Molecular regulation of necroptosis in inflammatory diseases.

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

Skin and joints inflammatory diseases affect billions of people worldwide annually. While they display a rather low mortality rate, they severely impair patients' quality of life. In 30% of patients, cutaneous lesions are associated with joints inflammation, in diseases such as psoriatic arthritis (PsA) or Systemic Lupus Erythematous (SLE). Psoriasis and arthritis are complex diseases, the pathogenesis of which involves genetic, immunological and environmental factors.

This project will examine new cellular mechanisms which we suspect might initiate inflammation. The so-called programmed necrosis pathway, also termed necroptosis, has been shown to trigger inflammation in numerous disease models. Necroptotic cell death is highly pro-inflammatory, due to the release upon cell death of intracellular molecules called alarmins. However, to date, necroptosis has been poorly investigated in human diseases due to the lack of reliable biomarkers. Our recent work focused on developing such markers and showed that necroptosis is activated in the severe drug-induced skin inflammatory disease Toxic Epidermal Necrolysis (TEN). As necroptosis appears as an early event in TEN, it could thus constitute both an early diagnosis marker and new therapeutic target.
This approach would enable to make more accurate diagnosis at early disease stages and to inhibit inflammation before it spreads, while current treatments target the amplification of the inflammatory response through molecules of critical importance for the immune system.

This project will study the role of necroptosis in skin inflammation, different forms of arthritis as well as pancreatitis and Alzheimer disease.
Having previously demonstrated the capacity of necroptosis to induce inflammation, we will also focus on deciphering how it is activated in our different diseases of interest. This will be achieved by means of:
1. Biochemistry: We will analyze the molecular organisation of the necrosome, the protein complex that regulates necroptosis, in skin cells as well as in joint cells, pancreatic cells or neurons.
2. Patients' study: We will compare the activation and mechanisms of necroptosis in different forms of skin and joint inflammatory diseases.
3. Cell biology: Identifying the molecules which trigger necroptosis in human inflammatory diseases.
4. Mouse genetics: Blocking these molecules in a mouse genetic model of necroptosis-induced skin inflammation to test the efficiency of this strategy to alleviate inflammation in vivo.

Ultimately, if this study is successful, not only will we have demonstrated the role of necroptosis in human inflammatory diseases, but we will also have unravelled new therapeutic approaches with a great potential in a wide range of diseases.

Technical Summary

This project will assess the role of programmed necrosis, also termed necroptosis, in inflammatory diseases and seek to identify necroptosis inducers in pathophysiological conditions. Necroptosis has previously been shown to trigger inflammation in multiple animal disease models. To date, the role of necroptosis has been poorly investigated in human diseases. Recently, I have identified alarmin-triggered necroptosis as a central mechanism in the pathophysiology of skin inflammation in Toxic Epidermal Necrolysis (TEN).
This Study will investigate the mechanisms of necroptosis activation by alarmins in epidermal keratinocytes. Experiments will, in a first place, use in vitro cell death assays (Wst1, CytoTox Glo, caspase assays), immunofluorescence and live imaging to monitor cell death. Then, we will seek to identify the components of the alarmin-induced necrosome, using proteomics and phosphoproteomics analysis.

The role of alarmins will then be assessed in in vivo models of necroptosis-induced skin inflammation, by means of genetic crosses or use of blocking antibodies. The improvement of skin inflammation will be evaluated by macroscopic observation and histological analysis. Finally, we will extend the study of the role of necroptosis and alarmins in inflammatory diseases to a broader spectrum of skin and joint inflammatory diseases, thanks to a collaboration with clinicians at University Hospital of Wales, as well as models of pancreatitis and Alzheimer disease, using the biomarkers that I have previously developed.

The results of this study are expected to demonstrate the role of necroptosis in inflammatory diseases and decipher its molecular regulation by alarmins in vitro and in vivo, opening the way to new therapeutic approaches for inflammation.

Planned Impact

Who will benefit from this research?

This project should generate important outputs for multiple beneficiaries:

-Academic beneficiaries: discussed in previous section.
-Clinicians: this project comprises an important part of translational research which should help developing new diagnostic tools and innovative therapeutic approaches.
-Pharmaceutical industries (including CROs and SMEs): will benefit from increased scientific knowledge, strong UK research capacity and availability of a highly skilled research group in the field of programmed necrosis. If this project is successful, pharmaceutical industry may also benefit from commercial exploitation of this research.
-Public sector: the NHS will benefit from enhanced quality of life, early diagnosis tools, allowing reduced healthcare costs and decreased side effects, formal links between clinicians and researchers. Schools will benefit from educational opportunities, such as public lecture and access to our lay articles.
-General Public: patients will benefit from earlier diagnosis, availability of advanced therapies, with reduced side effects and improved efficacy, decreased healthcare spending and enhanced quality of life. Members of the public will benefit from clear use of public funding and greater understanding of science through public engagement activities.

How will they benefit?

-Enhancing quality of life: Diagnosis tools developed in this program will offer opportunities to better discriminate between different forms of diseases and to allow diagnosis at early lesional stages in inflammatory diseases. This will allow to inhibit inflammatory diseases at early stages, increasing the chances of complete disease remission and preventing the chronicity of inflammation and, ultimately, to develop a personalized treatment approach based on the early detection of inflammatory mediators and mechanisms of inflammation, in order to improve the care of millions of individuals affected by inflammatory diseases annually.
-Workforce: The PDRA and the technician employed on this project will improve their technical skills in numerous scientific disciplines, which will support their career development and develop their expertise in the field of programmed necrosis and inflammation for further application and dissemination.
-Economic benefit: in addition to direct healthcare costs, loss of productivity and indirect costs are estimated at over £50 billion for inflammatory diseases annually in UK. The development of new therapeutic approaches targeting early inflammatory mediators and novel pro-inflammatory pathways constitute a significant opportunity to have more cost-effective and efficient therapies, and will trigger investment and future research in that field.
-Building UK Research Capacity: our consortium comprises scientists and clinicians from a broad range of discipline including, dermatology, rheumatology, neurology, gastroenterology, immunology, molecular and cellular biology. Our common purpose is to establish UK as a forefront of innovative strategies for inflammation biology.

Publications

10 25 50
 
Description Programmed necrosis and inflammation in barrier epithelia
Amount £251,692 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 02/2022
 
Description Characterization of the HMGB1-induced necrosome 
Organisation University of Bristol
Department Proteomics Facility
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shown that HMGB1 can induce MLKL phosphorylation in keratinocytes in a TNF-independent manner. We are aiming at characterizing the composition of the HMGB1-induced necrosome. We have successfully immunoprecipitated P-MLKL and RIP3 from HMGB1-stimulated keratinocytes. These immunocomplexes will be analysed by Dr. Kate Heesom and her team at the University of Bristol Proteomics Facility. In parallel, a whole phosphoproteomics analysis will be persormed on total cell extractys from keratinocytes treated with HMGB1 at different time points.Extracts or IPs from keratinocytes treated with TNF-SMAC mimetics-zVAD wil be used as a control.
Collaborator Contribution Dr. Kate Heesom and her team will perform prteomic analysis of our P-MLKL and RIPK3 complexes immunoprecipitated from HMGB1-stimulated keratinocytes by Liquid Chromatography-Mass Spectrometry (LC-MS). In addition, a whole phosphoproteomic analysis of HMGB1 treated keratinocytes will be performed also by LC-MS after enrichment for phosphopeptides using the Tandem-Mass Tagging technique.
Impact Experiments still ongoing.
Start Year 2018
 
Description Programmed necrosis markers in arthritis 
Organisation Cardiff University
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We established the techniques to study necroptosis biomarkers in human patients and mouse models samples of arthritis.
Collaborator Contribution Our partner Arthritis Research UK Biomechanis and Bioengineering Centre (ARUKBBC) provided us with patients samples from their Biobank (Osteoarthritis-OA and Rheumatoid Arthritis-RA) and tissue samples from their mouse and rat models of arthritis (ACL rupture in mice and MNX in rats for OA and Antigen-Induced Arthritis for RA).
Impact We have shown that both mouse models of OA and RA display an increased expression of RIPK3, the central regulator kinase of the necroptotic cascade, both in the cartilage and in the bone, while no significant increase in the apoptotic marker cleaved caspase-3 could be detected these tissues.
Start Year 2018
 
Description Role of HMGB1 and necroptosis in the pathophysiology of Toxic Epidermal Necrolysis 
Organisation Henri Mondor Hospital
Country France 
Sector Hospitals 
PI Contribution We have identified necroptosis and HMGB1 as major pathogenic factorsat early stages of Lesional Development in the life-threatening drug-induced skin inflammatory condition called Toxic Epidermal Necrolysis.
Collaborator Contribution The Dept of Dermatology and the Dept of Pathology of the Henri Mondor Hospital rovided us with skin samples from patients with characterized Toxic Epidermal Necrolysis from the French National Registry for Severe Cutaneous adverse drug Reactions (REGISCAR).
Impact This collaboration allowed us
Start Year 2017
 
Description Role of necroptosis in Graft versus Host Disease (GvHD) 
Organisation Hospital Saint-Louis
Country France 
Sector Hospitals 
PI Contribution My team will perform immunostainings for necroptosis biomarkers on skin sections from patients with acute versus chronic GvHD.
Collaborator Contribution My partners are currently establishing the Ethics protocol and recruiting patients to establish a collection of blood samples and skin biopsies of patients with acute and chronic GvHD. They will also perform analysis for serum markers of necroptosis and inflammation, as well as corresponding analysis of immune cell populations.
Impact This collaboration is just starting. I will receive the tissue samples for histological and immunostaining analysis by the summer of 2020. This study should provide us with innovative new biomarkers of GvHD as well as potential therapeutic targets.
Start Year 2020