Investigating the function of group 3 innate lymphoid cells in the renal tract

The kidneys play a critical role in cleaning the blood and controlling the body's water balance. Unfortunately, their function can be affected by a number of common diseases that can be life-threatening, including infection. Urinary tract infections (UTIs) are common, affecting up to half of women at some point in their lifetime. Recurrent infections can cause scarring that leads to permanent kidney damage and even kidney failure. All of the body's tissues house cells of the immune system. These sentinels stand guard in tissues and alert the body to infection by starting an inflammatory response. In the kidney, the response of immune sentinels to infection or tissue damage determines the extent of inflammation, and this can influence whether bacteria are cleared, whether there is collateral damage, and ultimately whether the function of the kidneys recovers or develops scarring. At present, we don't fully understand what type of tissue-resident immune cells live in human kidneys and bladder or their exact function. Addressing this challenge will help us to development better treatments for diseases that affect the kidney, including UTIs.

In the last 5 years, a new family of immune cells have been discovered, the innate lymphoid cells (ILCs). These cells act as tissue sentinels, producing chemical signals (cytokines) that activate the immune system. One member of the ILC family, the ILC3s, have been found to be enriched in the intestine where they prevent the invasion of bacteria from the gut and produce factors that maintain the health of tissue cells.
There is currently no information on whether there are ILC3s in human kidneys and bladder. We were interested in finding out if ILC3s could be important in these tissues and performed some preliminary experiments. Excitingly, we found ILC3s in mouse and human kidneys and bladder.
The objective of this project is to find out what exactly they are doing, as follows:
1. Where ILC3s are positioned in kidneys and bladder and whether they are situated near other immune sentinels.
2. What sort of immune signalling molecules (chemokines and cytokines) are produced by ILC3s at during health and in response to the bacteria that cause UTIs. We can use mouse models and human tissues that have been donated for transplant, but cannot be used.
3. How ILC3s patrol the kidney and bladder and whether they form interactions with other immune cells. To answer this question we will use a special sort of microscope and mice that have fluorescently labelled ILC3s. We have developed a protocol that allows us to image the cells in real time in the kidney and bladder, so we can begin to investigate the factors that control their movement and their interactions with other immune cells.
4. Whether outcome and disease severity in UTI is affected by an absence of ILC3s, using mice that lack ILC3s due to genetic deletion of a factor that is required for their development.

Elucidating the workings of ILC3s will help us to understand whether they contribute to helpful immune responses that clear bacteria in the renal tract. This will being to enable us to harness their useful functions to develop better therapeutic strategies that might reduce the frequency of UTIs or the scarring that can be associated with recurrent infections. In the long run, this could prevent patients from getting kidney failure and could help prolong the life of kidney transplants, which can also be damaged by recurrent infection.

Planned experiments:
1a. Confocal imaging of sections prepared from kidneys and bladder (tissues obtained from ROR-yt-GFP mice and human organ donors that have consented for research) to determine anatomical location and markers expressed (MHCII, CD80/86, IL22, IL17). Flow cytometric analysis of human kidneys, analyzing cortex and medulla samples separately, using flow panels already optimized (CD45, lineage (CD3/19/14/94), CD127, c-KIT, CRTH2 and NKp44, (Figure 3 of Case for Support)). Transcriptomic analysis of flow-sorted human kidney ILC3s will also be undertaken.
1b. Two-photon (2P) intravital imaging of RORyt-GFP/ID2-CFP double reporter mouse. ILC3 migratory speed and area patrolled will be measured using Imaris software. The ID2 reporter required will be generated during this project.
1c. Histological studies of kidney and bladder tissue (using CD3, CD4, CD11c, F4/80, CD11b and CD64 co-staining to identify ILC3s, Th17 cells, DCs and macrophages) to investigate ILC3 co-localisation with other tissue-resident cells. Dynamic interactions will be assessed using intravital, 2P imaging of a triple reporter mouse (RORyt-GFP/ID2-CFP/CD11c-EYFP).

2a. FACS analysis of kidney and bladder ILC3 following intravesical challenge with uropathogenic E coli (UPEC). Surface MHCII and CD80/86 and intracellular IL17 and GM-CSF of ILC3s will be investigated. Similar phenotyping of human kidney and bladder ILC3s will be performed following UPEC challenge in vitro.
2b. RORyt-deficient mice will be used in the UTI model and infection severity assessed (bacterial counts in kidney, bladder, blood and spleen as well as neutrophil and monocyte infiltration into the kidneys and bladder). Providing the ROR-yt deficient mice show a phenotype, we will use bone marrow chimeras to produce ILC3-specific knockouts and use them in the UTI model.
2c. 2P intravital imaging of bladder ILC3s, Th17 cells and DCs during UTI using triple reporter mice. Imaris used to analyse data.

Who might benefit from this research?
1. Academic beneficiaries
2. Translational/clinical beneficiaries
3. Patients, economic and societal benefits
4. The general public

How might they benefit from this research?
1. Academic beneficiaries.
Basic immunologists interested in innate lymphoid cell (ILC) biology, mucosal immunology and sterile inflammation will benefit from findings made in this project. Our experiments will provide some transcriptomic datasets for the community on ILC3s in human kidneys, which other groups can use to inform their own experiments. We are also committed to contributing to technological advances in biological imaging through our two-photon imaging experiments.
2. Translational and clinical beneficiaries
The data we generate will have translational implications in that it will expand our understanding of disease mechanisms in three clinical areas:
a. Urosepsis - Bacterial infection of the lower urinary tract (bladder) is common, affecting more than 6 million patients annually in the US. Ascending infection involving the kidneys (pyelonephritis) is a significant contributing factor to renal scarring.
b. Chronic kidney disease (CKD) - Pyelonephritis can contribute to the development of CKD. CKD represents an important global health problem with a recent study showing a prevalence of 14% in the UK.
c. Kidney transplantation - Infection in transplanted kidneys is common, due to immunosuppression and reflux up the transplanted ureter. This can reduce the lifespan of the graft.
Our data will therefore be of interest to nephrologists, intensivists, and renal transplant clinicians.
3. Patients, economic and societal benefits
The identification of new therapeutic strategies in urosepsis would have a significant health economic benefit. Up to half of women experience at least one episode of UTI in their lifetime, many requiring time away from work. Moreover, recurrent pyelonephritis can lead to CKD and end-stage renal failure (ESRF). These patient require dialysis, which is life-sustaining but consumes a disproportionate share of resources; 2% of NHS spending for <0.1% population.
Kidney transplantation saves approximately £20,000 per annum for the lifetime of that graft. Recurrent infections cause scarring and a decline in graft function. Therefore prolonging the half-life of grafts by preventing infection and scarring would ultimately reduce the number of patients returning to dialysis.
The avoidance of dialysis not only has economic benefits, but also has societal benefits, since many patients receiving dialysis are unable to work due to the time constraints imposed by the requirement for thrice weekly dialysis sessions. Transplantation restores patient independence and frequently facilitates a return to work.
4. The general public
We plan to engage the interested general public in our work through activities in the University of Cambridge annual Science Festival (http://www.med.cam.ac.uk/clatworthy/public-engagement/). Our imaging techniques will generate work which is of aesthetic value in addition to its scientific content, and we plan to exhibit these in in public collections, such as the Wellcome Image Gallery.