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Access Flow Cytometry Facilities within the Midlands Innovation Universities
Jill Johnson – School of Life & Health Sciences
Case study
Investigating pericyte phenotype and function under chronic inflammatory conditions
Scar formation is a vital mechanism of tissue repair following injury. However, healthy tissue repair can develop into pathological fibrosis, which ultimately leads to tissue destruction and organ failure. Fibrosis is associated with chronic inflammation, oxidative stress, and ageing. However, there are currently no treatment options for organ fibrosis, and these diseases impose a significant burden on public health care systems and have detrimental impacts on patient quality of life. Importantly, little is known about the factors that initiate fibrosis. Previous work in the research group of Dr Jill Johnson has identified pericytes, a type of tissue-resident mesenchymal stem cell, as the primary driver of fibrosis. Pericytes provide support to capillaries throughout the body and are particularly vital to maintaining healthy tissue structure. Importantly, pericytes are strongly associated with tissue fibrosis in the lung, liver, and kidney. Recent studies have shown that pericytes contribute to fibrosis by uncoupling from local blood vessels, followed by migration to the site of inflammation via the CXCL12/CXCR4 axis and differentiation into scar-forming myofibroblasts (Figure 1). Dr Johnson’s group has used flow cytometry to identify lung pericytes (CD45-/Ter119-/CD31-/CD146+/PDGFRb+) and shown that these cells upregulate markers of cell migration (CD13, podoplanin, CXCR4) in response to chronic airway inflammation driven by house dust mire (HDM) exposure.
Funding – UK Medical Research Council, MR/K011375/1.
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https://www.birmingham.ac.uk/university/colleges/mds/facilities/flow-cytometry.aspx
Research Facilities Manager: Adriana Flores-Langarica
Advanced Flow Cytometry Specialist: Guillaume Desanti
Flow Cytometry Specialist: Ferdus Sheik
Mass Cytometry Specialist: Shahram Golbabapour
Case study
Investigating the transcriptional networks regulating Innate Lymphoid Cell fate and function
Identification of the Innate Lymphoid Cell (ILC) family over 10 years ago revealed a new axis in supporting tissue homeostasis and coordinating the response to local infection or damage. Several types of ILC have now been described, however, many of these populations do not appear to be terminally differentiated, and rather, they appear able to extensively remodel their effector functions. How this cellular ‘plasticity’ is regulated post-development remains poorly understood, but remains a key question given the ability of these cells to coordinate the actions of many immune populations and that altered ILC frequencies correlates with several inflammatory conditions.
Using mouse models to inducibly delete different transcription factors, alone and in multiple combinations, we sought to unpick how different networks combine to control ILC plasticity. Crucially, we additionally ‘fate-mapped’ those cells in which cre-recombinase mediated gene-deletion was induced to accurately track the fate of these cells in vivo. Complex flow cytometry monitoring surface phenotype, cytokine and transcription factor expression in combination with fate-mapping (revealed by tdRFP expression) was fundamental to these studies.
Reference: Fiancette, R., Finlay, C.M., Willis, C. et al. Reciprocal transcription factor networks govern tissue-resident ILC3 subset function and identity. Nat Immunol 22, 1245–1255 (2021). https://doi.org/10.1038/s41590-021-01024-x
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https://www.cranfield.ac.uk/facilities/flow-cytometry
Academic Lead: Francis Hassard, 01234 750 111
Uses: Flow cytometry for assessing microbiological water quality Characterisation of bacteria, eukaryotes and virus from complex environmental samples. Biofilm characterisation.
Case study
Flow cytometry fingerprints to investigate bacterial water quality events in drinking water
Author – Dr Francis Hassard, Cranfield Water Science Institute, Cranfield University.
Summary
Deviations in the quality of final treated drinking water from Water Treatment Works (WTW) can result in problems such as regulatory fines, reputational damage, taste and odour complaints and, significantly, potential for increased public health risk. Traditional drinking water flow cytometry (FCM) parameters that measure intact and total cell populations (viability), the nucleic acid content of bacteria and the microbial fingerprint better reflect the inherent heterogeneity within drinking water bacterial communities compared to culture-based approaches. Here, daily inter-stage monitoring and flow cytometry data was undertaken at WTW and weekly analysis of hydraulically linked service reservoirs was undertaken over a 12-month period. Extra information provided by the flow cytometry fingerprint (e.g. fluorescence intensity distribution of cells) was assessed.
Methods
BD Accuri C6 flow cytometer (Becton Dickinson U.K. Ltd., U.K.) which was equipped with a 488 nm solid state laser. Green fluorescence was collected in the FL1 channel at 533 nm (FL1) and red fluorescence in the FL3 channel at 670 nm (FL3) after staining with SYBR GI and Propidium Iodide. The fingerprint analysis was performed using CHIC on FlowJo, ImageJ, and R software. A non-parametric analysis of similarities (ANOSIM) was performed using 9999 permutations (free) to test for significant difference between the interstage microbial fingerprints at WTW A and the fingerprints from different SR outlets.
Results
Changes to the distribution of bacteria within the microbial fingerprint (diversity quantified via Bray-Curtis dissimilarity index) provided a leading indicator for detecting events, such as poor microbial water quality and compliance exceedance.
Funding – Engineering and Physical Research Council and Southeast Water.
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Core Biotechnology Services – Main University Campus, Maurice Shock Building
Academic Lead: David Cousins
Enquiries: flowcytometry@leicester.ac.uk and Reshma Vaghela
Case Study
Investigating Type 2 cell subsets during clinical trials of novel biologics to treat Asthma and COPD
Summary
Both asthma and COPD involve inflammatory responses in the lung that impact disease severity and progression. Many patients have a disease phenotype that involves eosinophils and Type 2 immunity. Many novel biologic drugs targeting molecules in the Type 2 immunity pathway are either licenced or in development to treat these diseases. Understanding the mechanism of action of these drugs during clinical development is critical to inform future therapeutic approaches.
Using multicolour acoustic focussing flow cytometry we have developed a panel of antibodies to allow us to deep phenotype and enumerate several Type 2 immune response cells from 100 ml of whole blood. Cells that can be assessed include eosinophils, basophils, conventional and pathogenic Th2 cells and Type 2 innate lymphoid cells. Proof of concept was developed using samples from patients being treated with Mepolizumab (1). This approach is being used to examine immune responses in several clinical studies using novel biologics to target Type 2 immunity.
Reference – AKA Wright et al. (2019) Mepolizumab does not alter the blood basophil count in severe asthma Allergy 74(12):2488-2490. doi: 10.1111/all.13879.
Funding – Leicester Drug Discovery and Diagnostics (LD3) with financial contributions from MRC grant MC_PC_15045 and supported by the NIHR Leicester Biomedical Research Centre
School of Sport, Exercise and Health Sciences, National Centre of Sport and Exercise Medicine
Academic Lead: Professor Lettie Bishop
Case study
Aims:
To investigate the health implications of temperature interventions (e.g., hot water immersion, sauna, or exercise in the heat) in humans
To investigate the impact of exercising muscle mass on inflammatory responses and glycaemic control in humans
To investigate health benefits of exercise in chronic kidney patients
Tissues analysed:
Whole blood
Peripheral blood mononuclear cells (PBMCs)
Main markers of interest:
Monocyte subset distribution, leukocyte distribution
Intracellular heat shock protein 72
Toll like receptors
Microparticles
Flow cytometers used in research:
BD FACS Calibur (now retired…)
BD Accuri C6
Example gating strategy:
Selection of CD14 positive cells after exclusion of CD56 positive natural killer cells. Monocyte subsets are then defined based on CD16 and CD14 expression, and intracellular heat shock protein 72 (iHsp72) then determined for each of the subsets
Example papers:
Highton PJ, White AEM, Nixon DGD, Wilkinson TJ, Neale J, Martin N, Bishop NC, Smith AC. Influence of acute moderate- to high-intensity aerobic exercise on markers of immune function and microparticles in renal transplant recipients. Am J Physiol Renal Physiol. 2020 Jan 1;318(1):F76-F85. doi: 10.1152/ajprenal.00332.2019.
Hoekstra SP, Wright AKA, Bishop NC, Leicht CA. The effect of temperature and heat shock protein 72 on the ex vivo acute inflammatory response in monocytes. Cell Stress Chaperones. 2019 Mar;24(2):461-467. doi: 10.1007/s12192-019-00972-6.
Hoekstra SP, Westerman MN, Beke F, Bishop NC, Leicht CA. Modality-specific training adaptations – do they lead to a dampened acute inflammatory response to exercise? Appl Physiol Nutr Metab. 2019 Sep;44(9):965-972. doi: 10.1139/apnm-2018-0693.
Hoekstra SP, Bishop NC, Faulkner SH, Bailey SJ, Leicht CA. Acute and chronic effects of hot water immersion on inflammation and metabolism in sedentary, overweight adults. J Appl Physiol (1985). 2018 Dec 1;125(6):2008-2018. doi: 10.1152/japplphysiol.00407.2018.
Dungey M, Young HML, Churchward DR, Burton JO, Smith AC, Bishop NC. Regular exercise during haemodialysis promotes an anti-inflammatory leucocyte profile. Clin Kidney J. 2017 Dec;10(6):813-821. doi: 10.1093/ckj/sfx015.
Leicht CA, Paulson TA, Goosey-Tolfrey VL, Bishop NC. Arm and Intensity-Matched Leg Exercise Induce Similar Inflammatory Responses. Med Sci Sports Exerc. 2016 Jun;48(6):1161-8. doi: 10.1249/MSS.0000000000000874.
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Academic Lead: Professor Lee Machado
Technical Lead: Lewis Collins
Facility Manager (general enquiries and grant application support): David Onion
Facility Technician (for scheduling and billing enquiries): Nicola Croxall
An important challenge during the COVID-19 pandemic has been to understand asymptomatic disease as this may be a key source of transmission. Asymptomatic disease is by definition hard to screen for so there is currently a lack of clarity about this aspect of the COVID-19 spectrum. It is clear that adaptive immunity is strongly activated during asymptomatic infection but some features of the T cell and antibody response may differ from those in symptomatic disease.
Using spectral flow cytometry we have developed a panel of 22 antibodies to enable us to deep phenotype the cellular responses following either asymptomatic or symptomatic infection. Specifically, we have examined cytokine expression to Spike, Membrane and Nucleocapsid SARS-CoV2 (using overlapping peptide pools) and identified memory phenotypes related to clinical outcome.
Funding – National Core Studies : Immunity theme: “Asymptomatic Covid-19 In Education (ACE) Immunity Study”, L Fairclough, M Wills and A Godkin.
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https://warwick.ac.uk/fac/sci/lifesci/tic/flow
WISB Manager for enquiries: Sarah Bennett
Case study
High resolution cell cycle mapping based on flow cytometry
Summary
Regulation of the mammalian cell cycle and how it interacts with growth, cell size, transcription, translation, and metabolism is not clear; some mechanisms and factors have been identified, but details are murky. In particular, how variation on the single cell level and the noise this introduces into the system are handled, remains obscure.
Using flow cytometers at the Warwick School of Life Sciences allows us to resolve the cell cycle at high precision and single cell level (Fig. 1). We can combine this with measurement of transcription rates and other growth-related processes via different fluorescent channels to understand better the interplay of growth, cell cycle, and single cell variation. FACS sorting further allows us to isolate cells in specific cell cycle phases and subject them to transcriptomics and similar experimental techniques.
Fig. 1. Resolution of mammalian cell cycle phases (colours) based on four different fluorescent stains (axis labels) with a BD Fortessa flow cytometer.
Funding – Warwick Integrative Synthetic Biology Centre (WISB), BBSRC/EPSRC (BB/M017982/1).
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