Wendy J. van Zuylen

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow‐derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac‐4, 5, 7) in BMM, whereas Hdac‐1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS‐induced expression of the Cox‐2, Cxcl2, and Ifit2 genes. In the case of Cox‐2, this effect was also apparent at the promoter level. Overexpression of Hdac‐8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox‐2 mRNA. Another class of LPS‐inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.—Aung, H. T., Schroder, K., Himes, S. R., Brion, K., van Zuylen, W., Trieu, A., Suzuki, H., Hayashizaki, Y., Hume, D. A., Sweet, M. J., Ravasi, T. LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression. FASEB J. 20, 1315–1327 (2006)

High resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class I: implications for T-cell receptor engagement and T-cell immunodominance

Although HLA class I alleles can bind epitopes up to 14 amino acids in length, little is known about the immunogenicity or the responding T-cell repertoire against such determinants. Here, we describe an HLA-B*3508-restricted cytotoxic T lymphocyte response to a 13-mer viral epitope (LPEPLPQGQLTAY). The rigid, centrally bulged epitope generated a biased T-cell response. Only the N-terminal face of the peptide bulge was critical for recognition by the dominant clonotype SB27. The SB27 public T-cell receptor (TcR) associated slowly onto the complex between the bulged peptide and the major histocompatibility complex, suggesting significant remodeling upon engagement. The broad antigen-binding cleft of HLA-B*3508 represents a critical feature for engagement of the public TcR, as the narrower binding cleft of HLA-B*3501LPEPLPQGQLTAY, which differs from HLA-B*3508 by a single amino acid polymorphism (Arg156 → Leu), interacted poorly with the dominant TcR. Biased TcR usage in this cytotoxic T lymphocyte response appears to reflect a dominant role of the prominent peptide·major histocompatibility complex class I surface.

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Congenital cytomegalovirus infection in pregnancy and the neonate: consensus recommendations for prevention, diagnosis, and therapy

Congenital cytomegalovirus is the most frequent, yet under-recognised, infectious cause of newborn malformation in developed countries. Despite its clinical and public health importance, questions remain regarding the best diagnostic methods for identifying maternal and neonatal infection, and regarding optimal prevention and therapeutic strategies for infected mothers and neonates. The absence of guidelines impairs global efforts to decrease the effect of congenital cytomegalovirus. Data in the literature suggest that congenital cytomegalovirus infection remains a research priority, but data are yet to be translated into clinical practice. An informal International Congenital Cytomegalovirus Recommendations Group was convened in 2015 to address these questions and to provide recommendations for prevention, diagnosis, and treatment. On the basis of consensus discussions and a review of the literature, we do not support universal screening of mothers and the routine use of cytomegalovirus immunoglobulin for prophylaxis or treatment of infected mothers. However, treatment guidelines for infected neonates were recommended. Consideration must be given to universal neonatal screening for cytomegalovirus to facilitate early detection and intervention for sensorineural hearing loss and developmental delay, where appropriate. The group agreed that education and prevention strategies for mothers were beneficial, and that recommendations will need continual updating as further data become available.

Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice

We generated double‐transgenic mice carrying cointegrated tissue‐specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c‐fms) gene, containing a deletion of the trophoblast‐specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4‐dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4‐dependent promoter directed expression of that protein in mononuclear phagocytes of double‐transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression.

Potent T cell response to a class I‐binding 13‐mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein‐Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9‐mer (56LPQGQLTAY64), 11‐mer (54EPLPQGQLTAY64), and 13‐mer (52LPEPLPQGQLTAY64) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HLA allele was directed strongly and exclusively towards the 11‐mer peptide. In contrast, EBV‐exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13‐mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156Leucine; B*3508, 156Arginine). This minor difference was shown to enhance binding of the 13‐mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13‐mer epitope defined in this study represents the longest class I‐binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition.

Prevention of congenital cytomegalovirus complications by maternal and neonatal treatments: a systematic review.

Human cytomegalovirus is the leading non‐genetic cause of congenital malformation in developed countries. Congenital CMV may result in fetal and neonatal death or development of serious clinical sequelae. In this review, we identified evidence‐based interventions for prevention of congenital CMV at the primary level (prevention of maternal infection), secondary level (risk reduction of fetal infection and disease) and tertiary level (risk reduction of infected neonates being affected by CMV). A systematic review of existing literature revealed 24 eligible studies that met the inclusion criteria. Prevention of maternal infection using hygiene and behavioural interventions reduced maternal seroconversion rates during pregnancy. However, evidence suggested maternal adherence to education on preventative behaviours was a limiting factor. Treatment of maternal CMV infection with hyperimmune globulin (HIG) showed some evidence for efficacy in prevention of fetal infection and fetal/neonatal morbidity with a reasonable safety profile. However, more robust clinical evidence is required before HIG therapy can be routinely recommended. Limited evidence also existed for the safety and efficacy of established CMV antivirals (valaciclovir, ganciclovir and valganciclovir) to treat neonatal consequences of CMV infection, but toxicity and lack of randomised clinical trial data remain major issues. In the absence of a licensed CMV vaccine or robust clinical evidence for anti‐CMV therapeutics, patient education and behavioural interventions that emphasise adherence remain the best preventative strategies for congenital CMV. There is a strong need for further data on the use of HIG and other antivirals in pregnancy, as well as the development of less toxic, novel, antiviral agents. Copyright

Congenital cytomegalovirus infection in pregnancy: a review of prevalence, clinical features, diagnosis and prevention

Human cytomegalovirus (CMV) is under‐recognised, despite being the leading infectious cause of congenital malformation, affecting ~0.3% of Australian live births. Approximately 11% of infants born with congenital CMV infection are symptomatic, resulting in clinical manifestations, including jaundice, hepatosplenomegaly, petechiae, microcephaly, intrauterine growth restriction and death. Congenital CMV infection may cause severe long‐term sequelae, including progressive sensorineural hearing loss and developmental delay in 40–58% of symptomatic neonates, and ~14% of initially asymptomatic infected neonates. Up to 50% of maternal CMV infections have nonspecific clinical manifestations, and most remain undetected unless specific serological testing is undertaken. The combination of serology tests for CMV‐specific IgM, IgG and IgG avidity provide improved distinction between primary and secondary maternal infections. In pregnancies with confirmed primary maternal CMV infection, amniocentesis with CMV‐PCR performed on amniotic fluid, undertaken after 21–22 weeks gestation, may determine whether maternofetal virus transmission has occurred. Ultrasound and, to a lesser extent, magnetic resonance imaging are valuable tools to assess fetal structural and growth abnormalities, although the absence of fetal abnormalities does not exclude fetal damage. Diagnosis of congenital CMV infection at birth or in the first 3 weeks of an infants life is crucial, as this should prompt interventions for prevention of delayed‐onset hearing loss and neurodevelopmental delay in affected infants. Prevention strategies should also target mothers because increased awareness and hygiene measures may reduce maternal infection. Recognition of the importance of CMV in pregnancy and in neonates is increasingly needed, particularly as therapeutic and preventive interventions expand for this serious problem.

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.

The Wnt pathway: a key network in cell signalling dysregulated by viruses.

Viruses are obligate parasites dependent on host cells for survival. Viral infection of a cell activates a panel of pattern recognition receptors that mediate antiviral host responses to inhibit viral replication and dissemination. Viruses have evolved mechanisms to evade and subvert this antiviral host response, including encoding proteins that hijack, mimic and/or manipulate cellular processes such as the cell cycle, DNA damage repair, cellular metabolism and the host immune response. Currently, there is an increasing interest whether viral modulation of these cellular processes, including the cell cycle, contributes to cancer development. One cellular pathway related to cell cycle signalling is the Wnt pathway. This review focuses on the modulation of this pathway by human viruses, known to cause (or associated with) cancer development. The main mechanisms where viruses interact with the Wnt pathway appear to be through (i) epigenetic modification of Wnt genes; (ii) cellular or viral miRNAs targeting Wnt genes; (iii) altering specific Wnt pathway members, often leading to (iv) nuclear translocation of β‐catenin and activation of Wnt signalling. Given that diverse viruses affect this signalling pathway, modulating Wnt signalling could be a generalised critical process for the initiation or maintenance of viral pathogenesis, with resultant dysregulation contributing to virus‐induced cancers. Further study of this virus–host interaction may identify options for targeted therapy against Wnt signalling molecules as a means to reduce virus‐induced pathogenesis and the downstream consequences of infection. Copyright