Patricia C. Fulkerson, Ph.D. - US grants

DESCRIPTION (provided by applicant): Eosinophilia occurs in a variety of clinical disorders including parasitic infections, hypereosinophilic syndrome, cancer, and atopic diseases. Eosinophils are derived from lineage-committed hematopoietic progenitor cells expressing the transcription factor GATA-1 and interleukin-5 receptor alpha (IL-5Ra). Although the significance of GATA-1 in eosinophil development is well established, the regulatory pathways that direct the differentiation of the eosinophil lineage-committed progenitor to mature eosinophils are not well understood, especially when compared with other hematopoietic lineages. The long term goals of the candidate, Dr. Fulkerson, are to identify novel therapeutic targets to block eosinophil production and recruitment for the treatment of patients with eosinophilic asthma and other inflammatory disorders. Dr. Fulkerson is dedicated to a career in academic medicine and to becoming an independent research investigator. The training plan outlined in this proposal will provide Dr. Fulkerson an opportunity to establish a scientific niche and facilitate the maturation of Dr. Fulkerson into an independent physician scientist with submission of a R01 grant in year three of the proposal. The objective of this proposal is to delineate the regulatory activity of the transcription factor Spi-C in eosinophil maturation. The role of Spi-C in mature eosinophils or eosinophil differentiation is completely unknown. The proposed studies are expected to provide a detailed understanding of the pathways important for eosinophil differentiation, survival and effector function via regulation of IL-5Ra expression and expression of eosinophil granule proteins. Defining the molecular regulators of eosinophil function and IL-5 responsiveness will undoubtedly provide key information with clinical applications, especially given the IL-5Ra- and IL-5-directed therapies that are currently under avid development. The central hypothesis of this proposal is that the transcription factor Spi-C coordinates with the transcription factors PU.1 and GATA-1 to regulate eosinophil differentiation via regulation of expression of genes important for eosinophil survival, proliferation and effector function. We will test this hypothesis with three specific aims. First, we will identify the role of Spi-C in eosinophil maturation using a novel in vitro culture system developed by Dr. Fulkerson that results in phenotypically mature eosinophils. Second, we will characterize the regulation of IL-5Ra by Spi-C and PU.1. Finally, we will determine the role of Spi-C and PU.1 in regulating expression of eosinophil granule proteins. Together, the proposed studies will elucidate the role of Spi-C in eosinophil differentiation. Specifically, the studies will delineate the regulation of granule protein expression and IL-5 responsiveness by Spi-C and PU.1. As release of granule proteins is an important effector function of eosinophils and IL-5 signaling is critical to eosinophilia associated with disease, further defining their regulation could lead to new therapeutic targets in eosinophil-associated disorders. Project Narrative Eosinophil accumulation and activation in tissues occurs in a variety of clinical disorders including parasitic infections, hypereosinophilic syndrome, cancer, and atopic diseases and results in the release of cytotoxic granule proteins that activate and damage neighboring cells leading to tissue damage, organ dysfunction and patient morbidity. The proposed studies are expected to provide a detailed understanding of the pathways important for eosinophil differentiation, survival and effector function, particularly production of granule proteins. This proposal will provide key information with clinical applications, especially given the eosinophil- targeted therapies that are currently under avid development.

Project Summary Recruitment of eosinophils (Eos) from the bloodstream into tissues can occur under a variety of conditions and lead to the release of preformed and newly synthesized products, including cytokines, chemokines, lipid mediators and cytotoxic granule proteins, which can initiate and quickly escalate local inflammatory and remodeling responses. Notably, eosinophil-targeted therapy has been very effective in clinical trials at reducing mature eosinophils in the blood. However, tissue eosinophilia is only partially suppressed and likely results from accessory pathways that continue to promote Eos recruitment and survival. The residual tissue eosinophilia contributes to persistent symptoms and increased risk for tissue damage in patients with Eos-mediated diseases. Thus, new therapies designed with an improved understanding of the mechanism of tissue eosinophilia are needed and likely to have a significant clinical impact. Our preliminary studies implicate Aiolos as a potential regulator of Eos accumulation in eosinophilic asthma as Aiolos-deficiency results in impaired responses to CCR3 ligands and to IL-5, a key cytokine in the pathogenesis of eosinophilic asthma. The central hypothesis of this proposal is that Aiolos controls airway eosinophilia in asthma by two processes, 1) positive regulation of Ccr3 expression by direct transcriptional activation, and 2) a positive feedback loop involving Aiolos, Il5ra, and IL-5. In Aim 1, we will identify the Aiolos-dependent transcriptome in Eos and delineate the mechanism for Aiolos-dependent expression of CCR3. We will also determine the consequence of Aiolos deficiency on Eos-mediated tissue pathology in experimental asthma. In Aim 2, we will determine the mechanism for Aiolos-mediated regulation of IL-5-responsiveness by evaluating the consequence of Aiolos deficiency, haploinsufficiency and overexpression on stage-specific responses by EoPs, eosinophil precursors (preEos) and mature Eos to IL-5. We will also dissect the relationship between Aiolos expression, Il5ra expression and IL-5 stimulation during Eos development in the setting of experimental asthma. Finally, in Aim 3, we will determine the relationship between expression levels of Aiolos in human Eos and 1) a specific Eos gene signature, 2) functional response of Eos to IL-5 and CCL11, and 3) asthma disease severity and Eos phenotype. The high prevalence of eosinophilic inflammation in pediatric asthma and the recent FDA approval of IL-5-targeted therapy for eosinophilic asthma highlight the significance of this application which focuses on delineating the mechanistic relationship between Aiolos expression in Eos, Eos recruitment into the inflamed lung, and IL-5 responsiveness of Eos. Our proposed mechanistic studies using both mouse and human cell systems (Aims 1 and 2) supported by translational studies with Eos from patients with eosinophilic asthma (Aim 3) will provide compelling evidence to support our central hypothesis. The immediate significance of our study is its potential to uncover a dose-dependent relationship between Aiolos expression in Eos and response to IL-5 which would provide rationale for therapy selection for patients based on the Eos phenotype (e.g. Aiolos expression level).