Regulating granulocyte viability during emergency granulopoeisis

© 2014 Dr. Joanne Alice O'DonnellNeutrophils play key roles in responses to infection. While much is known about how neutrophils function and how granulopoiesis is stimulated during infection, little is known about how the viability of neutrophils is regulated in inflamed tissues. The regulation of neutrophil viability is crucial to ensure sufficient neutrophils are present to combat infection. Conversely, the timely resolution of inflammatory responses requires the death and subsequent clearance of neutrophils from tissues. It has been postulated that death receptor signalling in response to ligands that are abundant in an inflammatory milieu are involved in this process. This thesis first investigates the molecular regulators of death receptor signalling in neutrophils and then explores their role in neutrophil progenitors. A rapid high-throughput live cell imaging platform was developed to study the molecular signalling pathway of the death receptor Fas and to understand the negative regulators of this pathway in neutrophils. Fas death signalling in neutrophils was found to be negatively regulated by the intrinsic apoptosis pathway, specifically by Mcl-1 and Bcl-2, members of the pro-survival Bcl-2 family. Toll-like receptor ligands and the pro-inflammatory cytokine IL-18 were also found to inhibit Fas-induced death in neutrophils. Furthermore, Fas was found to regulate neutrophil lifespan during viral and bacterial infection in mice. This suggests that neutrophils encountering Fas may not be destined to die by Fas-mediated apoptosis during situations where neutrophils are still required to combat infection. The role of RIPK1 signalling downstream of Fas and TNF was also studied. It was intended that RIPK1 would be studied in the context of neutrophil lifespan ex vivo using chimeric mice generated through the transplantation of Ripk1-/- fetal liver cells, as Ripk1-/- mice die soon after birth. Unexpectedly, RIPK1 was found to be required for the establishment of hematopoiesis in the transplantation setting, prompting investigation of death receptor signalling in stem and progenitor cells. RIPK1 was found to modulate the survival of these cells by negatively regulating TNF-induced apoptosis and necroptosis. Together, the data presented in this thesis illustrate that death receptor signalling contributes to the viability of progenitors, stem cells and neutrophils during inflammation, and factors present in the inflammatory milieu can modulate the impact of this signalling. This suggests that death receptor signalling contributes to the regulation of neutrophil numbers at key stages of emergency granulopoiesis. Precisely how other signals present during inflammation and infection affect the biological consequences of death receptor signalling needs to be clarified in future studies