Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2008. / Includes vita. Includes bibliographical references (leaves 96-117).

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Pharmacology and Toxicology. Bibliography: leaves 185 - 197.

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Synthesis of polysaccharide intercellular adhesin (PIA), accumulation associated protein (Aap) and extracellular matrix binding protein (Embp) are major mechanisms used by Staphylococcus epidermidisto evade immunity through biofilm formation. These evasion strategies are particularly suited for colonisation of medical devices such as heart valves, joint prostheses and central venous catheters resulting in significant patient morbidity. Two biological activities of PIA, Aap and Embp contribute to their role as an evasion molecules. Firstly their ‘barrier’ function limiting penetration of immune cells and antibiotics. Secondly, their ‘immunomodulatory’ properties which influence cytokine responses. At present little is known about these functional activities in physiological media and biological fluids. This thesis uses a cell biology approach to study the environment necessary for PLA production. Specifically in vitromodelling of biofilm formation, PIA production and S. epidermidisleukocyte co-culture experiments have been used to assess conditions that are conducive for PIA production. This thesis has identified that:• Specific cell culture media cause unique profiles of biofilm accumulation with differential production of PIA, Aap and Embp. • Fetal bovine serum and pooled human serum support S. epidermidisgrowth but differentially affect biofilm formation by PIA, Aap and Embp. • Large scale production of PIA (~20mg) can be achieved by culturing in Iscove's Modified Dulbecco's Media which has allowed streamlining of current isolation procedures. • PIA induction of cytokines, including IL-8 and TNF is dependent on being tethered to the bacterial membrane. • Macrophages can penetrate into a S. epidermidisproduced PIA ‘barrier’. • Immunosuppression of whole blood with dexamethasone unmasks the pathogenic advantage of PIA in S. epidermidis expressing PIA compared to negative controls. • Whole blood killing of S. epidermidisis dependent on CD1 lb/CD 18. • PIA induces whole blood killing dysfunction which is likely related to C5a production. • PIA can be produced in a whole blood environment.• Inocula of -1 0 colony forming unit of S. epidermidisare required to form biofilms in whole blood. This study suggests the importance of studying clinically important biofilm production mechanisms under conditions that closely resemble those in human disease.

The a7 nicotinic acetylcholine receptor (a7nAChR) expressed on macrophages, a critical link between the neural and immune systems, provides a defense mechanism during inflammatory diseases. Release of acetylcholine in target tissues activates a7nAChR, a necessary element of the cholinergic anti-inflammatory pathway, inhibiting pro-inflammatory signaling pathways via NF-kB. However, other potential aspects of a7nAChR function are not well understood. The purpose of our project is to evaluate the role of a7nAChR activation in macrophage migration and accumulation at the site of inflammation. We first evaluated the survival of WT and a7nAChR-/- mice during LPS-induced endotoxemia. We found that a7nAChR-/- mice have significantly decreased survival compared to WT. We next examined differences in monocyte migration by tracking adoptively transferred, fluorescently labeled a7nAChR-/- and WT monocytes in the model of LPS-induced endotoxemia using flow cytometry and imaging flow cytometry. We found that a7nAChR-/- monocytes have significantly reduced migration to the lung, liver, and spleen during endotoxemia, in both a7nAChR-/- and WT recipient mice. Based on this result, we investigated if adoptive transfer of WT or a7nAChR-/- monocytes would decrease or improve survival in LPS-induced endotoxemia. Adoptively transferred, unlabeled WT monocytes improved survival in a7nAChR-/- recipient mice, though this effect did not reach significance. Survival was unaffected by adoptive transfer of a7nAChR-/- monocytes. Notably, this data coincides with the protective role of both macrophages and a7nAChR during sepsis that has been demonstrated in multiple recent publications. To reveal a potential mechanism, we tested the effect of a7nAChR-deficiency on the expression of adhesive and chemokine receptors at the macrophage surface. We selected 10 receptors to evaluate via qRT-PCR and flow cytometry. Our qRT-PCR experiments demonstrated a significantly reduced expression in CCR5, CCR2, integrin αMß2, and integrin αXß2 in a7nAChR-/- peritoneal macrophages, when compared to WT. The reduction in expression of CCR2 and αXß2 was corroborated by our flow cytometry results. Interestingly, the decrease in CCR5 and αMß2 was lower, but still detectable, and this discrepancy can be attributed to post transcriptional regulation of these receptors. The role of these receptors was further investigated in an in vitro 3D migration assay. Macrophages deficient in a7nAChR showed significantly decreased migration within a fibrin matrix (integrin αMß2 dependent) along a RANTES gradient (CCR5-mediated). The reduction in the migration of a7nAChR-/- macrophages toward MCP-1 (CCR2-mediated) did not reach significance, although still measurable. This experiment confirmed the chemokine-independent contribution of αMß2 to mesenchymal macrophage migration. These protective effects of αMß2 and CCR5 during sepsis were reported previously and are related to the regulation of monocyte recruitment and efflux at the site of inflammation. In summary, we discovered a new link between the parasympathetic nervous system and immune response based on a7nAChR-regulated macrophage migration during inflammation. The signaling pathway downstream of a7nAChR that modulates αMß2 and CCR5 expression is yet to be identified and is the objective of our ongoing investigation

The a7 nicotinic acetylcholine receptor (a7nAChR) expressed on macrophages, a critical link between the neural and immune systems, provides a defense mechanism during inflammatory diseases. Release of acetylcholine in target tissues activates a7nAChR, a necessary element of the cholinergic anti-inflammatory pathway, inhibiting pro-inflammatory signaling pathways via NF-kB. However, other potential aspects of a7nAChR function are not well understood. The purpose of our project is to evaluate the role of a7nAChR activation in macrophage migration and accumulation at the site of inflammation. We first evaluated the survival of WT and a7nAChR-/- mice during LPS-induced endotoxemia. We found that a7nAChR-/- mice have significantly decreased survival compared to WT. We next examined differences in monocyte migration by tracking adoptively transferred, fluorescently labeled a7nAChR-/- and WT monocytes in the model of LPS-induced endotoxemia using flow cytometry and imaging flow cytometry. We found that a7nAChR-/- monocytes have significantly reduced migration to the lung, liver, and spleen during endotoxemia, in both a7nAChR-/- and WT recipient mice. Based on this result, we investigated if adoptive transfer of WT or a7nAChR-/- monocytes would decrease or improve survival in LPS-induced endotoxemia. Adoptively transferred, unlabeled WT monocytes improved survival in a7nAChR-/- recipient mice, though this effect did not reach significance. Survival was unaffected by adoptive transfer of a7nAChR-/- monocytes. Notably, this data coincides with the protective role of both macrophages and a7nAChR during sepsis that has been demonstrated in multiple recent publications. To reveal a potential mechanism, we tested the effect of a7nAChR-deficiency on the expression of adhesive and chemokine receptors at the macrophage surface. We selected 10 receptors to evaluate via qRT-PCR and flow cytometry. Our qRT-PCR experiments demonstrated a significantly reduced expression in CCR5, CCR2, integrin αMß2, and integrin αXß2 in a7nAChR-/- peritoneal macrophages, when compared to WT. The reduction in expression of CCR2 and αXß2 was corroborated by our flow cytometry results. Interestingly, the decrease in CCR5 and αMß2 was lower, but still detectable, and this discrepancy can be attributed to post transcriptional regulation of these receptors. The role of these receptors was further investigated in an in vitro 3D migration assay. Macrophages deficient in a7nAChR showed significantly decreased migration within a fibrin matrix (integrin αMß2 dependent) along a RANTES gradient (CCR5-mediated). The reduction in the migration of a7nAChR-/- macrophages toward MCP-1 (CCR2-mediated) did not reach significance, although still measurable. This experiment confirmed the chemokine-independent contribution of αMß2 to mesenchymal macrophage migration. These protective effects of αMß2 and CCR5 during sepsis were reported previously and are related to the regulation of monocyte recruitment and efflux at the site of inflammation. In summary, we discovered a new link between the parasympathetic nervous system and immune response based on a7nAChR-regulated macrophage migration during inflammation. The signaling pathway downstream of a7nAChR that modulates αMß2 and CCR5 expression is yet to be identified and is the objective of our ongoing investigation.

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