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

Co-supervisor: Associate Prof. James Fullerton, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences

Commercial partner: DJS Antibodies

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; Khoyratty et al, Nature Immunology 2021]. 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.

Apply using course: DPhil in Molecular and Cellular Medicine