Understanding the molecular pathways that underlie dectin-1 mediated cytokine responses

Invasive fungal infections are uncommon in healthy individuals however several conditions can greatly increase the risk of developing these infections. For example patients undergoing immunosuppressive therapies, either following organ transplant or for the treatment of diseases such as cancer and autoimmune conditions, are much more likely to develop invasive fungal infections. People with a suppressed immune system either as a result of infections such as HIV or due to inherited mutations in key proteins that regulate immunity are also at much greater risk of acquiring fungal infections. In these patient groups when invasive fungal infections occur they are difficult to treat and have a high mortality rate. This represents an increasing problem as the numbers of people in high-risk groups grows and as resistance to existing antifungals increases. Understanding how the immune system detects and responds to fungal pathogens is therefore an important issue.
Following infection, one of the first cell types to detect fungal pathogens are specialized cells in the immune system termed antigen-presenting cells (APCs). Once these cells sense the presence of a fungal pathogen they undertake several functions, key amongst which is the production of cytokines, small proteins that are released from immune cells and that are required to co-ordinate the immune response. APCs are able to detect fungal pathogens as they express certain specialized receptors that are specific for molecules that are found on the surface of fungal pathogens but not on the body's own cells. One such receptor is a protein called dectin-1. The relevance of dectin-1 to fungal infection has been demonstrated by the observations that both dectin-1 knockout mice and people that carry an inactivating mutation in the dectin-1 gene are more sensitive to fungal infection.
In the research outlined in this proposal we will use a variety of techniques to study the way in which the activation of dectin-1 promotes the required cellular response and controls the production of cytokines. Key to this is understanding how dectin-1 affects intracellular signaling networks, many of which are controlled by a process known as protein phosphorylation. These processes are essential for the cell to translate the detection of a fungal pathogen by dectin-1 into the correct immune response. A major part of this study will be to use sate of the art mass spectrometry methods to map global changes in protein phosphorylation that occur inside the cell following the activation of dectin-1. This will allow us to build up a picture of the signaling networks activated. We will then examine the contribution of specific parts of this network to the control of cytokine production.
This work will increase our understanding of how the immune system deals with fungal infections, and may suggest ways in which the immune system could be targeted to increase its ability to combat fungal infections. In addition several of the cytokines that are induced by fungal pathogens can, in other circumstances, have pathological roles such as in autoimmune diseases like arthritis. Understanding how their production is controlled therefore also has wider implications for interpreting how the immune system functions and for future drug development programs for a number of immune related diseases.

The proposed research will examine the intracellular signaling mechanisms that control the production of cytokines downstream of the C-type lectin dectin-1 and investigate the cross talk between dectin-1 and Toll like receptor signaling. To do this a range of techniques will be used to examine these processes in primary macrophages and dendritic cells isolated from either mice or humans. The use of primary murine macrophages allows us to exploit relevant gene targeted mice to study protein function. This technique is essential to study the function of specific proteins in primary macrophages. While siRNA can be used for some of the experiments, it is frequently inefficient in macrophages and does not provided the same degree of experimental confidence in its results as gene targeting. Primary human macrophages will also be studied in order to ensure that our results translate into human systems. In addition to this small molecule kinase inhibitors, screened for selectivity by the International Center for Kinase Profiling in Dundee, will also be used to study these systems.
In order to study the APC phenotype a range of experimental techniques will be used. These will include multiplex ELISAs, qPCR and expression profiling techniques in addition to standard cell culture and biochemical techniques. The project will make extensive use of SILAC based phospho-proteomics to characterize intracellular signaling events in macrophages stimulated by dectin-1 in comparison to TLR2. This will allow us to create a map of the relevant signaling networks and provide the basis for further work to identify the critical events in allowing dectin-1 to strongly activate IL-10 induction while repressing IL-12.

The initial aim of this project is to investigate how dectin-1 mediates cytokine production and to map the signaling networks activated in response to dectin-1. While this has an immediate impact on our understanding of how the immune system reacts to fungal pathogens it also has wider implications.
Dectin-1 activation induces an interesting phenotype in dendritic cells and macrophages and understanding how this occurs has wider implications for our understanding of how the immune system functions. For example, simulation of macrophages with ligands that act via a combination of dectin-1 and TLRs induces a phenotype that is very similar to regulatory macrophages. These cells have been suggested to play anti-inflammatory roles and to promote the resolution of inflammation. Work from other labs has suggested that the reintroduction of regulatory macrophages into mice can protect from endotoxic shock and models of multiple sclerosis. Thus manipulating macrophage function may have a therapeutic benefit. Understanding the molecular mechanisms underlying the polarization of macrophages to a regulatory phenotype would be of a significant advantage in developing drugs designed for this purpose.
The ability of dectin-1 to repress TLR induced IL-12 but not IL-23 production by dendritic cells allows them to promote the development of Th17 cells, which play important roles in combating fungal infections. Increasingly IL-23 and Th17 cells also play an important role in the development of autoimmune diseases. While there has been considerable development of immunosuppressive drugs to treat autoimmunity, these compounds remain far from perfect and have a range of serious side effects. There is therefore a need to develop better therapeutic strategies or drugs that target the immune system. Drug development in this area is still a major focus in the pharmaceutical industry and understanding the molecular pathways that regulate the balance between IL-12 and IL-23 production will aid in the selection of novel cellular targets for drug development.
Thus in addition to contributing to the scientific knowledge on both anti-fungal responses as well as in a broader immunology setting, the work also has the potential to inform the selection of potential targets for drug development for a number of diseases whose pathology involves the inappropriate production of cytokines. As such it will be of interest not only to the academic community but also the pharmaceutical industry.

Our work will also have other wider benefits. In addition to providing research experience and training for postdoctoral researchers, they will also benefit from help with developing transferrable skills, such as presentation, communication and writing skills in addition to experience with project management and supervision of other researchers. This should allow them to pursue successful careers in science or in other disciplines.

Finally we will also continue to play an active role in informing the public about biomedical research and its importance. To do this we will continue our participation in science festivals in Dundee and Edinburgh that are focused on engaging children in science. We will also seek to expand this work via participation in the STEM Ambassador program, which facilitates the interaction between researchers and science programs in schools. My group also receives funding from Arthritis Research UK, and we have had several meetings with the fundraising division of this charity to talk to donors about the type of work we undertake and what its benefits are. This is something we are keen to continue with in the future.