Newborns are at increased risk of infection yet the mechanisms underlying their susceptibility are incompletely defined. Toll-like receptors (TLRs) play crucial roles in the recognition of microbial components including bacterial lipopeptides (BLPs), expressed by a wide-range of bacteria, that activate monocytes via a heterodimer of TLR1/2 (triacylated BLPs) or TLR2/6 (diacylated BLPs). Preliminary studies indicate that despite normal expression of TLRs and associated signaling components, human newborn blood monocytes demonstrate a 2- to 3-log impairment in BLP-induced synthesis of pro-inflammatory and Th1-polarizing cytokine tumor necrosis factor-alpha (TNF-alpha) with preservation of BLP-induced production of IL-6, a cytokine with anti-inflammatory and Th2-polarizing activities. Similar impairment in neonatal TNF-alpha production is evident in response to E. coli K1/r, a pathogenic bacterium that expresses BLPs. We have discovered that impaired BLP- and E. col1- induced TNF-alpha production from neonatal blood monocytes is attributable to the action of plasma adenosine, an endogenous purine metabolite with immunomodulatory properties that is released from cells under conditions of stress or hypoxia. Neonatal mononuclear cells are especially sensitive to adenosine-induced inhibition of TNF-alpha production and to adenosine-induced production of cyclic AMP, an intracellular metabolite that inhibits TNF-alpha synthesis while preserving IL-6 production. The overall goal of this project is to characterize the role of the neonatal adenosine system in modulating TLR-mediated innate immune responses in neonatal blood monocytes. This goal will be accomplished through three specific aims: (1) to determine the mechanism for the greater adenosine sensitivity of neonatal mononuclear cells, (2) to define the adenosine receptor sub-type(s) that mediate inhibition of BLP- and E. coli-induced TNF-alpha production from neonatal blood monocytes;and (3) to characterize the mechanism by which engagement of adenosine receptors results in diminished BLP- and E. coli-induced monocyte TNF-alpha production while preserving IL-6 production. Mechanistic analysis will define how adenosine alters neonatal TLR-mediated immune responses. As a whole, these studies will deepen our understanding of how the unique physiology of the human newborn fundamentally alters innate immunity at birth.