INTRACELLULAR CYTOKINE TRAFFICKING AND PHAGOCYTOSIS IN MACROPHAGES

Macrophages are a central player in the immune system. They are sentinels that phagocytose and degrade senescent cells and foreign invading microorganisms such as bacteria and yeast, and then present antigens derived from the foreign invaders to activate the adaptive immune system. Macrophages also regulate immunity through the release of cytokines that control the progression of an immune response. One of the pro-inflammatory cytokines secreted by macrophages, tumour necrosis factor alpha (TNF􀀁), has a central role in several acute and chronic diseases. While much is known about the physiological and pathological effects of secreted TNF􀀁, details on its intracellular trafficking and regulation of release from macrophages were minimal at the beginning of this study. This thesis aimed to build on the limited knowledge of TNF􀀁 trafficking to gain a more thorough understanding of these processes. TNF􀀁 accumulates in the Golgi complex before release. A surprising and novel result from these studies was that TNF􀀁 does not traffic directly from the Golgi complex to the cell surface. It is instead delivered to the cell surface via an endosomal compartment, the recycling endosome (RE). This was additionally interesting because delivery of the RE to the nascent phagocytic cup is required for efficient phagocytosis. Work performed during this thesis showed that TNF􀀁 is in fact delivered, along with the RE, to the phagocytic cup. Thus, an unexpected link between TNF􀀁 secretion and phagocytosis was discovered. To examine the full extent of TNF􀀁 and RE delivery to the phagocytic cup, phagocytosis of a variety of particles and microorganisms was compared. It was found that TNF􀀁 is not delivered to the phagocytic cup in all instances of phagocytosis. This was also found to be the case for the RE. Both TNF􀀁 and the RE are delivered together to the phagocytic cup during phagocytosis of yeast, Gram-positive bacteria and IgGopsonised particles, but not during the phagocytosis of Gram-negative bacteria or complement-opsonised particles. These findings therefore revealed that delivery of TNF􀀁 and the RE to nascent phagocytic cups is not a universal feature of phagocytosis. Lipid rafts are a prominent feature of immune cells, and were therefore examined in this project in determining the control mechanisms of TNF􀀁 delivery. The trafficking of TNF􀀁 to the cell surface via the RE was shown to require the cell surface SNAREs syntaxin 4 and SNAP-23 being located in cholesterol-dependent lipid rafts. Lipid rafts were also required for delivery of TNF􀀁 to the nascent phagocytic cup during the phagocytosis of yeast. Removal of cholesterol from the cells, thereby dissociating the lipid rafts, prevented both TNF􀀁 delivery to the phagocytic cup and the efficient phagocytosis of yeast because it also blocked the delivery of RE membrane to the phagocytic cup. Finally, to determine if the delivery of TNF􀀁 to the nascent phagocytic cup along with RE during certain phagocytic events is unique, or if other macrophage proteins are also delivered to the cup, the trafficking of three additional proteins was examined during concomitant phagocytosis. It was found that apolipoprotein E is not trafficked via the RE nor delivered to the phagocytic cup, while both interleukin-6 and transferrin receptor are delivered to the cell surface via the RE but are not focally delivered to the nascent phagocytic cup. Preliminary results also suggested that the RE is capable of compartmentalising and thereby selectively targeting cargo. Overall, this thesis provides novel and important findings on intracellular trafficking in macrophages. A link between TNF􀀁 secretion and phagocytosis was demonstrated, as was the lipid raft dependence of cytokine secretion and phagocytosis and differential cargo and RE recruitment to the phagocytic cup. Together, the results significantly advance our understanding of macrophage function as an immune mediator.