Investigating Novel Roles of Integrins in Type 2 Immunity and Asthma

Asthma is an inflammatory disease of the airways. Asthma affects many people, 5.4 million people in the UK alone. In addition, exacerbations of asthma result in 3-4 deaths each day in the U.K. Recent progress in asthma has led to the understanding that there are different types of asthma, called phenotypes. These different types of asthma have differing underlying disease mechanisms, which are termed endotypes. This understanding has resulted in novel therapies, which target the immune and inflammatory mechanisms of some patients with asthma.

However, there are some types of asthma which we are unable to treat with new medications. In addition, some patients fail to respond to recently developed therapies.

We hence need to develop an improved understanding of the immune mechanisms underlying asthma, aiming to develop new therapies to help those patients for whom there is currently no effective treatment.

Aim 1

There is a subgroup of patients with asthma, in whom asthma is obesity associated. This represents a particularly difficult to treat type of asthma. A recently identified cell type called innate lymphoid cell (ILC) produces many of the mediators associated with asthma. Interestingly, these cells have not only been associated with asthma but also with obesity. In particular, an ILC subtype called ILC2 are thought to be important in orchestrating the mechanisms underlying asthma. In addition, the same cells (ILC2) are thought to promote a lean body type. Preliminary data from the McKenzie lab has identified a new molecule on these cells (called leukocyte-function associated antigen-1 - LFA-1) which activates many of the inflammatory processes associated with asthma. Interestingly, this interaction appears to be particularly important in fatty tissue.

Given the link between LFA-1 and ILC2 in obesity, I believe there may be a link between LFA-1 on ILC2 and asthma. While I expect LFA-1 to be elevated in the blood of normal weight patients with asthma, I expect that this will be relatively under-expressed on ILC2 from blood from patients with asthma and obesity.

In order to do this, patients with severe asthma and healthy controls will be invited to donate a small amount of blood (20-40 mls - about the volume of an espresso). In addition, I will also study the role of LFA-1 using mice. Mice exist in which LFA-1 is not existent. I will compare mice with and without LFA-1 in mouse models of asthma. I expect that we will see less severe asthma in mice lacking LFA-1.

Aim 2
New technologies have enabled us to study the development of specific immune responses in more detail than ever before. New, preliminary data from the McKenzie lab has identified a certain molecule (called CD51) as potentially being very important on cells causing inflammation (T-cells) in patients with asthma. Interestingly, CD51 is known to interact with another protein called periostin. Periostin itself is a marker for inflammation underlying asthma. However, nothing is known about the expression and role of CD51 in patients with asthma. I propose to study CD51 expression on T-cells from the blood of volunteers with and without asthma. I believe that we will find more CD51 expressed on T-cells from patients with asthma.
In addition, there are mice available which lack CD51. We can delete CD51 in T-cells specifically by generating a new mouse strain. We will then study these mice in which CD51 is lacking in T-cells in mouse models of asthma. I expect that the mice lacking CD51 will have less severe asthma than those who have CD51.

Summary
I am proposing to study two recently identified molecules (CD51 and LFA-1) with potential roles in asthma. I believe that studying these may identify them as new therapeutic targets, resulting in novel therapies.

Aim 1 - Characterisation of the function of LFA-1 on ILC2 in Asthma

We will study ILC2 from the blood of volunteers with and without asthma. Volunteers with asthma will be recruited from the Cambridge severe asthma service. We will use flow cytometry to analyse ILC2 populations and LFA-1 expression on ILC2, comparing patients with severe asthma and obesity (BMI > 30), severe asthma and no obesity (BMI < 30) and matched controls. We will also sort ILC2 to study expression of LFA-1 and other adhesion molecules using PCR.
In order to study the function of LFA-1 in asthma, we will pursue a translational approach studying human volunteers while exploring potential mechanisms in murine models. In order to study the murine models, we will generate bone marrow chimeric mice which lack expression of LFA-1 in ILC2. We will compare these mice to wild type mice in murine models of asthma.


Aim 2 - Characterisation of CD51 in T-cells in asthma

We will employ a similar strategy to explore the function of CD51 in T-cells in asthma.
We will isolate T-cells form volunteers with and without asthma and differentiate them into Th2 cells in vitro. We will then compare expression of CD51 on the T-cells differentiated in vitro using flow cytometry and PCR.
In order to explore its role in mouse models, we will generate a mouse line which will lack CD51 in CD4+ T-cells (CD4 driven Cre crossed into a floxed CD51 line).
Should these studies be successful, we will also seek to study T-cells from the lungs of patients, comparing broncho-alveolar lavage T-cells from patients with asthma and volunteers.

The most important aspect of the proposed work is to identify novel potential targets for drugs which may help patients with asthma. This will be achieved by generating impact in several key areas. The most important means of achieving this impact will be through peer-reviewed publications and conference presentations. However, where there are other means I will detail this in the specific section.

Academic
The academic community will benefit from the proposed research. The link between type 2 immune processes and obesity and type 2 immune processes and asthma is clear. However, there is currently no mechanistic work linking the two. The proposed research will contribute to an enhanced understanding of this link.

Industry
Development of novel asthma treatments is a key priority for the pharmaceutical industry. Asthma treatments, such as inhalers, regularly feature amongst the most sold products. With the emergence of biological treatments, such as monoclonal antibodies to IL-5 and IL-13, novel revenue streams for pharma are emerging in asthma. There are numerous other links between Cambridge University and industry, for example to Glaxo-Smith-Kline through the Varsity programs. In addition, the McKenzie lab has existing links with MedImmune/Astra Zeneca, who are moving onto site in Cambridge. Should this application be successful, I will look to further these collaborations. This will be made easier by the fact that I have already collaborated with some of these, and can engage in face-to-face discussions about the project and its potential impacts.

Patients:
There is no direct benefit to patients taking part in this project. However, it has the potential to contribute to improved patient care.
One of the main improvements in asthma care has been the understanding that there are different asthma phenotypes and underlying inflammatory endotypes. This project aims to improve understanding of asthma that remains difficult-to-treat. By targeting people with phenotypes that are poorly understood such as obesity-associated asthma, I seek to generate the maximal impact in those patients for whom there is currently no effective treatment.
In addition, the project seeks to identify novel therapeutic targets on ILC and T-cells, key inflammatory players in the asthma phenotypes studied. The time-span for development of novel therapies is 10+ years, so this impact may only be realised over the long term.

Public:
To maintain the publics trust and confidence, it is important that it is engaged in the research process. For example, the ethics for my previous project were reviewed by a panel, which included lay members, prior to submission to the ethics committee. I took on board several suggestions, including taking bronchoscopy samples from people who were undergoing bronchoscopy for clinical reasons. Should this application be successful, I would seek to engage the public again in a similar manner when applying for ethical approval.
I also engaged in the public engagement programs of the MRC-LMB, for example I contributed to the next LMB open day. I look forward to continue to do so in future years and explain my research to a wider audience.
Furthermore, if successful, I will also make use of social media (such as facebook and twitter) to disseminate news about the impact of this research and other research projects of relevance.