Kevin Robertson

The Transcription Factor STAT-1 Couples Macrophage Synthesis of 25-Hydroxycholesterol to the Interferon Antiviral Response

Summary Recent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.

Absence of splenic latency in murine gammaherpesvirus 68-infected B cell-deficient mice

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of mice which causes an acute lung infection and establishes a latent infection in B lymphocytes. In this paper we describe the infection in transgenic B cell-deficient (muMT) mice, to determine whether a latent infection can be established in a mouse lacking circulating B lymphocytes. Little difference was observed in the acute lung infection, although there was a slight delay in virus clearance in the muMT mice. This indicates that antiviral antibody is of little importance in the resolution of the lung infection. Neither free nor latent virus could be detected in the spleen in the muMT mice. In addition, these mice did not develop MHV-68-induced splenomegaly. These data suggest that within the lymphoid compartment B lymphocytes are the sole reservoir for MHV-68 infection in vivo, confirming earlier work which identified B cells as the site of latent infection based on cell fractionation studies. In addition, our study shows that CD4-driven lymphocyte expansion leading to splenomegaly is dependent on the presence of MHV-68-infected B cells in the spleen. Although no free virus was detected (using conventional biological assays) in the lung after the resolution of the acute infection, MHV-68 genome was detected in the lungs of both control and muMT mice by PCR analysis. This suggests that cells in the lung may act as a reservoir of latent virus which is independent of the B lymphocyte infection.

Increased Human Immunodeficiency Virus Type 1 (HIV-1) env Compartmentalization in the Presence of HIV-1-Associated Dementia

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) surface Env protein has been implicated in the development of HIV-1-associated dementia (HAD). HIV-1 env diversity was analyzed by heteroduplex tracking assay in 27 infected subjects with various neurological statuses. env compartmentalization between the blood and cerebral spinal fluid (CSF) was apparent with all neurological categories. However, in subjects with HAD, significantly more CSF virus was represented by CNS-unique env variants. Variants specialized for replication in the CNS may play a larger role in the development of HAD. Alternatively, HAD may be associated with a more pronounced state of immunosuppression that permits more extensive replication and independent evolution within the CNS compartment.

Cross-sectional characterization of HIV-1 env compartmentalization in cerebrospinal fluid over the full disease course.

Objectives:To characterize HIV-1 env compartmentalization between cerebrospinal fluid (CSF) and peripheral blood plasma over all stages of the HIV-1 disease course, and to determine the relationship between the extent of CSF HIV-1 env compartmentalization and clinical neurologic disease status. Design:Paired blood plasma and CSF specimens were collected from 66 HIV-infected patients cross-sectionally representing all major clinical stages relating to HIV-associated neurologic disease, including primary infection, asymptomatic chronic infection, chronic infection with minor global impairment, and immune deficiency with HIV-associated dementia. Methods:Heteroduplex tracking assays and bulk sequence analysis targeting the V1/V2, C2-V3, and V4/V5 regions of env were performed to characterize the genetic makeup of complex HIV-1 populations in the cross-sectional blood plasma and CSF specimens. The levels of blood plasma/CSF env compartmentalization were quantified and compared across the different clinical stages of HIV-1 neurologic disease. Results:Blood plasma/CSF env compartmentalization levels varied considerably by disease stage and were generally consistent across all three regions of env characterized. Little or no compartmentalization was observed in non-impaired individuals with primary HIV-1 infection. Compartmentalization levels were elevated in chronically infected patients, but were not significantly different between mildly impaired and non-impaired patients. Patients with HIV-associated dementia showed significantly greater blood plasma/CSF env compartmentalization relative to other groups. Conclusion:Increased CSF compartmentalization of the HIV-1 env gene, which may reflect independent HIV-1 replication and evolution within the central nervous system, is specifically associated with HIV-associated dementia and not the less severe forms of HIV-1 neurologic disease.

Discrete Clusters of Virus-Encoded MicroRNAs Are Associated with Complementary Strands of the Genome and the 7.2-Kilobase Stable Intron in Murine Cytomegalovirus

ABSTRACT The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5′ splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.

A logic-based diagram of signalling pathways central to macrophage activation

BackgroundThe complex yet flexible cellular response to pathogens is orchestrated by the interaction of multiple signalling and metabolic pathways. The molecular regulation of this response has been studied in great detail but comprehensive and unambiguous diagrams describing these events are generally unavailable. Four key signalling cascades triggered early-on in the innate immune response are the toll-like receptor, interferon, NF-κB and apoptotic pathways, which co-operate to defend cells against a given pathogen. However, these pathways are commonly viewed as separate entities rather than an integrated network of molecular interactions.ResultsHere we describe the construction of a logically represented pathway diagram which attempts to integrate these four pathways central to innate immunity using a modified version of the Edinburgh Pathway Notation. The pathway map is available in a number of electronic formats and editing is supported by yEd graph editor software.ConclusionThe map presents a powerful visual aid for interpreting the available pathway interaction knowledge and underscores the valuable contribution well constructed pathway diagrams make to communicating large amounts of molecular interaction data. Furthermore, we discuss issues with the limitations and scalability of pathways presented in this fashion, explore options for automated layout of large pathway networks and demonstrate how such maps can aid the interpretation of functional studies.

Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis

ABSTRACT Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).

Construction of a large scale integrated map of macrophage pathogen recognition and effector systems

BackgroundIn an effort to better understand the molecular networks that underpin macrophage activation we have been assembling a map of relevant pathways. Manual curation of the published literature was carried out in order to define the components of these pathways and the interactions between them. This information has been assembled into a large integrated directional network and represented graphically using the modified Edinburgh Pathway Notation (mEPN) scheme.ResultsThe diagram includes detailed views of the toll-like receptor (TLR) pathways, other pathogen recognition systems, NF-kappa-B, apoptosis, interferon signalling, MAP-kinase cascades, MHC antigen presentation and proteasome assembly, as well as selected views of the transcriptional networks they regulate. The integrated pathway includes a total of 496 unique proteins, the complexes formed between them and the processes in which they are involved. This produces a network of 2,170 nodes connected by 2,553 edges.ConclusionsThe pathway diagram is a navigable visual aid for displaying a consensus view of the pathway information available for these systems. It is also a valuable resource for computational modelling and aid in the interpretation of functional genomics data. We envisage that this work will be of value to those interested in macrophage biology and also contribute to the ongoing Systems Biology community effort to develop a standard notation scheme for the graphical representation of biological pathways.

Regression of a Murine Gammaherpesvirus 68-Positive B-Cell Lymphoma Mediated by CD4 T Lymphocytes

ABSTRACT Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.

Human cytomegalovirus UL7, a homologue of the SLAM-family receptor CD229, impairs cytokine production

Human cytomegalovirus (HCMV), the β‐herpesvirus prototype, has evolved a wide spectrum of mechanisms to counteract host immunity. Among them, HCMV uses cellular captured genes encoding molecules capable of interfering with the original host function or of fulfilling new immunomodulatory tasks. Here, we report on UL7, a novel HCMV heavily glycosylated transmembrane protein, containing an Ig‐like domain that exhibits remarkable amino acid similarity to CD229, a cell‐surface molecule of the signalling lymphocyte‐activation molecule (SLAM) family involved in leukocyte activation. The UL7 Ig‐like domain, which is well‐preserved in all HCMV strains, structurally resembles the SLAM‐family N‐terminal Ig‐variable domain responsible for the homophilic and heterophilic interactions that trigger signalling. UL7 is transcribed with early‐late kinetics during the lytic infectious cycle. Using a mAb generated against the viral protein, we show that it is constitutively shed, through its mucine‐like stalk, from the cell‐surface. Production of soluble UL7 is enhanced by PMA and reduced by a broad‐spectrum metalloproteinase inhibitor. Although UL7 does not hold the ability to interact with CD229 or other SLAM‐family members, it shares with them the capacity to mediate adhesion to leukocytes, specifically to monocyte‐derived DCs. Furthermore, we demonstrate that UL7 expression attenuates the production of proinflammatory cytokines TNF, IL‐8 and IL‐6 in DCs and myeloid cell lines. Thus, the ability of UL7 to interfere with cellular proinflammatory responses may contribute to viral persistence. These results enhance our understanding of those HCMV‐encoded molecules involved in sustaining the balance between HCMV and the host immune system.