14 research outputs found
Construction of a large scale integrated map of macrophage pathogen recognition and effector systems
<p>Abstract</p> <p>Background</p> <p>In an effort to better understand the molecular networks that underpin macrophage activation we have been assembling a map of relevant pathways. Manual curation of the published literature was carried out in order to define the components of these pathways and the interactions between them. This information has been assembled into a large integrated directional network and represented graphically using the modified Edinburgh Pathway Notation (mEPN) scheme.</p> <p>Results</p> <p>The diagram includes detailed views of the toll-like receptor (TLR) pathways, other pathogen recognition systems, NF-kappa-B, apoptosis, interferon signalling, MAP-kinase cascades, MHC antigen presentation and proteasome assembly, as well as selected views of the transcriptional networks they regulate. The integrated pathway includes a total of 496 unique proteins, the complexes formed between them and the processes in which they are involved. This produces a network of 2,170 nodes connected by 2,553 edges.</p> <p>Conclusions</p> <p>The pathway diagram is a navigable visual aid for displaying a consensus view of the pathway information available for these systems. It is also a valuable resource for computational modelling and aid in the interpretation of functional genomics data. We envisage that this work will be of value to those interested in macrophage biology and also contribute to the ongoing Systems Biology community effort to develop a standard notation scheme for the graphical representation of biological pathways.</p
The in silico macrophage: toward a better understanding of inflammatory disease
Macrophages function as sentinel, cell-regulatory hubs capable of initiating,
perpetuating and contributing to the resolution of an inflammatory response,
following their activation from a resting state. Highly complex and varied gene
expression programs within the macrophage enable such functional diversity. To
investigate how programs of gene expression relate to the phenotypic attributes
of the macrophage, the development of in silico modeling methods is needed.
Such models need to cover multiple scales, from molecular pathways in
cell-autonomous immunity and intercellular communication pathways in tissue
inflammation to whole organism response pathways in systemic disease. Here, we
highlight the potential of in silico macrophage modeling as an amenable and
important yet under-exploited tool in aiding in our understanding of the immune
inflammatory response. We also discuss how in silico macrophage modeling can
help in future therapeutic strategies for modulating both the acute protective
effects of inflammation (such as host defense and tissue repair) and the
harmful chronic effects (such as autoimmune diseases).Comment: 7 pages plus 1 figur
Visualisation of BioPAX Networks using BioLayout Express (3D).
BioLayout Express (3D) is a network analysis tool designed for the visualisation and analysis of graphs derived from biological data. It has proved to be powerful in the analysis of gene expression data, biological pathways and in a range of other applications. In version 3.2 of the tool we have introduced the ability to import, merge and display pathways and protein interaction networks available in the BioPAX Level 3 standard exchange format. A graphical interface allows users to search for pathways or interaction data stored in the Pathway Commons database. Queries using either gene/protein or pathway names are made via the cPath2 client and users can also define the source and/or species of information that they wish to examine. Data matching a query are listed and individual records may be viewed in isolation or merged using an 'Advanced' query tab. A visualisation scheme has been defined by mapping BioPAX entity types to a range of glyphs. Graphs of these data can be viewed and explored within BioLayout as 2D or 3D graph layouts, where they can be edited and/or exported for visualisation and editing within other tools
Controlling the Response: Predictive Modeling of a Highly Central, Pathogen-Targeted Core Response Module in Macrophage Activation
We have investigated macrophage activation using computational analyses of a compendium of transcriptomic data covering responses to agonists of the TLR pathway, Salmonella infection, and manufactured amorphous silica nanoparticle exposure. We inferred regulatory relationship networks using this compendium and discovered that genes with high betweenness centrality, so-called bottlenecks, code for proteins targeted by pathogens. Furthermore, combining a novel set of bioinformatics tools, topological analysis with analysis of differentially expressed genes under the different stimuli, we identified a conserved core response module that is differentially expressed in response to all studied conditions. This module occupies a highly central position in the inferred network and is also enriched in genes preferentially targeted by pathogens. The module includes cytokines, interferon induced genes such as Ifit1 and 2, effectors of inflammation, Cox1 and Oas1 and Oasl2, and transcription factors including AP1, Egr1 and 2 and Mafb. Predictive modeling using a reverse-engineering approach reveals dynamic differences between the responses to each stimulus and predicts the regulatory influences directing this module. We speculate that this module may be an early checkpoint for progression to apoptosis and/or inflammation during macrophage activation
DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells
BACKGROUND: The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs). RESULTS: Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules. CONCLUSIONS: The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies
The mEPN scheme: an intuitive and flexible graphical system for rendering biological pathways
<p>Abstract</p> <p>Background</p> <p>There is general agreement amongst biologists about the need for good pathway diagrams and a need to formalize the way biological pathways are depicted. However, implementing and agreeing how best to do this is currently the subject of some debate.</p> <p>Results</p> <p>The modified Edinburgh Pathway Notation (mEPN) scheme is founded on a notation system originally devised a number of years ago and through use has now been refined extensively. This process has been primarily driven by the author's attempts to produce process diagrams for a diverse range of biological pathways, particularly with respect to immune signaling in mammals. Here we provide a specification of the mEPN notation, its symbols, rules for its use and a comparison to the proposed Systems Biology Graphical Notation (SBGN) scheme.</p> <p>Conclusions</p> <p>We hope this work will contribute to the on-going community effort to develop a standard for depicting pathways and will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest.</p
Modelling and analysis of macrophage activation pathways
Macrophages are present in virtually all tissues and account for approximately 10% of
all body mass. Although classically credited as the scavenger cells of innate immune
system, ridding a host of pathogenic material and cellular debris though their
phagocytic function, macrophages also play a crucial role in embryogenesis,
homeostasis, and inflammation. De-regulation of macrophage function is therefore
implicated in the progression of many disease states including cancer, arthritis, and
atherosclerosis to name just a few. The diverse range of activities of this cell can be
attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its
physiology depending on its environment; for instance in response to different types of
pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by
the macrophages capacity to adjust rapidly its transcriptional profile in response to a
given stimulus. This includes interferons which are a group of cytokines capable of
activating the macrophage by interacting with their cognate receptors on the cell. The
different classes of interferons activate downstream signalling cascades, eventually
leading to the expression (as well as repression) of hundreds of genes.
To begin to fully understand the properties of a dynamic cell such as the macrophage
arguably requires a holistic appreciation of its constituents and their interactions.
Systems biology investigations aim to escape from a gene-centric view of biological
systems. As such this necessitates the development of better ways to order, display,
mine and analyse biological information, from our knowledge of protein interactions
and the systems they form, to the output of high throughput technologies. The
primary objectives of this research were to further characterise the signalling
mechanisms driving macrophages activation, especially in response to type-I and type-
II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to
data analysis and modelling. In order to achieve this end I have explored and
developed methods for the executing a ‘systems-level’ analysis. Specifically the
questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the
similarities and differences between the macrophage response to different types of
interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the
macrophage transcriptome affected by siRNA targeting of key regulators of the
interferon pathway? (d) To what extent does a model of macrophage signalling aid
interpretation of the data generated from functional genomics screens?
There is general agreement amongst biologists about the need for high-quality
pathway diagrams and a method to formalize the way biological pathways are
depicted. In an effort to better understand the molecular networks that underpin
macrophage activation an in-silico model or ‘map’ of relevant pathways was
constructed by extracting information from published literature describing the
interactions of individual constituents of this cell and the processes they modulate
(Chapter-2). During its construction process many challenges of converting pathway
knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be
addressed. The final model comprised 2,170 components connected by 2,553 edges,
and is to date the most comprehensive formalised model of macrophage signalling.
Nevertheless this still represents just a modest body of knowledge on the cell. Related
to the pathway modelling efforts was the need for standardising the graphical
depiction of biology in order to achieve these ends. The methods for implementing this
and agreeing a ‘standard’ has been the subject of some debate. Described herein (in
Chapter-3) is the development of one graphical notation system for biology the
modified Edinburgh Pathway Notation (mEPN). By constructing the model of
macrophage signalling it has been possible to test and extensively refine the original
notation into an intuitive, yet flexible scheme capable of describing a range of
biological concepts. The hope is that the mEPN development work will contribute to
the on-going community effort to develop and agree a standard for depicting
pathways and the published version will provide a coherent guide to those planning to
construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse
macrophages to interferon stimulation, genome wide expression profiling was
performed and an explorative-network based method applied for analysing the data
generated (Chapter-4). Although transcriptomics data pertaining to interferon
stimulation of macrophages is not entirely novel, the network based analysis of it
provided an alternative approach to visualise, mine and interpret the output. The
analysis revealed overlap in the transcriptional targets of the two classes of interferon,
as well as processes preferentially induced by either cytokine; for example MHC-Class
II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by
IFN-β. To further investigate the contribution of individual proteins towards generating
the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress
the expression of selected target genes. However in macrophages and other cells
equipped with pathogen detection systems the act of siRNA trasfection can itself
induce a type-I interferon response. It was therefore necessary to contend with this
autocrine production of IFN-β and optimise an in vitro assay for studying the
contribution of siRNA induced gene-knock downs to the interferon response
(described in Chapter-5). The final assay design incorporated LPS stimulation of the
macrophages, as a means of inducing IFN-β autonomously of the transfection induced
type-I response. However genome-wide expression analysis indicated the targeted
gene knock-downs did not perturb the LPS response in macrophages on this occasion.
The optimisation process underscored the complexities of performing siRNA gene
knockdown studies in primary macrophages. Furthermore a more thorough
understanding of the transcriptional response of macrophages to stimulation by
interferon or by LPS was required. Therefore the final investigations of this thesis
(Chapter-6) explore the transcriptional changes over a 24 hour time-course of
macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage
pathway model in interpreting the response to the three stimuli.
Taken together the work described in this thesis highlight the advances to be made
from a systems-based approach to visualisation, modelling and analysis of macrophage
signalling
Identification of novel candidate genes for regulation of follicle selection in the avian ovary
Selective breeding of chickens for high growth rate and other production traits has led
to the modern commercial broiler, a bird that has the genetic potential for reaching an
average body weight of 2.7kg within 6 weeks of hatch. However, the breeding stock for
modern broilers has to be feed controlled in order to lay large numbers of viable
hatching eggs. Broiler breeders, when fed ad libitum, have a propensity to produce
internal ovulations, double-yolked, misshapen or shell-less eggs. This is due to the
release of multiple ova at ovulation, which results in a significant loss of production.
Feed control has been shown to mitigate this effect but welfare concerns have been
raised as to the side-effects for the birds. The main objective of this research was to
determine the genetic basis for the regulation of ovarian follicle selection and its
dysfunction in ad libitum-fed broiler breeders, and how this might be addressed by
genetic selection to limit the impact on the management and welfare of future broiler
breeders.
A multi-layered statistical, expression profiling and cluster analysis of ovarian gene
expression data from a microarray study was carried out to identify candidate genes for
further study.Key stages of development were investigated for feed restricted and ad
libitum-fed broiler breeders. Several gene candidate genes were validated by qPCR in a
comparison of different ovarian tissues in layer type hens for subsequent analysis in
broiler breeders. Sequencing of the founders of an Advanced Intercross Line (AIL) of
commercial broiler breeders and White Leghorn layers was performed covering 3
regions of each of the primary candidate genes in order to identify genetic variation that
could account for differences in follicle number between broilers and layers.
Expression data from a microarray study highlighted a number of potential candidate
genes for regulation of follicle development. One of these genes, Platelet Derived
Growth Factor Receptor Like (PDGFRL), shares significant sequence homology with
the active domains of Platelet Derived Growth Factor Receptor β. Expression profiling
in layers showed peak PDGFRL expression in 5-6 mm follicles and the F2 follicle (P
<0.001). PDGFRL was also up-regulated in response to ad libitum feeding in broiler
breeders in 6-8 mm follicles (P<0.016), the point at which follicle selection and
recruitment is considered to occur. In addition to this, while PDGFRL expression
remains relatively constant between tissues under ad libitum conditions, it shows a clear
reduction in expression (P <0.001) in prehierarchical follicles relative to the stroma and
the F1 follicle under feed restriction. This observation is consistent with results from the
original microarray study. Sequencing of the AIL Founders highlighted several SNPs in
the broiler that have the potential to be used as markers for incorporation into
commercial selection programs. EST alignment in preparation for targeted sequencing
of PDGFRL also highlighted three potential forms of the protein, each with a different
5’ starting sequence. Initial investigation has shown all three to be expressed in ovarian
follicles. QPCR in a panel of 13 tissues shows marked differences between the 3
variants, implying different and perhaps specialised roles for each. The PDGFR family
has a potential role in steroidogenesis, and the expression profiling, combined with the
clear effect on expression from ad libitum feeding in broiler breeders, suggest that
PDGFRL is a strong candidate for involvement in the regulation of follicle development
GDF9, shown to be associated with multiple ovulation in sheep, and FSH receptor, a
mediator of neuroendocrine signalling to the ovary, were also investigated. They
behaved as expected in layer type birds but both showed significant differential
expression (P = 0.005 and 0.018 respectively) as a result of ad libitum feeding in broiler
breeders. Though these two genes have been extensively investigated, these are
previously unobserved effects. SNPs have also been identified in these genes which
have the potential to be used as markers for incorporation into commercial selection
programs. To fully exploit these results, additional investigation is recommended to
confirm these results in commercial populations and to determine how they can be
employed to best effect