How does inflammation regulate CLEC-2-mediated thrombosis after infection?

A common consequence of serious infections is sepsis. This is where infection causes a profound inflammation around the body. Unfortunately, death is a common outcome and the fatality rate is often as high as 30%, and current treatments are sub-optimal. In most cases the pathogen causing sepsis is not cultured from the blood. Sepsis is a syndrome with many presentations, not least amongst these is thrombosis, where clots form in the vessels. Even in non-septic patients with a serious infection, thrombosis occurs frequently. In some cases these clots can break off, or embolise, and cause problems elsewhere in the body such as the lungs. How thrombi form after infection is largely unknown. It is also unclear whether these clots are always harmful or if they can have beneficial roles too. Moreover, it is not even clear whether the treatments used against thrombi work. A major reason for this is that the mechanisms that underlie thrombosis during infection are largely unknown.

Using a model of Salmonella infection in mice we have identified a previously unknown mechanism that regulates thrombosis after infection. This involves a key cell type in the blood, called platelets, which are critical for normal blood coagulation and the control of bleeding. We have found that after infection these cells get activated through a molecule on their surface called CLEC-2. Despite their being activated in the blood, the cells that activate them are in tissues and so they and possibly even their products are not readily be detected by blood tests. These cells appear to interact with platelets through breaks in the blood vessels. What is important too is what type of cell it is activating the platelets. They are immune cells responding to the inflammation in the tissues. Therefore, it is inflammation in the tissues that leads to thrombosis in the vessels. Also, whilst this form of thrombosis induction requires infection to start, it continues long after the infection is under control. This helps answer a number of the key unknown questions about thrombosis, such as why normal treatments do not work well against this form of thrombosis and also why in sepsis a pathogen is commonly not cultured. The mechanism through which thrombosis starts is also one that is attractive for targeting for therapies since, unlike other ways of inducing thrombosis, it is not likely that it will affect the balancing act needed to maintain normal blood functioning. Other treatments, if they work at all, can have unpleasant side-effects. Hopefully, targeting this route of platelet activation will avoid many of these.

Like all new discoveries, we need to understand more so that we can identify targets for therapy and that is what we wish to do in this project. In particular, we wish to address some major questions surrounding this. Although many questions arise from our findings the key ones we wish to answer are focused around i) Whether the same mechanism for thrombosis is active in all parts of the body; ii) What are the inflammatory triggers for the process and for how long are they needed and iii) How is the "upper limit" of thrombosis reached and how is thrombosis maintained. Our model of Salmonella infection in the mouse is unique as it is able to answer all of these. The features that make it so appropriate to study thrombosis include the persistence of thrombosis for weeks and there appears to be a role for a type of immunity (adaptive) that has not been widely studied before in this context. Therefore, this project will address and answer these key clinical problems.

Thrombosis is a common consequence after infection and associated with poor outcome. Despite its fundamental importance in infection, it is poorly understood. In this project we want to develop our findings that have led to the identification of a novel mechanism underpinning how thrombosis is induced after infection. To identify this role for CLEC-2 we have used a murine model of invasive salmonellosis where the infection and thrombosis persists for weeks and where infection is required for the induction of thrombosis but not its maintenance. At its heart is CLEC-2-mediated activation of platelets via podoplanin ligation. This occurs through the TLR4 and IFNg-dependent recruitment of podoplanin-expressing monocytic cells to the liver. In conjunction with other peri-vascular podoplanin-expressing cells these cells appear to contact platelets through damaged regions of the vasculature resulting in their activation. The thrombi form in these damaged sites. What is significant is that targeting this mode of platelet activation is unlikely to have significant effects on other areas of haemostasis and so is attractive from a therapeutic view-point. The work raises multiple key questions. Although we cannot answer all of them in one grant we wish to focus on a number of key elements that can only be addressed in this model. These fundamental questions include: i) Can thrombosis have beneficial as well as harmful effects? ii) Do CLEC-2 and inflammation only play roles in the induction of thrombosis; iii) Does adaptive immunity promote or inhibit the longevity and extent of thrombosis; iv) How does thrombocytopenia affect thrombus development?

Within the MRC document: Research Changes Lives: The MRC Strategic Plan 2014-2019, this project aligns with:

Strategic Aim ONE
Research Priority Theme one: Resilience, repair and replacement (Natural protection and Repair and replacement)

Strategic Aim THREE
Going global: Accelerating progress in international health research (Global Health)

There are a number of groups within the UK and international research base and on a wider level within society that this work will impact upon. These are described below.

Clinical medicine and society

The potential impact of this research on clinical medicine is enormous. Collectively in all its forms thrombosis is one of the leading causes of death, responsible for more than 10 million deaths yearly. Whilst current treatments are often helpful, there are many sub-groups where this is not the case. Our work provides a new opportunity to target where these treatments are less effective and therefore reduce this number of deaths. The treatments that derive from this project will also help patients overcome non-fatal complications from thrombosis. Since sepsis has a fatality rate in different settings in the range of 20-40% and a causative pathogen is not identified in the blood in up to 60% of instances, our project is likely to provide novel avenues to explore how sepsis can be modulated. Resulting from this appreciation will be the potential benefits of this project in aiding the diagnosis of complications from sepsis and thrombosis. The identification that thrombocytopenia and thrombosis can be distinct processes will lead to improved diagnostics for different forms of thrombosis during infection and also to help understand the quite dramatic impact and consequence that thrombocytopenia has on the host.

Academia and Industry

This project will have impact in this area because it takes a multidisciplinary approach to tackle a key problem; how does infection cause thrombosis after infection. We have identified a new mechanism of infection-associated thrombosis that links inflammation and platelet activation without affecting haemostasis. This will lead to other researchers in academia and industry focusing on this area as the pathway involved is already one that is being investigated in other diseases and will invigorate research in this area. The current project will develop this further by directing our focus onto the key unanswered questions in this area that it has not been possible to answer previously. These are: whether thrombosis is beneficial, whether targeting podoplanin alone is sufficient to interfere in this process and lastly, what is the role of adaptive immunity in this process? Since these are issues that are directly relevant to patients across the world it will attract industrial interest and the generation of additional funds for the UK economy by attracting additional inward investment. Targeting podoplanin for other diseases has received significant interest in recent years, thus identifying its role in thrombosis is likely to extend the potential benefit of any therapeutics in development, meaning these may provide patient benefit within a much shorter time-frame than would normally be envisaged.