Macrophages are primary mediators of innate immunity, providing surveillance in every organ against potential pathogens. They bridge the innate and acquired immune systems, as both affecter and effecter cells of T and B lymphocyte directed immunity. The destructive potential of activated macrophages generates much of the pathology of acute inflammatory states such as sepsis. Macrophages are widely distributed and contribute to normal metabolic processes such as tissue remodelling and repair, and are implicit to chronic autoimmune diseases such as Crohns Disease and arthritis. The innate immune system provides the first line of defence against infection, but the traditional focus of immunogenetic studies has been the acquired immune system. This thesis explores the transcriptional potential of macrophage populations under a single pathogenic stimulus - Lipopolysaccharide (LPS) - and examines the influence of genotype on the response generated using the inbred mouse strains C57B16/J, BALB/c, DBA/2J, C3H/HeJTlr4/Lps-d and C3H/ARCLpsn.
The first chapter of this thesis provides a background to the model system and tools used in this study, and sets these in the context of LPS signalling. It offers a comprehensive overview of LPS signalling mechanisms in macrophages, and provides a review of mouse models of infectious and inflammatory disease. Finally, the impact of genomic technologies on gene discovery in the immune setting is discussed.
The second chapter provides an overview of the international transcriptome effort, Functional- Annotation-of-The-Mouse-2 (FANT0M2). This chapter consists of three publications. The first is a description of the generation, curation, sequencing and prioritisation of the full-length cDNA libraries that fed into the FANT0M2 pipeline. The second is the major publication arising from FANT0M2, summarising analysis of transcriptional units, particularly in regard to genome annotation, classes of transcripts and alternative splicing. The third publication focuses on the Gnas imprinted genomic region, where a set of overlapping coding and non-coding transcripts are oppositely regulated. This study demonstrates the clarity that the FANT0M2 project has brought to complicated regions such as the Gnas complex, and provides experimental validation for the predictions made about this region through the FANTOM annotation process.
The third chapter focuses on the discovery of transcripts in activated macrophage populations. This work was supplementary to the FANT0M2 project, and provides new theoretical and functional insights into macrophage biology, and into the depth and complexity of the genome itself.
The fourth chapter used a series of inbred mouse lines to look for genetic networks underlying macrophage function. The data indicate that inbred strains differ greatly in their inducible gene expression profiles, suggesting that the basis for idiosyncratic strain responses and susceptibility to pathogen challenge. Paradoxically this diversity highlighted a conserved set of transcripts that is proposed to be dependant on a canonical receptor-mediated signalling pathway. This set allowed in-depth sequence analysis of transcription factor binding to identify core sequence motifs in macrophage activation. Finally, this chapter investigates novel susceptibility loci, predicted from the genomic clustering of transcriptional differences between two of the strains, BALB/c and C57B1/6J.
The fifth chapter compares Embryonic Stem (ES) cells and BMM from the same mouse strain, SV129/01a, This study demonstrated that ES cells were non-responsive to LPS, and that this was due to a lack of CD 14, one of components of the LPS-receptor complex. ES cells were also predicted to have poor responses to LPS because low levels of Tlr4 and the essential signalling molecule, Myd88, led to insufficiencies in the LPS signalling cascade, A comparison of transcriptional differences between the two cell types provided an insight into their respective phenotypes and growth characteristics. Examination of the transcriptional networks in comparison to an undifferentiated ES cell line, as well as in a LPS-activated time course, provided insights into the mechanisms of macrophage plasticity and inflammatory programs.
The sixth and final chapter of this thesis reviewed the genomic maps for two of the susceptibility loci identified from expression studies in the previous chapters. The Interferon (Ifn)□ locus is tightly linked with the Tlr4/Lps-d and Ifn□ loci, and the gene products of this region are predicted to alter the activation of the innate immune system. This region was identified from differences in the temporal induction of a set of genes, distinguishing "primed" C57B1/6J from "delayed" BALB/c mice. Chromosome 11 has been identified in at least three independent studies of pathogen susceptibility, and is a modifier of the response to gram-negative bacteria (Salmonella) and gram-positive bacteria (Bacillus). The transcriptional studies of the previous region have refined the region into two loci of oppositely regulated transcript clusters. This analysis overlaid the physical maps of the RIKEN clones with the genetic maps of the regions, as well as the MiT haplotype maps that predict regions of high and low polymorphism between the mouse strains. The bioinformatics approach taken in this chapter provides the foundation for future mapping and epigenetic studies of the regions.