Molecular mechanisms retaining macrophages at sites of inflammation
Lead Research Organisation:
University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease
Abstract
How cells move within our bodies and reach their appropriate destination is a fundamental topic in biology. We have identified many cues that cells follow to reach their necessary locations in the body. However, far less is known about the signals that keep them in those places. The retention of cells at their correct site of action has relevance across biology, being critical during development, for normal maintenance of our bodies, and in ageing and disease. Retention is particularly key during immune responses: following recruitment of cells, e.g. to sites of tissue damage, failing to retain cells could undermine immune responses to infection or negatively affect repair. At the same time, excessive retention could cause tissue damage due to the negative effects some immune cells exert within our bodies. Understanding these processes can provide points of intervention to stimulate or dampen immune responses as appropriate, as well as provide a more general understanding of how we keep cells in their correct locations. Immune cell dysfunction contributes to a wide range of human diseases including chronic inflammatory conditions (e.g. COPD), cancer and neurodegeneration. Fortunately, immune cells are amenable to pharmacological interventions so identification of new drug targets is of particular use in this area of medicine.
Fruit flies (Drosophila) contain a population of blood cells very similar to the white blood cells known as macrophages within our own bodies. Due to their genetic redundancy, superlative genetics and imaging capabilities, fruit flies they make an excellent (and less ethically challenging) model compared to other multicellular organisms to understand regulation of the immune system. Our recent work has uncovered a novel molecule able to retain macrophages at wounds called Simu. Simu interacts with molecules present on the surfaces of damaged cells at sites of damage. We propose Simu helps immune cells to adhere to wounds, retaining them at these critical locations until it is time for them to depart.
In this proposal we wish to understand how Simu facilitates retention of macrophages at sites of damage. We will use fly genetics in combination with real-time imaging of macrophage responses to injury in developing embryos. Using high-powered lasers we can reproducibly wound the surface of fly embryos, towards which macrophages mount a rapid response. Using fluorescent markers, we can visualise fly macrophages as they migrate to wounds and use our assays of recruitment and dispersal from wounds to understand which genes are important in this process and how they interact with each another. In parallel, we aim to find new molecular players in an unbiased fashion: we will identify which proteins are increased or decreased following challenge of healthy cells with dying cells of the type found at wounds. This will also enable us to see which signalling proteins are altered via a modification called phosphorylation. We will then return to our wounding assays to determine which new regulators are important in governing retention at wounds. We now know that there can be negative consequences to failed retention of immune cells at sites of inflammation, therefore we will also capitalise on the identification of these new molecules to examine their impact on subsequent macrophage behaviour.
Taken together this work will enable us to assemble a map of key regulators that retain immune cells at injuries. Our unbiased approaches will also enable identification of new and exciting regulators of this process, while our characterisation of how contact between dying cells and immune cells can change the behaviour of the latter will provide an insight into the importance of these mechanisms. Ultimately this research will improve our understanding of a fundamental area of biology and has the potential to uncover new drug targets for the manipulation of immune cell function within human patients.
Fruit flies (Drosophila) contain a population of blood cells very similar to the white blood cells known as macrophages within our own bodies. Due to their genetic redundancy, superlative genetics and imaging capabilities, fruit flies they make an excellent (and less ethically challenging) model compared to other multicellular organisms to understand regulation of the immune system. Our recent work has uncovered a novel molecule able to retain macrophages at wounds called Simu. Simu interacts with molecules present on the surfaces of damaged cells at sites of damage. We propose Simu helps immune cells to adhere to wounds, retaining them at these critical locations until it is time for them to depart.
In this proposal we wish to understand how Simu facilitates retention of macrophages at sites of damage. We will use fly genetics in combination with real-time imaging of macrophage responses to injury in developing embryos. Using high-powered lasers we can reproducibly wound the surface of fly embryos, towards which macrophages mount a rapid response. Using fluorescent markers, we can visualise fly macrophages as they migrate to wounds and use our assays of recruitment and dispersal from wounds to understand which genes are important in this process and how they interact with each another. In parallel, we aim to find new molecular players in an unbiased fashion: we will identify which proteins are increased or decreased following challenge of healthy cells with dying cells of the type found at wounds. This will also enable us to see which signalling proteins are altered via a modification called phosphorylation. We will then return to our wounding assays to determine which new regulators are important in governing retention at wounds. We now know that there can be negative consequences to failed retention of immune cells at sites of inflammation, therefore we will also capitalise on the identification of these new molecules to examine their impact on subsequent macrophage behaviour.
Taken together this work will enable us to assemble a map of key regulators that retain immune cells at injuries. Our unbiased approaches will also enable identification of new and exciting regulators of this process, while our characterisation of how contact between dying cells and immune cells can change the behaviour of the latter will provide an insight into the importance of these mechanisms. Ultimately this research will improve our understanding of a fundamental area of biology and has the potential to uncover new drug targets for the manipulation of immune cell function within human patients.
Technical Summary
How cell migration is controlled is a fundamental question in biology. While we have identified many chemotactic cues regulating this process, the retention of cells has received far less attention. Retention of cells is relevant to development, homeostasis, ageing and in particular immune responses. Failing to retain immune cells such as macrophages at sites of injury can have damaging consequences for the organism, while the same is true of excessive retention.
Using Drosophila embryos to model inflammatory responses we have identified a multifunctional receptor, Simu, that is necessary to retain macrophages (plasmatocytes of the hemocyte lineage) at wounds (Roddie et al., PLoS Biol). Our preliminary data indicates that components of integrin-mediated adhesion and the CED-1 family member Draper exhibit a similar phenotype with premature departure of macrophages from embryonic wounds. Combining live imaging with a genetic approach, we will dissect the downstream mechanisms keeping macrophages at wound sites in the developing Drosophila embryo, using genetic interaction experiments to understand whether components of integrin-mediated adhesion and signalling molecules work in the same or a parallel pathway to Simu.
As an unbiased approach we will use mass spectrometry to characterise changes in the proteome and phosphoproteome on contact with necrotic cells, which represent the predominant form of cell death found at Drosophila wounds. We will then validate and characterise the functional importance of the novel molecular players that we find and integrate them with our earlier findings to map out the network of molecules regulating macrophage retention. Simu appears to be a central regulator of interactions with dying cells, exhibiting roles in clearance of both apoptotic and necrotic cells. We will make use of recent reporters of macrophage state (Coates et al., 2021) to reveal the importance of Simu-mediated interactions with dying cells in vivo.
Using Drosophila embryos to model inflammatory responses we have identified a multifunctional receptor, Simu, that is necessary to retain macrophages (plasmatocytes of the hemocyte lineage) at wounds (Roddie et al., PLoS Biol). Our preliminary data indicates that components of integrin-mediated adhesion and the CED-1 family member Draper exhibit a similar phenotype with premature departure of macrophages from embryonic wounds. Combining live imaging with a genetic approach, we will dissect the downstream mechanisms keeping macrophages at wound sites in the developing Drosophila embryo, using genetic interaction experiments to understand whether components of integrin-mediated adhesion and signalling molecules work in the same or a parallel pathway to Simu.
As an unbiased approach we will use mass spectrometry to characterise changes in the proteome and phosphoproteome on contact with necrotic cells, which represent the predominant form of cell death found at Drosophila wounds. We will then validate and characterise the functional importance of the novel molecular players that we find and integrate them with our earlier findings to map out the network of molecules regulating macrophage retention. Simu appears to be a central regulator of interactions with dying cells, exhibiting roles in clearance of both apoptotic and necrotic cells. We will make use of recent reporters of macrophage state (Coates et al., 2021) to reveal the importance of Simu-mediated interactions with dying cells in vivo.
