Reducing organ fibrosis by targeting a novel pro-fibrotic CLEC4D expressing myeloid subset.
Lead Research Organisation:
University of Edinburgh
Department Name: Centre for Cardiovascular Science
Abstract
Patients with kidney and liver disease lose their normal organ tissue as it is progressively replaced by scar tissue, resulting in a reduction in organ function. Eventually, this may lead to organ failure and the need for patients to undergo chronic dialysis therapy or kidney/liver transplantation. Despite these life-saving therapies, kidney and liver disease are the 11th and 12th leading causes of death. Unfortunately, the number of patients with kidney and liver disease is continuing to rise and treatment options remain limited, therefore it is vitally important that researchers identify novel therapies that slow progression of disease.
Regardless of the underlying cause of the injury, damage to the kidneys and liver results in recruitment of white blood cells to the injured organs. These cells are part of the body's immune system that is important for fighting infection, however during organ injury they may promote further damage and scarring. We know that drugs that prevent recruitment of these immune cells to injured organs can slow, but not stop, progression of liver and kidney disease. However, some subsets of the immune cells may be beneficial as they are required for normal tissue functions and to promote repair. Hence, blocking all immune cells from reaching the damaged organ is not advisable and a more targeted approach to inhibit specifically those subsets that promote injury is likely to be more beneficial.
In order to identify the specific immune cell subsets that promote organ injury, we have employed a state-of-the art technology called single-cell RNA sequencing in models of kidney and liver disease. This enabled us to determine which genes were switched on or off in individual immune cells. This revealed that there were many more immune cell subsets than we were previously aware of. By analysing the genes that are activated in each cell, we have identified a subset of immune cells that expresses many genes that promote inflammation and scarring, leading us to suspect that this subset may promote kidney and liver injury. The immune cells in this subset have a specific protein called CLEC4D on their surface and we believe that targeting this CLEC4D molecule may prevent organ injury in a more precise manner than current therapies.
In this project we will use surplus tissue from biopsies from patients with liver and kidney disease to examine which molecular pathways are activated in the immune cells that express CLEC4D. This knowledge will help us develop new drugs that can prevent these cells causing inflammation and scarring.
Our collaborators have developed an antibody that binds to and neutralises the action of CLEC4D. We will use pre-clinical models of kidney and liver injury to test whether this antibody prevents the activation of immune cells, thereby preventing development of scar tissue in the organs. If successful, this could be used to prevent the development of scarring in the organs of patients with kidney and liver disease.
In addition, we have found a specific biochemical pathway is activated in the CLEC4D+ immune cells, which we believe contributes to inflammation and scarring. We will use genetic tools to block this pathway in the immune cells to test this hypothesis in models of kidney and liver disease. This will indicate whether blockade of this pathway by administration of a drug might be an alternative method of preventing kidney and liver scarring.
In summary, our studies will determine whether a number of complementary strategies focused on inhibition of a specific subset of immune cells could reduce inflammation and scarring in organs including kidney and liver to prevent organ failure.
Regardless of the underlying cause of the injury, damage to the kidneys and liver results in recruitment of white blood cells to the injured organs. These cells are part of the body's immune system that is important for fighting infection, however during organ injury they may promote further damage and scarring. We know that drugs that prevent recruitment of these immune cells to injured organs can slow, but not stop, progression of liver and kidney disease. However, some subsets of the immune cells may be beneficial as they are required for normal tissue functions and to promote repair. Hence, blocking all immune cells from reaching the damaged organ is not advisable and a more targeted approach to inhibit specifically those subsets that promote injury is likely to be more beneficial.
In order to identify the specific immune cell subsets that promote organ injury, we have employed a state-of-the art technology called single-cell RNA sequencing in models of kidney and liver disease. This enabled us to determine which genes were switched on or off in individual immune cells. This revealed that there were many more immune cell subsets than we were previously aware of. By analysing the genes that are activated in each cell, we have identified a subset of immune cells that expresses many genes that promote inflammation and scarring, leading us to suspect that this subset may promote kidney and liver injury. The immune cells in this subset have a specific protein called CLEC4D on their surface and we believe that targeting this CLEC4D molecule may prevent organ injury in a more precise manner than current therapies.
In this project we will use surplus tissue from biopsies from patients with liver and kidney disease to examine which molecular pathways are activated in the immune cells that express CLEC4D. This knowledge will help us develop new drugs that can prevent these cells causing inflammation and scarring.
Our collaborators have developed an antibody that binds to and neutralises the action of CLEC4D. We will use pre-clinical models of kidney and liver injury to test whether this antibody prevents the activation of immune cells, thereby preventing development of scar tissue in the organs. If successful, this could be used to prevent the development of scarring in the organs of patients with kidney and liver disease.
In addition, we have found a specific biochemical pathway is activated in the CLEC4D+ immune cells, which we believe contributes to inflammation and scarring. We will use genetic tools to block this pathway in the immune cells to test this hypothesis in models of kidney and liver disease. This will indicate whether blockade of this pathway by administration of a drug might be an alternative method of preventing kidney and liver scarring.
In summary, our studies will determine whether a number of complementary strategies focused on inhibition of a specific subset of immune cells could reduce inflammation and scarring in organs including kidney and liver to prevent organ failure.
Technical Summary
Kidney and liver disease are major causes of morbidity/mortality, however, therapeutic options remain limited.
Both organs are sensitive to fibrosis, therefore to identify injurious scar-associated monocyte derived macrophages (SAMac) for precision targeting, we performed scRNA-seq in pre-clinical models of kidney and liver disease. We identified a subset found only during organ injury, which is tagged by Clec4d expression, found in areas of fibrosis, and are enriched for pro-inflammatory, pro-fibrotic genes. We observed that CLEC4D is expressed by pro-inflammatory CD14++CD16- Mo-Mac recruited to injured human kidneys, suggesting these could be the source of SAMac; hence we hypothesise that targeting the CLEC4D+ subset may inhibit kidney/liver disease in humans.
Intriguingly, the Clec4d+ SAMac subset is enriched for genes in the arginine-ornithine biosynthesis pathway, including Ass1 and Arg1. Ass1 promotes a pro-inflammatory macrophage phenotype, while the products of this pathway act as substrates for cell proliferation and collagen synthesis; hence we hypothesise that blocking the arginine-ornithine biosynthesis pathway in SAMac will inhibit inflammation and fibrosis.
We will use Nanostring CosMx SMI technology to spatially resolve and characterise the transcriptome of CLEC4D+ SAMac in FFPE sections from patients with kidney and liver disease. We will examine if the presence of these cells will predict patients who have more rapid progression of their disease.
We will utilise a Clec4d neutralising antibody in models of kidney/liver disease to provide proof of principle that inhibiting CLEC4D prevents activation of the pro-inflammatory SAMac phenotype and ameliorates organ fibrosis.
Finally, we will knockout Arg1 and Ass1 in human monocytes and mice monocytes using a transgenic mouse, perform metabolic studies to determine how immunometabolic changes affect the SAMac phenotype and the resultant effect on organ fibrosis in pre-clinical models.
Both organs are sensitive to fibrosis, therefore to identify injurious scar-associated monocyte derived macrophages (SAMac) for precision targeting, we performed scRNA-seq in pre-clinical models of kidney and liver disease. We identified a subset found only during organ injury, which is tagged by Clec4d expression, found in areas of fibrosis, and are enriched for pro-inflammatory, pro-fibrotic genes. We observed that CLEC4D is expressed by pro-inflammatory CD14++CD16- Mo-Mac recruited to injured human kidneys, suggesting these could be the source of SAMac; hence we hypothesise that targeting the CLEC4D+ subset may inhibit kidney/liver disease in humans.
Intriguingly, the Clec4d+ SAMac subset is enriched for genes in the arginine-ornithine biosynthesis pathway, including Ass1 and Arg1. Ass1 promotes a pro-inflammatory macrophage phenotype, while the products of this pathway act as substrates for cell proliferation and collagen synthesis; hence we hypothesise that blocking the arginine-ornithine biosynthesis pathway in SAMac will inhibit inflammation and fibrosis.
We will use Nanostring CosMx SMI technology to spatially resolve and characterise the transcriptome of CLEC4D+ SAMac in FFPE sections from patients with kidney and liver disease. We will examine if the presence of these cells will predict patients who have more rapid progression of their disease.
We will utilise a Clec4d neutralising antibody in models of kidney/liver disease to provide proof of principle that inhibiting CLEC4D prevents activation of the pro-inflammatory SAMac phenotype and ameliorates organ fibrosis.
Finally, we will knockout Arg1 and Ass1 in human monocytes and mice monocytes using a transgenic mouse, perform metabolic studies to determine how immunometabolic changes affect the SAMac phenotype and the resultant effect on organ fibrosis in pre-clinical models.
