Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human.

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4(+) Th cells upon activation, whereas the 'standard' proteasome is up-regulated in Tregs only upon activation.

Into the unknown: expression profiling without genome sequence information in CHO by next generation sequencing.

The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.

Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3.

BACKGROUND: Normalization of microarrays is a standard practice to account for and minimize effects which are not due to the controlled factors in an experiment. There is an overwhelming number of different methods that can be applied, none of which is ideally suited for all experimental designs. Thus, it is important to identify a normalization method appropriate for the experimental setup under consideration that is neither too negligent nor too stringent. Major aim is to derive optimal results from the underlying experiment. Comparisons of different normalization methods have already been conducted, none of which, to our knowledge, comparing more than a handful of methods. RESULTS: In the present study, 25 different ways of pre-processing Illumina Sentrix BeadChip array data are compared. Among others, methods provided by the BeadStudio software are taken into account. Looking at different statistical measures, we point out the ideal versus the actual observations. Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data. Taking together all different kinds of measures, the ideal method for our dataset is identified. CONCLUSIONS: Pre-processing of microarray gene expression experiments has been shown to influence further downstream analysis to a great extent and thus has to be carefully chosen based on the design of the experiment. This study provides a recommendation for deciding which normalization method is best suited for a particular experimental setup.

Effect of RNA quality on transcript intensity levels in microarray analysis of human post-mortem brain tissues.

BACKGROUND: Large-scale gene expression analysis of post-mortem brain tissue offers unique opportunities for investigating genetic mechanisms of psychiatric and neurodegenerative disorders. On the other hand microarray data analysis associated with these studies is a challenging task. In this publication we address the issue of low RNA quality data and corresponding data analysis strategies. RESULTS: A detailed analysis of effects of post chip RNA quality on the measured abundance of transcripts is presented. Overall Affymetrix GeneChip data (HG-U133_AB and HG-U133_Plus_2.0) derived from ten different brain regions was investigated. Post chip RNA quality being assessed by 5'/3' ratio of housekeeping genes was found to introduce a well pronounced systematic noise into the measured transcript expression levels. According to this study RNA quality effects have: 1) a "random" component which is introduced by the technology and 2) a systematic component which depends on the features of the transcripts and probes. Random components mainly account for numerous negative correlations of low-abundant transcripts. These negative correlations are not reproducible and are mainly introduced by an increased relative level of noise. Three major contributors to the systematic noise component were identified: the first is the probe set distribution, the second is the length of mRNA species, and the third is the stability of mRNA species. Positive correlations reflect the 5'-end to 3'-end direction of mRNA degradation whereas negative correlations result from the compensatory increase in stable and 3'-end probed transcripts. Systematic components affect the expressed transcripts by introducing irrelevant gene correlations and can strongly influence the results of the main experiment. A linear model correcting the effect of RNA quality on measured intensities was introduced. In addition the contribution of a number of pre-mortem and post-mortem attributes to the overall detected RNA quality effect was investigated. Brain pH, duration of agonal stage, post-mortem interval before sampling and donor's age of death within considered limits were found to have no significant contribution. CONCLUSION: Basic conclusions for data analysis in expression profiling study are as follows: 1) testing for RNA quality dependency should be included in the preprocessing of the data; 2) investigating inter-gene correlation without regard to RNA quality effects could be misleading; 3) data normalization procedures relying on housekeeping genes either do not influence the correlation structure (if 3'-end intensities are used) or increase it for negatively correlated transcripts (if 5'-end or median intensities are included in normalization procedure); 4) sample sets should be matched with regard to RNA quality; 5) RMA preprocessing is more sensitive to RNA quality effect, than MAS 5.0.

Expression profiling of epithelial plasticity in tumor progression.

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.

Gene silencing with STAT6 specific siRNAs blocks eotaxin release in IL-4/TNFalpha stimulated human epithelial cells.

Small interfering RNAs have evolved as effective tools for the study of gene functions. Here, we demonstrate the use of different siRNAs for the specific knock down of the STAT6 transcription regulator and the complete silencing of the downstream signaling pathway. The knock down of STAT6 resulted in a complete loss of STAT6 specific DNA binding activity and blocked the release of eotaxin-3 in human epithelial cells (BEAS-2B) stimulated with IL-4 and TNFalpha with no signs of unspecific gene silencing. Other signaling pathways like the EGF stimulated release of IL-8 were still active in BEAS-2B cells treated with STAT6 specific siRNAs, demonstrating the specificity of these molecules.

Off-target analysis of control siRNA molecules reveals important differences in the cytokine profile and inflammation response of human fibroblasts.

The use of RNA interference for the manipulation of gene expression has seen great applications from basic science to clinical investigations. However, limited selectivity and the induction of off-target effects by double stranded RNA molecules have been analyzed and discussed since the discovery of this gene expression regulation mechanism. In this study, the specificity of 13 commercially available control siRNA molecules is addressed by the analysis of gene expression profiles in 2 human cell lines HT1080 and HaCaT and in the mouse cell line 3T3-L1. The off-target signatures of the transfected siRNA molecules differ greatly between the cell lines and only a small overlap was seen for the 2 human cell lines. In particular, the HT1080 cell line showed the highest number of detected gene expression differences. In these cells, several different control siRNA molecules activated a common profile of 79 deregulated genes including a reduced interleukin-1beta (IL-1beta) and IL-24 expression. Functional analysis of MMP1 secretion and tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release revealed a reduction of NFkappaB signaling caused by at least 2 out of the 13 tested control siRNA molecules. Our findings strongly argue for a careful analysis of the control siRNA molecules for any given RNAi experiment.

Phenocopy--a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization.

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor ß Receptor I (TGF-ßR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-ß biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.

miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.

BACKGROUND: In humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance. PRINCIPAL FINDINGS: DNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression. CONCLUSION: Investigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression.

FPRL-1 induces modifications of migration-associated proteins in human neutrophils.

Human polymorphonuclear neutrophils (PMNs) are an important cell population of the innate immune system, which migrates following concentration gradients of chemokines or chemoattractants to locations of infection and inflammation in order to eliminate invading microorganisms and cell debris. For both migration and adhesion of PMNs to various tissues, the dynamic remodeling of the cytoskeleton is key prerequisite. In this context, the formyl peptide receptor-like 1 (FPRL-1) is an important chemoattractant receptor expressed on PMNs. In this study, we show that a short stimulation of FPRL-1 with either a synthetic peptide ligand (W-peptide) or a natural ligand (sCKbeta8-1) changes the protein pattern of PMNs as assessed by 2-D-DIGE. MS analysis of selected deregulated protein species resulted in the identification of proteins that are involved in the remodeling process of the actin- and tubulin-based cytoskeleton, such as L-plastin, moesin, cofilin, and stathmin. Subsequent validation experiments performed either by Western blotting or phosphoprotein-specific gel staining (Pro-Q Diamond) revealed that L-plastin is phosphorylated, whereas moesin, cofilin, and stathmin are dephosphorylated in PMNs upon FPRL-1 stimulation. These findings suggest that FPRL-1 signaling targets proteins that regulate the motility of PMNs and moreover show that 2-D-DIGE is a technique capable of detecting and quantifying differently modified (e.g., phosphorylated) protein variants.

Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response.

BACKGROUND: Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR). METHODS: The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated. RESULTS: Our data demonstrate that poly(I:C), a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-alpha, GM-CSF, GRO-alpha, TARC, MCP-1, MIP-3alpha, RANTES, IFN-beta, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C) as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C) induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C) decreased the expression of TLR5, TLR6 and TOLLIP. CONCLUSION: Poly(I:C), an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C) on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type-1 and type-2 cytokines is important considering the impact of exogenous and endogenous TLR ligands on Th1 or Th2 driven pulmonary inflammations like COPD or asthma, respectively.

Proteomic study of human bronchoalveolar lavage fluids from smokers with chronic obstructive pulmonary disease by combining surface-enhanced laser desorption/ionization-mass spectrometry profiling with mass spectrometric protein identification.

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate cellular and molecular changes in the course of lung disorders. The pattern of soluble proteins in BALF obtained from patients at different stages of respiratory disorders may provide deeper insights in the molecular mechanisms of the disease. We used surface-enhanced laser desorption/ionization mass spectrometry (MS) for differential protein display combined with reversed-phase chromatography and subsequent matrix-assisted laser desorption/ionization-MS or nanoliquid chromatography MS/MS analysis for protein identification to compare the protein pattern of BALF samples obtained from ten smokers suffering from chronic obstructive pulmonary disease (COPD), eight clinically asymptomatic smokers, and eight nonsmokers without pulmonary disease. In this context, we were able to identify small proteins and peptides, either differentially expressed or secreted in the course of COPD or in a direct response to cigarette smoke. The concentrations of neutrophil defensins 1 and 2, S100A8 (calgranulin A), and S100A9 (calgranulin B) were elevated in BALFs of smokers with COPD when compared to asymptomatic smokers. Increased concentrations in S100A8 (Calgranulin A), salivary proline-rich peptide P-C, and lysozyme C were detected in BALFs of asymptomatic smokers when compared to nonsmokers, whereas salivary proline-rich peptide P-D and Clara cell phospholipid-binding protein (CC10) were reduced in their concentration. The identified proteins and peptides might be useful in the future as diagnostic markers for smoke-induced lung irritations and COPD.

Elevated expression of TARC (CCL17) and MDC (CCL22) in models of cigarette smoke-induced pulmonary inflammation.

TARC (CCL17) and MDC (CCL22) are well-known chemoattractants for Th2 cells. Here, we evaluated the role of both chemokines for cigarette smoke-induced airway inflammation. The expression profiles of MDC, TARC, and their receptor CCR4 were analyzed in models of acute and chronic cigarette smoke-induced airway inflammation that is characterized by a Th1 immune response. The results were compared to the expression of both chemokines in models of idiopathic pulmonary fibrosis and acute asthma, which are associated with a Th2 immune response. The expression of MDC and TARC was found to be elevated in all lung inflammation models. In contrast to the findings in the asthma and lung fibrosis models, the increased expression of MDC and TARC in the cigarette-smoke model was not associated with an increased infiltration of Th2 cells into smoke-treated lungs. Our data indicate that instead of Th2 cells, airway epithelial cells expressing CCR4 might be the principal targets for MDC and TARC released from alveolar macrophages during cigarette smoke-induced airway inflammation.