Characterisation of human and murine renal myeloid immune sentinels and investigation of their role in sterile inflammation

What is the scientific or medical context of the research?
When cells in the body are damaged, the immune system (which normally fights infection) can become inappropriately activated. This is called "sterile inflammation". Unfortunately, immune system activation in this context can cause collateral tissue damage, compounding the original problem. Most of the body's tissues have resident immune sentinels which can sense damaged tissue and are involved in initiating inflammation. We are interested in a subset of these sentinels known as dendritic cells and macrophages which are thought to play a role in initiating this inflammation.

To which diseases/ conditions is the research relevant?
The research will provide information that is relevant to any disease in which tissue damage induces sterile inflammation. This includes important and prevalent problems like heart attacks, strokes, acute kidney failure (known as acute kidney injury (AKI)) and autoimmune diseases like systemic lupus erythematosus.

What is the research trying to achieve?
We aim to identify and characterise the immune cells which reside permanently in tissues (compared to immune cells which pass through them in a transient fashion) that are responsible for sensing tissue damage. We want to know which surface markers these cells express and how they can respond to damage signals, and integrate these signals with other immune activating signals. In particular we aim to assess how damage signals affect the ability of immune sentinels to migrate out of tissues to lymph nodes, where they can propagate an inflammatory response. Most of the information available at present relates to mouse tissue cells; we would like to examine human tissue so that we can begin to identify treatment targets which will work in human disease. We will also use a novel mouse model to examine the behaviour of these cells (in real time during tissue damage) via a cutting edge form of imaging known as 2-photon microscopy.

Why is this important?
The information is particularly relevant to AKI, which complicates up to 10% of all hospital admissions and 40% of patients in intensive care units. AKI frequently occurs in the setting of critical illness when the blood flow to the kidney is disturbed. It is also responsible for the delay in function seen in up to one half of kidneys transplanted from deceased donors. A number of patients with AKI require temporary (and some even long term) haemodialysis to "clean" the blood, removing toxins which would normally be excreted by the kidney. Haemodialysis is a life-sustaining treatment, but is associated with a number of complications which can be life-threatening. Despite being both serious and common, there are as yet no targeted treatments for AKI.

Who is carrying out the research?
The research will be carried out by Dr Miriam Berry, a renal trainee (Specialist Registrar), in the laboratory of Dr Menna Clatworthy (University Lecturer in Transplantation Medicine and Honorary Consultant Nephrologist). Both have had first hand experience of the clinical importance of AKI in native and transplant kidneys. Dr Clatworthy is a young investigator with an excellent academic track record, having won a number of young investigator awards during her PhD. She recently completed a period of post-doctoral research at the National Institutes of Health, Bethesda, USA, in a laboratory with a specialist interest in 2-photon imaging. After a stellar undergraduate career at the University of Cambridge, Dr Berry has completed most of her clinical training and is now committed to a basic science PhD.

How, briefly, is the research to be conducted?
The central component of the study will involve using human nephrectomy specimens (which would otherwise be discarded) or using kidneys donated for transplantation, but subsequently deemed unsuitable for use in view of scarring. The immune processes occurring within these damaged kidneys will be examined.

Aims and objectives:
Parts 1-3: To characterise human kidney myeloid cells and assess their response to tissue damage.
Part 4: To examine the dynamic behaviour of murine renal dendritic cells (DCs) in sterile inflammation.

Methodology:
1: Surface markers and anatomical location. We will:
a. Use a panel of surface markers (including CD11c, CD14, CD16, CD32A/B, CD68, BDCA1, BDCA2, HLA-DR, CD123, CD141, DC-SIGN, DC-LAMP) in flow cytometric studies and compare subsets to peripheral blood DCs/monocytes.
b. Perform gene expression profiling using microarray analysis on renal DCs/macrophages and compare expression patterns with those observed in peripheral blood DCs/monocytes.
c. Determine the anatomical location of human renal DCs and macrophages in the cortex and medulla using immunohistochemistry and confocal microscopy.
2: Functional studies
The large volume of renal tissue available will allow us to isolate sufficient numbers of cells to perform functional studies on renal DCs/macrophages. We will determine:
a. The response to a panel of DAMPs (including HMGB-1, HSP, ATP, uric acid, formyl peptides). We will also assess whether other immune signals can modulate inflammasome activation, particularly IgG immune complexes.
b. The effect of DAMPs on DC chemotaxis. We wish to determine if DAMPs can prime DCs to migrate from kidneys to draining lymph nodes by assessing chemokine receptor expression (CCR7) in renal DCs and to utilise a novel 3-D chemotaxis assay with a defined chemokine (CCL19) gradient.
3: Ex vivo assessment of human kidney myeloid cells
Kidneys donated for transplant (but unsuitable for use) will be reperfused using a normothermic perfusion circuit. Core biopsies will be processed for histology/confocal microscopy and flow cytometry.
4: Assess the dynamic behaviour of renal DCs in sterile inflammation using 2-photon microscopy and CD11cEYFP mice.

Scientific potential:our work will generate a unique dataset on human renal DCs.

In addition to the academic beneficiaries listed in the previous section, in the longer term we hope that our work will provide a model in which potential therapeutic agents for sterile inflammation may be screened. Cambridge Immunology has been fostering links with a number of pharmaceutical companies, for example the first joint meeting with GSK was held in Cambridge earlier this year. This on-going dialogue should facilitate the application of any agents in development at these companies to our model of sterile inflammation in human kidneys.

The identification of therapeutic agents in this area will have a significant impact on our national health economy. In the specialty of nephrology, a drug that might ameliorate acute tubular necrosis (ATN) and reduce the need for acute dialysis could potentially save the NHS hundreds of thousands of pounds per year; ATN affects up to 10% of hospital inpatients. Around 10-15% of these patients will require at least one session of dialysis costing £150 per session (this figure does not include the costs associated with line insertion and its attendant complications nor the protracted hospital admission that acute dialysis entails). Any intervention which lessens the severity of AKI could potentially reduce the significant cost of providing acute renal replacement therapy which exceeds £10 million per year.
The therapeutic benefits of agents for AKI in renal transplantation would also achieve a significant cost saving; in the UK there are currently more than 6000 patients on the renal transplant waiting list. The majority of these receive 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 year per transplant for the lifetime of that graft. However demand continues to greatly outstrip supply, with only 700 deceased donor transplants and 300 living donor transplants performed in the UK each year. Any strategy to increase the pool of potential donor organs will have far reaching economic benefits. One solution has been to use grafts obtained from deceased cardiac death donors; however, ATN is a significant problem in these kidneys, especially in those with a prolonged cold ischaemic time. Any therapy which might reduce the severity of this problem would allow more marginal kidneys with longer ischaemic times to be considered, expanding the organ pool and reducing the number of patients on dialysis. In addition to the reduction in haemodialysis costs as a result of successful transplantation, recipients of kidney transplants also enjoy a better quality of life, live longer and are more likely to undertake paid work representing a significant societal benefit.

On a more aesthetic level, the microscopy images generated by the two-photon imaging part of the project will be submitted to public displays intended to bring science to the general public, including the Wellcome Trust Image gallery.

The timescales over which these benefits might be seen vary; benefits to fellow academics in the field will be rapid, and likely to occur within the timeframe of the fellowship, and similarly for dissemination of the images obtained to improve the presentation of basic science to the general public. Translational application of the data to test new therapeutics for sterile inflammation is likely to take longer, in the region of 5-10 years after completion of the fellowship. The economic and societal benefits that we project are likely to occur within our professional life-time.