Identification of the Receptor Used by the Ecotropic Mouse GLN Endogenous Retrovirus.

Approximately 10% of the mouse genome is composed of endogenous retroviruses belonging to different families. In contrast to the situation in the human genome, several of these families correspond to recent, still-infectious elements capable of encoding complete viral particles. The mouse GLN endogenous retrovirus is one of these active families. We previously identified one fully functional provirus from the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identified receptors for mouse ecotropic retroviral envelope proteins. In this study, we have identified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope is due to the presence or absence of an N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains of the receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses a member of the solute carrier superfamily as a receptor.IMPORTANCE Endogenous retroviruses are genomic traces of past infections present in all vertebrates. Most of these elements degenerate over time and become nonfunctional, but the mouse genome still contains several families with full infection abilities. The GLN retrovirus is one of them, and its members encode particles that are able to infect only mouse cells. Here, we identified the cellular protein used as a receptor by GLN for cell entry. It is SLC19A1, the reduced folate carrier. We show that GLN infection is limited to mouse cells due to both a mutation in the mouse gene preventing the glycosylation of SLC19A1 and also other residues conserved within the rat but not in the hamster and human proteins. Like all other gammaretroviruses whose receptors have been identified, GLN uses a member of the solute carrier superfamily for cell entry, highlighting the role of these proteins for retroviral infection in mammals.

A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion.

Endogenous retroviruses are cellular genes of retroviral origin captured by their host during the course of evolution and represent around 8% of the human genome. Although most are defective and transcriptionally silenced, some are still able to generate retroviral-like particles and proteins. Among these, the HERV-K(HML2) family is remarkable since its members have amplified relatively recently and many of them still have full length coding genes. Furthermore, they are induced in cancers, especially in melanoma, breast cancer and germ cell tumours, where viral particles, as well as the envelope protein (Env), can be detected. Here we show that HERV-K(HML2) Env per se has oncogenic properties. Its expression in a non-tumourigenic human breast epithelial cell line induces epithelial to mesenchymal transition (EMT), often associated with tumour aggressiveness and metastasis. In our model, this is typified by key modifications in a set of molecular markers, changes in cell morphology and enhanced cell motility. Remarkably, microarrays performed in 293T cells reveal that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETV5 and EGR1, which are downstream effectors of the MAPK ERK1/2 and are associated with cellular transformation. We demonstrate that HERV-K(HML2) Env effectively activates the ERK1/2 pathway in our experimental setting and that this activation depends on the Env cytoplasmic tail. In addition, this phenomenon is very specific, being absent with every other retroviral Env tested, except for Jaagsiekte Sheep Retrovirus (JSRV) Env, which is already known to have transforming properties in vivo. Though HERV-K Env is not directly transforming by itself, the newly discovered properties of this protein may contribute to oncogenesis.

HIV-1 infection of macrophages dysregulates innate immune responses to Mycobacterium tuberculosis by inhibition of interleukin-10.

Human immunodeficiency virus (HIV)-1 and Mycobacterium tuberculosis (M. tuberculosis) both target macrophages, which are key cells in inflammatory responses and their resolution. Therefore, we tested the hypothesis that HIV-1 may modulate macrophage responses to coinfection with M. tuberculosis. HIV-1 caused exaggerated proinflammatory responses to M. tuberculosis that supported enhanced virus replication, and were associated with deficient stimulus-specific induction of anti-inflammatory interleukin (IL)-10 and attenuation of mitogen-activated kinase signaling downstream of Toll-like receptor 2 and dectin-1 stimulation. Our in vitro data were mirrored by lower IL-10 and higher proinflammatory IL-1ß in airway samples from HIV-1-infected patients with pulmonary tuberculosis compared with those with non-tuberculous respiratory tract infections. Single-round infection of macrophages with HIV-1 was sufficient to attenuate IL-10 responses, and antiretroviral treatment of replicative virus did not affect this phenotype. We propose that deficient homeostatic IL-10 responses may contribute to the immunopathogenesis of active tuberculosis and propagation of virus infection in HIV-1/M. tuberculosis coinfection.

Transcriptional profiling of innate and adaptive human immune responses to mycobacteria in the tuberculin skin test.

The tuberculin skin test (TST) is a model of integrated innate and adaptive human immune responses to Mycobacterium tuberculosis, but the component processes that are involved in this model have not previously been defined in vivo. We used transcriptional profiling to study these responses within the TST at molecular and system levels. Skin biopsies from TST injection sites were examined in subjects classified as TST(+) or TST(-) by clinical and histological criteria. Genome-wide expression arrays showed evolution of immune responses reflecting T-cell activation and recruitment with uniquely Th1-polarized responses and cytotoxic T cells (CTLs). In addition, distinct innate immune and IFN-γ-stimulated gene expression signatures were identified, under the regulation of NF-κB and STAT1 transcriptional control. These were highly enriched for chemokines and MHC class II molecules providing a potential mechanism for paracrine amplification of inflammatory responses in the TST, by supporting cellular recruitment and enhancing antigen presentation. The same repertoire of innate and adaptive immune responses was evident in TST(+) and TST(-) subjects alike, clinically positive TSTs being distinguished only by quantitatively greater differences. These data provide new insights into complex multifaceted responses within the TST, with much greater sensitivity than previous clinical or histological assessments.

Genome-wide innate immune responses in HIV-1-infected macrophages are preserved despite attenuation of the NF-kappa B activation pathway.

Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs, and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defense is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling, and cytokine secretion. We show that in an ex vivo model, M-CSF-differentiated monocyte-derived macrophages uniformly infected with replicating CCR5-tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-kappaB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to LPS stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS-responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-kappaB signaling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.

Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays.

BACKGROUND: Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. RESULTS: We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that interarray variability is small (only around 2% of the mean log signal), while interarray variability from replicate array measurements has a standard deviation (SD) of around 0.5 log(2) units ( 6% of mean). The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. CONCLUSIONS: This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of the SD arising from experimental (non biological) intra- and interarray variability, and for a lower threshold for determining whether an individual gene is expressed. The study provides a reliable basis for further more extensive studies of the systems biology of eukaryotic cells.

Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages.

Quantitative measurement of NF-kappaB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-kappaB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-kappaB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-kappaB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-kappaB activation reporter cell line.

HIV-1 infection of macrophages is dependent on evasion of innate immune cellular activation.

OBJECTIVE: The cellular innate immune response to HIV-1 is poorly characterized. In view of HIV-1 tropism for macrophages, which can be activated via pattern recognition receptors to trigger antimicrobial defences, we investigated innate immune responses to HIV-1 by monocyte-derived macrophages. DESIGN: In a model of productive HIV-1 infection, cellular innate immune responses to HIV-1 were investigated, at the level of transcription factor activation, specific gene expression and genome-wide transcriptional profiling. In addition, the viral determinants of macrophage responses and the physiological effect of innate immune cellular activation on HIV-1 replication were assessed. RESULTS: Productive HIV-1 infection did not activate nuclear factor-kappaB and interferon regulatory factor 3 transcription factors or interferon gene expression (IFN) and caused remarkably small changes to the host-cell transcriptome, with no evidence of inflammatory or IFN signatures. Evasion of IFN induction was not dependent on HIV-1 envelope-mediated cellular entry, inhibition by accessory proteins or reverse transcription of ssRNA that may reduce innate immune cellular activation by viral RNA. Furthermore, IFNbeta priming did not sensitize responses to HIV-1. Importantly, exogenous IFNbeta or stimulation with the RNA analogue poly I:C to simulate innate immune activation invoked HIV-1 restriction. CONCLUSION: We conclude that macrophages lack functional pattern recognition receptors for this virus and that HIV-1 tropism for macrophages helps to establish a foothold in the host without triggering innate immune cellular activation, which would otherwise block viral infection effectively.

HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor.

The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.

Natural cathepsin E deficiency in the immune system of C57BL/6J mice.

Cathepsin E is an aspartic endosomal proteinase, expressed at high levels in some epithelial and haemopoetic cells. The enzyme has been implicated in a variety of functions, including antigen processing. This study documents strain-specific variation in expression of cathepsin E in mice. The levels of cathepsin E protein and message are profoundly decreased in haemopoetic cells from C57BL/6J mice, compared to levels in 129S2/Sv or Balb/c. The deficiency is cell-type-specific, as protein levels in gut are not affected. Deficiency affects B cell, T cells, macrophages and dendritic cells. The low cathepsin E phenotype cosegregates with the C57BL/6J genotype in a panel of C57BL/6J x 129S2/Sv F2 mice. Analysis of the promoter region of cathepsin E reveals a polymorphism which destroys a previously described functional PU.1 transcription binding consensus sequence in the C57BL/6J genome. Antigen processing of ovalbumin by dendritic cells, which has previously been shown to require cathepsin E, is impaired in C57BL/6J-derived dendritic cells. C57BL/6J mice thus exhibit a profound tissue-specific deficiency in cathepsin E expression, which may have important implications for the immune phenotype of this mouse strain.