Acute dendritic cell responses to TLR ligands

Several important cells in our immune system collaborate when we are infected with a microbe or when we are vaccinated against one to activate an ?immune response?. In most cases the first cells to encounter the invader or vaccine are so called dendritic cells (DC). These cells act as messengers which ferry a processed form of the microbe or vaccine from the site of vaccination to the specialised sites in the body where other immune cells, particularly lymphocytes, are waiting to be activated. The coming together of dendritic cells and so called T lymphocytes is a key event that ultimately leads to elimination of the invader and/or to the creation of a ?memory? to allow a faster response to the same insult in the future.

Many immunologists believe that better understanding of the biology of dendritic cells will help in the design of new and better performing vaccines. We are working on the early events that occur when dendritic cells encounter foreign material whether it be a vaccine or the actual pathogen. We have found that DC ?wake up? from a resting state when they sense bacterial or viral products and boost their ability to capture and process foreign material so that they can better display it to T cells. We are also studying intriguing structures called podosomes that are thought to be relevant to the journey that dendritic cells make out of the tissues towards their encounter with T cells. We have been studying the detailed biochemical signalling events that are triggered in dendritic cells by microbial material and how podosomes might be involved in aiding migration of dendritic cells. We think that more detailed knowledge will allow vaccines to be designed that are taken up more efficiently by dendritic cells and which boost their migration from the tissues to the sites where collaborations with lymphocytes take place.

Dendritic cells (DC) are now recognised as key immune cells that detect infectious microorganisms through various pattern recognition receptors and translate these ?innate? immune signals into an ?adaptive? immune response. Much recent work has focussed on responses made by Toll-like receptors (TLRs) whose signalling leads to increased expression of cytokines and co-stimulatory molecules. However TLR signalling also triggers a number of striking cell biological changes in DC, some within minutes. We recently showed that LPS and other TLR ligands induce an acute upregulation of actin dependent endocytosis leading to enhanced antigen capture and presentation and simultaneously, a transient loss of F-actin rich podosomes. In this proposal we wish to follow up our latest findings that activation of the MAP kinase activated kinase RSK is crucial for this endocytic response and surprisingly, that the ?wiring? of the MAP kinase pathway is different in DC . In DC we found that RSK can be activated by Erk, as in most cells, but also by a novel p38 MAP kinase pathway. We propose projects which aim to discover what other DC responses RSK is involved in, what its key substrates are, how p38 is able to activate RSK, what the significance of this might be and what roles the other MAP kinase activated kinases (MSK1/2, MNK1/2 and MK2/3) may play in DC. We also propose studies to probe the significance of podosomes in DC and their regulation by TLR signalling. Podosomes are intriguing structures that are thought to be involved in cell migration and invasiveness. A key feature of podosomes and related structures called invadopodia is their ability to organise matrix metalloprotease (MMP) activity. We have recently found that DC podosomes engage in focussed pericellular proteolysis and that this is due to the enzyme MT1-MMP. Intriguingly, TLR-ligand induced podosome disassembly requires MMP activity, but this is due to a second unidentified enzyme. Because TLR signalling regulated MMP activity is likely to be important for DC migration though tissue and matrix barriers we propose to identify the second MMP activity and assess what roles these two MMPs play in DC migration in vivo. Finally, we propose various cell biological studies on DC podosomes and in particular an analysis of their structure at the EM level which we think will both clarify their relationship to invadopodia and help us to assess their role in DC in vivo.