Imaging Functionally Distinct Eosinophil Subtypes Within Tissue Biopsies Using Gene Expression Profiling and an In Situ Hybridization Approach Based

Imaging Functionally Distinct Eosinophil Subtypes Within Tissue Biopsies Using Gene Expression Profiling and an In Situ Hybridization Approach Based Imaging Functionally distinct eosinophil subtypes within tissue biopsies using gene expression profiling and In Situ hybridization approach based on a concurrent multiple RNA targeting strategy Allergic diseases are characterized by the local tissue infiltration of unique inflammatory leukocytes that are often dominated by eosinophils. These infiltrating eosinophils are currently perceived as a single monolithic population, although recent studies suggest that unique eosinophil subtypes that potentially display distinct activities co-exist. Unfortunately, efforts to stratify eosinophils into functionally distinct groups has thus far proven to be difficult and time consuming, leaving the role of individual cells largely unknown. This proposal integrates growing definitions of eosinophil subtype-specific gene expression with a novel multi-RNA targeting approach, allowing single cell gene expression imaging within tissue biopsies by reiterative cycles of in situ hybridization. The central hypothesis of these studies are that the immune polarization of tissue infiltrating eosinophils is disease specific, diagnostic of ongoing local immune responses, and potentially a prognostic indicator of disease outcomes. The immediate objective is to demonstrate the utility of this strategic in situ approach as a way to identify and image individual eosinophils within an available tissue biopsy or cell cytospin preparation. Strategically, these studies will initially use eosinophils and established mouse models of disease with which we have previously reported mechanistic studies exploring the role of eosinophils in these settings. Our intermediate/long term goal is to translate this strategic approach to human subjects by developing signature eosinophil gene expression biomarkers that will allow physicians to image patient biopsies as part of novel diagnostic approaches. The objectives will be accomplished by the completion of the following Specific Aims: (1) To determine the identity and spatial distribution of eosinophil subtypes within biopsies using a multiple RNA targeting approach with cleavable fluorescent probes and reiterative in situ hybridization imaging. Our immediate objective will be to exploit our characterization of eosinophil-specific gene expression and use of mouse models of allergic respiratory inflammation to demonstrate a novel ability to define and localize functionally distinct immune polarized eosinophil subtypes via a signature pattern of gene expression; (2) To define the stratification of immune polarized eosinophil subtypes resident in tissues linked with established mouse models of human disease, including respiratory viral infection, cancer, organ transplant, and atopic dermatitis. It is noteworthy that as part of these studies we will also define the distribution and location of eosinophil subtypes resident at homeostatic baseline (e.g., gastrointestinal tract, uterus, adipose tissue, and the bone marrow); and (3) To perform the needed bioinformatics and translational pilot studies demonstrating the utility of multiple-target in situ technologies to image human eosinophil subtypes using clinically available tissue sections/cell preparations from disease patients with an associated eosinophilia.