CISBIC Sub-project 3: Innate Immune Signalling

This project addresses issues related to the innate immune response. In a fundamental process that is conserved between plants, insects and mammals, the initial encounter between host and pathogen involves recognition of conserved microbial components by a set of pattern recognition receptors (PRRs; including the "toll-like receptors", mannose receptor, and scavenger receptors) on the surface of the host cell. Signals from these receptors are integrated by the host cell, triggering an innate response customised to reflect the degree of “danger” posed by the particular pathogen.

Importantly, it is becoming clear that any one pathogen triggers both pro- and anti-inflammatory cascades by ligating different PRRs, possibly even through differentially glycosylated forms of the same signalling ligand. By altering their repertoire of surface-expressed ligands, therefore, pathogens can influence the nature of the immune response in a way that may allow them to evade effective immunity.

An important additional component of the infection-associated signalling cascade has been demonstrated at Imperial College and by others. The Notch receptor and Notch ligand families are expressed by cells of the immune system and fundamentally influence the outcome of immune stimulation. Triggering of Notch receptors on T cells by the Delta-like1 ligand expressed on antigen-presenting cells converts a normally IFNg-dominated response to an IL-10-dominated response, for example, and the pattern of Notch ligands on antigen-presenting cells is in turn regulated by TLR signalling. The Notch pathway therefore represents a direct link between TLR signalling and the adaptive immune response. There are 4 mammalian Notch receptors and 5 ligands; little is known about the differential regulation of their expression on cells of either innate or adaptive immune systems.

This sub-project will use a systems biology approach to study how signals from multiple PRRs are combined within an antigen-presenting cell, and how this is integrated with Notch signalling to shape the subsequent adaptive response. A limited amount of modelling has been done for TLR signalling and transcription factors such as NFkB. We will build on this by a combination of experimental and modelling approaches. The experimental approach undertaken within the current exemplar will focus on a mammalian cell model, but the project will be closely linked with parallel research efforts in other systems. Comparative biology across plant, insect and animal kingdoms has been highly informative in investigation of innate immune mechanisms and we anticipate synergy between this project and the strong research programme of Profs Fotis Kafatos and Bob Sinden in the area of insect immunity and the plant pathogenesis group at Imperial College.

Organisms:

  • Mus musculus

Funding:

For more information about the outputs from this project or to discuss data reuse please contact bsshelp@imperial.ac.uk

CISBIC Sub-project 3

Project Members
  • Prof Michael Stumpf
  • Prof Maggie Dallman
  • Prof Jaroslav Stark
  • Dr Anna Rose (biology)
  • Dr Simon Moon (modelling)
  • Dr Frances Turner (statistics)
  • Daniel Silk (modelling)
Software
  • Gene Genie (Turner, Tomlinson)
    Online tool for analysis of public microarray data sets, enabling selection of gene sets that exhibit differential expression under specified conditions
  • ABC-SysBio: A tool for parameter inference and model selection (Stumpf)
    ABC-SysBio is a tool for parameter inference and model selection used in the analysis of Hes1 qRT-PCR data from sub-project 3
Datasets
  • Time-course analysis of Notch and TLR signalling cross-talk in dendritic cells (Rose, prepublication)
    Murine dendritic cells were differentiated from the bone marrow of 15 C57Bl/6 mice (8-12 weeks old) for 8 days in the presence of GM-CSF. On day 8, cells were harvested, pooled and 1.5x106 cells were seeded into 6-well plates pre-coated with the Notch ligand Jagged-1 (Fc fusion protein) or IgG1 (control) in the presence or absence of the TLR4 ligand LPS (100ng/ml). Cells from 3 wells per condition were harvested every 30 mins for 4 hrs.
  • Notch signalling via Delta-like-1 and cross-talk with TLR signalling in dendritic cells (Rose,prepublication)
    Murine dendritic cells were differentiated from the bone marrow of 20 C57Bl/6 mice (8-12 weeks old) for 8 days in the presence of GM-CSF. On day 8, cells were harvested, pooled and 1.5x106 cells were seeded into 6-well plates pre-coated with the Notch ligand Delta-like 1 or Jagged-1 (both were Fc fusion proteins) or IgG1 (control) in the presence or absence of the TLR4 ligand LPS (100ng/ml). Cells from 3 wells per condition were harvested at 0h, 1h, 2h and 4h.
  • Hes1 quantitative real-time PCR (Rose)
    Dendritic cells were differentiated from bone marrow. Rat Jgd1/humanFc fusion protein (R&D Systems) or human IgG1 (Sigma Aldrich) (control samples) were immobilized onto tissue culture plates (10 μg ml−1 in PBS) overnight at 4 °C. Dendritic cells were spun onto the plate and cells were collected at the appropriate time. Total RNA was isolated using the Absolutely RNA micro prep kit (Stratagene). Complementary DNA was generated from 125 ng of total RNA using an archive kit (Applied Biosystems). 1 μl of cDNA was used with PCR Mastermix and TaqMan primer and probes (both Applied Biosystems) and analysed on an Applied Biosystems 7500 PCR system. Cycle thresholds were normalized to 18S and calibrated to a PBS-treated control sample for relative quantification.
Publications

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