A pathway regulating neutrophil integrin inactivation and its contribution to inflammation

The body's immune cells generate inflammation as part of their defence against infections. But sometimes, immune cells generate inflammation even in the absence of infection. Chronic inflammatory conditions, such as rheumatoid arthritis or multiple sclerosis cause much suffering and pose a massive drain on health services world-wide. To generate inflammation, a type of white blood cell called the neutrophil is essential. Neutrophils are the most common type of white blood cell. Neutrophils are recruited from the blood stream to sites of infection or injury. In order to leave the bloodstream, neutrophils become sticky: they adhere to the inside of the wall of the blood vessel before they transmigrate. This process relies on particular cell surface molecules, the integrins. Integrins exist in a "sticky" (active) and a "non-sticky" (inactive) conformation. It's already known that absence of integrins, or their inability to become activated interferes with the recruitment of neutrophils to sites of infection. A small number of studies analysed integrins that were experimentally rendered permanently active. These activated integrins, too, did not support neutrophil recruitment. We recently showed that a cellular regulator called ARAP3 is important for the regulation of neutrophil integrins, allowing activated integrins to become inactive. Loss of ARAP3 changes the activity profile of the neutrophil. For example, it interferes with migration in the test tube, as neutrophils adhere too firmly to the underlying support. We think that neutrophils need to be able to activate, and inactivate their integrins, "fine-tuning" integrin activity for optimal migration through the body's tissues. Therefore, loss of ARAP3 may also interfere with neutrophil recruitment, and ultimately inflammation. We will study this in the test tube and in models for neutrophil recruitment and for inflammation. We think that ARAP3 is a good target for combating inflammation. This work will build the foundations for new therapeutic approaches targeting chronic inflammation.

The proposed work explores modulation of the activity status of neutrophil integrins, specifically integrin inactivation, and how this critically regulates neutrophil transendothelial migration, neutrophil recruitment and inflammation. The work builds onto our recent in vitro work, that demonstrated that the GTPase activating protein (GAP) ARAP3 is a negative regulator of beta2 integrin activity and integrin-dependent processes in the neutrophil. The proposed work will analyse primary murine and human neutrophils, as well as (human) PLB-985 cells, which are amenable to lentiviral transduction and can be induced to behave neutrophil-like. Three aims will be addressed. (1) To characterise which neutrophil integrins are regulated by ARAP3 in terms of their activity status, the mechanism by which this occurs (surface availability and/or activity status), and whether this activity is conserved between murine and human neutrophils. (2) To identify how this modulation of integrins affects neutrophil transendothelial migration in vitro, and neutrophil recruitment to sites of sterile inflammation in vivo. (3) To address the effects of ARAP3-dependent regulation of neutrophil integrins on the induction, persistence and resolution of inflammation in models of acute and chronic inflammation. The proposed work will contribute to our understanding of the regulation of neutrophil integrins, analysing their inactivation, a previously underexplored step. This work will furthermore characterise the importance of integrin inactivation to neutrophil recruitment and inflammation. The 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 at a number of levels.

This is fundamental research aimed to understand the molecular control of neutrophils, which are involved in the generation of chronic inflammation. The primary immediate beneficiaries will be the wider academic biomedical science community in the UK and abroad. Visibility will be achieved by publishing our work in peer-reviewed journals with a broad readership. In agreement with MRC policies, our work will be published as open access articles, broadening the readership further still.

The main impact of the proposed research lies in the generation of new knowledge and scientific advancements, an established strength of the UK economy. Tackling a mechanism that underpins chronic inflammation, our scientific research will provide the basis for future translational research. The work will be of immediate benefit for the wider academic community, of indirect benefit for the pharmaceutical sector, and ultimately benefit patients.

The private sector will benefit from the proposed work. We are already exploring shared interests in neutrophil biology and inflammation with colleagues at Blueberry Therapeutics. This resulted in the recent submission of a joint CASE studentship application to MRC.

The general public will also benefit from the proposed work, thanks to public engagement activities aimed at both adults and children. We target local schools and present at the Edinburgh International Science Festival, which is held annually and draws in a very large audience.