Architectural structure and regulation of TOLLIP in IPF

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Project Summary Idiopathic pulmonary fibrosis (IPF) kills 50,000 patients annually. Pathogenesis involves an aberrant repair response after various causes of lung injury, mediated by innate immunity involving toll-like receptors (TLRs). TLRs undergo negative inhibition by toll interacting protein (TOLLIP). Single nucleotide polymorphisms (SNPs) in TOLLIP associated with susceptibility, and survival in IPF. The genetic variants regulating TOLLIP activity and their effect on the innate immune response to lung injury in IPF is currently unknown. In our preliminary data, we sequenced TOLLIP by next generation sequencing (NGS) in 192 patients with IPF and identified; multiple functional SNPs capable of regulating TOLLIP expression in vitro, a simple repeating region highly associated with susceptibility, as well as several SNPs that correlate with survival. We hypothesize that genetic variants within TOLLIP modulate the innate immune response, thereby influencing IPF susceptibility and survival. Our overall goal is to understand the role of TOLLIP in the susceptibility and disease course of IPF through determining the architectural diversity of its locus and its regulation by genetic variation. Our approach is to sequence the TOLLIP gene, and perform a case-control analysis in a subset of 1,000 IPF cases along with 1,000 matched healthy controls. We will validate and replicate our findings using the remaining cohort and perform regression analyses to determine susceptibility- associated SNPs that predict survival. Uncommon variants will be aggregated by predicted function and analyzed for susceptibility and survival. SNPs will also be assessed for association with mRNA lung expression and other lung molecular markers and will be prioritized for further study. We will determine which SNPs regulate mRNA expression levels using luciferase assays. In our preliminary data, we have identified several candidate SNPs within regulatory regions (i.e. 3'UTR, 5'UTR, or exonic) using in silico methods, confirmed in cell culture models. This suggests multiple potential modes of regulation. Stimulation with TLR ligands will also identify potential TLRs involved. Lastly, we will determine whether TOLLIP genotypes correlate with Tollip protein levels, in blood monocytes and B cells in a prospective cohort of patients with IPF. We will also determine if TOLLIP genotypes correlate with Tollip protein levels in human lung macrophages from a large cohort of non-IPF donor lungs and compare these results to samples from IPF explant lungs. Lastly, we will determine the effect of TOLLIP variants on TLR signaling by stimulating blood monocytes and lung macrophages with TLR ligands. TOLLIP variant-dependent differences in Tollip protein levels after TLR stimulation, in both the blood and the organ of disease, will address the role of TOLLIP variant functional effects on the uncontrolled fibrotic response in IPF. The proposed work will identify functional TOLLIP variants associated with IPF and delineate the mechanisms by which they regulate critical innate TLR responses in IPF. Understanding the genetic variation of TOLLIP may provide direct clinical assessment, for both harm and benefit, in developing treatments for IPF.