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2014 Research Grant Program Winning Abstract Illuminating Neonatal Sepsis as Age-Specific Systemic Inflammatory Response Syndromes Playing Up between Neonatal Monocytes and Endothelia By Dorothee Viemann Despite enormous progress in the field of neonatology sepsis is still the leading cause of death. Preterm babies are especially at high risk for fatal courses that directly correspond to severe sepsis associated with so called cytokine storms in adults. Clinically, the hallmark of the neonatal patient group is hyperinflammatory immune responses. These are poorly explained and typically cited as immaturity reporting functional deficiencies in neonatal immune cells. In a murine model of neonatal sepsis, it was shown that the main difference between the neonatal and adult course of sepsis is set up in the innate immune system. However, the exact causes are still unclear. Our group intensively investigated signaling pathways of myeloid and endothelial cells (ECs). We could show that contrary to general assumptions not monocytes but endothelia are responsible for unlimited fatal cytokine responses in a virtually complete NF-kappaB-dependent manner. Especially pathogens causing fatal systemic inflammatory response syndromes in humans induced cytokine storms in ECs and rather suppressed the immune response of myeloid cells. In neonates endothelia are totally unexplored and their role in the pathogenesis of neonatal sepsis is unknown. In preliminary studies we determined soluble endothelial marker in serum probes of healthy term and preterm babies. Our first data show surprising results with an overwhelming birth-associated activation of the endothelium in preterm but not term newborns. A similar massive inflammatory response can be induced in vitro when EC monolayers are incubated with supernatants (SNTs) of overnight cultivated unstimulated neonatal monocytes. Effects correlate most strongly with the TNF-production of neonatal monocytes. Subsequently, we found that in contrast to adult monocytes, ECs cannot be tolerized against bacterial activation by low dose LPS and are primed by even low doses of TNF for subsequent stimulation with lipopolysaccharid (LPS) from gram-negative bacteria. The essential clue is that neonatal monocytes in contrast to adult monocytes are not yet able to regulate their immune responses down. Soon to be published data of our group will demonstrate molecular mechanisms that explain why neonatal myeloid cells fail down-regulating their immune response to LPS. Our analyses of the myeloid-specific serum marker S100A8/A9 showed that serum levels in healthy term babies are massively increased after birth but dramatically decrease during the first 5 days of life. In preterms, however, S100A8/A9 remains increased during the first two weeks of life reflecting an ongoing inflammatory activation of the myeloid cell system. Altogether we have evidence that demonstrates that in neonates at risk for sepsis an ongoing monocyte-mediated priming of endothelia lasts for the first 2 critical weeks of life. This age-specific vicious circle playing up between neonatal monocytes and endothelia exposes neonates to the dangers of hyperinflammatory reactions when encountering bacteria after birth. Objectives: 1) Confirmation of the role of endothelia for the pathogenesis of neonatal sepsis. 2) Analysis of the immunological maturation of neonatal ECs and monocytes. Serum probes of term and preterm babies will be examined during the first 2 weeks of life. Levels of IL-8 and EC marker are determined as surrogate for endothelial activation and TNF and S100A8/A9 for the activation status of monocytes. Results will be correlated with gestational age (GA)-dependent risk profiles, incidence and severity grade of sepsis in this patient group. For the maturation studies ECs are isolated from umbilical cord veins of babies of different GA. Monocytes are enriched from residues of routine blood drawings in preterm and term babies during the first three months of life. Age-dependent changes in the LPS response of both cell types are determined by analyzing gene expression, IL-8 and TNF in culture SNTs and cell calcium influx. For the cytokine studies in serum probes we will apply Human IL-8 and TNF Enhanced Sensitivity Flex Sets from BD Bioscience. Monocytes will be isolated from blood samples using the Monocyte Enrichment Set. The purity of isolated monocytes and ECs is checked by flow cytometry (FACSCanto II) upon staining with BD Bioscience lineage marker antibodies. Cytokine analyses in SNTs are performed using the TNF- and IL-8 ELISA and calcium influx is measured with the Calcium-Assay Kit (all BD Bioscience). Understanding neonatal sepsis as hyperinflammatory vicious circle playing up between monocytes and endothelium will open complete novel preventive and therapeutic approaches for the field of neonatology.