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

Development and Evaluation of Mouse Monoclonal Antibodies Against Human C1q

The complement protein C1q plays an important role in breast cancer susceptibility, development, and progression. Mice genetically deficient in C1qA exhibit an eighty-five percent decrease in mammary tumour incidence when administered the chemical carcinogen DMBA. Similarly, in the aggressive MMTV-PyMT model of mouse mammary cancer, C1qA null mutant mice exhibit two thirds of the tumour burden seen in wild type mice and a third of the late-stage carcinoma at eighteen weeks of age. There may also be a role for C1q in human breast cancer as women genetically deficient in C1q have a decreased incidence of breast cancer. C1q plays a key role in macrophage-mediated efferocytic uptake of dying mammary epithelial cells, thereby preventing apoptotic cells from becoming necrotic and releasing pro-inflammatory cytoplasmic components. Persistence of necrotic cells alters the immune response during cancer initiation, as evidenced by an increase in cytotoxic T cells mobilised to the mammary gland of C1q null mutant mice in response to carcinogen DMBA. The over-arching goal of this research project was to generate and characterise an inhibitor of C1q-mediated efferocytosis. A monoclonal antibody was chosen as the C1q inhibitor, to further explore the role of C1q in breast cancer and as a potential first step in development of a new breast cancer therapeutic. Human C1q was inoculated into C1qA null mutant mice to generate an antibody-mediated immune response to C1q. Three fusions of immune splenocytes from these mice yielded a total of 2,776 cultures of which 2,017 contained viable hybridomas. Antibody binding by enzyme-linked immunosorbent assay (ELISA) included both linear epitopes (contiguous amino acids) and conformational epitopes (binding to a three-dimensional structure), making this an ideal screening strategy. Screening of these cultures by ELISA identified eight hybridomas that bound C1q. Of these, four were successfully expanded and cultures established from single cells. Thus, four candidate monoclonal antibodies were generated: BHI1-1G4, BHI1-4D3, BHI3-3F6, and BHI3-8B9. Characterisation of these antibodies was performed to determine the specificity of their binding conditions. Binding of the monoclonal antibodies in assays that involve denaturation of proteins and presentation of linear epitopes was not observed. These assays included western blotting, immunohistochemistry on normal breast sections, and immunocytochemistry on a fixed macrophage cell line. An assay with potential to display conformational epitopes, immunocytochemistry on unfixed macrophages, also did not demonstrate monoclonal binding. The antibodies were also tested for binding to C1q by immunoprecipitation. This assay can detect conformational epitopes, and all four monoclonal antibodies were demonstrated to bind soluble C1q by this method. Combined, these studies suggested that all four candidate monocloncal antibodies bind only to intact native C1q and do not bind to denatured antigen. Identification of a monoclonal antibody that inhibits C1q-mediated efferocytosis required development of a bioassay that quantifies the functional activity of candidate antibodies. An in vitro efferocytosis assay was developed involving fluorescent green-labelled macrophages co-cultured with fluorescent red-labelled MDA-MB-231 breast cancer cells induced to undergo apoptosis by cross-linking the TRAIL receptor 2. Co-localisation of red and green fluorescence as an indicator of efferocytosis was investigated in bone marrow-derived macrophages from C1qA replete and null mice. Reduced efferocytosis was observed in macrophages where C1q was absent and was quantified using ImageJ software involving digital masking of images and Boolean calculation of overlap. Hybridoma supernatants from candidate antibodies BHI1-1G4 and BHI1-4D3 were tested in the assay using the mouse macrophage cell line RAW 264.7 labelled green and dying breast cancer cells labelled red. Commercially available anti-C1q antibody 9A7 and hybridoma supernatant from non-C1q binding cell line BHI3-2C12 were also assessed. The antibodies exhibited variable capacity to affect C1q-mediated phagocytosis however whether a specific candidate monoclonal antibody could effectively inhibit C1q was inconclusive due to a high degree of variability in the timing of apoptotic cell uptake. This research led to the generation of four candidate monoclonal antibodies with potential for further pre-clinical research. Future work should concentrate on purification of the antibodies in order to improve investigation of their inhibitory capacity in C1q-mediated efferocytosis bioassays. Studies in mouse mammary cancer models would also provide valuable data on the potential of these candidate antibodies for downstream clinical applications in breast cancer patients.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

Using systems biology to investigate how age-related changes in TGFβ signalling alter pro-inflammatory stimuli

PhD ThesisOsteoarthritis (OA) is a degenerative condition caused by dysregulation of multiple molecular signalling pathways. This dysregulation results in damage to cartilage, a smooth and protective tissue that enables low friction articulation of synovial joints. Matrix metalloproteinases (MMPs), especially MMP13, are key enzymes in the cleavage of type II collagen which is a vital component for cartilage integrity. Various stimuli have been identified as inducers of MMP expression such as excessive load, injury and inflammation. Although previously considered a non-inflammatory arthritis, recent research has shown that inflammation may play an important role in OA development. A novel meta-analysis of microarray data from OA patients was used to create a cytoscape network representative of human OA. This enabled the identification of key processes in OA development, of which inflammation was prominent. Examining various different signalling pathways highlighted a role for transforming growth factor beta (TGFβ) in protecting against pro-inflammatory cytokine-mediated MMP expression. Indeed, TGFβ plays key roles in all facets of cartilage biology including development and maintenance of cartilage integrity. With age there is a change in the ratio of two TGFβ type I receptors (ALK1/ALK5), a shift that results in TGFβ losing its protective role in cartilage homeostasis. Instead, TGFβ promotes cartilage degradation and this correlates with the spontaneous development of OA in murine models. However, the mechanism by which TGFβ protects against pro-inflammatory responses and how this changes with age has not been extensively studied. Mathematical modelling has previously revealed how stochastic changes in TGFβ signalling during ageing led to the upregulation of MMPs. I have expanded the TGFβ section of this model to incorporate the pro-inflammatory stimulus interleukin-1 (IL-1) + oncostatin M (OSM) in order to investigate how TGFβ mediates MMP repression, specifically MMP-13. TGFβ signalling appears to interact with the activator protein 1 (AP-1) complex, which has an important role in MMP upregulation. However, the model indicates this interaction alone is insufficient to mediate the full effect of TGFβ, predicting it may also reduce MMP-13 mRNA stability. Furthermore, the model enabled me to predict how age alters these interactions; it suggested TGFβ would provide limited repression with a prolonged inflammatory response. Combining the modelled genes with the microarray network provided a global overview of how alterations in one pathway can affect others and lead to OA development. This study therefore demonstrates the power of combining computational biology with experimentally-derived data to provide insight into the importance of TGFβ signalling, and how age-related changes can lead to cartilage damage and OA development.Centre of Integrated Research into Musculoskeletal Ageing (CIMA), Arthritis research UK and the Medical Research Counci

The role of the aryl hydrocarbon receptor (AHR) in immune regulation in the human intestine

Environmental factors play a major role in the development of inflammatory bowel disease (IBD). However, current understanding about how environmental factors influence disease is limited and there is a major unmet need for advice to allow patients and at-risk individuals to modify their risk of disease. The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor and functions as an environmental sensor. Diverse murine studies have shown activating this receptor has immunoregulatory consequences and is beneficial in models of IBD. However, few studies have examined this pathway in the human intestine. It has been proposed that low AHR activity may play a role in IBD aetiopathology and AHR stimulation is beneficial. In this study a quantitative measure of AHR activation, CYP1A1 gene expression, is optimised and validated. This assay is used show that the AHR pathway is not less active in IBD than health. Unexpectedly the AHR pathway is more active and sensitive to stimulation in non-haematopoietic cells than intestinal immune cells and this difference is greater in Crohn’s disease than health. I demonstrate an important consequence of this difference could be an increased ability of intestinal stromal cells to degrade AHR ligands. The impact of AHR activation on intestinal immune cells is not known. This is of particular importance now medication targeting AHR has entered clinical trials. In this study, single cell sequencing is used to precisely characterise the immune cells expressing AHR. AHR expression is seen in a minority of cells (6.4%) and the majority of these cells are lymphocytes, although AHR expression is seen in antigen presenting cells and other cell types. I determine the impact of AHR stimulation in intestinal immune cells using RNASeq. This work newly identifies a number of genes associated with IBD as regulated by AHR. In addition, novels of effects of AHR signalling including pH sensing, cytoskeletal and microtubule arrangement and Rap 1 Signalling are reveal and provide many avenues for future research

Initial Analysis of Tumour Necrosis Factor alpha Promotor Activation by Interferon gamma using Boolean Logic Modelling

<p>A report produced as part of the MSc in Genomics and Pathway Biology course at the Division of Pathway Medicine, University of Edinburgh. (http://www.ed.ac.uk/schools-departments/pathway-medicine).  Note, that this has not been peer-reviewed.</p