Neutrophil subset behaviour in vascular and tissue microenvironment: organ-on-a-chip*

Project No: MRC-1
Intake: 2024 KIR Non Clinical

PROJECT OVERVIEW

Neutrophils represent a major arm of the innate immune defence system that can tailor their behaviour to support organ homeostasis and mount tissue specific and transcriptionally regulated inflammatory response [Ballesteros et al, Cell 2020]. Recent developments in the field emphasised the fact that during inflammation neutrophils in circulation and tissue are presented as functionally, morphologically, and behaviourally heterogeneous cells [Wang et al, Nature Reviews Rheumatology 2022]. Increased neutrophil production driven by infection, injury, inflammation or cancer (emergency granulopoiesis) leads to the mobilisation of both mature neutrophils and immature neutrophil precursor cells into the blood and affected sites, thus increasing the phenotypic and functional diversity. We identified multiple neutrophil subsets in the human blood, based on both nuclear morphology and cell surface receptor expression, in patients with giant cell arteritis (GCA), a common form of primary systemic vasculitis in adults [Wang et al, JCI Insight 2020], severe COVID-19 [Covid-19 Multi-omics Blood Atlas (COMBAT) Consortium, Cell 2022] and sepsis [Kwok et al, Nature Immunology 2023]. One neutrophil state depicted cells with an unusual nuclear morphology, characteristic of immature neutrophils, extended life span, high level of reactive oxygen species (ROS) production and ability to cause damage to vascular wall [Wang et al, JCI Insight 2020].

This project is set up to dissect whether these previously identified human and mouse neutrophil subsets exhibit distinct behaviour in the vasculature (mimicking arterial, venular and capillary endothelium) and tissue microenvironment and whether there are common inter-species signatures. We will develop a 3D microvessel/tissue-on-a-chip system, using primary neutrophil subsets from healthy participants, in which an endothelial vessel is perfused with neutrophils in a membrane-free and tubular manner against a collagen hydrogel with a chemotactic trigger (chemokines or other tissue cells). Using this system and confocal microscopy, we will (1) monitor the interactions of neutrophils at different maturation stages with the vessels; (2) examine the effect of neutrophil ROS and NET generation on vascular damage; (3) examine recruitment and migration of neutrophils through an extracellular matrix; (4) assess the effect of existing and new drugs under development which inhibit the identified molecular regulators for their effect on neutrophil function. Demographic differences (e.g. sex, age) may be specifically explored. To validate the in vitro design and data with live tissue settings, we will use 3D imaging on tissue ex-plants assisted by the Light Sheet Microscopy (LSM), with scope to undertake in vivo validation using human immune challenge paradigms.

The outcome of this study is expected to contribute to our understanding of the heterogeneity of neutrophil responses in vasculature and tissue and present a novel organ-on-a-chip model for assessing the effect of new drugs and/or molecular perturbations in both mouse and human settings. It will provide a unique opportunity to bridge the discovery and translation fields, integrating both academic and biotech perspectives on the project development, milestones and outcomes, as well as an opportunity to test newly developed reagents in the resultant tissue-on-a-chip system, which contributes to real-world decision making.

KEYWORDS (5 WORDS)

Immunology, experimental medicine, inflammation, molecular biology

TRAINING OPPORTUNITIES

The NDORMS Kennedy Institute is a world-renowned research centre and is housed in a brand new state-of-the-art research facility. Training will be provided in multiple techniques that will constitute a comprehensive immunological tool kit (cell isolation, FACS, ELISA, primary cell culture) with additional exposure to advanced imaging (confocal microscopy, LSM) approaches. This rare opportunity to develop a novel organ-on-a-chip model will include cell isolation, handling and culture. For this proposal, two types of endothelial cells (arterial and microvascular) will be used to generate the vasculature. The candidate can benefit from the hands-on experience with these techniques in the Udalova lab, and from access to clinical samples and expertise in their immune analysis in the Fullerton group (the NDORMS Botnar Institute).

A core curriculum of lectures will be taken in the first term to provide a solid foundation in a broad range of subjects including genomics, epigenetics, immunology and data analysis. Students will attend weekly seminars within the department and those relevant in the wider University. Students will be expected to present data regularly to the department, to attend external conferences to present their research globally. Importantly, the student will have access to, and the full support of, DJS Antibodies’s, team, including their expertise in target identification, validation and translational assays. Training will be provided, particularly in areas of drug discovery and development. Finally, students will undertake a placement at the DJS Antibodies to learn about their discovery platform for antibodies to complex protein targets and gain an understanding of company operations.