Biological Networks

Networks of coordinated interactions among biological entities govern a myriad of biological functions that span a wide range of both length and time scales—from ecosystems to individual cells and from years to milliseconds. For these networks, the concept “the whole is greater than the sum of its parts” applies as a norm rather than an exception. Meanwhile, continued advances in molecular biology and high-throughput technology have enabled a broad and systematic interrogation of whole-cell networks, allowing the investigation of biological processes and functions at unprecedented breadth and resolution—even down to the single-cell level. The explosion of biological data, especially molecular-level intracellular data, necessitates new paradigms for unraveling the complexity of biological networks and for understanding how biological functions emerge from such networks. These paradigms introduce new challenges related to the analysis of networks in which quantitative approaches such as machine learning and mathematical modeling play an indispensable role. The Special Issue on “Biological Networks” showcases advances in the development and application of in silico network modeling and analysis of biological systems

The Endothelial Cell Response to Inflammation, the Functional Role of the Endothelial-enriched Protein KANK3 and the Adipose Tissue Transcriptome

A compilation of three complementary projects explores various facets of endothelial cell biology and transcriptomics, illuminating the intricate dynamics underlying cellular responses to specific stimuli across different tissues. The first project examines how endothelial cells react to the inflammatory molecule tumour necrosis factor (TNF), by studying these cells over time after TNF exposure. We identified distinct gene expression patterns and revealed two central temporal phases of gene upregulation in the endothelial response. The induction of interferon response genes, without de novo interferon production, was further investigated. An online resource was developed for comprehensive data exploration (www.endothelial-response.org). The second project analysed adipose tissue to define cell type enriched transcripts and differences between the sexes and depot types. We found mesothelial cells to be the main driver for heterogeneity between subcutaneous and visceral adipose tissue. This data is accessible through the Human Protein Atlas. The third project focuses on KANK3, which was predicted to be an endothelial enriched gene in the previous study, and others from the group. Our findings show that KANK3 is endothelial specific in multiple tissues through the body, inhibition of KANK3 in endothelial cells affects cell motility, expression of blood clotting proteins on gene and protein level, and thrombin generation. Together, these projects enhance our understanding of endothelial cell responses to inflammation and detail the functional investigation of an uncharacterised endothelial protein. Each project offers a different perspective, by examining temporal responses, functional changes, and tissue-wide patterns. This multifaceted approach deepens our insights into cell biology and furthers our understanding of critical health processes

A systems based approach to neutrophil gene expression

Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, expressing an array of destructive molecules and antimicrobial processes that characterise the cells as front-line defenders of the innate immune system, thus neutrophils are crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune response. The molecular changes that underlie this regulation are poorly defined, yet represent an attractive area of research to fully elucidate the role and regulatory capacity of neutrophils within the immune response. RNA-Seq provides an accurate and robust mechanism for global characterisation of cellular transcripts. Neutrophils were isolated from healthy donors and incubated with or without inflammatory cytokines for 1 h. RNA was extracted and analysed by RNA-Seq using the SOLiD or Illumina platforms. Raw data was quantified using a number of software packages which formed a bioinformatic pipeline for data analysis which was developed during the course of the research. Results were validated by a selection of traditional laboratory functional assays. Priming of neutrophils by GM-CSF and TNFα was found to induce differential gene expression and activation of transcription factors, which led to differential regulation of apoptotic pathways. Stimulation of neutrophils with inflammatory cytokines/chemokines (IL-1β, IL-8, G-CSF, IFNγ) resulted in expression of discrete gene sets and differential activation of signalling pathways. Stimulation of neutrophils with IL-6 did not induce any significant expression of genes but result in activation of STAT signalling. Comparison of gene expression of neutrophils isolated by density gradient and magnetic bead preparation revealed significant differences in gene expression and function, in part attributable to levels of contamination associated with each isolation method. Bead isolation was found to enrich a more heterogeneous neutrophil population including a subpopulation of neutrophils expressing transcripts previously associated with low density granulocytes. Thus, RNA-Seq and bioinformatic analysis has provided a full characterisation of neutrophil gene expression under inflammatory conditions and identified several new areas of research that could lead to targeted drug design for the treatment of inflammatory disease

Measuring blood flow and pro-inflammatory changes in the rabbit aorta

Atherosclerosis is a chronic inflammatory disease that develops as a consequence of progressive entrapment of low density lipoprotein, fibrous proteins and inflammatory cells in the arterial intima. Once triggered, a myriad of inflammatory and atherogenic factors mediate disease progression. However, the role of pro-inflammatory activity in the initiation of atherogenesis and its relation to altered mechanical stresses acting on the arterial wall is unclear. Estimation of wall shear stress (WSS) and the inflammatory mediator NF-κB is consequently useful. In this thesis novel ultrasound tools for accurate measurement of spatiotemporally varying 2D and 3D blood flow, with and without the use of contrast agents, have been developed. This allowed for the first time accurate, broad-view quantification of WSS around branches of the rabbit abdominal aorta. A thorough review of the evidence for a relationship between flow, NF-κB and disease was performed which highlighted discrepancies in the current literature and was used to guide the study design. Subsequently, methods for the measurement and colocalization of the spatial distribution of NF-κB, arterial permeability and nuclear morphology in the aorta of New Zealand White rabbits were developed. It was demonstrated that endothelial pro-inflammatory changes are spatially correlated with patterns of WSS, nuclear morphology and arterial permeability in vivo in the rabbit descending and abdominal aorta. The data are consistent with a causal chain between WSS, macromolecule uptake, inflammation and disease, and with the hypothesis that lipids are deposited first, through flow-mediated naturally occurring transmigration that, in excessive amounts, leads to subsequent inflammation and disease.Open Acces

Redox-Active Molecules as Therapeutic Agents

Oxidative stress and altered redox signaling have been described in a plethora of pathological conditions. Redox-active molecules can thus potentially be used to modulate the etiology/progression of such diseases. Recent advances in molecular biology and pharmacology have strengthened this area of research by providing novel mechanistic insights. This book compiles a collection of 13 articles, covering a range of topics from in vitro studies to clinical research, focused on the potential therapeutic effects of either natural or synthetic compounds, applicable to different redox-related diseases

Analysis of the epigenetic landscape in murine macrophages

Macrophages are cells of the innate immune system and play essential roles in the regulation of inflammatory responses in all parts of the body. Furthermore, macrophages are also involved in different tissue–specific functions and maintenance of the tissue homeostasis. These functions are controlled by the epigenetic landscape, consisting of promoters and enhancers that together regulate gene expression. Enhancers are stretches of regulatory genomic sequences in the non–coding regions of the genome that can be bound by lineage– determining transcription factors. These enhancers can loop in three–dimensional space to be in close proximity to promoters and contribute to the regulation of gene expression. Previous studies suggest that there are about 1 million enhancers in the mammalian genome, of which only about 30,000 – 40,000 are selected in each specific cell type. This dissertation studies the regulation of the epigenetic landscape of murine macrophages by utilizing different tissue macrophages, different complex and simple stimuli, as well as natural genetic variation as a mutagenesis screen. The overarching research question of this dissertation is to understand how the enhancer landscape in macrophages gets selected and regulated in order to control gene expression. In more detail, the main questions answered in this dissertation are: What are the epigenetic mechanisms that are responsible for tissue–specific functions? How do complex stimuli change the epigenetic landscape of macrophages in comparison to simple stimuli? How does natural genetic variation influence the epigenetic landscape and gene expression in murine macrophages? In Chapter 1 (Gosselin, D., Link, V. M., Romanoski, C. E. et al. (2014) appeared in Cell) we investigate the influence of the tissue environment on the epigenetic landscape in mouse macrophages. We compare macrophages residing in the brain (microglia) with macrophages from the peritoneal cavity by measuring mRNA expression, as well as enhancer activation (H3K4me2, H3K27ac, and PU.1). We find highly expressed genes unique to one population of macrophages, which correlates well with the activity signature at enhancers in the corresponding cells. By analyzing the enhancer landscape, we find that the macrophage lineage–determining transcription factor PU.1 plays a key role in establishing the enhancer repertoire, creating a common, macrophage–specific enhancer landscape. Furthermore, expression of tissue–specific transcription factors in collaboration with PU.1 drives a subset of tissue–specific enhancers regulating the differences in gene expression between different tissue–specific macrophage populations. In Chapter 2 (Eichenfield, D. Z., Troutman, D. T., Link, V. M. et al. (2016) appeared in eLife) we investigate the effect of complex stimuli onto the epigenetic landscape in macrophages on the example of wounds. Stimulation of macrophages with homogenated tissue to mimic a wound environment shows a unique pattern of gene expression, which is different from gene expression patterns found after single stimuli (e.g. LPS, IL–4 etc.). To gain insight into the regulation of the enhancer landscape after complex stimuli, we compare the epigenome after single stimuli and tissue homogenate and find substantial differences in enhancer selection and activation. We find that the complex damage signal promotes co–localization of several signal–dependent transcription factors to enhancers not observed under the single stimuli. Therefore, more complex polarizations of cells lead to new combinations of signal–dependent transcription factors and an epigenetic landscape different than observed with single stimuli. In Chapter 3 (Link et al. (2018b) appeared in bioRxiv) MARGE (Mutation Analysis for Regulatory Genomic Elements) is presented, a new method to analyze the effect of natural genetic variation on transcription factor binding and open chromatin. MARGE provides a suite of software tools that integrates genome–wide genetic variation data (including insertions and deletions) with epigenetic data. It provides software to create custom genomes based on a reference genome and variation data, to shift coordinates between different custom genomes, as well as do downstream ChIP–seq analysis. The main algorithm in MARGE analyzes if mutations in transcription factor binding motifs are significantly affecting transcription factor binding or open chromatin. MARGE provides a pairwise comparison, in which the significance of each motif is calculated with a student’s t–test. It compares the transcription factor binding distribution of each mutated motif in individual one with the distribution in individual two. For a more general approach that allows comparisons of many individuals MARGE implements a linear mixed model, modeling transcription factor binding with fixed effects motif existence and random effects locus and genotype. The development of this software allows in depth analysis of genetic variation data in combination with epigenetic data. In Chapter 4 (Link et al. (2018a) under review in Cell) we analyze the effect of natural genetic variation in five diverse strains of mice on the epigenetic landscape. We choose three well–known laboratory inbred mouse strains, as well as two very diverse wild–derived inbred mouse strains. We investigate the enhancer landscape, open chromatin and binding of the most important macrophage lineage–determining transcription factors. We observe substantial strain–specific differences in gene expression of which the majority can be explained by cis–regulatory elements. Application of MARGE onto the transcription factor binding data reveals roles of about 100 transcription factors in establishing the enhancer repertoire in macrophages. Unexpectedly, we find that a substantial fraction of strain– specific DNA binding of transcription factors cannot be explained by local mutations. Investigation of this phenomenon in more detail shows highly interconnected clusters of transcription factors that reside within topologically associating domains. These interconnected clusters are highly correlated with activation of enhancers and gene expression of the nearest gene, uncovering a new layer of transcriptional regulation. In Chapter 5, I briefly discuss additional contributions to the field of macrophage biology I made during my Ph.D. Namely, I was involved in two additional projects. In the first project (Pirzgalska et al. (2017) appeared in Nature Medicine) we identify sympathetic neuron–associated macrophages (SAM) that import and degrade norepinephrine via expression of solute carrier family 6 member 2 (Slc6a2) and monoamine oxidase A (MAOa). We demonstrate that SAM–mediated clearance of extracellular norepinephrine contributes to obesity and we show the relevance of this finding in humans, as we found that SAMs are also present in human tissues. The second project (Oishi et al. (2017) appeared in Cell Metabolism) studies the role of nuclear receptors (LXR and SREBP) in induction of anti–inflammatory fatty acids. We find that right after stimulation of TLR4 (during the induction phase) NF–kB dependent genes are upregulated, whereas LXR dependent genes are repressed. This leads to activation of SREBP1, which drives the expression of enzymes involved in mono–unsaturated and omega–3 polyunsaturated fatty acid biosynthesis. The fatty acids produced by these enzymes repress inflammatory genes under the control of NF–kB and the inflammatory signal gets resolved. In summary, my studies used a combination of experimental and computational approaches to investigate the effect of tissue–environment and factors, complex stimuli and natural genetic variation on the epigenetic landscape in macrophages. These studies broadened our understanding of the regulation of gene expression by the epigenetic landscape substantially. We showed that there is a core set of lineage–determining transcription factors in macrophages, which require diverse signal–dependent transcription factors to establish the enhancer landscape. Not only did we show that transcription factors regulated by the local environment play essential roles in establishing and maintaining tissue–specific functions of macrophages, but also that more complex stimuli can re–direct and combine signal–dependent transcription factors to establish new enhancers, not observed under the single stimuli. Using natural genetic variation as a mutagenesis screen allowed us to estimate the involvement of about 100 transcription factors in shaping the enhancer landscape, as well as to uncover a new layer of transcription regulation due to highly interconnected clusters of concordantly bound transcription factors

Natural Medicine in Therapy

Enter For a long time, natural medicine has been used as a therapeutic therapy based on generations of indigenous practices. Today the rise in natural remedies has been largely driven by public demand and billions of dollars are spent annually on herbal medicines. It is therefore important to document the effectiveness of natural medicine, its potential side effects, and potential interactions