Eosinophil and macrophage crosstalk defines cell survival and modulates local tissue niche.

Human bodies are made up from tissues, such as heart or skin, which consist of multiple different cells types: the cells of the tissue and immune cells. Immune cells are the cells that protect the body from, for example, bacteria and viruses, but they also play a role in keeping our tissues healthy. Different types of immune cells communicate and instruct each other in this process. Interruption in this communication often makes people ill or make an injury take longer to heal. In this research, I explore the messages sent between two types of immune cells, macrophages and eosinophils and how that communication changes tissue health.

Macrophages are large immune cells in your body that sense problems in your tissue, such as damage and germs. Eosinophils are best known to protect us from parasites, but they have number of other very important functions in the body. Eosinophils carry large amount of different signalling molecules in their special compartments, called granules. Depending on the messages from other cells, eosinophils release back selected signalling molecule to perform their functions. For example, during liver damage, liver cells send message to eosinophils to come to the liver. Once they arrive, eosinophils communicate with the liver cells to help them heal. Similarly, we know that eosinophils talk to macrophages and this communication is important for maintaining healthy tissues and even a healthy body weight. So, communication between cells is essential to keep healthy tissues and body. If this conversation is interrupted, cells are misdirected and often eosinophils gather together in high numbers in one tissue. This overcrowd pushes eosinophils to send locally wrong signals, which then cause tissue damage, like during asthma. In most cases, we do not understand the messages causing eosinophil gathering in healthy tissues.

Over 10 million people in UK suffer from eosinophil related diseases, some life-threatening and most life-altering. Hospital admissions for anaphylaxis, severe allergic reactions, increased by 6-fold in the last 20 years. The rising frequency of eosinophil-mediated diseases requires quick and new diagnostic and treatment methods. Understanding basic rules of cells communication is the first and fundamental step towards these goals.

I have a model in which macrophages in one tissue are defective and, in this model, I have observed notable changes in eosinophils numbers and survival. This is a very novel observation. We know that eosinophil talk directly to macrophages, but macrophages replies or initial messages to eosinophils are not often seen. I will use this model to understand how these cells communicate, specifically:
1. How do defects in macrophages affects local tissue and eosinophil numbers and survival?
2. What messages are used to communicate, and can they I use of them to control eosinophils in the body?
3. Can I manipulate this communication to prevent tissue damage?

My data shows macrophage-eosinophil communication to be at the heart of normal tissue function. I have identified a candidate signal that might be responsible for high numbers of eosinophil in the tissue. This is very exciting because such messages between these cells are not recognised. The aim is to understand how this communication maintains normal tissue functioning and how it can lead to disease and tissue damage.

This project investigates the communication networks between peritoneal macrophages (pMO) and local cells, particularly eosinophils. In my model, change to a single gene of pMO results in significant alterations to the microenvironment and interestingly, prolonged survival of eosinophils. This phenotype is seen in tissues affected by eosinophilic diseases but shows no consequent damage to local tissue.
Using cutting-edge methodologies, such as single cell sequencing (10xChromium) and imaging flow cytometry (Image Streamx MkII) and lipidomics, I will define detailed cell populations in my model to understand how pMO control this tissue niche. My pilot data implicated lipid mediators in pMO and eosinophil crosstalk and in the unique maintenance of these cells in the peritoneal cavity. I will confirm this novel finding in in vivo and in vitro settings using adoptive transfer system and signalling inhibitors/mimics. Global and cell focused lipidomics (Sciex 6500 Qtrap) analysis, complemented with in vivo lentivirus-mediated overexpression of enzymes involved in lipid synthesis in macrophages to validate the mediators supporting eosinophil survival. Bioinformatic comparison of RNA-seq of eosinophils with transcriptome profiles deposited in NCBI will identify the resemblance of these cells to these in other tissues. Consequently, the role of lipid mediators could be linked to specific eosinophil phenotypes in multiple tissues and diseases for the benefits of wider scientific audience.
These studies will decipher the complex communication network between macrophages, eosinophils and their microenvironment and will be further explored in the context of acute and chronic inflammatory stimuli, Staphylococcus epidermis-induced fibrosis. Knowledge of cell biology resulting from this research has a potential to be translated and applied to other tissues, providing strong fundamentals for understanding of the complex nature of intercellular communication and interdependence.

Immediate impacts will be realised from this proposal by the continued development of a highly trained researcher and students in collaboration with the host laboratory. The proposal will use innovative technologies to derive novel research outcomes and will represent a step change in the immediate field.

Proposed research will aid the understanding of 1. how genetic make-up of macrophages affects local tissue. This will be achieved by comprehensive comparison of transcriptomics of multiple cell types in the model system with phenotypically identified alterations to the local niche tissue; 2. what are the signals sent between macrophages and eosinophils. The pilot data suggests interdependence of macrophages and eosinophils in the context of lipid mediator synthesis and consequent cell survival. These findings will have broader applicability to other tissues and possibly disease pathologies, thereby providing benefits to a range of internationally acclaimed researchers outside of my professional circle; and 3. the impact of those signals on living system functions, will be investigated during homeostasis and inflammatory challenge. Highly established method of staphylococcus epidermis-induced fibrosis will be employed for this purpose in collaboration with the laboratory that pioneered the method. This could not only unveil the mechanisms leading to development of eosinophilic diseases, but future research streaming from these findings may also highlight avenues that can be exploited for modulation of immune or inflammatory responses to prevent or resolve eosinophilia in patients.

Together, these studies will support the excellence of basic bioscience research in the United Kingdom; they will drive innovation in the research areas involving cell communication and will advance discovery of how living systems function. Large amounts of "omic" data will be generated and made available for other researchers worldwide via public repositories. Due to universal application of cell communication systems, the knowledge and data exchange from this study will encourage discoveries in the multiple research areas.

The model system used in this proposal resembles that of tissue eosinophilia observed in patients suffering with eosinophilic diseases. Triggers leading to eosinophil accumulation in tissues remain unknown and the phenotype and functions of these cells are poorly understood. Thus, research outcomes from this unique model will aid identification of the mechanism of eosinophil accumulation in tissues, including the molecules and other cells important for the process. These aspects would be of interest in the long term to Industry, identifying possible development of therapeutics and potentially leading to substantial benefits for patients suffering with eosinophilic diseases.
Knowledge exchange is at the heart of this project and it will be delivered by engagement with researchers during international conferences and through production of high impact publications. The general public and pupils will be reached through several established schemes, such as Science in Health Live!, Green Man festival, public science talks and popular science publication in ReMEDy newsletter published by Cardiff University. Since well-known events will be utilised for the public engagement, social benefits will be evident within the first year of the fellowship.

Eosinophils roles and functions are only recently fully appreciated and investigated. Novel interdependence axis of macrophages and eosinophils indicated from this proposal will become a leading study in the understanding of those cells and their interaction with the local microenvironment. Transferability of this communication mechanism to other tissues and cells emphasises imperative merit of the topic. This study complements other innovative research in the BBSRC portfolio and strongly aligns with the BBSRC theme: "advancing the frontiers of bioscience discovery".