Investigating the thermal biology of inflammasomes - is NLRP3 a thermosensor?

Inflammation is a natural response to infection and injury and is an essential part of our immune response which protects us from invading microbes. For example, when we get a cut on our finger, the redness, heat and pain that we feel actually helps us clear the infection and heal the wound. Inflammation is therefore generally beneficial to us. However, it is becoming clear that inflammation also plays a negative role in many diseases, particularly those associated with ageing and metabolism. In disorders such as arthritis, liver disease, and Alzheimer's disease, this damaging inflammation causes disease progression. It is therefore imperative that we study the molecular mechanisms of inflammation as this informs our understanding of these prevalent diseases which are a huge burden on our health service and our society.

The main objective of our research proposal is to discover how a protein complex called the inflammasome is regulated or fine-tuned during the immune response. Inflammasomes, and specifically one type of inflammasome called NLRP3, are one of the most potent causes of inflammation in our immune cells. Our preliminary experiments have shown that increased temperatures, which are similar to those which occur during fever, seem to specifically block the inflammation caused by inflammasomes. This suggests that the immune response has developed a way to limit excessive or harmful inflammation triggered by these inflammasomes. We want to understand how this happens and the first aim of our study is to define how heat shock proteins (HSPs), which are naturally induced by high temperatures, influence inflammasome activity. To do this we will perform experiments using immune cells called macrophages which express high levels of inflammasome proteins. We will use chemical inhibitors of HSPs to study how HSP function affects inflammasome-dependent inflammation and the interactions of inflammasome proteins.

Our next focus will be the inflammasome sensor protein NLRP3. We have developed a new idea that NLRP3 itself can sense changes in temperature. Changes in temperature are a physiological stress faced by all organisms and as NLRP3 is able to sense many types cellular stress it could thus also be a thermosensor. To investigate this idea, we will use advanced techniques such as quantitative mass spectrometry to determine how NLRP3 behaves when cells are heated to fever range temperatures. We suspect that how NLRP3 interacts with other proteins will change with changes in temperature.

Our third objective is to understand how mutant forms of NLRP3 are activated by cold temperatures. Mutations in NLRP3 cause a rare inherited disease called familial cold autoinflammatory syndrome (FCAS). FCAS patients experience inflammatory symptoms such as skin rashes and fever when they are exposed to cold temperatures. No one yet understands how this unusual response occurs and so we will develop new cell models to study the mechanisms of this disease. We will then study the behaviour of FCAS NLRP3 in these cells using mass spectrometry and other biochemical techniques.

Ultimately, new knowledge about the function and regulation of inflammasomes and NLRP3 could help the development of new drugs for people suffering with inflammatory diseases.

Inflammation is an essential host response to infection and injury, but unregulated inflammation is highly damaging to the host and must be limited by negative feedback signalling. Inflammasomes are intracellular protein complexes that control the production of the pro-inflammatory cytokines IL-1beta and IL-18 and a lytic cell death programme known as pyroptosis. Inflammasome signalling is thus an extremely inflammatory process and inflammasome-dependent inflammation is associated with the pathogenesis of many common diseases including arthritis, Alzheimer's Disease and NASH. While clinical trials for NLRP3 inflammasome inhibitors were initiated in 2019, there remains a deficit in our knowledge of inflammasome regulation, that urgently needs to be addressed. The endogenous mechanisms that limit inflammasome activity are not understood and this research proposal will address this gap in our knowledge. Our novel preliminary data demonstrate that fever range temperatures can specifically limit inflammasome activity in mouse and human macrophages. Furthermore, we have identified that NLRP3 itself is highly sensitive to temperature and thus may function as a thermosensor. We will investigate the temperature-dependent mechanisms of inflammasome regulation using a range of pharmacological and biochemical approaches. We will employ cellular thermal shift assays and cutting-edge advanced quantitative mass spectrometry techniques to study NLRP3 protein-protein interactions as a function of temperature. Mutations in NLRP3 cause familial cold autoinflammatory syndrome (FCAS) but how cold temperature triggers NLRP3 activation in FCAS is not understood. We will develop FCAS cell models including patient-derived iPSCs that will allow us to characterise FACS NLRP3 using a range of methods. This innovative proposal addresses fundamental questions in the biology of inflammation and our insights will help to advance inflammasome targeted therapies for human health.