Unravelling key regulators of neutrophil behaviour in live tissue using advanced microscopy technology

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 response1. 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 2,3,4. A continuum of neutrophil states was uncovered in the mouse model of induced vascular inflammation, comprising a state characterised by an oblate morphology, low speed and association with endothelial junction3. We identified multiple neutrophil subsets in the blood of patients with systemic vasculitis, based on both nuclear morphology and cell surface receptor expression4. One neutrophil state depicted cells with an unusual nuclear morphology, characteristic of immature neutrophils, extended life span, high level of reactive oxygen species production and ability to cause damage to vascular wall and was unequivocally associated with both the clinical phenotype and response to treatment4.

This project is set up to dissect whether the identified molecular signatures can be represented by corresponding behaviour in a signal-driven live tissue microenvironment using advanced microscopy technology5.

First, we will use an innovative in vitro system of a 3D collagen type IV matrix and ZEISS Lattice Lightsheet Microscopy (LLSM), to investigate the patterns of neutrophil behaviour in the matrix under controlled conditions both intrinsically (using HoxB8 neutrophils with defined state of differentiation; with knock-outs of key regulators of neutrophil differentiation and/or activation1 etc) and extrinsically (in the presence of activating stimuli; in the presence of other immune cells etc).

Second, we will use tissue ex-plants to study primary neutrophil dynamics in tissue and correlate with the previously observed in vitro patterns of behaviour and/or cell-cell interactions associated with different molecular perturbations. This part of the project will be supported by the use of the custom-built super resolution LLSM of the BioCOP project at the Rosalind Franklin Institute.

Finally, we will investigate spatial interactions of neutrophils at different states with other immune cells in the selected tissue/organs (e.g. knee joint).

KEYWORDS

Live cell imaging, super-resolution microscopy, neutrophils, inflammation, molecular signatures.

TRAINING OPPORTUNITIES

The Kennedy Institute is a world-renowned research centre and is housed in a state-of-the-art research facility. Training will be provided in techniques in a wide range of cutting edge imaging (immunofluorescence, live imaging, organ imaging, super-resolution etc) approaches, as well as immunological and spatial single cell platforms (10x, Nanostring CosMx). Recently developed novel in vitro and in vivo models of neutrophil biology will be extensively used and new models will be generated. A core curriculum of lectures will be taken in the first term to provide a solid foundation in a broad range of subjects including musculoskeletal biology, inflammation, epigenetics, translational 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, the Genomics of Inflammation Laboratory, the Biophysical Immunology Laboratory, Imaging Forum and to attend external conferences to present their research globally. Students will also have the opportunity to work closely with members of the Rosalind Franklin Institute, Rheumatoid Arthritis Pathogenesis Centre of Excellence (Glasgow/Birmingham/Newcastle/Oxford) as well as Novonordisk Immunometabolism consortium (Oxford/Karolinska Institute/University of Copenhagen).

KEY PUBLICATIONS

1. Khoyratty T*, Ai Z*, Ballesteros I, Mathie S, Eames HL, Martín-Salamanca S, Wang L, Hemmings A, Willemsen N, von Werz V, Zehrer A, Walzog B, van Grinsven E, Hidalgo A, Udalova IA. Distinct transcription factor networks control neutrophil-driven inflammation. Nature Immunology, 2021 Sep;22(9):1093-1106.
2. Wang L, Luqmani R, Udalova IA. The role of neutrophils in rheumatic disease-associated vascular inflammation. Nature Reviews Rheumatology. 2022 Mar;18(3):158-170.
3. Crainiciuc G, ....., Hidalgo A. Behavioural immune landscapes of inflammation. Nature. 2022 Jan;601(7893):415-421
4. Wang L, Ai Z, Khoyratty T, Zec K, Eames HL, van Grinsven E, Hudak A, Morris S, Ahern D, Monaco C, Eruslanov EB, Luqmani R, Udalova IA. ROS producing immature neutrophils are linked to GCA vascular pathologies. Journal of Clinical Investigations Insight. 2020 Oct 15;5(20):e139163.
5. Fritzsche M. Live microscopy: cracking the challenge to image biology unfolding in cells, tissues, and organs. Communications Biology. 2022 Jul 7;5(1):665.

THEMES

Innate Immunity, Cell Dynamics, Microscopy, Computational Biology