-containing intracellular compartments. It has been previously reported that HIV-1-infected macrophages have reduced functions associated with the mannose receptor, including impaired Pneumocystis carinii phagocytosis and mannosylated albumin uptake. Several HIV-1-derived proteins including the Tat protein have been shown to transcriptionally repress host cell genes. The present study was undertaken to define the role of the HIV-1-derived protein Tat in HIV-mediated mannose receptor down-regulation. Cotransfection of the human macrophage cell line U937 with a Tat expression vector and a mannose receptor promoter-luciferase reporter construct resulted in down-regulation of mannose receptor promoter activity. This repression was targeted to the basal promoter. Expression of either one- or two-exon Tat resulted in decreased promoter activity. The addition of the transactivation response element (TAR) sequence enhanced the Tat-mediated repression. Down-regulation was also seen when transfected cells were treated with exogenously added Tat protein. These results are consistent with a mechanism whereby Tat reduces mannose receptor promoter activity by interfering with the host transcriptional initiation machinery, potentially resulting in decreased levels of surface mannose receptor available for Ag or pathogen capture.

PU.1 and USF are required for macrophage-specific mannose receptor promoter activity.

In the current study we report the isolation of 854 base pairs of the rat mannose receptor promoter. Analysis of the sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the monocytic cell line RAW. No activity was observed in pre-monocytic cell lines or epithelial cell lines. Mutation of the TTTAAA sequence to TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound to the mannose receptor promoter, but only PU.1 and USF contributed to activation. Transient transfections using a dominant negative construct of USF resulted in a 50% decrease in mannose receptor promoter activity, further establishing the role of USF in activating the rat mannose receptor promoter. Comparison of the rat, mouse, and human sequence demonstrated that some binding sites are not conserved. Gel shifts were performed to investigate differences in protein binding between species. USF bound to the rat and human promoter but not to the mouse promoter, suggesting that different mechanisms are involved in regulation of mannose receptor expression in these species. From these results we conclude that, similar to other myeloid promoters, transcription of the rat mannose receptor is regulated by binding of PU.1 and a ubiquitous factor at an adjacent site. However, unlike other myeloid promoters, we have identified USF as the ubiquitous factor, and demonstrated that the promoter contains a functional TATA box.