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Viral respiratory infections are a common cause of severe disease, especially in infants, people who are immunocompromised, and in the elderly. Neutrophils, an important innate immune cell, infiltrate the lungs rapidly after an inflammatory insult. The most well-characterized effector mechanisms by which neutrophils contribute to host defense are largely extracellular and the involvement of neutrophils in protection from numerous bacterial and fungal infections is well established. However, the role of neutrophils in responses to viruses, which replicate intracellularly, has been less studied. It remains unclear whether and, by which underlying immunological mechanisms, neutrophils contribute to viral control or confer protection against an intracellular pathogen. Furthermore, neutrophils need to be tightly regulated to avoid bystander damage to host tissues. This is especially relevant in the lung where damage to delicate alveolar structures can compromise gas exchange with life-threatening consequences. It is inherently less clear how neutrophils can contribute to host immunity to viruses without causing immunopathology and/or exacerbating disease severity. In this review, we summarize and discuss the current understanding of how neutrophils in the lung direct immune responses to viruses, control viral replication and spread, and cause pathology during respiratory viral infections.

Replication defective viral genomes (DVGs) generated during virus replication are the primary triggers of antiviral immunity in many RNA virus infections. However, DVGs can also facilitate viral persistence. Why and how these two opposing functions of DVGs are achieved remain unknown. Here we report that during Sendai and respiratory syncytial virus infections DVGs selectively protect a subpopulation of cells from death, thereby promoting the establishment of persistent infections. We find that during Sendai virus infection this phenotype results from DVGs stimulating a mitochondrial antiviral-signaling (MAVS)-mediated TNF response that drives apoptosis of highly infected cells while extending the survival of cells enriched in DVGs. The pro-survival effect of TNF depends on the activity of the TNFR2/TRAF1 pathway that is regulated by MAVS signaling. These results identify TNF as a pivotal factor in determining cell fate during a viral infection and delineate a MAVS/TNFR2-mediated mechanism that drives the persistence of otherwise acute viruses. Replication defective viral genomes (DVGs) can facilitate persistence of paramyxoviruses, but the underlying mechanisms are unclear. Using FISH, Xu et al. here analyze the cellular response to DVGs on a single cell level and show that a MAVS-mediated TNF response specifically extends survival of cells enriched in DVGs.

Human parainfluenza virus 3 is a highly abundant RNA virus that primarily affects young children, the elderly, and immunocompromised individuals, leading to severe lower respiratory infections and pneumonia. Despite an urgent need of treatment options for these high-risk patients, neither a vaccine nor specific antiviral are currently approved. Blocking viral entry by targeting the viral surface glycoprotein haemagglutinin-neuraminidase (HN) has shown promising results in vitro and, to some extent, in vivo. However, to further evaluate these antiviral approaches for potential human application, a detailed understanding of early hPIV-3 infection and drug treatment mechanisms in human lung tissue is needed. In this study, we established a model for early hPIV-3 infection in human precision-cut lung slices (PCLS). We demonstrate specific infection of small airway epithelial cells followed by a distinct antiviral and inflammatory response marked by expression and secretion of type I, II and III interferons, chemokines such as IP-10 and ITAC, and pro-inflammatory markers IL-6 and TNF-α, but only limited induction of cytokines associated with high clinical severity, such as IL-8. Prophylactic treatment with two viral entry HN-inhibitors significantly reduced hPIV-3 viral load and inflammatory response after infection in the human lung tissue slices, demonstrating the high usability of the PCLS infection model for pharmacological assessment of novel antiviral drugs.

Respiratory infections with RNA viruses such as respiratory syncytial virus (RSV) and influenza lead to significant morbidity and mortality. Using a natural rodent pathogen, Sendai virus (SeV), which is similar to RSV, mice made atopic with house dust mite survived a normally lethal SeV infection. One protein that we found markedly elevated in the lungs and bronchoalveolar lavage fluid of atopic mice was neuregulin-1 (NRG1). Administration of NRG1 protected naïve (non-atopic) mice from death with both SeV and mouse adapted influenza A virus (IAV). Survival was associated with reduced alveolar epithelium permeability and reduced phosphorylation of mixed lineage kinase domain-like (MLKL) protein indicating inhibition of necroptosis. In vitro , treatment of mouse lung epithelial cells with NRG1 inhibited SeV induced necroptosis, and NRG1 administration to differentiated human bronchial epithelial cells infected with RSV reduced transepithelial fluid leak and expression of necroptosis associated genes RIPK3 and MLKL , while regulating genes associated with homeostatic maintenance, suggesting stabilized epithelial integrity. In conclusion, our data demonstrate a unique function of NRG1 in respiratory viral infections by reducing alveolar leak, inhibiting epithelial necroptosis, and promoting homeostatic regulation of airway epithelium, all of which associate with markedly reduced mortality to the respiratory viral insult.

Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus–host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air–liquid interface, organoids, or ‘on-chip’ technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium—the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.

Influenza A virus is responsible for annual epidemics and occasional pandemics leading to significant mortality and morbidity in human populations. Parainfluenza viruses also contribute to lung infections and chronic lung disease. In this study, we investigated the effect of high glucose on the productivity of influenza A and Sendai (murine parainfluenza type 1) viruses in A549 immortalized cells. A glycolytic pattern of infection was determined by monitoring the release of lactate and phosphofructokinase (PFK) activity in infected and uninfected cells. qRT-PCR was used to analyze the expression of viral and cellular cytokine mRNA levels in cultured cells. The data show that the productivity of both influenza and Sendai viruses was reduced in A549 cells cultured in high-glucose conditions. This was accompanied by increased lactate production and altered PFK activity profile. Endogenous or virus infection-induced interferon β (IFN-β) mRNA expression was significantly decreased in high glucose compared to normal glucose status during early times of infection. Unlike in Sendai virus-infected cells, H1N1 virus reversed the significant increase in transforming growth factor β1 (TGF-β1) mRNA expression due to increased glucose concentration during early infection times. In conclusion, high glucose may have a negative effect on influenza and parainfluenza productivity in vitro. This effect may be considered when evaluating personalized therapeutic/diagnostic markers in infection-accompanied hyperglycemic status.

Mammalian organs consist of diverse, intermixed cell types that signal to each other via ligand-receptor interactions – an interactome – to ensure development, homeostasis and injury-repair. Dissecting such intercellular interactions is facilitated by rapidly growing single-cell RNA sequencing (scRNA-seq) data; however, existing computational methods are often not readily adaptable by bench scientists without advanced programming skills. Here, we describe a quantitative intuitive algorithm, coupled with an optimized experimental protocol, to construct and compare interactomes in control and Sendai virus-infected mouse lungs. A minimum of 90 cells per cell type compensates for the known gene dropout issue in scRNA-seq and achieves comparable sensitivity to bulk RNA sequencing. Cell lineage normalization after cell sorting allows cost-efficient representation of cell types of interest. A numeric representation of ligand-receptor interactions identifies, as outliers, known and potentially new interactions as well as changes upon viral infection. Our experimental and computational approaches can be generalized to other organs and human samples.

Background During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear. Results We report here that RLR activation triggers MAVS ubiquitination on lysine 7 and 10 by the E3 ubiquitin ligase TRIM25 and marks it for proteasomal degradation concomitantly with downstream signaling. Inhibition of this MAVS degradation with a proteasome inhibitor does not affect NF-κB signaling but it hampers IRF3 activation, and NEMO and TBK1, two essential mediators in type I IFN production, are retained at the mitochondria. Conclusions These results suggest that MAVS functions as a recruitment platform that assembles a signaling complex involving NEMO and TBK1, and that the proteasome-mediated MAVS degradation is required to release the signaling complex into the cytosol, allowing IRF3 phosphorylation by TBK1.

Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM-/- mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM-/- mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM-/- mice during SeV infection. Additionally, phosphorylation of STAT1 and NFκB p65 was lower in NKLAM-/- than wild type mice. The dysregulated phosphorylation profile of STAT1 and NFκB in NKLAM-/- mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NFκB. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM-/- mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM-/- mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.

It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.