Project Summary / Abstract Neutropenia, a deficiency in certain white blood cells, is a common side effect of chemotherapy that exposes patients to a high risk of death from opportunistic infections. Existing methods to restore neutrophils, including G-CSF administration and granulocyte infusions, have not produced a clear improvement in outcome, and new approaches are needed. White blood cells are the product of bone marrow hematopoietic stem and progenitor cells (HSPCs) that can be triggered to divide and differentiate by circulating inflammatory signals. Our prior work shows that the inflammatory cytokine interferon gamma (IFNg) is a potent stimulus for hematopoietic stem cell (HSC) division and myeloid differentiation. Yet persistent IFNg exposure inhibits HSC self-renewal, eventually leading to bone marrow failure. Whether HSCs or their progeny, the multipotent progenitors (MPPs), are equally responsive to IFNg is unknown. Furthermore, a lack of molecular understanding of signaling pathways induced by IFNg to promote HSPC differentiation poses a barrier to utilizing proimmune functions of IFNg while preventing deleterious effects. In preliminary work we identify genes induced in HSCs upon IFNg stimulation, and we show by gain- and loss-of-function studies that an exemplary gene can critically regulate HSPC differentiation. Thus we hypothesize that IFNg-induced transcriptional changes can be used to activate quiescent HSCs to produce more neutrophils, resulting in improved recovery from infection. The rationale is that regulated induction of specific IFNg targets in HSCs may enhance short-term myelopoiesis without disrupting long-term bone marrow function. Three aims are designed to characterize the kinetics, mechanism, and outcome of IFNg-dependent HSPC differentiation. In Aim 1, we will measure the contribution of HSCs versus MPPs to circulating granulocytes after IFNg stimulation using lineage-tracing, thereby revealing which of these HSPC subtypes is most potent as a source of neutrophils. In Aim 2, we will evaluate IFNg-inducible factors for their role in HSPC differentiation using loss of function, xenotransplant, and functional biochemical studies. Finally, in Aim 3, we will test the utility of IFNg-induced HSPC myeloid differentiation in pathogen clearance using a mouse model of Group A Streptococcal myositis. Studies in Aim 3 will be informed by, but not dependent on, results of Aims 1 and 2. These studies will provide critical mechanistic data needed to design novel stem cell-based therapies for neutropenic fever and may reveal new insights into bone marrow failure syndromes that result from excessive inflammation.