Leukocyte guidance and gradient interpretation in vivo

Our immune system is designed to protect us from infections. For any immune response to occur, white blood cells (leukocytes) need to exit the blood stream and then navigate within tissues in order to find and eliminate invading microbes. This process absolutely relies on their ability to accurately interpret guidance signals produced within tissues. Any malfunction in this process can be the basis of inflammatory diseases (such as allergy, asthma, arthritis and various conditions ending with the suffix '-itis') or insufficient responses to infection (for example in immuno-compromised patients with leukocyte migration defects, such as 'WHIM syndrome' patients). For this reason, manipulating leukocyte navigation for treating inflammatory diseases is a major biomedical target.
This target has yet to be met, as we still have little understanding of how leukocytes are guided within the body. Although many types of guidance cues have been identified over the past decades, their actual mode of action and interpretation by leukocytes are poorly understood. If we can understand this, we can be in a much better position of designing effective and selective treatments for inflammatory diseases.
I have set up a model system in which we can directly visualize how leukocytes are guided towards infected tissues and dissect the genes and mechanisms that underlie this process. For this I have taken advantage of the zebrafish model, as it is optically transparent, allowing us to easily monitor leukocyte migration, and genetically tractable, allowing us to uncover the underlying genetic mechanisms. Using this unique model system, I have so far discovered that certain guidance cues produced at sites of infection - small proteins called 'chemokines' - need to interact with complex sugars in the tissue environment in order to accurately guide the migration of leukocytes. This has many implications and fundamentally changes our understanding of how chemokines operate. In my proposed plan I intend to elucidate further the mechanism of action of chemokines. We (myself, a research assistant and a team of collaborators with complementary expertise) will investigate how sugars of the tissue environment change the signaling properties of chemokines and how exactly leukocytes process this information into directed movement. We will also expand this approach to other important guidance signals, beyond chemokines. These studies will provide a better understanding of how leukocytes 'read' guidance signals so as to navigate correctly in the body, which can ultimately open new avenues for treating inflammatory diseases.

Neutrophil migration to sites to infection or injury is a major driving force of inflammation. However, little is known about how neutrophils sense inflammatory guidance cues in vivo. We will address this knowledge gap by exploiting the zebrafish model, which uniquely combines amenability to in vivo imaging and genetic manipulation. In previous work, we identified a zebrafish homologue of a well-known human chemokine, CXCL8. Using a combination of in vivo imaging, quantitative and molecular approaches, we discovered that this chemokine forms tissue-bound gradients by binding extracellular proteoglycans and that neutrophis pursue these gradients in a complex manner by adjusting 'gears'. My objective is to further elucidate neutrophil guidance mechanisms by using and extending the tools I established. First, we will explore how neutrophils interpret chemokine gradients in vivo. For this we will probe intracellular gradient sensing events using appropriate fluorescent probes and high-resolution imaging. This will provide unprecedented insight into the information processing underlying complex neutrophil migration behaviours. Secondly, we will investigate how tissue proteoglycans regulate chemokine signaling and concentration at sites of inflammation. For this we will functionally assess chemokine mutants, as well as identify and genetically perturb candidate proteoglycans. This will illuminate the molecular and environmental requirements for functional gradient formation. Finally, we will explore the bigger picture of neutrophil inflammatory responses and ask how different cues, beyond chemokines, contribute to neutrophil guidance. This will provide insight into the function of poorly understood guidance cues and help obtain a more integrated understanding of neutrophil inflammatory responses. Thus, our studies will provide a better understanding of the molecular mechanisms underlying neutrophil guidance, which may open new avenues for treating inflammatory diseases.

I anticipate my research project to have a broad impact, beyond academia. The likely beneficiaries are:

Pharmaceutical industry. Chemokines are attractive drug targets due to their diverse functions in cell migration processes as well as their contribution to HIV infection as viral co-receptors. The FDA has already approved a number of molecules targeting chemokines and chemokine receptors in the last 5 years. Despite this success, many chemokine drug trials have met significant hurdles (Viola et al. Ann. Rev. of Pharmacol. and Toxicol. 2008; Schall et al., Nat. Rev. Immunol. 2011). This is due to a general lack of understanding of how chemokines operate in vivo. Our in vivo study of a zebrafish homologue of human CXCL8 (IL-8) and the receptors CXCR1 and CXCR2 may thus have a big long-term impact in the design of chemokine drugs. Our study will provide insight into the molecular and environmental requirements for chemokine gradient formation as well as the signal processes that allow neutrophils to read these gradients in vivo. Any of these aspects of my project can lead to new ideas for drug design. It is also noteworthy that unlike teleost fish (van der Aa et al. 2010, Sarris et al. 2012, Deng et al. 2013), rodents lack homologs of CXCL8 and CXCR1 (the closest equivalent in rodents are the chemokines CXCL1 and CXCL2)(Stadtman et al. Front. Immunol. 2012). Therefore the zebrafish model may prove particularly competitive for studies of CXCL8/CXCR1/CXCR2.
Besides chemokines, we plan to investigate other inflammatory cues, including cannabinoids whose role in inflammation is recognised but remains poorly understood. Pharmaceutical companies (e.g. GSK) are currently investing considerable research in the potential of cannabinoids to be used as immunomodulatory agents. The ease with which we can test chemical inhibitors and assess their effects on neutrophil homeostasis/function in zebrafish can form the basis of collaborations with industry to test candidate drugs and help improve their design. I am aware of funding schemes to establish collaborations with industry (such as the MRC Industry Collaboration Agreement) and plan to pursue such schemes once our project is at a mature enough stage. To this end, I will team up with other zebrafish researchers in the UK and beyond in order to pursue such schemes as a collective effort.

Biotech companies. Our in vivo studies will reveal the physiological characteristics of chemokine gradients and this knowledge can be used to design new in vitro chemotaxis systems that better mimic 'real' gradients. I will be communicating with companies commercialising such systems (e.g. Ibidi GmbH, Germany, 'Thermoscientific', UK and 'Gradientech', Sweden) so as to promote the design of 'haptotactic' gradient systems, which may expand their range of products and competitiveness. Our studies will also benefit companies commercializing image analysis softwares (Bitplane) and microscopy equipment (e.g. Leica, Olympus, Zeiss). I have contributed images in the past to such companies for marketing purposes. Moreover, our experiences and dialogue with these companies will help improve their product design and implementation. Finally, several companies (such as Anaspec, USA) are investing in producing antibodies and recombinant proteins for the growing zebrafish community. I have already contributed to Anaspec's production line by testing their antibodies in zebrafish or contributing gene clones. We will continue to be in contact with Anaspec, as well as other companies specialized in chemokine reagents (Almac, UK), to promote the generation of new antibodies and recombinant proteins.

General public. Inflammatory diseases (such as asthma, allergy and arthritis) are widespread and can be life threatening. By contributing to the understanding of neutrophil migration, a key driving force of inflammation, our studies may ultimately improve the life of an enormous number of people in the UK and beyond.