PROJECT SUMMARY Microbial infections are a major cause of infant mortality worldwide. For particularly vulnerable populations such as pre-term and low birthweight babies, the risk of invasive infections further escalates. The neonatal period is defined by a distinct, often described as immature, immune system. Many features of a protective host response to infection are deficient as compared with older children and adults. Our laboratory has identified that expression of the immune suppressive cytokine interleukin (IL)-27 is elevated in human and murine neonates. Other recent studies have shown IL-27 to be a biomarker for early onset neonatal sepsis. This suggests that elevated IL-27 may represent a risk factor and when further increased during bacterial challenge, compromise the host immune response. The overall premise of the current proposal is that IL-27 is a host molecule that represents a target for immune intervention to improve the host response and reduce susceptibility to bacterial infection early in life. We present strong evidence in a mouse model that the absence of IL-27 signaling translates to increased survival, improved weight gain, and enhanced clearance of bacteria during neonatal sepsis. To advance our knowledge of how IL-27 regulates the immune response during neonatal sepsis, we need to identify the complete repertoire of cell types responsible for IL-27 production, understand how these population may change over the course of infection, and further define their functionality. We will address this gap in understanding using an IL-27 reporter mouse that expresses a fluorescent protein under control of the IL-27p28 promoter. Using whole-animal imaging of the reporter mouse coupled with luminescent bacteria, this will allow us to identify IL-27 producers, sort them for further functional analysis, and correlate their presence in infected tissues with the bacterial burden. We also seek to understand cellular signaling pathways required for IL-27-mediated suppressive activity and compromised control of the bacterial burden. We hypothesize that signal transducer and activator of transcription (Stat)-3 signals downstream of IL-27 receptor binding to interfere with lysosomal trafficking and acidification. The net result is compromised bacterial clearance. Lastly, a primary objective is to investigate the outcomes of antagonizing IL- 27 during neonatal sepsis with the aim of establishing an immunotherapeutic approach for an infectious disease for which we can currently only offer antibiotics and supportive care. Antibiotic resistance confounds our reliance on this approach. Administration of a neutralizing antibody conjugated to a fluorescent tag will allow for visualization of tissue penetration in real time and directly correlate the presence of the antagonist with control of bacterial growth. At the completion of this project, we expect to have performed preclinical validation of a promising immunotherapeutic approach to improve i...