Investigating the Role of Phosphoinositide 3-Kinase in Cytokine Trafficking and Secretion

Cytokines are secreted by cells of the immune system to coordinate immune responses. Lipopolysaccharide (LPS) activation of macrophages elicits the de novo biosynthesis and secretion of inflammatory cytokines, including tumor necrosis factor-α (TNF). TNF is an early-response, proinflammatory cytokine with essential roles in immunity; but paradoxically, excessive secretion of TNF has clinically important sequelae in inflammatory diseases, such as rheumatoid arthritis. Whilst the physiological effects of TNF and many other cytokines are well known, we have only begun to understand the intracellular trafficking pathways that lead to their secretion. Yet, these pathways potentially harbor molecular targets that could rationalize new avenues for control of cytokines in disease. Previous efforts from this laboratory have chartered the key organelles for constitutive trafficking of TNF to the macrophage cell surface and have set the stage for this PhD project, which further investigates the molecules and carriers involved in TNF trafficking and secretion. Based on very preliminary data at the outset, this project has focused on exploring the involvement of PIs and PI 3-kinases (PI3Ks) in TNF trafficking. The PI3K family and their products are classically involved in signal transduction, and evidence to support their regulation in constitutive secretory pathways were minimal at the beginning of the study. A central goal of this thesis was to investigate a role for PI3K in the secretory pathway of TNF. Initial studies surveyed the distribution of different phosphoinositides (PIs) on the subcellular membranes in macrophages. By expression of various fluorescent PI-binding modules as probes, results in Chapter 3 provide the first map of PIs associated with macrophage organelles. Our findings also suggest candidate PIs with possible physiological roles in TNF trafficking. In a series of investigations using PI3K broad-spectrum and isoform-selective inhibitors, small-interfering RNAs, and genetic inactivation in mice, the class I PI3K p110δ was identified as the predominant isoform in regulating TNF trafficking and secretion. Ablation of p110δ function selectively blocked the secretion of TNF and accumulated the cytokine at the trans-Golgi network (TGN). These findings led to the use of live-cell imaging to study tubular carriers emerging from the TGN for TNF transport. It was then also revealed that LPS acutely induces the recruitment of p110δ to the Golgi membranes where it is kinase active. Furthermore, this activity of p110δ was shown to be essential for dynamic fission of membrane tubules carrying TNF at the TGN. These findings in Chapter 4 have not only provided the first demonstration of a class I PI3K and its activity at the Golgi, but also excitingly unravelled p110δ as a new component of tubule-fission machinery at the TGN. Finally, work was extended beyond these novel discoveries to explore the influence of p110δ activity in fission of different tubular carriers at the TGN and their roles in cargo export. TNF exits the TGN in tubules that were identified by the GRIP-golgin p230, but not those demarked by golgin-97. By comparing the requirement of p110δ activity in fission of these TGN GRIP tubules, data in Chapter 5 have revealed a selectivity of p110δ for fission of p230 tubules, but not golgin-97 tubules. Preliminary studies with a soluble IL-6 and other cargo shed light on cargo-sorting at the TGN and the selectivity of tubular carriers and their fission machinery. Overall, this thesis provides evidence to support a role for PI3K p110δ in TNF trafficking and secretion. A new function for p110δ in fission of a selective set of tubular carriers for TNF at the TGN is revealed. In a broader view, findings from this work have highlighted the potential of p110δ inhibition as a means to modulate TNF secretion in the future in treatment of inflammatory diseases