Genome-wide association (GWA) studies have identified over 300 regions associated with more than 70 common diseases1. However, identifying causal genes within an associated region remains a major challenge1,2. One approach to resolving causal genes is through the dissection of gene-phenotype correlations. Here we use polychromatic flow cytometry to show that differences in surface expression of interleukin-2 (IL-2) receptor alpha-chain (IL-2RA, or CD25) protein are restricted to particular immune cell types and correlate with several haplotypes in the IL2RA region that have previously been associated to the autoimmune diseases type 1 diabetes (T1D) and multiple sclerosis2-4. We confirm our strongest gene-phenotype correlation at the RNA level by allele-specific expression (ASE). We also define key parameters for the design and implementation of post-GWA gene-phenotype investigations, and demonstrate the usefulness of a large bioresource of genotype-selectable normal donors from whom fresh, primary cells can be analyzed.

Background— Activated macrophages contribute to the pathogenesis of inflammatory diseases such as atherosclerosis. Although Notch signaling participates in various aspects of immunity, its role in macrophage activation remains undetermined. Methods and Results— To explore the role of Notch signaling in inflammation, we examined the expression and activity of Notch pathway components in human primary macrophages in vitro and in atherosclerotic plaques. Macrophages in culture express various Notch pathway components including all 4 receptors (Notch1 to Notch4). Notch3 selectively increased during macrophage differentiation; however, silencing by RNA interference demonstrated that all receptors are functional. The ligand Delta-like 4 (Dll4) increased in macrophages exposed to proinflammatory stimuli such as lipopolysaccharide, interleukin-1β, or minimally-modified low-density lipoprotein in a Toll-like receptor 4– and nuclear factor-κB–dependent fashion. Soluble Dll4 bound to human macrophages. Coincubation of macrophages with cells that expressed Dll4 triggered Notch proteolysis and activation; increased the transcription of proinflammatory genes such as inducible nitric oxide synthase, pentraxin 3 and Id1; resulted in activation of mitogen-activated protein kinase, Akt, and nuclear factor-κB pathways; and increased the expression of Dll4 in macrophages. Notch3 knockdown during macrophage differentiation decreased the transcription of genes that promote inflammation, such as inducible nitric oxide synthase, pentraxin 3, Id1, and scavenger receptor-A. These in vitro findings correlate with results of quantitative immunohistochemistry, which demonstrated the presence of Dll4 and other Notch components within macrophages in atherosclerotic plaques. Conclusion— Dll4-triggered Notch signaling may mediate inflammatory responses in macrophages and promote inflammation.