Understanding VRAC-dependent regulation of the NLRP3 inflammasome

Inflammation is our bodies' response to infection and injury. It is characterised by redness, swelling, and heat and requires recruitment of cells from the blood to an infected or injured area. Typically inflammation is a beneficial response. However, a failure to switch off an inflammatory response, or for it to resolve, can lead to chronic or inappropriate inflammation. This can result in the death of healthy cells and tissue. As we age the bodies control of how it regulates inflammation begins to diminish. Therefore when our bodies are challenged by an injury or an infection they fail to cope efficiently. Much research on mechanisms of inflammation has been carried out with respect to many types of infection and injury in many organs and great progress has been made. However, there are still processes that we just do not understand. The main aim of this proposal is to understand the regulation of the formation of an important inflammatory complex - the NLRP3 inflammasome. We think we have identified new parts of this pathway which we will now test by generating cells and animals in which we have removed specific genes. This will give us information on the specific reactions that occur and their consequence. Through understanding these complex processes we aim to identify new ways of treating disease.

Inflammation that occurs in response to tissue injury or disease can contribute to damage. Members of the interleukin-1 (IL-1) family of cytokines are often implicated in disease processes, namely the pro-inflammatory members IL-1a and IL-1b. IL-1 release from activated immune cells is regulated by a protein complex called the inflammasome. IL-1 is expressed in response to pathogen or damage associated molecular patterns (PAMPs or DAMPs) that stimulate pattern recognition receptors (PRRs) on cells of the innate immune system. Once expressed, pro-IL-1 remains intracellular until an additional PAMP or DAMP stimulation activates inflammasomes. Inflammasomes activate caspase-1 which in turn processes pro-IL-1 allowing release of the mature cytokine. The inflammasome most implicated in non-communicable diseases is formed by the PRR NLRP3. The NLRP3 inflammasome is also the best studied inflammasome, yet there are still large gaps in knowledge concerning its activation. We have discovered that the volume-regulated anion channel (VRAC), a Cl- channel composed of LRRC8 subunits, could be an important regulator of the NLRP3 inflammasome. We now aim to verify the importance of VRAC to NLRP3-dependent inflammatory responses using a number of CRISPR based strategies to knockout the VRAC essential sub-unit LRRC8A.

Knocking out LRRC8A in vitro will allow us to verify the importance of VRAC in NLRP3 inflammasome activation in response to a range of well-characterised NLRP3 activating stimuli. We will also knock out LRRC8A in macrophages specifically and demonstrate its relevance in an in vivo model of NLRP3-dependent inflammation. This, combined with our repurposing of existing medicines as VRAC inhibitors, will establish VRAC as a potential novel therapeutic target for the treatment of inflammatory disease.

- Who will benefit from this research?
The immediate beneficiaries of this research will be the Faculty here at the University of Manchester. The development of methodologies, the training of students and of postdoctoral researchers will bring many benefits that will ultimately enrich the research environment. The potential identification of drug targets will benefit our local commercialisation team (UMIP) and may, in the longer term, provide economic benefits. Beyond this the presentation/publication of papers at conferences and in international high impact peer reviewed journals will benefit the wider scientific community and the University in general. The data generated through the course of this proposal may inform strategy boards of funding bodies (UK and overseas) on the growing recognition of the importance of inflammation to disease in general. Clinicians may be informed of new strategies or treatments for the management of inflammatory disease that will ultimately lead to benefits for patients. The wider public will also benefit from increased understanding of and exposure to science through activities run by our labs. We also participate in careers fairs with local schools benefiting local school children to learn about future careers in science.

- How will they benefit from this research?
Most immediately research from this proposal will help inform researchers in the field and in the scientific community in general about key, up to now, unknown mechanisms of inflammation. The creative experiments we have designed, and the discoveries we make, will inspire other researchers to investigate similar mechanisms and thus our levels of knowledge on these process in general will increase substantially. The new areas of research that our discoveries will open will lead to the recruitment and training of students in this area which will spawn further developments. Many of these future developments will likely involve the identification of targets that may eventually lead to interventions that inhibit inflammation in animal models of disease. These will inform clinical studies and may lead to new treatments for disease. Due to the nature of this research there is scope for the commercialisation of the discoveries and potential economic benefits to the University and to the economy in general. Inflammatory disease has a massive impact on human suffering and thus research into the mechanisms of inflammation has potentially a direct impact on the health and well-being of people in the UK and worldwide.