Towards systems biology analysis of multi-scale inflammatory signalling
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
Since the discovery of the first vaccine by Edward Jenner over 200 years ago, a major challenge in immunology has been to understand how an organism can protect itself against infections caused by foreign microorganisms such as viruses or parasites. We know now that the first line of defence against such threats is served by an innate immune system, which fights against invaders. However, a prolonged immune response is not beneficial for the organism and often contributes to various chronic diseases such as rheumatoid arthritis or asthma. Therefore, understanding the balance between the ability of the immune system to rapidly detect and remove foreign threats and to efficiently stop immune signalling is a fundamental yet not fully understood aspect of immunology. Inflammatory processes have been studied for years at the organism level (for example periodic fevers during malarial infections). However, it is critical to appreciate that inflammation starts from single cells that make up tissues. A cell has a range of sensors and intracellular mechanisms, which may recognise signals and respond by activating protective programmes. These aim to defend the infected cell, but also to warn neighbouring cells about incoming threats and shielding whole tissues from damage. Communication between such individual cells is controlled by group of small protein molecules, called cytokines that are produced and released into the extracellular environment. My recent theoretical work has predicted that even small changes in the timing of single-cell cytokine production might substantially affect the tissue-level inflammatory signalling. This perhaps suggests a new and exciting way of treating inflammatory disease. However in order to achieve it, a better understanding of the connection between single-cell behaviour and apparent tissue level outcome (e.g., in the inflamed arthritic joint) is required. The main goal of this Fellowship programme is to develop novel theoretical and experimental approaches for the analysis of such multi-scale systems. These methods will build up an understanding of tissue-level behaviour by developing new computational models that will allow the generation of computer predictions. (These new approaches will be relevant to other biological systems.) In order to build a more complete picture of inflammation, I will incorporate various cell signalling pathways into these models. I will measure cytokine levels in order to characterise the most important factors during the inflammatory response. I will quantify and visualise inflammatory processes in living single cells under the microscope. These movies will be used to develop accurate and realistic single-cell and tissue-level mathematical models that will be analysed to understand the control of inflammatory signalling (e.g. by investigating how specific connections in the cytokine network contribute to the tissue-level response). As a result, potential targets can be identified and selected for experimental confirmation. The work in this project will contribute to the current understanding of the mechanisms that underlie the control of inflammatory signals. This will assist with the identification of more efficient treatments of inflammatory diseases.
Technical Summary
I will develop an integrated systems biology programme aiming towards multi-scale analyses of complex biological systems. I previously applied mathematical modelling and live-cell imaging approaches to investigate the single-cell dynamics and function of the NF-kappaB system, a critical early mediator of inflammation. This work predicted that the tissue-level inflammatory signalling can be controlled through the subtle changes in single-cell dynamics through underlying genetic networks. Failure to resolve inflammation is often associated with diseases such as rheumatoid arthritis and asthma. Therefore, a key question is to quantitatively understand the control of inflammatory signalling in cells and tissues. In this Fellowship programme I will develop and apply a set of novel theoretical and experimental approaches based on my own experience, and the collaborators, expertise, and cutting-edge technologies available to me in Manchester. The theoretical work will develop: 1) Dynamical mathematical models of the cellular paracrine/autocrine network. 2) Assembly of single cell dynamical models describing interactions between key inflammatory transcription factors (such as STATs, IRFs and NF-kappaB) that can be used to interpret and direct experimental strategy, and provide experimentally-testable predictions. 3) State-of-the-art algorithms for stochastic spatial simulation and multi-scale models of inflammation. The experimental work will include quantitative cytokine profiling (for inference of paracrine/autocrine networks) and multi-parameter live-cell imaging (for visualisation of inflammatory interactions using custom-made microfluidic devices). Modelling and experimental approaches will be used iteratively to optimize experimental design. My goal is to understand this complex and non-linear system, in order to control inflammation and to treat inflammatory disease.
Planned Impact
I am fully committed to increase the likelihood of potential impact of this Fellowship project. I have indentified the following areas of future impact: 1. Academics and Clinicians: This project is an interdisciplinary systems biology project that studies key regulatory steps associated with propagation and resolution of inflammation. By its very nature it is of interest to immunologists, biomedical scientists and clinicians who are working in the field of inflammatory diseases. Developed novel multi-scale models will allow work across scales from molecules, cells and tissue and investigate complex regulation of diverse biological systems. These approaches will improve currently available models due to increased depth of molecular description of single cell regulation. These approaches will be of interest to systems biologists and mathematicians. I will engage with academics through high impact publications, talks at major conferences and professional group website. Whenever possible models will be made widely available. 2. Engagement with identified beneficiaries: In this project I have several named key external and internal collaborators. The data and knowledge that we generate in this project will be made available to them to assist with further development of the current understanding of inflammatory processes. I also hope to be able to incorporate data available through these collaborations into models which will benefit all groups. 3. Instrumentation companies: My aspiration is to strongly engage with instrumentation companies in order to develop experimental platforms and processing algorithms for efficient visualisation of tissue-level dynamical processes. 4. Pharmaceutical industry: The project is important for understanding how drugs may better control the resolution of inflammatory signalling. I have had active contacts with J. Unitt from AstraZeneca who was a co-author on our recent Science paper. The development of modelling approaches that predict how new anti-inflammatory drugs may functionally change the dynamics and function of inflammatory responses is an important objective. 5. Public Understanding of Science and external public relations: The use of microscopy provides a visual platform for the development of public understanding of science (PUS) and systems biology approaches. I will engage with the University of Manchester Press Office to coordinate media publicity and will develop a personal professional website to publicize results to scientists and also to the general public. Within FLS in Manchester there is excellent support for PUS activities. 6. Training: I will contribute to increasing the depth of systems biology training. I was already involved in training a group of biologists in mathematical modelling in Liverpool and realize the need for interdisciplinary expertise in systems biology. I will closely work with the project's PDRA to transfer my expertise in interdisciplinary research and develop his career in systems biology. I also believe that I can make personal contributions to development of mammalian cell systems biology by driving strategic interdisciplinary initiatives, such as the 'Insults and Inflammation' initiative (led by W. Müller) that aims towards rational clinical intervention strategies to help treat inflammatory diseases. 7. Exploitation: I will continuously monitor for commercially exploitable information and knowledge. In this respect, University of Manchester has a defined procedure for assessment of opportunities. I was already approached by Dr. A. Carcon (Commercialisation Executive, Manchester's Intellectual Property Limited) and I'm now well aware of appropriate routes.
People |
ORCID iD |
Pawel Paszek (Principal Investigator) |
Publications
Adamson A
(2016)
Signal transduction controls heterogeneous NF-?B dynamics and target gene expression through cytokine-specific refractory states.
in Nature communications
Assas MB
(2017)
Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor a neutralization.
in Clinical and experimental immunology
Bagnall J
(2015)
Quantitative dynamic imaging of immune cell signalling using lentiviral gene transfer
in Integrative Biology
Bagnall J
(2018)
Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation.
in Science signaling
Bagnall J
(2020)
Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling.
in Cell systems
Bagnall J.
(2012)
Single cell dynamics of macrophage activation and signalling
in IMMUNOLOGY
Brignall R
(2017)
Integration of Kinase and Calcium Signaling at the Level of Chromatin Underlies Inducible Gene Activation in T Cells.
in Journal of immunology (Baltimore, Md. : 1950)
Daniels MJ
(2016)
Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer's disease in rodent models.
in Nature communications
Diamond C
(2016)
Investigating IL-1ß Secretion Using Real-Time Single-Cell Imaging.
in Methods in molecular biology (Clifton, N.J.)
Description | 1) We have developed new understanding of how cells process inflammatory cues and make decisions. Our work (published in Nature Communications, 2016) showed that cells use dynamics and timing to make sense of different environmental inflammatory signals. In particular we show thet the key transcription factor NF-kappaB can be selective tuninned to different signals, to allow more information to be transferred. We described that inflammatory cell responses are inherently variable, but how cells respond (in terms of inflammatory NF-kappaB signalling) might be imprinted. 2) We developed new understanding of how Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue (Since signalling, 2018). In macrophage populations, TLR4 stimulates the dose-dependent transcription of NF-kappaB target genes. However, we found that, when assessed with single RNA counting, individual cells exhibited a wide range (3 orders of magnitude) of expression of the gene encoding pro-inflammatory cytokine tumor necrosis factor (TNFa). The TLR4-induced TNFa transcriptional response correlated with the magnitude of the activation of NF-kappaB signaling and cell size. We compared the rates of TNFa production and uptake in macrophages and mouse fibroblasts and generated a mathematical model to explore macrophage TLR4 response heterogeneity and TNFa signal propagation in the tissue. The model predicted that the local propagation of TLR4-dependent TNF cytokine response and cellular NF-kappaB signaling is limited to small distances of few cell diameters between neighboring tissue-resident macrophage cells. In our predictive model, TNFa propagation was constrained by competitive TNFa uptake, rather than heterogeneous production. We proposed that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues, while avoiding out-of-context responses at long range. 3) We demonstrated that heterogeneity of TLR-dependent gene expression is determined by gene specific constrains (Cell Systems, pending submission). Using single cell transcriptomics and single molecule mRNA counting we demonstrate that key pro-inflammatory cytokines Interleukin 1a and 1ß (IL1a and IL1ß) are robustly expressed in only a subset of cells. In contrast, other genes, including Tumour Necrosis Factor alpha (TNFa), exhibit more ubiquitous expression patterns. By mathematically modelling mRNA count distributions, we show that the transcription of IL1ß (in contrast to TNFa ) does not conform to a standard stochastic burst model. Critically, the heterogeneous expression of IL1a and IL1ß is coordinated in individual cells via shared transcription sites. In agreement, chromatin modulation alters mRNA distributions, resulting in quantitative changes to underlying kinetic rates. The mRNA mean/variance relationships are constrained along linear trends. This demonstrates that gene specific architecture determines the heterogeneity of the TLR4-mediated gene expression responses. 4) We developed new molecular biology tools for live-cell imaging analyses of immune cell signalling. We have optimised a lentiviral system for generation of stable immune cell lines expressing fluorescent and luminescent reporters/fusions for time-lapse, multi-parameter live-cell imaging and developed an automated workflow for quantification of number of molecules using fluorescence correlation spectroscopy (Bagnall et al, Integrative Biology). This is a key enabling technology in the lab and a step change in the field, which forms a basis for collaborations resulting in a number of high impact papers (Elife, Nature Communications, Cell Death Differentiation). |
Exploitation Route | Our work provides fundamental new understanding of inflammatory signalling, which will be required for better understanding of immune response and infection biology. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | https://www.research.manchester.ac.uk/portal/pawel.paszek.html |
Description | This project offers a great potential for a better understanding of cell signalling and decision-making pathways involved in inflammation and innate immunity. This in turn creates the opportunity for the identification of better drug targets and more efficacious modes of treatment of disease. This is important for applied research and is of relevance to healthcare and to the pharmaceutical industry. By its very nature this project will be of specific interest to immunologists, biomedical scientists and clinicians who are working in the field of normal inflammatory control and disease. Datasets and mathematical models developed are shared with the community via relevant repositories (E.g., ArrayExpress or Pride). The new genetic constructs and cell lines generated are of use to the wider bio-imaging community. In Manchester, there is considerable interest in using and developing our resources for neuro-inflammation, immunology, gut physiology, circadian clock, stem cell, cancer and cell signalling research. Our constructs (developed for the recent paper, Bagnall et al., 2015) have already been shared with >20 different groups locally in UK as well internationally including the NF-kappaB community in the USA. The use of microscopy generates movies and images that are colourful and visual. They represent an excellent resource for the development of public understanding of science. Group members have participated in several public engagement activities including "The Worm Wagon", "Fabulous Physics" and FLS Community Open Day. We are planning to contribute to similar exhibitions, and talking to schools and other groups. |
First Year Of Impact | 2013 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Cultural,Societal |
Description | A*Star PhD Studenship |
Amount | £15,000 (GBP) |
Organisation | University of Manchester |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2017 |
End | 09/2021 |
Description | BBSRC DTC PhD Studentship |
Amount | £10,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2012 |
End | 09/2015 |
Description | BBSRC DTP PhD Studentship |
Amount | £32,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2012 |
End | 09/2016 |
Description | BBSRC project grant (responsive mode) |
Amount | £505,000 (GBP) |
Funding ID | BB/R007691/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2018 |
End | 03/2021 |
Description | BBSRC sLoLa Grant (co-investigator) |
Amount | £4,160,524 (GBP) |
Funding ID | BB/K003097/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2013 |
End | 04/2017 |
Description | DTP PhD studentship |
Amount | £100,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2018 |
End | 10/2022 |
Description | ISSF Research Grant |
Amount | £35,604 (GBP) |
Funding ID | 097820/Z/11/B |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2013 |
End | 09/2014 |
Description | ISSF Research Training Grant |
Amount | £2,158 (GBP) |
Funding ID | 097820/Z/11/B |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2013 |
End | 09/2014 |
Description | ISSF Research grant |
Amount | £12,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2015 |
End | 09/2016 |
Description | MRC Discovery award: "Capacity Building in Single Cell Inflammation Discovery: Developing the Next Generation of Scientists" |
Amount | £750,000 (GBP) |
Funding ID | MC_PC_15072 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2017 |
Description | Royal Society Research Grant |
Amount | £15,000 (GBP) |
Funding ID | RG120445 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2013 |
End | 04/2014 |
Description | Understanding heterogeneity of immune cell responses through analysis of transcriptional bursting |
Amount | £120,000 (GBP) |
Funding ID | 220025/Z/19/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2019 |
End | 09/2022 |
Description | Wellcome Trust PhD studentship programme in Quantitative and Biophysical Biology |
Amount | £1,000,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2020 |
Title | Lentiviral system for delivery of fluorescent/luminescence probes for live-cell imaging |
Description | Developed a flexible lentiviral system for transduction of fluorecently/luminoscet fusion/reporter for studies of cellular signalling system in living single-cells. |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | Resarch enabling technology, development of new research ideas and extending collaborations. |
Title | Analysis of transcriptional bursting of TLR-dependent genes |
Description | Models and algorithms for the theoretical argument in the J. Bagnall, W. Rowe, N. Alachkar, J. Roberts, H. England, C. Clark, M Platt, D. A .Jackson, M. R. Muldoon and P. Paszek, Gene-Specific Linear Trends Constrain Transcriptional Variability of the Toll-like Receptor Signaling, Cell Systems 11, 1-15, 2020 |
Type Of Material | Computer model/algorithm |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Sharing tools in the community |
URL | https://github.com/ppaszek/transcriptionalBursting |
Title | Data from: Dynamic NF-?B and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation |
Description | |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Title | Integration of kinase and calcium signaling at the level of chromatin underlines inducible gene activation in T cells |
Description | GEO submission of ATAC-seq and RAN-seq analysis of T-cell signalling |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | Resource |
Title | Mathematical model of NF-kB signalling |
Description | Mathematical mode to simulate dynamical NF-kB signalling |
Type Of Material | Computer model/algorithm |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | Sharing tools with other researchers |
URL | https://www.nature.com/articles/ncomms12057 |
Title | Secretome analysis of macrophage signalling |
Description | Secretome analysis of RAW264.7 cells in response to TLR4 agonist |
Type Of Material | Database/Collection of data |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Resources for use to other researchers |
URL | http://www.ebi.ac.uk/pride/archive/projects/PXD001905 |
Title | Single-cell transcriptomics analysis of TRL-induced macrophages |
Description | Single-cell transcriptomics analysis of Lipid A stimulated RAW264.7 cell line using Fluidigm C1 system. |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Sharing data with community |
URL | https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9219/ |
Title | Transcriptome analysis of macrophage signalling |
Description | Gene expression analysis of TLR4 signalling in RAW 264.7 macrophage cell line |
Type Of Material | Database/Collection of data |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | Incasing depth of knowledge |
URL | https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3155/ |
Description | Collaboration with the SysmedIBD consortium |
Organisation | SysMedIBD Consortium |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration with SysmedIBD, a £12m EU Systems Medicine of Chronic Inflammatory Disease Consortium (12/2012-2017) lead by Manchester, involving 12 European institutions. I formally joined the project as Co-I in Manchester with management responsibilities for 1 PDRA (from 1st September 2013). As a part of the collaboration we are providing reagents and training. |
Collaborator Contribution | Development of transgenic mice for NF-kB system |
Impact | Multidisciplinary involving mathematicians, biologists and clinicians. |
Start Year | 2013 |
Description | Fabulous Physics |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | A PhD student in my lab helped at an event called Fabulous Physics in the Physics Department at Loreto College, this was in collaboration with the Institute of Physics and The Ogden Trust. This event was an opportunity for GCSE students who might be considering studying physics beyond year 11 to explore the subject even more |
Year(s) Of Engagement Activity | 2014 |
Description | Personal invitation to International Meeting on Computational Immunology |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Personal Invitation to a panel discussion on Computaional Immunology approaches. Specifically, this meeting discussed the upcoming challenges to the integration of computational and mathematical modelling into the experimental process. The meeting hadtwo main foci: (1) Combing experimentation with computational approaches (2) Advancing acceptance of modelling by the wider immunology community. Outcomes of the meeting we hope to see are: 1 - An understanding, from an experimental perspective, what modelling approaches have to offer and clear identification of their limitations 2 - An understanding of how to develop models and simulations which can provide a certain level of confidence in their validity and how the models map to the underlying immunological system 3 - A new network of researchers in the area of computational immunology and provide new, or improved collaborations and sharing of technologies |
Year(s) Of Engagement Activity | 2014 |
URL | https://www.york.ac.uk/computational-immunology/events/ |
Description | Physics Olympics |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | A PhD student I'm my lab helped at an event called Physics Olympics in the Physics Department at Loreto College. This event was an opportunity for GCSE students who might be considering studying physics beyond year 11 to explore the subject even more. |
Year(s) Of Engagement Activity | 2014 |