The first wave of the inducible gene network up-regulated by pathogen-stimulated mononuclear cells encodes a variety of effector proteins with pleitropic biological activities. This class of primary immediate early (IE) genes codes for potent pro-inflammatory cytokines and chemokines that play a prominent role during the manifestation of inflammatory response. In an attempt to better understand induction mechanisms for such genes, I have focused on those coding for human interleukin-1β (IL1B) and tumor necrosis factor α (TNF), which exhibit both transient IE induction as well as cell-type restriction. Employing a combined approach using cell lines and primary cells, reporter transient transfection, chromatin conformational capture and immunoprecipitation, evaluation of transcript integrity, ectopic expression in a non-competent cell type, and comparison to mouse orthologs, I have determined that a complex array of mechanisms interplay in order to distinctly regulate the Toll-like receptor (TLR) signaling-dependent induction of these two important pro-inflammatory genes whose deregulation provides the etiology for numerous diseases. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), which are the hallmarks of poised IE genes. In contrast, IL1B is stringently regulated by long-distance chromosome gyrations, multistep activation through a unique doubly-paused Pol II which, in association with the monocyte lineage factor Spi1/PU.1 (Spi1), maintains a low TBP and Pol II occupancy prior to activation. Activation and DNA binding of the transcription factors C/EBPβ and NF-κB resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of the positive elongation factor b (P-TEFb). This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Nucleosome occupancy and chromatin modification analyses of the IL1B and TNF promoters, revealed activation-specific changes in chromatin marks that are supportive for nucleosome clearance and formation of nucleosome free regions (NFR). Furthermore, ectopic expression of Spi1, along with a TLR surrogate (over-expressed TNF receptor associated factor 6, TRAF6), in a cell line incompetent for IL1B transcription, is observed to prime the cell's endogenous genome for IL1B induction by appropriately phasing promoter nucleosomes and recruiting paused Pol II in a manner reminiscent of that observed in competent monocytes. Here I report a novel connection between the metabolic state of cells and HIF-1α in regulating murine Il1b gene expression. With regard to the lipopolysaccharide (LPS) unresponsive state known as endotoxin tolerance, my data revealed that following transient induction, IL1B and TNF remained marked with paused Pol II complexes for up to 24 hours post-stimulation. Upon subsequent LPS exposure, tolerized TNF remained in an unresponsive paused state, while IL1B resumed transcription due to recruitment of positive elongation kinase P-TEFb. Emerging evidence suggests that inflammatory responses of LPS/TLR4 activated macrophages are interconnected with metabolic pathways, resulting in the shift of energy utilization by the cells. Here I report that inhibition of either phosphoinositide 3-kinase (PI3K) or glucose metabolism had a greater affect on the transcriptional response of Il1b than of Tnf. The differences between these two genes, especially for endotoxin tolerance, suggest that il1b may play a distinct role from tnf in chronic inflammation. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

Background: Psoriasis is a complex disease with a genetic component contributing to disease pathogenesis. Chronic plaque psoriasis can be dichotomised into two subtypes according to age of onset; type 1 (early-onset; <40 years) and type 2 (late-onset; ≥40 years). Despite clinical and biological differences between the two subtypes, the genetics underpinning late-onset psoriasis remains poorly characterised compared to early-onset psoriasis. Aims: The aim of this project was to identify genetic loci associated with late-onset psoriasis, to assess the overlap of loci with early-onset psoriasis and to elucidate the functional role of the identified variants. Methods: The study had three parts; the first was a candidate-gene association study of the IL1B gene. A total of 16 SNPs from the region were genotyped in 595 late-onset and 1,137 early-onset psoriasis samples and compared to 4,770 controls from the European population. The second was a large-scale study conducted in 543 late-onset psoriasis and 4,373 controls using the Immunochip array. The third was a functional study using bioinformatics data mining, chromatin immunoprecipitation and electrophoretic mobility shift assay techniques to analyse the role of a disease-associated variant at the biological level. Results: The candidate-gene study replicated a previously reported association at a promoter polymorphism, rs16944 (P<0.05), within the IL1B gene and discovered a novel association at a second variant, rs11687624 (P<3.12x10-3), in late-onset psoriasis. None of the variants analysed were significantly associated with early-onset psoriasis. Bioinformatic eQTL data suggests the two variants and their proxies are associated with the expression of IL1A, IL1B, IL38 and PAX8. The Immunochip study identified 6 non-HLA loci (P<2.3x10-5) previously associated with early-onset psoriasis to also be associated with late-onset psoriasis (IFIH1, IL12B, IL23A, IL23R, TRAF3IP2 and ZNF313). Conditional analysis of the MHC region also identified two loci (HLA-C and HLA-A). A novel locus, IL1R1, was associated with late-onset psoriasis, but not early-onset psoriasis. Bioinformatic data mining found no role for the IL1R1 variants as eQTLs and prioritised the IL1B variant rs2708914 for functional analysis. The transcription factor STAT3 was found to be enriched at rs2708914 in keratinocyte and CD8+ T-lymphocyte cell lines. Allele-specific binding could not be established. Conclusions: This project is the largest genetic study of late-onset psoriasis to date and provides evidence that it shares susceptibility loci with early-onset psoriasis as well as having specific susceptibility loci. These findings provide further evidence for the dichotomisation of chronic plaque psoriasis, firstly to facilitate better understanding of the pathogenesis of the two subtypes and secondly to enable tailored therapy to be developed. Both have potential benefits for patients in the future. The genetic and functional studies conducted have provided a platform from which further studies can be carried out.