Dynamic regulation of human immunity by Interferon Regulatory Factor 8 (IRF8)

The immune system is our major defence against bacterial infections, viruses and cancer. It is also responsible for inflammatory diseases and plays a major role in age-related degenerative diseases. The immune system is made of many different cells including granulocytes, monocytes, dendritic cells and lymphocytes. All of these are created in the bone marrow from stem cells through the process of blood formation, known as haematopoiesis. This research project aims to understand how haematopoiesis controls immune responses.

Haematopoiesis is a complex process. The production of different cells depends upon specific genes that are turned on and off by proteins that bind to DNA, known as transcription factors. Production is finely tuned to allow the immune system to respond to specific threats, a process known as demand-adaptation. Interferon Regulatory Factor 8 (IRF8) is a key transcription factor in haematopoiesis but how it is regulated and how it controls other genes is not well understood. I have previously found that people with a faulty IRF8 gene are unable to make dendritic cells, and in some cases also monocytes, but have a large excess of neutrophils. The level of imbalance depends upon how much IRF8 activity is present, strongly suggesting that IRF8 is the master control switch. If there are insufficient dendritic cells, then it is not possible to develop immune memory to past viral infections and vaccines. Lack of neutrophils lead to frequent and often fatal bacterial infections, while too many can cause inflammation in the lungs and suppress natural immune responses to cancer. Correctly balanced haematopoiesis is therefore critical to maintaining health and responding effectively to disease.
In this project I will study how IRF8 is regulated and how it maintains the balance of haematopoiesis by controlling the activity of other genes. I will use cutting-edge techniques that reveal these processes within single cells. I will also use genetic variation in the IRF8 gene to study how differences between individuals affect immunity.

The project has four Aims. In the first Aim, I will use single cell techniques to map the developmental pathways of immune cells, identify regions of DNA important for regulating IRF8 in these pathways, and define the genes that IRF8 controls. In Aim 2, I will extend these techniques to analyse haematopoietic stem cells from patients with sepsis caused by infection, or those with breast cancer, conditions that are both associated with extreme imbalance of immune cells. This will allow me to identify the mechanisms that drive the imbalance and find ways that these could be controlled for medical benefit. In Aim 3, I will gather samples from people with severe mutations in the IRF8 gene in order to understand how different parts of IRF8 are required for it to function correctly. Finally, in Aim 4, I will study natural genetic variation in the region of the IRF8 gene to create a detailed map of how the activity of the IRF8 gene is controlled. It is likely that as a population, we have a wide range of IRF8 responses and that genetic variation gives us diversity as a species in the face of infection and other challenges.

Together, this work will define the precise role of IRF8 in human haematopoiesis and how this controls immune cell development. I will discover the factors that control IRF8 and map the network of genes influenced by IRF8 in at each stage of immune cell development. This will identify ways to modify haematopoiesis to achieve better immune responses to infections, vaccines and immune therapy, to suppress harmful immune reactions, and to augment immunity to cancer.

Human immunity critically depends upon the coordinated production of functionally specialised cells by haematopoiesis, under the control of lineage-specific transcription factors (TFs). Rapid adaptation of haematopoiesis is required to meet the demands of immunity, especially in the production of innate myeloid cells; neutrophils, monocytes and dendritic cells (DCs). Interferon regulatory factor 8 (IRF8) is a pioneer TF for myeloid cells and critical lineage-determining TF for DCs. IRF8 plays a pivotal role in demand adaptation as illustrated by predominant granulopoiesis and monocyte/DC depletion in IRF8 deficiency, the association of IRF8 variants with blood parameters and immune diseases, and suppression of IRF8 in sepsis and cancer.
In Aim 1 I will use single cell multiomic genetic and epigenetic approaches to achieve an integrated analysis of the chromatin architecture of the IRF8 locus, genome-wide chromatin occupancy by IRF8 and associated regulatory networks, mapped in developmental time and across myeloid cell lineages. Extending these approaches to Aim 2, I will determine the changes in IRF8 expression, chromatin occupancy and enhancer activation driving abnormal haematopoiesis in patients with sepsis and breast cancer. This will identify regulatory networks and mechanisms that are modified by disease. In Aim 3, I will define the effects of rare, loss-of-function mutations in IRF8 on downstream activities and analyse protein structure-function relationships as potential therapeutic targets. Finally in Aim 4, I will determine the effect of variations in regulatory regions on the lineage-specific and dynamic expression of IRF8 by eQTL analysis to fine-map the architecture of IRF8 regulation.
Together these approaches will define the dynamic role and regulatory mechanisms of IRF8 in immune cell haematopoiesis with the ultimate goal of targeting IRF8 to manipulate immune responses for therapeutic gain.