Gene editing for latent herpes simplex virus infection reduces viral load and shedding in vivo.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.

Gene editing and elimination of latent herpes simplex virus in vivo.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.

The effects of genotype × phenotype interactions on silver nanoparticle toxicity in organotypic cultures of murine tracheal epithelial cells.

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no in vitro studies to date have used organotypic cultures as a high-content in vitro model of the conducting airway to characterize the effects of interactions between host genetic and acquired factors, or gene × phenotype interactions (G × P), on AgNP toxicity. In the present study, we derived organotypic cultures from primary murine tracheal epithelial cells (MTEC) to characterize nominal and dosimetric dose-response relationships for AgNPs with a gold core on barrier dysfunction, glutathione (GSH) depletion, reactive oxygen species (ROS) production, lipid peroxidation, and cytotoxicity across two genotypes (A/J and C57BL/6J mice), two phenotypes ('Normal' and 'Type 2 [T2]-Skewed'), and two exposures (an acute exposure of 24 h and a subacute exposure of 4 h, every other day, over 5 days [5 × 4 h]). We characterized the 'T2-Skewed' phenotype as an in vitro model of chronic respiratory diseases, which was marked by increased sensitivity to AgNP-induced barrier dysfunction, GSH depletion, ROS production, lipid peroxidation, and cytotoxicity, suggesting that asthmatics are a sensitive population to AgNP exposures in occupational settings. This also suggests that exposure limits, which should be based upon the most sensitive population, should be derived using in vitro and in vivo models of chronic respiratory diseases. This study highlights the importance of considering dosimetry as well as G × P effects when screening and prioritizing potential respiratory toxicants. Such in vitro studies can be used to inform regulatory policy aimed at special protections for all populations.

The Effects of Genotype × Phenotype Interactions on Transcriptional Response to Silver Nanoparticle Toxicity in Organotypic Cultures of Murine Tracheal Epithelial Cells.

The airway epithelium is critical for maintaining innate and adaptive immune responses, and occupational exposures that disrupt its immune homeostasis may initiate and amplify airway inflammation. In our previous study, we demonstrated that silver nanoparticles (AgNP), which are engineered nanomaterials used in multiple applications but primarily in the manufacturing of many antimicrobial products, induce toxicity in organotypic cultures derived from murine tracheal epithelial cells (MTEC), and those differentiated toward a "Type 2 [T2]-Skewed" phenotype experienced an increased sensitivity to AgNP toxicity, suggesting that asthmatics could be a sensitive population to AgNP exposures in occupational settings. However, the mechanistic basis for this genotype × phenotype (G × P) interaction has yet to be defined. In this study, we conducted transcriptional profiling using RNA-sequencing to predict the enrichment of specific canonical pathways and upstream transcriptional regulators to assist in defining a mechanistic basis for G × P effects on AgNP toxicity. Organotypic cultures were derived from MTEC across 2 genetically inbred mouse strains (A/J and C57BL/6J mice), 2 phenotypes ("Normal" and "T2-Skewed"), and 1 AgNP exposure (an acute 24 h exposure) to characterize G × P effects on transcriptional response to AgNP toxicity. The "T2-Skewed" phenotype was marked by increased pro-inflammatory T17 responses to AgNP toxicity, which are significant predictors of neutrophilic/difficult-to-control asthma and suggests that asthmatics could be a sensitive population to AgNP exposures in occupational settings. This study highlights the importance of considering G × P effects when identifying these sensitive populations, whose underlying genetics or diseases could directly modify their response to AgNP exposures.

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus.

Infection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography-mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in PR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell protein profiles will elucidate cellular processes in IAV pathogenesis, which may provide insight into potential therapies to modulate disease severity.

Neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology.

Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMN-depleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.

Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses.

Severe respiratory viral infections are associated with spread to the alveoli of the lungs. There are multiple murine models of severe respiratory viral infections that have been used to identify viral and host factors that contribute to disease severity. Primary cultures of murine alveolar epithelial cells provide a robust in vitro model to perform mechanistic studies that can be correlated with in vivo studies to identify cell type-specific factors that contribute to pathology within the alveoli of the lung during viral infection. In this study, we established an in vitro model to compare the responses of type I (ATI) and type II (ATII) alveolar epithelial cells to infection by respiratory viruses used in murine models: mouse-adapted severe acute respiratory syndrome-associated coronavirus (SARS-CoV, v2163), murine coronavirus MHV-1, and influenza A (H1N1) virus, strain PR8. Murine alveolar cells cultured to maintain an ATII cell phenotype, determined by expression of LBP180, were susceptible to infection by all three viruses. In contrast, ATII cells that were cultured to trans-differentiate into an ATI-like cell phenotype were susceptible to MHV-1 and PR8, but not mouse-adapted SARS-CoV. Epithelial cells produce cytokines in response to viral infections, thereby activating immune responses. Thus, virus-induced cytokine expression was quantified in ATI and ATII cells. Both cell types had increased expression of IL-1ß mRNA upon viral infection, though at different levels. While MHV-1 and PR8 induced expression of a number of shared cytokines in ATI cells, there were several cytokines whose expression was induced uniquely by MHV-1 infection. In summary, ATI and ATII cells exhibited differential susceptibilities and cytokine responses to infection by respiratory viruses. This in vitro model will be critical for future studies to determine the roles of these specialized cell types in the pathogenesis of respiratory viral infection.

Virus-infected alveolar epithelial cells direct neutrophil chemotaxis and inhibit their apoptosis.

The alveolar epithelium is a critical target for pulmonary viruses and can produce proinflammatory cytokines and chemokines upon viral infection. However, the molecular interactions between virus-infected alveolar epithelial cells and inflammatory cells, including polymorphonuclear leukocytes (PMNs), have not been thoroughly characterized. Rat coronavirus (RCoV) is used as a model to study the immune response to viral infection in the lung of the natural host. We have developed an in vitro model to characterize the response of PMNs to RCoV-infected type I-like alveolar epithelial (AT1) cells, the primary target for RCoV infection in the alveoli. Multiple CXC chemokines that signal through CXCR2 were required for PMN chemotaxis toward medium from RCoV-infected AT1-like cells (RCoV-AT1). Furthermore, RCoV-AT1 inhibited spontaneous PMN apoptosis, including activation of effector caspase 3 and initiator caspases 8 and 9. Use of a selective inhibitor of CXCR2, SB265610, demonstrated that CXCR2 signaling was required for RCoV-AT1-mediated inhibition of PMN apoptosis. These data suggest that CXC chemokines produced by RCoV-infected AT1-like cells inhibit PMN apoptosis during infection. These studies provide new insight into the molecular mechanisms whereby alveolar epithelial cells direct the functions of PMNs during viral infection of the lung.

The interplay between hMLH1 and hMRE11: role in MMR and the effect of hMLH1 mutations.

Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction. Our in vitro MMR assay demonstrated that the dominant-negative hMRE11(452-634) mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 3' excision and 3'-directed MMR activities. Furthermore, the expression of hMRE11(452-634), and to a lesser extent hMRE11(1-634) (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (>50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants.