Chronic Toll-like Receptor Ligation and Hematopoietic Stem Cell Aging

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DESCRIPTION (provided by applicant): Whereas the hematopoietic system is the most characterized of any stem-cell driven tissue; and given the astronomical output requirements of hematopoietic stem cells (HSC) over a lifespan, this system has become an optimal target for studies on aging and regenerative medicine. In light of the recent discovery that microbial toll-like receptor (TLR) ligands can directly impact HSC homeostasis, research aimed at protecting HSC from the stress of infection is becoming increasingly relevant. Here we introduce a new experimental approach to determine whether chronic infection-related conditions hasten HSC aging. Through the use of an established model, we are primed to answer new questions concerning the effects of chronic TLR ligation on the aging and long-term physiology of HSC. The experimental plan is outlined by two Specific Aims, which are:(1) Determine the numbers, phenotype, differentiation potential, and cell cycle status of HSC and primitive progenitors in chronically-stimulated marrow, and (2) Rigorously test the integrity of HSC recovered from chronic TLR ligand-stimulated mice. Our strategy consists of investigating both the immediate and long-term effects of chronic TLR ligation on HSC aging. Daily intraperitoneal injections with LPS or Pam3CSK4 will be used to model chronic infection. We will determine if chronic TLR stimulation alters HSC integrity by evaluating bone marrow population dynamics, surface phenotypes, and progenitor cell cycle status during treatment, and throughout age-accelerating conditions. Multi-color flow cytometry and molecular assays such as western blot and RT-PCR will reveal whether chronic TLR stimulation induces lineage biases in aging HSC. Novel application of F1 hybrid recipients will also allow precise quantification of HSC fitness during serial transplants. Well-defined culture systems will further assess HSC differentiation potential. Chronic infections require significant efforts in patient treatment each year. Whether chronic infection-related deficiencies lead to accelerated HSC aging is an important question we are poised to address in the studies outlined here. Diminished immune system function due to exhaustion of bone marrow stem cells is a common consequence of aging. Because of this, even mild infections pose a serious threat to elderly or immune-compromised patients. The studies outlined here present a new approach for identifying potential means of protecting bone marrow stem cells from the ravages of infection and aging. This information may lead to clinical applications that prolong life in aged or immune-compromised patients by preserving the integrity of the immune system during challenging circumstances.