Theo J. Moraes

Identification of nucleolin as a cellular receptor for human respiratory syncytial virus

Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.

Neutrophil granule contents in the pathogenesis of lung injury.

Purpose of reviewThis review summarizes recent literature on the role of neutrophil granule contents in acute lung injury and the mechanisms by which these contribute to inflammatory tissue injury. Recent findingsNeutrophil products such as elastase, reactive oxygen species, and antimicrobial peptides can alter pulmonary cell function in a nondegradative fashion through activation of cell surface receptors or modulation of signal transduction pathways. These effects can be either beneficial or detrimental to the host. SummaryThe primary function of neutrophils in the innate immune response – to contain and kill invading microbial pathogens – is achieved through a series of rapid and coordinated responses culminating in phagocytosis and intracellular killing of the pathogens. Neutrophils have a potent antimicrobial arsenal that includes oxidants, proteinases, and cationic peptides. Reactive oxygen species such as oxygen are produced by the phagocyte NADPH oxidase and are microbicidal. Granules within the neutrophil cytoplasm contain potent proteolytic enzymes and cationic proteins that can digest a variety of microbial substrates. These compounds are released directly into the phagosome, compartmentalizing both the pathogen and the cytotoxic products. Under pathological circumstances, however, unregulated release of microbicidal compounds into the extracellular space can paradoxically damage host tissues. Nonspecific inhibition of neutrophils is not clinically realistic, as it would leave the host vulnerable to infection. As the mechanisms of action of neutrophil granule contents are elucidated, therapeutic targets will be identified that will allow for suppression of neutrophils’ detrimental effects while avoiding inhibition of their beneficial effects.

Proteases and lung injury.

ObjectiveAcute respiratory distress syndrome (ARDS) represents an inflammatory process that is initiated by diverse systemic and/or pulmonary insults, resulting in a clinical syndrome of severe respiratory distress and refractory hypoxemia. Neutrophils and their cytotoxic products, including oxidants and proteases, such as elastase, have been implicated as playing a key role in the pathophysiology of ARDS. This article reviews some of the physiologic actions of proteases, specifically elastase, the evidence for neutrophil elastase involvement in ARDS, and the potential therapeutic use of neutrophil elastase inhibitors in lung injury. Data SourceA review of published literature (original articles and reviews) in English from 1965 to 2002. ConclusionAlthough the data support a key role for neutrophil elastase in the pathogenesis of ARDS, further study is needed to fully define the actions of neutrophil elastase, and how these actions affect host functions, before we can exploit this knowledge for therapeutic benefit.

A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity

Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12−/− but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrates gene exons and by MMP-12–mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3–infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

T‐cell intrinsic effects of GITR and 4‐1BB during viral infection and cancer immunotherapy

Summary:  GITR [glucocorticoid inducible tumor necrosis factor receptor (TNFR)‐related protein] and 4‐1BB are costimulatory TNFR family members that are expressed on regulatory and effector T cells as well as on other cells of the immune system. Here we discuss the role of GITR and 4‐1BB on T cells during viral infections and in cancer immunotherapy. Systemic treatment with agonistic anti‐4‐1BB antibody leads to a number of immune system abnormalities, and clinical trials of anti‐4‐1BB have been terminated. However, other modes of 4‐1BB ligation may be less toxic. To date, similar toxicities have not been reported for anti‐GITR treatment of mice, although anti‐GITR antibodies can exacerbate mouse autoimmune models. Intrinsic effects of GITR and 4‐1BB on effector T cells appear to predominate over their effects on other cell types in some models. Despite their similarities in enhancing T‐cell survival, 4‐1BB and GITR are clearly not redundant, and both pathways are required for maximal CD8+ T‐cell responses and mouse survival following severe respiratory influenza infection. GITR uses TNFR‐associated factor (TRAF) 2 and TRAF5, whereas 4‐1BB recruits TRAF1 and TRAF2 to mediate survival signaling in T cells. The differential use of signaling adapters combined with their differential expression may explain the non‐redundant roles of GITR and 4‐1BB in the immune system.

Influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5.

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.

The Canadian Healthy Infant Longitudinal Development (CHILD) Study: examining developmental origins of allergy and asthma

The Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort study recruited 3624 pregnant women, most partners and 3542 eligible offspring. We hypothesise that early life physical and psychosocial environments, immunological, physiological, nutritional, hormonal and metabolic influences interact with genetics influencing allergic diseases, including asthma. Environmental and biological sampling, innate and adaptive immune responses, gene expression, DNA methylation, gut microbiome and nutrition studies complement repeated environmental and clinical assessments to age 5. This rich data set, linking prenatal and postnatal environments, diverse biological samples and rigorous phenotyping, will inform early developmental pathways to allergy, asthma and other chronic inflammatory diseases.

Associations between bacterial communities of house dust and infant gut

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Incorporation of 4-1BB ligand into an adenovirus vaccine vector increases the number of functional antigen-specific CD8 T cells and enhances the duration of protection against influenza-induced respiratory disease.

T cell based influenza vaccines offer the potential for cross protective immunity to multiple clades of influenza virus. Here we explored the effect of increasing CD8 T cell responses during intranasal vaccination by incorporating a T cell costimulator, 4-1BBL. Inclusion of 4-1BBL in an influenza nucleoprotein (NP)-containing adenoviral vector increased the number of NP-specific CD8 T cells and lowered the vaccine dose required for short-term protection from influenza-induced disease in mice. At higher vaccine doses, the inclusion of 4-1BBL increased the duration of protection of mice from influenza-induced mortality. Bone marrow chimera experiments revealed that the major effects of 4-1BBL were directly on αβ T cells with minor additional effects through cells other than αβ T cells. The implications of these findings are that including 4-1BBL or adjuvants that induce 4-1BBL expression may be of benefit in a vaccine setting for enhancing the magnitude and duration of T cell responses to influenza virus.