TGF-beta2 and IL-4Ralpha Pathway Interactions in Asthma

National Jewish Health, Denver CO

Investigators

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Abstract

DESCRIPTION (provided by applicant): The overall goal of this proposal is to better understand the interactions between IL-4Ralpha related pathways and TGF-beta2 at the level of the epithelial cell and fibroblast, focusing on the importance of these interactions to eosinophilia and mucus production. We will specifically address the interactions and associated mechanisms, which influence mucus production at the epithelial cell level and the synergistic increases in eotaxin-1 production at the fibroblast level. We will utilize primary human cells from normal and diseased subjects. As our studies are done almost entirely in the "out-bred" human system, it is of paramount importance to incorporate genetic elements that may be directly involved in the interactions, specifically polymorphisms in IL-4Ralpha. In Specific Aim #1, we will expand on preliminary data, which show IL-4Ralpha stimulation increases mucus, TGF-beta2 and 15 LO 1 levels in epithelial cells. We will then pursue an improved understanding of the interactions between IL-4Ralpha and TGF-beta2 pathways and whether 15S HETE or 13S HODE play intermediary roles. Additionally, as we propose to use human cells, all subjects will be genotyped for single nucleotide polymorphisms (SNPs) in IL-4Ralpha. These data will confirm at the in vitro level our in vivo and clinical findings which suggest polymorphisms in I4R or STAT-6 binding sites impact functional outcomes. Specific Aim #2 will directly explore the signaling and transcriptional interactions behind the synergy of TGF-beta2 with IL- 13/ IL-4Ralpha utilizing primary human fibroblasts (and later epithelial cells). As there are technical limits to human primary cells, yet many of the findings we observe are found only in human cells, our initial mechanistic studies will be limited to two approaches. The first will evaluate the impact of TGF-beta2 stimulation on the I4R binding motif and its subsequent effect on STAT-6 activation and binding. The second approach will determine the promoter-binding pattern of the nuclear proteins generated following IL-13+TGF-beta2 stimulation. When one or more potential protein binding patterns are confirmed, the proteins will be identified and their ability to replicate the synergy confirmed. Finally, similar to Aim #1, as primary human cells will be evaluated, we anticipate variability in the response will occur. Therefore, the impact of the same polymorphisms in IL-4Ralpha identified in Aim # 1 will be related to changes in signal transduction and transcriptional binding. These approaches should lead to improvement in our understanding of interactions between these innate and adaptive immune response elements at the tissue and cellular level, as well as their potential impact on human asthma.