Investigating the role of Siglec-G and innate B cells in sterile inflammation associated with acute kidney injury

Acute kidney injury (AKI) is a common and serious medical problem affecting 10% of all hospital inpatients. Severe AKI, which usually requires dialysis (a form of blood-cleaning treatment), is associated with a 10-20% chance of death within one year. As well as this, virtually all kidneys retrieved for transplantation experience a degree of AKI. This often means that some transplanted kidneys do not work initially. It also limits the use of less-than-perfect organs and can contribute to scarring in the long term. Unfortunately, at the moment, there are no specific therapies for AKI.

Recent research suggests that the immune system, which normally fights infections, may be activated when tissues are injured, inadvertently causing 'collateral damage'. This process of injury-induced inflammation is thought to play a role in a number of serious medical conditions, including heart attacks and AKI. One major culprit of collateral tissue damage in these conditions are immune cells called neutrophils, which normally live in the bone marrow and blood but are recruited to injured tissues where they release chemicals (designed to kill microbes) which exacerbate tissue damage. The factors controlling neutrophil recruitment are unclear, but we have evidence to suggest that another type of immune cell, B cells, orchestrate this. This would be important to confirm because if we can control these B cells, then we may be able to reduce the severity of AKI. We think we may be able to achieve this using a molecule expressed by these B cells - Siglec-G. Siglec-G acts as an 'off-switch' for B cells, so we wish to use some therapeutic agents which trigger this off-switch. Potentially these agents could switch off the B cells which orchestrate neutrophil recruitment to damaged kidneys. This would reduce collateral damage and provide a novel treatment for AKI and other conditions in which damaged tissues trigger immune inflammation.
Our specific experimental plans are as follows:
1. The death of cells in AKI causes kidneys to release molecules called damage-associated molecular patterns (DAMPs), which trigger the immune system. To examine this process more closely, we will isolate and grow human B cells obtained from the bloodstream from healthy donors, as well as mouse B cells. We will assess whether B cells stimulated with DAMPs in a culture dish produce chemicals which mobilise neutrophils.
2. We will assess whether the B cells themselves are recruited to damaged kidneys, and if they are, we will investigate their movement into and around the kidney. To do this, we will need to use a mouse model of AKI and a special type of imaging - 2 photon microscopy. This is a relatively novel technique of visualising cells, in which our laboratory principal investigator, Dr Menna Clatworthy, has training and expertise.
3. We will test whether B cells are the important producers of the neutrophil mobilising chemical GM-CSF. To do this, we will make a mouse in which B cells cannot produce GM-CSF. We anticipate that these mice will have less severe AKI due to reduced neutrophil mobilisation.
4. We will investigate the therapeutic potential of agents which trigger Siglec-G, the B cell 'off-switch'. We can do this by culturing B cells (both human and mouse) with Siglec-G-triggering agents and assessing whether there is a reduction in the neutrophil-mobilising chemicals produced. We can also look in our mouse model of AKI. We will do this in two ways; firstly we will make a mouse with B cells which lack Siglec-G - we would expect them to develop very severe AKI. In contrast we will test whether Siglec-G stimulators can prevent or treat AKI in mice with normal Siglec-G. If these experiments work, it will provide proof of principle that this could be a useful therapy in human AKI and pave the way for human studies.

Experimental plans:
1. To determine if B cells stimulated with DAMPs produce neutrophil/monocyte chemokines. Human and murine B cells will be stimulated with a panel of DAMPs (HMGB1, HSPs, Formyl peptides, ATP). GM-CSF, CCL1, CXCL2, CCL2, CCL7 and CX3CL1 levels in culture supernatants will be measured. Cells will be analysed by FACS (B cell markers: B220, IgM, CD5, CD1d, GL7, CD138, CD27 and intracellular staining for GM-CSF) and qRT-PCR. Using an in vivo model of AKI (cisplatin-induced ATN), we will determine whether B cells in the spleen or bone marrow produce chemoattractants. For human B cells, we will also perform gene expression profiling following DAMP-stimulation.
2. Assessment of kinetics and characteristics of B cells recruited to kidney in AKI. In a murine model of AKI, we will determine by FACS the kinetics of B cell entry into kidneys, markers expressed, and chemokines produced. We will use 2 photon microscopy to assess dynamic behaviour of B cells in AKI.
3. To investigate the importance of B cell production of GM-CSF in renal neutrophil recruitment in AKI. Bone marrow chimeras will be generated, reconstituting irradiated wild type (WT) mice with 80% uMT and either 20% WT bone marrow or 20% GM-CSF-/- bone marrow. ATN will be induced and disease severity and immune response assessed by FACS analysis of bone marrow, spleen, blood and kidneys.
4. To determine if Siglec-G controls B-cell production of neutrophil/monocyte chemokines. WT and Siglec-G deficient B cells will be stimulated with DAMPs in vitro. We will also generate bone marrow chimeras (as above) in which only B cells are Siglec-G deficient and use in a murine model of ATN.
5. To assess the therapeutic potential of Siglec-G agonists in AKI. We will use Siglec ligands in vitro in murine and human B cell cultures to assess effects on DAMP-mediated activation. Reagents shown to be effective in vitro will be tested in vivo in a murine model of ATN.

Who might benefit from this research?
1. Academic beneficiaries.
Basic immunologists interested in B cell biology and sterile inflammation will benefit from findings made in this project. Our experiments on acute kidney injury (AKI) will provide information relevant to sterile inflammation in other organ systems.

2. Translational and clinical beneficiaries
The data we generate will expand our understanding of the mechanisms involved in sterile inflammation generally, and in AKI in particular, and will therefore be of relevance and interest to clinicians managing AKI in native kidneys (nephrologists and intensivists), transplant clinicians managing delayed graft function in kidney allografts and clinicians involved in treating other diseases in which sterile inflammation is thought to play a role (eg, myocardial infarction, stroke).

3. Patient, economic and societal benefits
The identification of new therapeutic strategies in sterile inflammation would have a significant health economic impact. 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 millions of pounds per year as 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 annum for the lifetime of that graft. However demand continues to outstrip supply greatly. One strategy to increase the pool of potential donor organs 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 used, expanding the organ pool and reducing the number of patients on 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 organised by the University of Cambridge (such as its annual Science Festival). We anticipate that our imaging techniques will generate work 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.
The timescales over which these benefits might be seen vary; benefits to fellow academics in the field and the general public will be rapid, and likely to occur within the timeframe of the fellowship. 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.