A High-throughput Screening Approach Identifies Selective Regulators of Il12b Transcription

Detection of pathogens by cells of the innate immune system lead to the expression of a subset of inflammatory mediators that will support the clearance of the pathogen present. Pro-inflammatory gene products display a very distinct and controlled pattern of expression. It is hypothesized that there are signaling pathways that, singly or in combination, are responsible for the selective expression of pro-inflammatory factors seen in different physiological settings. It is critical that factors are only induced in the context of an active assault by microbial pathogens and that resolution of this response is complete. Many human pathologies are thought to be caused or exacerbated by the presence of inflammatory mediators expressed outside of a biological need. Two of these tightly regulated inflammatory mediators that play key roles in inflammation, inflammatory auto-immune disease, and cancer are the cytokines interleukin 12 (IL-12) and interleukin 23 (IL-23). These cytokines share a common subunit, IL12p40, which is encoded by the Il12b gene. Transcription of Il12b can be induced in macrophages through recognition of pathogen associated molecules by Toll-like Receptors (TLRs). To better understand the signaling pathways and factors involved in the selective regulation of Il12b transcription, two cell based, high-throughput screens were performed. These screens were designed to test the hypothesis that treatments capable of modulating expression of a simple promoter reporter plasmid will generally target pathways that regulate proinflammatory gene expression in a non-selective manner, such as the NF-κB and AP-1 pathways; in contrast, treatments that modulate Il12b transcription only in the context of native chromatin will target pathways involved in the selective regulation of Il12b. To test this hypothesis, we have generated macrophage lines containing an Il12b bacterial artificial chromosome (BAC) transgene driving expression of an EGFP reporter. A basic Il12b enhancer-promoter-reporter plasmid driving expression of a DsRed gene was stably integrated into the same line. The LPS induced expression of the BAC reporter was directly compared to the expression of the basic DsRed reporter after exposure to the small molecule or siRNA libraries. The hypothesis is we will be able to enrich the pool of hits with treatments likely to be selective for Il12b by selecting hits that are selective for the chromatinized BAC. Here we present results from both the small molecule screen and the siRNA screen. One hit from the small molecule screen, β2-adrenergic agonists, is further established as a selective inhibitor of Il12b transcription, dependent on the transcription factor NFIL3