CHARACTERISING ARAP3-MEDIATED INTEGRIN INACTIVATION IN THE NEUTROPHIL

Immune cells need to generate inflammation to kill pathogens when they fight infections. This is a tightly regulated process, but occasionally the regulation goes wrong and immune cells generate inflammation even in the absence of infection. This happens in chronic inflammatory conditions, such as rheumatoid arthritis or chronic obstructive pulmonary disease. Chronic inflammation causes much suffering and poses an important drain on health services world-wide. No known drugs can cure chronic inflammation. Neutrophils, the most common immune cells in the blood, are important players in the generation of inflammation. Neutrophils leave the blood stream to travel to sites of inflammation. They can only do this if they can leave the blood stream by crawling through the vessel wall into the surrounding tissue. This relies on specialised adapters on their molecules, the integrins. Integrins exist in a "sticky" (active) and a "non-sticky" (inactive) state. Many labs have studied how integrins become active: this depends on several intracellular regulators. Not having integrins, or not being able to activate them stops neutrophil recruitment to inflammatory sites. This stops inflammation but also immunity and leads to recurrent infections. Only few studies have analysed integrins that are always active. It turns out that this also reduces neutrophil recruitment to inflammatory sites. We have shown that an intracellular regulator called 'ARAP3' is required for inactivating neutrophil integrins. Without ARAP3 neutrophils are not efficiently recruited to inflammatory sites, but recruitment is not abolished. ARAP3 is itself regulated by at two other regulators (called PI3-kinase and Rap), and it in turn regulates two further regulators (called Arf6 and RhoA). We will study how Rap, RhoA and Arf6 are working together with ARAP3 in the neutrophil to regulate integrin inactivation. We will do this by introducing small molecular changes into ARAP3 so that it can no longer talk to one of the other regulators in turn. We will use stem cells from the bone marrow of mice that have no ARAP3, and engineer them to have one of the slightly changed ARAP3s instead. The modified stem cells will then be placed into culture. When we change their culture medium, they will transform into neutrophils. These neutrophils' integrins will be analysed in the test tube. This will allow us to learn how the intracellular regulators work together with ARAP3 to regulate integrin inactivation. This work may build the foundations for new therapeutic approaches that target chronic inflammation.

The proposed work explores the mechanism by which neutrophil integrins are inactivated. It builds upon our current work, which demonstrates that the GTPase activating protein (GAP) ARAP3 is required for neutrophil integrin inactivation, thereby promoting neutrophil recruitment to sites of inflammation. ARAP3 is a PI3-kinase and Rap regulated GAP for Arf6 and RhoA. Rap, RhoA and Arf6 are all known regulators of leukocyte integrins. The proposed work will address how signalling through Rap-ARAP3-Arf6/RhoA regulates integrin inactivation in the neutrophil. We will employ conditionally HoxB8-immortalized hematopoietic progenitors, which will be differentiated to neutrophils in vitro. Unlike primary neutrophils, HoxB8 progenitor cells are amenable to genetic modification. Arap3-/- HoxB8 neutrophils will be engineered to express physiological amounts of ARAP3, or of ARAP3 incorporating individual point mutations that uncouple it from activation by Rap or that render it ArfGAP and/or RhoGAP dead. In particular we will elucidate (i) any role of Rap-ARAP3-RhoA signalling in integrin inactivation and (ii) any role of the ARAP3-Arf6 axis in integrin trafficking. In addition, integrin-dependent effector functions including migration and transendothelial migration in vitro as well as neutrophil recruitment in an adoptive transfer model in vivo will be analysed. The proposed work will contribute to our understanding of the regulation of integrin inactivation, a poorly defined, though important facet of integrin regulation, which is already recognized as a therapeutic target. The proposed work may lay the foundations to the development of new therapeutic approaches targeting neutrophil integrin inactivation in chronic inflammation.

The proposed work will generate impact by knowledge creation within academia, with the private sector and with the general public.

The proposed research is fundamental in nature. It aims to understand a molecular mechanism that underpins the inactivation of neutrophil integrins, a prerequisite for the generation of inflammation. The primary immediate beneficiaries of this work will be the wider biomedical academic community in the UK and elsewhere. To make our work as accessible as possible, it will be published as open-access research articles in peer-reviewed journals with a broad readership in keeping with RCUK policy.

The major impact of the proposed research will lie in the generation of new knowledge and scientific advance, two established strengths of the UK economy. Integrins are already recognized as promising therapeutic targets in inflammation. By researching a molecular signaling pathway that underpins the generation of inflammation, this research will build a platform for future research, both basic and translational. This work will therefore directly benefit the wider academic community and indirectly the pharmaceutical sector. The ultimate long-term beneficiaries will be patients.

Those within the private sector who are interested in mechanisms regulating the generation of inflammation, will also benefit from the proposed work. This is exemplified by the fact that we have already commenced a joint research project with colleagues at GlaxoSmithKline.

Last but not least, the general public who fund our work through the taxes they pay will benefit from public engagement activities aimed at both adults and children .