Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis.

The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.

Proteomic Profiling of Mouse Helper T Cell Differentiation.

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high-throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.

Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection.

Gene expression profiling to predict and assess the consequences of therapy-induced virus eradication in chronic hepatitis C virus infection.

UNLABELLED: Systems biology has proven to be a powerful tool to identify reliable predictors of treatment response in chronic hepatitis C virus (HCV) infection. In the present study, we studied patients with chronic HCV infection who responded to interferon (IFN)-based therapy, as evidenced by an absence of HCV RNA at the end of treatment, and focused on two issues that have not received much attention. First, we evaluated whether specific genes or gene expression patterns in blood were able to distinguish responder patients with a viral relapse from responder patients who remained virus negative after cessation of treatment. We found that patients with chronic HCV infection who were sustained responders and relapsers after IFN-based therapy showed comparable baseline clinical parameters and immune compositions in blood. However, at baseline, the gene expression profiles of a set of 18 genes predicted treatment outcome with an accuracy of 94%. Second, we examined whether patients with successful therapy-induced clearance of HCV still exhibited gene expression patterns characteristic of HCV or whether normalization of their transcriptome was observed. We observed that the relatively high expression levels of IFN-stimulated genes (ISGs) in patients with chronic HCV infection prior to therapy were reduced after successful IFN-based antiviral therapy (at 24 weeks of follow-up). These ISGs included the CXCL10, OAS1, IFI6, DDX60, TRIM5, and STAT1 genes. In addition, 1,428 differentially expressed non-ISGs were identified in paired pre- and posttreatment samples from sustained responders, which included genes involved in transforming growth factor beta (TGF-ß) signaling, apoptosis, autophagy, and nucleic acid and protein metabolism. Interestingly, 1,424 genes with altered expression levels in responder patients after viral eradication were identified, in comparison to normal expression levels in healthy individuals. Additionally, aberrant expression levels of a subset of these genes, including the interleukin-32 (IL-32), IL-16, CCND3, and RASSF1 genes, were also observed at baseline. Our findings indicate that successful antiviral therapy for patients with chronic HCV infection does not lead to normalization of their blood transcriptional signature. The altered transcriptional activity may reflect HCV-induced liver damage in previously infected individuals. IMPORTANCE: Tools to predict the efficacy of antiviral therapy for patients with HCV infection are important to select the optimal therapeutic strategy. Using a systems biology approach, we identify a set of 18 genes expressed in blood that predicts the recurrence of HCV RNA after cessation of therapy consisting of peginterferon and ribavirin. This set of genes may be applicable as a useful biomarker in clinical decision-making, since the number of genes included in the predictor is small and the correct prediction rate is high (94%). In addition, we observed that the blood transcriptional profile in patients with chronic HCV infection who were successfully treated is not normalized to the status observed in healthy individuals. Even 6 months after therapy-induced elimination of HCV RNA, gene expression profiles in blood are still altered in these patients with chronic HCV infection, strongly suggesting long-term modulation of immune parameters in previously infected patients.

Early divergence of Th1 and Th2 transcriptomes involves a small core response and sets of transiently expressed genes.

Th cells can adopt a number of different phenotypes. We performed microarray-assisted mRNA profiling on antigen-stimulated, TCR transgenic murine splenocytes that were cultured in the presence of cytokines. Transcriptome snapshots of Th cells differentiating into Th1 and Th2 phenotypes were obtained at various time points. Principal component analysis shows that time since activation and Th skewing are the largest sources of variance (i.e. the largest contributing factors) in our profiling experiments. Divergence between the Th1 and Th2 phenotypes is established early and does not increase in terms of number of differential genes from day 1 to day 4 after stimulation. Notwithstanding the lack of further divergence between the Th1 and Th2 lineages, we show that gene expression is best described by a 'turnover' rather than a 'core response' model, although we find evidence for both. We identify clusters of skewed genes associated with early persistent ('core response') and late ('turnover') Th1 and Th2 gene expression. In addition to the classical Th genes, members of the Batf transcription factor family are differentially expressed in particular helper phenotypes, suggesting an important role for this family in Th-cell phenotype differentiation.

Alveolar barrier disruption in varicella pneumonia is associated with neutrophil extracellular trap formation.

Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and is associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia, and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10, and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that alveolar barrier disruption in varicella pneumonia is associated with NET formation.

Analysis of Mouse Brain Transcriptome After Experimental Duvenhage Virus Infection Shows Activation of Innate Immune Response and Pyroptotic Cell Death Pathway.

Rabies is an important neglected disease, characterized by invariably fatal encephalitis. Several studies focus on understanding the pathogenic mechanisms of the prototype lyssavirus rabies virus (RABV) infection, and little is known about the pathogenesis of rabies caused by other lyssaviruses. We sought to characterize the host response to Duvenhage virus infection and compare it with responses observed during RABV infection by gene expression profiling of brains of mice with the respective infections. We found in both infections differentially expressed genes leading to increased expression of type I interferons (IFNs), chemokines, and proinflammatory cytokines. In addition several genes of the IFN signaling pathway are up-regulated, indicating a strong antiviral response and activation of the negative feedback mechanism to limit type I IFN responses. Furthermore we provide evidence that in the absence of significant neuronal apoptotic death, cell death of neurons is mediated via the pyroptotic pathway in both infections. Taken together, we have identified several genes and/or pathways for both infections that could be used to explore novel approaches for intervention strategies against rabies.

Transcriptomic Analyses Reveal Differential Gene Expression of Immune and Cell Death Pathways in the Brains of Mice Infected with West Nile Virus and Chikungunya Virus.

West Nile virus (WNV) and chikungunya virus (CHIKV) are arboviruses that are constantly (re-)emerging and expanding their territory. Both viruses often cause a mild form of disease, but severe forms of the disease can consist of neurological symptoms, most often observed in the elderly and young children, respectively, for which the mechanisms are poorly understood. To further elucidate the mechanisms responsible for end-stage WNV and CHIKV neuroinvasive disease, we used transcriptomics to compare the induction of effector pathways in the brain during the early and late stage of disease in young mice. In addition to the more commonly described cell death pathways such as apoptosis and autophagy, we also found evidence for the differential expression of pyroptosis and necroptosis cell death markers during both WNV and CHIKV neuroinvasive disease. In contrast, no evidence of cell dysfunction was observed, indicating that cell death may be the most important mechanism of disease. Interestingly, there was overlap when comparing immune markers involved in neuroinvasive disease to those seen in neurodegenerative diseases. Nonetheless, further validation studies are needed to determine the activation and involvement of these effector pathways at the end stage of disease. Furthermore, evidence for a strong inflammatory response was found in mice infected with WNV and CHIKV. The transcriptomics profile measured in mice with WNV and CHIKV neuroinvasive disease in our study showed strong overlap with the mRNA profile described in the literature for other viral neuroinvasive diseases. More studies are warranted to decipher the role of cell inflammation and cell death in viral neuroinvasive disease and whether common mechanisms are active in both neurodegenerative and brain infectious diseases.

Transcriptome assists prognosis of disease severity in respiratory syncytial virus infected infants.

Respiratory syncytial virus (RSV) causes infections that range from common cold to severe lower respiratory tract infection requiring high-level medical care. Prediction of the course of disease in individual patients remains challenging at the first visit to the pediatric wards and RSV infections may rapidly progress to severe disease. In this study we investigate whether there exists a genomic signature that can accurately predict the course of RSV. We used early blood microarray transcriptome profiles from 39 hospitalized infants that were followed until recovery and of which the level of disease severity was determined retrospectively. Applying support vector machine learning on age by sex standardized transcriptomic data, an 84 gene signature was identified that discriminated hospitalized infants with eventually less severe RSV infection from infants that suffered from most severe RSV disease. This signature yielded an area under the receiver operating characteristic curve (AUC) of 0.966 using leave-one-out cross-validation on the experimental data and an AUC of 0.858 on an independent validation cohort consisting of 53 infants. A combination of the gene signature with age and sex yielded an AUC of 0.971. Thus, the presented signature may serve as the basis to develop a prognostic test to support clinical management of RSV patients.

Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo.

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.

DC immunotherapy in HIV-1 infection induces a major blood transcriptome shift.

OBJECTIVE: This study aimed to evaluate the effect of dendritic cell (DC) vaccination against HIV-1 on host gene expression profiles. DESIGN: Longitudinal PBMC samples were collected from participants of the DC-TRN trial for immunotherapy against HIV. Microarray-assisted gene expression profiling was performed to evaluate the effects of vaccination and subsequent interruption of antiretroviral therapy on host genome expression. Data from the DC-TRN trial were compared with results from other vaccination trials. METHODS: We used Affymetrix GeneChips for microarray gene expression analysis. Data were analyzed by principal component analysis and differential gene expression was assessed using linear modeling. Gene ontology enrichment and gene set analysis were used to characterize differentially expressed genes. Transcriptome analysis included comparison with PBMCs obtained from DC-vaccinated melanoma patients and of healthy individuals who received seasonal influenza vaccination. RESULTS: DC-TRN immunotherapy in HIV-infected individuals resulted in a major shift in the transcriptome. Longitudinal analysis demonstrated that changes in the transcriptome sustained also during interruption of antiretroviral therapy. After DC-vaccination, the transcriptome was enriched for cellular immunity associated genes that were also induced in healthy adults who received live attenuated influenza virus vaccination. These beneficial responses were accompanied by detrimental signals of general immune activation. CONCLUSIONS: The DC-TRN induced changes in the transcriptome were profound, lasting, and consisted of both protective signals and signatures of inflammation and immune exhaustion, with a net result of decreased viral load, without clinical benefit. Thus transcriptome analysis provides useful information, dissecting both positive and negative effects, for the evaluation of safety and efficacy of immunotherapeutic strategies.

Time since onset of disease and individual clinical markers associate with transcriptional changes in uncomplicated dengue.

BACKGROUND: Dengue virus (DENV) infection causes viral haemorrhagic fever that is characterized by extensive activation of the immune system. The aim of this study is to investigate the kinetics of the transcriptome signature changes during the course of disease and the association of genes in these signatures with clinical parameters. METHODOLOGY/PRINCIPLE FINDINGS: Sequential whole blood samples from DENV infected patients in Jakarta were profiled using affymetrix microarrays, which were analysed using principal component analysis, limma, gene set analysis, and weighted gene co-expression network analysis. We show that time since onset of disease, but not diagnosis, has a large impact on the blood transcriptome of patients with non-severe dengue. Clinical diagnosis (according to the WHO classification) does not associate with differential gene expression. Network analysis however, indicated that the clinical markers platelet count, fibrinogen, albumin, IV fluid distributed per day and liver enzymes SGOT and SGPT strongly correlate with gene modules that are enriched for genes involved in the immune response. Overall, we see a shift in the transcriptome from immunity and inflammation to repair and recovery during the course of a DENV infection. CONCLUSIONS/SIGNIFICANCE: Time since onset of disease associates with the shift in transcriptome signatures from immunity and inflammation to cell cycle and repair mechanisms in patients with non-severe dengue. The strong association of time with blood transcriptome changes hampers both the discovery as well as the potential application of biomarkers in dengue. However, we identified gene expression modules that associate with key clinical parameters of dengue that reflect the systemic activity of disease during the course of infection. The expression level of these gene modules may support earlier detection of disease progression as well as clinical management of dengue.

Small hydrophobic protein of human metapneumovirus does not affect virus replication and host gene expression in vitro.

Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.