abstract: Eosinophils are innate immune cells that are most commonly associated with parasite infection and allergic responses. Recent studies, though, have identified eosinophils as cells with diverse effector functions at baseline and in disease. Eosinophils in specific tissue immune environments are proposed to promote unique and specific effector functions, suggesting these cells have the capacity to differentiate into unique subtypes. The studies here focus on defining these subtypes using functional, molecular, and genetic analysis as well as using novel techniques to image these subtypes in situ. To characterized these subtypes, an in vitro cytokine induced type 1 (E1) and type 2 (E2) eosinophil model was developed that display features and functions of eosinophils found in vivo. For example, E1 eosinophils secrete type 1 mediators (e.g., IL-12, CXCL9 and CXCL10), express iNOS and express increased levels of the surface molecules PDL1 and MHC-I. Conversely, E2 eosinophils release type 2 mediators (e.g., IL4, IL13, CCL17, and CCL22), degranulate and express increased surface molecules CD11b, ST2 and Siglec-F. Completion of differential expression analysis of RNAseq on these subtypes revealed 500 and 655 unique genes were upregulated in E1 and E2 eosinophils, respectively. Functional enrichment studies showed interferon regulatory factor (IRF) transcription factors were uniquely regulated in both mouse and human E1 and E2 eosinophils. These subtypes are sensitive to their environment, modulating their IRF and cell surface expression when stimulated with opposing cytokines, suggesting plasticity. To identify and study these subtypes in situ, chromogenic and fluorescent eosinophil-specific immunostaining protocols were developed. Methods were created and optimized, here, to identify eosinophils by their granule proteins in formalin fixed mouse tissues. Yet, eosinophil-specific antibodies alone are not enough to identify and study the complex interactions eosinophil subtypes perform within a tissue. Therefore, as part of this thesis, a novel highly-multiplexed immunohistochemistry technique was developed utilizing cleavable linkers to address these concerns. This technique is capable of analyzing up to 22 markers within a single biopsy with single-cell resolution. With this approach, eosinophil subtypes can be studied in situ in routine patient biopsies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020