Activation and Stabilization of the Caspase-8 Survivalsome is Indispensable for Functional Toll-like Receptor 4 Dependent Signaling in Septic Neutrophils

Sepsis, defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, is a complex, heterogenous, and redundant syndrome that is the leading cause of mortality in intensive care units worldwide. We investigated signalling mechanisms that mediate activation and termination of the response to infection by neutrophils – circulating white blood cells that are the primary agents of the early host response. While a failure of activation impairs antibacterial defenses, failure in terminating that response through a process called apoptosis contributes to systemic inflammation and tissue injury in septic patients. By investigating the intracellular biology that controls neutrophil activation and termination, we sought to identify potential targets to treat clinical sepsis. Using healthy human and septic patient neutrophils, we first showed the function of caspase-8, the apical caspase in the extrinsic cell death pathway, is modified by its phosphorylation. Caspase-8 tyrosine phosphorylation at tyrosine-380 established an anti-apoptotic signaling module we termed the survivalsome that promoted neutrophil III survival. Conversely, caspase-8 dephosphorylation at tyrosine-380 prevented survivalsome assembly and induced caspase-3 dependent neutrophil death. By inducing sepsis in a caspase-8 deficient mouse model, we demonstrated that in vivo caspase-8 deletion significantly reduces mortality, accelerates bacterial clearance, and limits lung injury. Serendipitously, we found an association between the variability of neutrophil response to lipopolysaccharide with caspase-8 variant expression. Neutrophils coexpressing both caspase-8 A and B isoforms demonstrated lower rates of apoptosis from baseline following lipopolysaccharide stimulation versus neutrophils expressing only A or B. Expression of caspase-8 isoforms further correlated with multiple organ dysfunction score and white blood cell count in septic patients. Since caspase-8 is phosphorylated in response to lipopolysaccharide, we investigated whether caspase-8 tyrosine phosphorylation at tyrosine-380 is required for functional Toll Like Receptor 4 (TLR-4) signal transduction. Using healthy human and septic neutrophils, we showed th ipopolysaccharide-induced TLR-4 activation increases caspase-8 tyrosine phosphorylation and MyD88 receptor-mediated signaling to trigger NF-B translocation and pro-inflammatory gene transcription. Finally, this PhD thesis uncovered a mechanism that stabilizes Src-induced caspase-8 tyrosine phosphorylation for phosphoinositide 3-kinase activation: heat shock protein 90 prevents ubiquitin-mediated degradation of caspase-8 and c-Src to prolong inflammatory neutrophil survival.Ph.D