Despite advances in critical care, mortality among patients with massive burns still remains high due to sepsis-induced systemic inflammatory response syndrome (SIRS) and multisystem organ failure. Patients with severe burns often manifest uncontrolled overproduction of proinflammatory cytokines and prostanoids due to continued monocyte/macrophage activation. Many microbial infections and inflammatory insults result in increased monocyte production driven by a concomitant elevation in monocytic lineage specific colony-stimulating factor, M-CSF. Yet, little research has been done on the regulation of monocyte production in thermal injury. Furthermore, the availability of increasing number of monocytes for activation as an underlying cause for cytokine overproduction leading to the development of SIRS in severe burn injury has never been explored. The hypothesis of this propsal is that severe burn injury and sepsis result in a bone marrow milieu conducive to increased monocytopoiesis. This continued availa-bility of increasing number of monocytes/macrophages for activation can potentially drive inflammation through dysregulated cytokine production. We will test this premise in a murine dorsal scald burn model in which sepsis will be induced at clinically relevant early and late time points after the initial burn injury. In the first aim, we will a) establish that bone marrow monocyte production is enhanced through flowcytometric analysis of monocytopoietic developmental antigens (ER-MP12 and 20) and clonogenic assays; b) measure plasma and bone marrow M-CSF levels; c) mRNA and cell surface expression of M-CSF receptor (c-fms) in total bone marrow cells and in different compartments of monocytic development; d) document the expression pattern of monocyte development associated transcription factors PU.1 and Egr-1 in different monocytopoietic compartments in burn and sepsis. In the second aim, using an in vitro monocytic differentiation of M1 cells, we will evaluate how different cytokines such as IL-1, IL-6 and IL-10 and the prostanoid PGE2 that are elevated in burn injury influence a) monocytic differentiation: b) c-fms expression; c) PU.l and Egr-1 activation and d) cytokine production and functions. In the third aim, we will determine if abrogation of the immunosuppressive and myelopoiesis regulating prostanoid, PGE2, can normalize monocytopoiesis, c-fms expression, PU.1 and Egr-1 expression and kinetics of monocyte release in our murine model. These studies will directly test our hypothesis and provide crucial information on the status and the mechanisms of monocytopoiesis in burn injury and sepsis. More importantly, these studies will help explain the aberrant cytokine production in severely injured burn patients and provide targets for rational therapies to improve their survival.
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