MICA: Modulation of IL-33-dependent responses using parasite products

This project will investigate how we can use proteins derived from a parasitic worm to either amplify or suppress "IL-33", a protein used for communication within the immune system. IL-33 is released on damage to various barrier sites (lung, skin, gut), and can lead to either allergic or inflammatory responses, depending on the context. These responses can be beneficial, e.g. they can efficiently clear bacterial infections, or they can be detrimental, e.g. they can lead to the development of allergic or inflammatory damage such as in asthma or acute respiratory distress syndrome. Therefore being able to effectively "tune" the IL-33 pathway up or down would be a powerful technique for treating a wide range of diseases including asthma, eczema, fungal or bacterial infections, and acute respiratory distress syndrome.

We have identified two proteins derived from a single parasitic worm (Heligmosomoides polygyrus) which act to suppress IL-33 responses; we have named these proteins HpARI and HpBARI. Suppression of IL-33 responses is advantageous to parasites, as it allows them to avoid triggering immune responses which could lead to their ejection or damage to their host. When we produced a mutant form of one of these IL-33-suppressive proteins, we found the mutant form had the surprising effect of amplifying (rather than suppressing) IL-33 responses due to stabilisation of the IL-33 molecule. Therefore we can use these proteins and mutants to either increase or decrease IL-33 responses and potentially treat a long list of diseases in which IL-33 has a causative, or curative, role.

This project will investigate the use of these proteins and their derivatives in mouse systems where IL-33 drives allergic responses (such as in asthma), damaging inflammatory responses (such as in acute lung injury or acute respiratory distress syndrome) or beneficial anti-bacterial responses (such as in pneumonia).

We will translate findings from the mouse towards human responses by using genetically-engineered mice which express the human form of the IL-33 molecule, and stimulating human blood cells with human IL-33 in the lab, and testing whether our proteins affect the responses of these cells.

Furthermore, we will engineer hybrid molecules, taking the active regions of our proteins, and combining them with proteins normally present in our blood. This will have the advantage that the resulting proteins will be largely ignored by the immune system as they look like one of the body's own proteins. This avoids a common problem (known as "immunogenicity") of using foreign proteins as medicines, where the immune system rejects the protein, preventing it from carrying out its function. These engineered proteins will be further assessed for activity against IL-33 responses and for immunogenicity (the level of recognition by the immune system) in mouse and human tests, as described above.

In summary, this project will investigate and characterise new parasite-derived proteins which can suppress or amplify the immune response, with the potential to be used as new medicines or tools for research in a range of allergic, inflammatory and infectious diseases.

In this project, we will characterise modulation of IL-33-dependent eosinophilic or neutrophilic responses by HpARI, HpBARI (and derivatives of these), parasite-derived modulators of the IL-33 pathway.

Objectives:
1. Show therapeutic efficacy of HpARI, HpARI_CCP1/2 and HpBARI in eosinophilic and inflammatory responses.
HpARI, HpARI_CCP1/2 and HpBARI will be administered into mouse airways with Alternaria, LPS, HCl or S. pneumoniae. Mice will be randomised to experimental groups, and cage-blocked. Power calculations have been used to ensure statistical significance is attained at expected effect sizes. Inflammation and damage will be measured by histology, flow cytometry, and cytokine/chemokine measurements. Human IL-33 knock-in, mouse IL-33 knock-out mice, and culture of human PBMCs with recombinant cytokines and HpARI/HpBARI derivatives will be used to translate results to human systems.

2. Elucidate the molecular mechanism of action of HpARI/HpBARI, and design minimally immunogenic mimetics.
Binding partner identification will be carried out using biotin label transfer and streptavidin pull-down, with targets identification by tandem mass spectrometry. Binding to murine and human targets will be characterised by SPR. Binding sites will be identified using HDX-MS. This will allow us to characterize the binding partners and molecular mechanism of action of HpARI and HpBARI, and to design humanized proteins, in which active sites are cloned into a host CCP protein scaffold (FBa).

3. Investigate the role of IL-33 in the recruitment of neutrophils.
We will assess the role of the IL-33 pathway in models of acute lung injury (HCl administration) or pneumonia (S. pneumoniae infection). The IL-33 pathway will be blocked using ST2-KO mice, mAbs, or HpARI/HpBARI administration. We will assess the IL-33-mediated activation and cytokine production of innate immune cells, chemokine responses, and neutrophil chemokine receptor expression.

This proposal will test the potential of immune modulatory proteins derived from a helminth parasite to modulate allergic and inflammatory responses. It will develop them as therapeutic agents for human disease and as research tools.

Successful development of therapeutic agents from these proteins could lead to treatments for asthma, allergy, sepsis, acute lung injury, and bacterial/fungal infections.

These syndromes affect a large proportion of the UK population: asthma affects 1/11 people in the UK and kills around 1,400 people; acute respiratory distress syndrome (ARDS) affects around 45,000 people per year, and kills almost half of these; and pneumonia (lung infection) kills around 29,000 people a year. Therefore the human cost of these diseases, in both morbidity and mortality, cannot be underestimated. Furthermore, these diseases put significant financial strain on the health services - asthma costs the NHS around £1 billion per year, while ARDS costs around £1.7 billion per year.

Current treatments for both asthma and ALI are largely supportive, while in pneumonia, antibiotic resistance is a growing and intractable problem. Therefore, interventions that prevent immune response hyperactivity (allergy, inflammatory pathology) or amplify effective anti-bacterial/fungal responses, would represent a huge step forward in treatment. These treatments would not only lead to an increase in a healthy lifespan, but also a decrease in illness-related work absences. The economic impacts in the form of increased productivity would be compounded by pharmaceutical industry gains: therapeutic sales in the nine major markets (9MM; US, France Germany, Italy, Spain, UK, Japan, China and India) are forecast to increase to $23.1 billion for asthma alone by 2023. Thus there is a large market potential for the outcomes of this research.

Furthermore, IL-33 is implicated, either detrimentally or beneficially, in a list of other diseases such as atherosclerosis, Alzheimers, atopic dermatitis and parasitic infections. Active research of the role of IL-33 in these indications is underway, and the molecules being developed here could be extremely useful as tool compounds in facilitating this research.

This proposal is highly interdisciplinary, with academic beneficiaries in the fields of immunology, parasitology, protein biology, protein engineering, biophysics and respiratory pathology, as detailed in the "Academic Beneficiaries" section. Advances made during this project will therefore advance understanding in many other scientific fields.

This project will benefit the University of Edinburgh and the researchers involved by producing high-impact publications, maintaining the reputation of the University as a world-leading research institution. Further development of the potential therapeutic agents developed in this project could lead to further commercialisation funding and licensing of treatments, with economic benefits to the University.

It will also benefit the lead investigator, Dr McSorley, as he transitions to full independence, increases his research standing and grows his research group. Furthermore, a Postdoctoral Research Associate will be recruited for this position, advancing their career within the MRC CIR, and giving them experience in immunological and molecular fields of research.