Transcriptomic analysis of primary nasal epithelial cells reveals altered interferon signalling in preterm birth survivors at one year of age.

Introduction: Many survivors of preterm birth (<37 weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1 year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-ß [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid.

In recent years the study of the commensal microbiota is driving a remarkable paradigm shift in our understanding of human physiology. However, intrinsic technical difficulties associated with investigating the Microbiomics of some body niches are hampering the development of new knowledge. This is particularly the case when investigating the functional role played by the human microbiota in modulating the physiology of key organ systems. A major hurdle in investigating specific Microbiome communities is linked to low bacterial density and susceptibility to bias caused by environmental contamination. To prevent such inaccuracies due to background processing noise, harmonized tools for Microbiomic and bioinformatics practices have been recommended globally. The fact that the impact of this undesirable variability is negatively correlated with the DNA concentration in the sample highlights the necessity to improve existing DNA isolation protocols. In this report, we developed and tested a protocol to more efficiently recover bacterial DNA from low volumes of bronchoalveolar lavage fluid obtained from infants and adults. We have compared the efficiency of the described method with that of a commercially available kit for microbiome analysis in body fluids. We show that this new methodological approach performs better in terms of extraction efficiency. As opposed to commercial kits, the DNA extracts obtained with this new protocol were clearly distinguishable from the negative extraction controls in terms of 16S copy number and Microbiome community profiles. Altogether, we described a cost-efficient protocol that can facilitate microbiome research in low-biomass human niches.

Rhinovirus Infection Drives Complex Host Airway Molecular Responses in Children With Cystic Fibrosis.

Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

The Detection of Bile Acids in the Lungs of Paediatric Cystic Fibrosis Patients Is Associated with Altered Inflammatory Patterns.

Background: Cystic fibrosis (CF) is a hereditary disorder in which persistent unresolved inflammation and recurrent airway infections play major roles in the initiation and progression of the disease. Little is known about triggering factors modulating the transition to chronic microbial infection and inflammation particularly in young children. Cystic fibrosis respiratory disease starts early in life, with the detection of inflammatory markers and infection evident even before respiratory symptoms arise. Thus, identifying factors that dysregulate immune responsiveness at the earliest stages of the disease will provide novel targets for early therapeutic intervention. Methods: We evaluated the clinical significance of bile acid detection in the bronchoalveolar lavage fluid of clinically stable preschool-aged children diagnosed with CF. Results: We applied an unbiased classification strategy to categorize these specimens based on bile acid profiles. We provide clear associations linking the presence of bile acids in the lungs with alterations in the expression of inflammatory markers. Using multiple regression analysis, we also demonstrate that clustering based on bile acid profiles is a meaningful predictor of the progression of structural lung disease. Conclusions: Altogether, our work has identified a clinically relevant host-derived factor that may participate in shaping early events in the aetiology of CF respiratory disease.

Developmental control of hypoxia during bud burst in grapevine.

Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.

Perturbation of polyamine catabolism affects grape ripening of Vitis vinifera cv. Trincadeira.

Grapes are economically the most important fruit worldwide. However, the complexity of biological events that lead to ripening of nonclimacteric fruits is not fully understood, particularly the role of polyamines' catabolism. The transcriptional and metabolic profilings complemented with biochemical data were studied during ripening of Trincadeira grapes submitted to guazatine treatment, a potent inhibitor of polyamine oxidase activity. The mRNA expression profiles of one time point (EL 38) corresponding to harvest stage was compared between mock and guazatine treatments using Affymetrix GrapeGen(®) genome array. A total of 2113 probesets (1880 unigenes) were differentially expressed between these samples. Quantitative RT-PCR validated microarrays results being carried out for EL 35 (véraison berries), EL 36 (ripe berries) and EL 38 (harvest stage berries). Metabolic profiling using HPLC and (1)H NMR spectroscopy showed increase of putrescine, proline, threonine and 1-O-ethyl-ß-glucoside in guazatine treated samples. Genes involved in amino acid, carbohydrate and water transport were down-regulated in guazatine treated samples suggesting that the strong dehydrated phenotype obtained in guazatine treated samples may be due to impaired transport mechanisms. Genes involved in terpenes' metabolism were differentially expressed between guazatine and mock treated samples. Altogether, results support an important role of polyamine catabolism in grape ripening namely in cell expansion and aroma development.

Adaptation of tobacco etch potyvirus to a susceptible ecotype of Arabidopsis thaliana capacitates it for systemic infection of resistant ecotypes.

Viral pathogens continue to emerge among humans, domesticated animals and cultivated crops. The existence of genetic variance for resistance in the host population is crucial to the spread of an emerging virus. Models predict that rapid spread decreases with the frequency and diversity of resistance alleles in the host population. However, empirical tests of this hypothesis are scarce. Arabiodpsis thaliana--tobacco etch potyvirus (TEV) provides an experimentally suitable pathosystem to explore the interplay between genetic variation in host's susceptibility and virus diversity. Systemic infection of A. thaliana with TEV is controlled by three dominant loci, with different ecotypes varying in susceptibility depending on the genetic constitution at these three loci. Here, we show that the TEV adaptation to a susceptible ecotype allowed the virus to successfully infect, replicate and induce symptoms in ecotypes that were fully resistant to the ancestral virus. The value of these results is twofold. First, we showed that the existence of partially susceptible individuals allows for the emerging virus to bypass resistance alleles that the virus has never encountered. Second, the concept of resistance genes may only be valid for a well-defined viral genotype but not for polymorphic viral populations.

Upper-limit mutation rate estimation for a plant RNA virus.

It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3x10(-5) per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.

The pleiotropic cost of host-specialization in Tobacco etch potyvirus.

Host-range expansion is thought to allow viruses to broaden their ecological niches by allowing access to new resources. However, traits governing the infection of multiple hosts may decrease fitness in the original one due to the pleiotropic effect of adaptive mutations that may give rise to fitness tradeoffs across hosts. Here, we have experimentally examined the consequences of host-specialization in the plant pathogen Tobacco etch potyvirus (TEV). Replicate populations of TEV were allowed to evolve for 15 serial undiluted passages on the original tobacco host or on pepper, a novel host. Virulence and biologically active viral load were evaluated during the course of the experiment for each lineage on both potential hosts. In agreement with the tradeoff hypothesis, lineages evolved in the novel host experienced substantial increases in virulence and virus accumulation in its own host, but suffered reduced virulence and accumulation on the original host. By contrast, lineages evolved on the ancestral host did not increase virulence or viral load on either host. Genomic consensus sequences were obtained for each lineage at the end time point. The potential relevance for the evolution of virulence and virus fitness of the characterized mutations is discussed.

Changes in the gene expression profile of Arabidopsis thaliana after infection with Tobacco etch virus.

BACKGROUND: Tobacco etch potyvirus (TEV) has been extensively used as model system for the study of positive-sense RNA virus infecting plants. TEV ability to infect Arabidopsis thaliana varies among ecotypes. In this study, changes in gene expression of A. thaliana ecotype Ler infected with TEV have been explored using long-oligonucleotide arrays. A. thaliana Ler is a susceptible host that allows systemic movement, although the viral load is low and syndrome induced ranges from asymptomatic to mild. Gene expression profiles were monitored in whole plants 21 days post-inoculation (dpi). Microarrays contained 26,173 protein-coding genes and 87 miRNAs. RESULTS: Expression analysis identified 1727 genes that displayed significant and consistent changes in expression levels either up or down, in infected plants. Identified TEV-responsive genes encode a diverse array of functional categories that include responses to biotic (such as the systemic acquired resistance pathway and hypersensitive responses) and abiotic stresses (droughtness, salinity, temperature, and wounding). The expression of many different transcription factors was also significantly affected, including members of the R2R3-MYB family and ABA-inducible TFs. In concordance with several other plant and animal viruses, the expression of heat-shock proteins (HSP) was also increased. Finally, we have associated functional GO categories with KEGG biochemical pathways, and found that many of the altered biological functions are controlled by changes in basal metabolism. CONCLUSION: TEV infection significantly impacts a wide array of cellular processes, in particular, stress-response pathways, including the systemic acquired resistance and hypersensitive responses. However, many of the observed alterations may represent a global response to viral infection rather than being specific of TEV.