Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

Manipulation of T cell function and conditional gene targeting in T cells.

Cre-Lox recombination is known as a site-specific recombinase technology, and is widely used to carry out modification at specific sites in the DNA of cells. The system consists of an enzyme, Cre recombinase, that recombines a pair of short target sequences, the lox sites. The Cre-Lox system can be used to activate or repress a gene depending on the placement of the lox sites. Placing the Cre recombinase under the control of a cell-specific promoter allows expression only in specific cells or cellular subsets, thus providing a powerful tool for analysis of gene function at specific developmental or physiological niches. Nowadays almost every aspect of T cell biology can be approached by a specific Cre model. This powerful tool allows scientists to overcome the limitations of gene-deficient animals and target a gene of interest specifically in T cell or T cell subsets by appropriate placement of the lox sites. Here we describe the main Cre lines that enable gene targeting in T helper cells or CD4 T cell subsets, and the most common methods of assessing the recombination efficiency.

TGF-ß signalling is required for CD4⁺ T cell homeostasis but dispensable for regulatory T cell function.

TGF-ß is widely held to be critical for the maintenance and function of regulatory T (T(reg)) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-ß receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-ß-driven peripheral tolerance is not regulated by TGF-ß signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T(reg) cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-ß signalling and the TR2-deficient T(reg) cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-ß signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.

The impact of breed and tissue compartment on the response of pig macrophages to lipopolysaccharide.

BACKGROUND: The draft genome of the domestic pig (Sus scrofa) has recently been published permitting refined analysis of the transcriptome. Pig breeds have been reported to differ in their resistance to infectious disease. In this study we examine whether there are corresponding differences in gene expression in innate immune cells RESULTS: We demonstrate that macrophages can be harvested from three different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing twenty-five animals from five different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, FLT1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. CONCLUSIONS: Pig macrophages more closely resemble human, than mouse, in their set of macrophage-expressed and LPS-inducible genes. Future research will address whether inter-individual variation in macrophage gene expression is heritable, and might form the basis for selective breeding for disease resistance.

Comparative analysis of monocyte subsets in the pig.

Human and mouse monocyte can be divided into two different subpopulations based on surface marker expression: CD14/16 and Ly6C/CX3CR1, respectively. Monocyte subpopulations in the pig were identified based on reciprocal expression of CD14 and the scavenger receptor CD163. The two populations, CD14(hi)-CD163(low) and CD14(low)-CD163(hi), show approximately equal abundance in the steady-state. Culture of pig PBMCs in CSF1 indicates that the two populations are a maturation series controlled by this growth factor. Gene expression in pig monocyte subpopulations was profiled using the newly developed and annotated pig whole genome snowball microarray. Previous studies have suggested a functional equivalence between human and mouse subsets, but certain genes such as CD36, CLEC4E, or TREM-1 showed human-specific expression. The same genes were expressed selectively in pig monocyte subsets. However, the profiles suggest that the pig CD14(low)-CD163(high) cells are actually equivalent to intermediate human monocytes, and there is no CD14(-) CD16(+) "nonclassical" population. The results are discussed in terms of the relevance of the pig as a model for understanding human monocyte function.

A gene expression atlas of the domestic pig.

BACKGROUND: This work describes the first genome-wide analysis of the transcriptional landscape of the pig. A new porcine Affymetrix expression array was designed in order to provide comprehensive coverage of the known pig transcriptome. The new array was used to generate a genome-wide expression atlas of pig tissues derived from 62 tissue/cell types. These data were subjected to network correlation analysis and clustering. RESULTS: The analysis presented here provides a detailed functional clustering of the pig transcriptome where transcripts are grouped according to their expression pattern, so one can infer the function of an uncharacterized gene from the company it keeps and the locations in which it is expressed. We describe the overall transcriptional signatures present in the tissue atlas, where possible assigning those signatures to specific cell populations or pathways. In particular, we discuss the expression signatures associated with the gastrointestinal tract, an organ that was sampled at 15 sites along its length and whose biology in the pig is similar to human. We identify sets of genes that define specialized cellular compartments and region-specific digestive functions. Finally, we performed a network analysis of the transcription factors expressed in the gastrointestinal tract and demonstrate how they sub-divide into functional groups that may control cellular gastrointestinal development. CONCLUSIONS: As an important livestock animal with a physiology that is more similar than mouse to man, we provide a major new resource for understanding gene expression with respect to the known physiology of mammalian tissues and cells. The data and analyses are available on the websites http://biogps.org and http://www.macrophages.com/pig-atlas.

Pig bone marrow-derived macrophages resemble human macrophages in their response to bacterial lipopolysaccharide.

Mouse bone marrow-derived macrophages (BMDM) grown in M-CSF (CSF-1) have been used widely in studies of macrophage biology and the response to TLR agonists. We investigated whether similar cells could be derived from the domestic pig using human rCSF-1 and whether porcine macrophages might represent a better model of human macrophage biology. Cultivation of pig bone marrow cells for 5-7 d in presence of human rCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, and CD172a), were potent phagocytic cells, and produced TNF in response to LPS. Pig BMDM could be generated from bone marrow cells that had been stored frozen and thawed so that multiple experiments can be performed on samples from a single animal. Gene expression in pig BMDM from outbred animals responding to LPS was profiled using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely resembled the known responses of human than mouse macrophages, sharing with humans the regulation of genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20, CXCL9, CXCL11, CXCL13), and the vitamin D3-converting enzyme, Cyp27B1. Conversely, in common with published studies of human macrophages, pig BMDM did not strongly induce genes involved in arginine metabolism, nor did they produce NO. These results establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages.

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

The mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease.

The biology of cells of the mononuclear phagocyte system has been studied extensively in the mouse. Studies of the pig as an experimental model have commonly been consigned to specialist animal science journals. In this review, we consider some of the many ways in which the innate immune systems of humans differ from those of mice, the ways that pigs may address the shortcomings of mice as models for the study of macrophage differentiation and activation in vitro, and the biology of sepsis and other pathologies in the living animal. With the completion of the genome sequence and the characterization of many key regulators and markers, the pig has emerged as a tractable model of human innate immunity and disease that should address the limited, predictive value of rodents in preclinical studies.