Anya C. Jones

25-hydroxyvitamin D3 status is associated with developing adaptive and innate immune responses in the first 6 months of life

Vitamin D (25[OH]D3) status in early life has been linked to the risk of allergic disease in multiple observational studies. While immunomodulating properties are well recognized, there are few longitudinal studies of 25(OH)D3 status, immune function and allergic disease in infants.

The Effects of In Utero Vitamin D Deficiency on Airway Smooth Muscle Mass and Lung Function

We have previously demonstrated increased airway smooth muscle (ASM) mass and airway hyperresponsiveness in whole-life vitamin D-deficient female mice. In this study, we aimed to uncover the molecular mechanisms contributing to altered lung structure and function. RNA was extracted from lung tissue of whole-life vitamin D-deficient and -replete female mice, and gene expression patterns were profiled by RNA sequencing. The data showed that genes involved in embryonic organ development, pattern formation, branching morphogenesis, Wingless/Int signaling, and inflammation were differentially expressed in vitamin D-deficient mice. Network analysis suggested that differentially expressed genes were connected by the hubs matrix metallopeptidase 9; NF-κ light polypeptide gene enhancer in B cells inhibitor, α; epidermal growth factor receptor; and E1A binding protein p300. Given our findings that developmental pathways may be altered, we investigated if the timing of vitamin D exposure (in utero vs. postnatal) had an impact on lung health outcomes. Gene expression was measured in in utero or postnatal vitamin D-deficient mice, as well as whole-life vitamin D-deficient and -replete mice at 8 weeks of age. Baseline lung function, airway hyperresponsiveness, and airway inflammation were measured and lungs fixed for lung structure assessment using stereological methods and quantification of ASM mass. In utero vitamin D deficiency was sufficient to increase ASM mass and baseline airway resistance and alter lung structure. There were increased neutrophils but decreased lymphocytes in bronchoalveolar lavage. Expression of inflammatory molecules S100A9 and S100A8 was mainly increased in postnatal vitamin D-deficient mice. These observations suggest that in utero vitamin D deficiency can alter lung structure and function and increase inflammation, contributing to symptoms in chronic diseases, such as asthma.

Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers.

Background The timing and mechanisms of asthma inception remain imprecisely defined. Although epigenetic mechanisms likely contribute to asthma pathogenesis, little is known about their role in asthma inception. Objective We sought to assess whether the trajectory to asthma begins already at birth and whether epigenetic mechanisms, specifically DNA methylation, contribute to asthma inception. Methods We used the Methylated CpG Island Recovery Assay chip to survey DNA methylation in cord blood mononuclear cells from 36 children (18 nonasthmatic and 18 asthmatic subjects by age 9 years) from the Infant Immune Study (IIS), an unselected birth cohort closely monitored for asthma for a decade. SMAD3 methylation in IIS (n = 60) and in 2 replication cohorts (the Manchester Asthma and Allergy Study [n = 30] and the Childhood Origins of Asthma Study [n = 28]) was analyzed by using bisulfite sequencing or Illumina 450K arrays. Cord blood mononuclear cell–derived IL‐1&bgr; levels were measured by means of ELISA. Results Neonatal immune cells harbored 589 differentially methylated regions that distinguished IIS children who did and did not have asthma by age 9 years. In all 3 cohorts methylation in SMAD3, the most connected node within the network of asthma‐associated, differentially methylated regions, was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. Moreover, SMAD3 methylation in IIS neonates with maternal asthma was strongly and positively associated with neonatal production of IL‐1&bgr;, an innate inflammatory mediator. Conclusions The trajectory to childhood asthma begins at birth and involves epigenetic modifications in immunoregulatory and proinflammatory pathways. Maternal asthma influences epigenetic mechanisms that contribute to the inception of this trajectory. Graphical abstract Figure. No Caption available.

Network analysis of immunotherapy-induced regressing tumours identifies novel synergistic drug combinations

Cancer immunotherapy has shown impressive results, but most patients do not respond. We hypothesized that the effector response in the tumour could be visualized as a complex network of interacting gene products and that by mapping this network we could predict effective pharmacological interventions. Here, we provide proof of concept for the validity of this approach in a murine mesothelioma model, which displays a dichotomous response to anti-CTLA4 immune checkpoint blockade. Network analysis of gene expression profiling data from responding versus non-responding tumours was employed to identify modules associated with response. Targeting the modules via selective modulation of hub genes or alternatively by using repurposed pharmaceuticals selected on the basis of their expression perturbation signatures dramatically enhanced the efficacy of CTLA4 blockade in this model. Our approach provides a powerful platform to repurpose drugs, and define contextually relevant novel therapeutic targets.

Genomic Responses during Acute Human Anaphylaxis Are Characterized by Upregulation of Innate Inflammatory Gene Networks

Background Systemic spread of immune activation and mediator release is required for the development of anaphylaxis in humans. We hypothesized that peripheral blood leukocyte (PBL) activation plays a key role. Objective To characterize PBL genomic responses during acute anaphylaxis. Methods PBL samples were collected at three timepoints from six patients presenting to the Emergency Department (ED) with acute anaphylaxis and six healthy controls. Gene expression patterns were profiled on microarrays, differentially expressed genes were identified, and network analysis was employed to explore underlying mechanisms. Results Patients presented with moderately severe anaphylaxis after oral aspirin (2), peanut (2), bee sting (1) and unknown cause (1). Two genes were differentially expressed in patients compared to controls at ED arrival, 67 genes at 1 hour post-arrival and 2,801 genes at 3 hours post-arrival. Network analysis demonstrated that three inflammatory modules were upregulated during anaphylaxis. Notably, these modules contained multiple hub genes, which are known to play a central role in the regulation of innate inflammatory responses. Bioinformatics analyses showed that the data were enriched for LPS-like and TNF activation signatures. Conclusion PBL genomic responses during human anaphylaxis are characterized by dynamic expression of innate inflammatory modules. Upregulation of these modules was observed in patients with different reaction triggers. Our findings indicate a role for innate immune pathways in the pathogenesis of human anaphylaxis, and the hub genes identified in this study represent logical candidates for follow-up studies.

Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection

BackgroundMycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah) are environmental mycobacteria and significant opportunistic pathogens. Mycobacterium avium infections in humans and pigs are mainly due to Mah. It is not known whether this is caused by a difference in virulence or difference in exposure to the two subspecies. The aim of the present study was to investigate the ability of the M. avium subspecies to replicate intracellularly and to characterise the gene expression program triggered by infection of human primary macrophages.ResultsAll isolates were able to invade and persist within human macrophages. However, intracellular replication was only evident in cells infected with the two Maa isolates. Transcriptional responses to the isolates were characterized by upregulation of genes involved in apoptosis, immune- and inflammatory response, signal transduction and NF-kB signaling, cell proliferation and T-cell activation. Although similar pathways and networks were perturbed by the different isolates, the response to the Maa subspecies was exaggerated, and there was evidence of increased activation of type I and II interferon signaling pathways.ConclusionMycobacterium avium isolates of different genetic characteristics invaded monocytes and induced different degree of macrophage activation. Isolates of Maa were able to replicate intracellularly suggesting that differences in exposure, uptake or induction of adaptive immunity are more likely explanations for the difference in prevalence between M. avium subspecies.

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics

Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDMS) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDMS-wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics.

Using Network Analysis to Understand Severe Asthma Phenotypes

Asthma is a highly complex and heterogeneous disease that comprises multiple subphenotypes. In most patients, the disease is characterized by reversible airflow limitation; however, a subset of patients suffer from a more severe form of the disease, which is associated with symptoms that are less controllable; they are unresponsive to treatment with conventional glucocorticoid (GC) therapy and require increasingly high doses of inhaled and/or oral GC in addition to combination therapy. Other characteristics associated with severe asthma include atopy, rhinosinusitis, reduced lung function, frequent and/or severe exacerbations, and variations in age of onset and airway inflammatory profiles (eosinophilic, neutrophilic, paucigranulocytic) (1). Understanding the pathogenesis of severe asthma is a daunting task, given that numerous candidate susceptibility genes have been identified and that these genes interact in complex ways with environmental cofactors (2). The application of high-throughput molecular profiling technologies (e.g., microarray, RNA-Seq) to the study of gene expression patterns in asthma has produced vast amounts of data that are now available in the public domain. These data can be downloaded and reanalyzed with the latest algorithms, unveiling new biological insights (3). Previous gene expression studies in asthma have primarily focused on identification of differentially expressed genes. However, this approach is limited, as genes do not exist or function in isolation; instead, they work together. A significant advance in this context was the application of network graph theory to genomic data analysis (4). The underlying concept here is that a functioning biological system can be represented as a “ball-and-stick” diagram, where “balls” represent genes, and “sticks” or links represent coexpression relationships between genes. Rather than focusing on gene expression levels, network analysis interrogates coexpression relationships across the samples to infer the wiring diagram of the underlying gene networks. Coexpression network analysis reveals two fundamental organizing principles of biological systems: the presence of highly connected “hub” genes, which dominate the structure of biological networks, and modules, which are sets of coexpressed genes that function in the same biological pathway or process (4). In this issue of the Journal, Modena and colleagues (pp. 1449– 1463) used coexpression network analysis to interrogate the molecular mechanisms underlying severe asthma (Figure 1) (5). They reanalyzed data they previously published and deposited in the Gene Expression Omnibus repository (6), which consisted of microarray profiles of gene expression derived from epithelial cell brushings collected from adult patients with asthma (n = 129) and healthy controls (n = 26) from the Severe Asthma Research Program cohort. Of note, around one third of the patients met the American Thoracic Society criteria for severe asthma, and two thirds of the patients with severe asthma were using oral corticosteroids. A coexpression network was constructed from all expressed genes (30,889) employing weighted gene coexpression network analysis (WGCNA) (7), resulting in the identification of 64 modules. The expression of each module was summarized using principal component analysis, and the first principal component was correlated with all available clinical traits, unveiling module–trait associations. The clinical traits most significantly associated with gene network patterns were body mass index, ethnicity, chronic rhinosinusitis, fractional exhaled nitric oxide, asthma severity, Asthma Quality of Life Questionnaire scores, FEV1% predicted, emergency room visits/hospitalization, and use of inhaled or oral corticosteroids. Notably, a module of chromosome Y-linked genes was strongly associated with male sex, thus demonstrating the plausibility of the overall approach. The authors found that four modules (color coded as magenta, mediumpurple3, red, and thistle1) were up-regulated in association with American Thoracic Society–defined asthma severity. Of these modules, magenta and red were most strongly and positively correlated with disease severity, emergency presentations/hospitalization, and use of inhaled and oral corticosteroids. They were also inversely related to lung function and quality-of-life scores (note that low Asthma Quality of Life Questionnaire scores indicate more severe symptoms). Magenta was enriched with proteins involved in mitosis and cell division. The red module was enriched with Th2-associated genes (e.g., IL-13, CCL26, CST1, CHI3L1, CLCA1, MUC5AC, and POSTN) (8), and expression of this module was strongly correlated with fractional exhaled nitric oxide and body mass index and was elevated in African Americans compared with subjects of European ancestry. Thistle1 was enriched with histones and related proteins, and mediumpurple3 was enriched with GC-induced genes. The authors also identified a Th1/interferon-associated module (CXCL9, CXCL10, CXCL11, IDO1, IL-15, PARP9, PSMB8, PSMB9, PSMB10, and STAT1), and although this module was up-regulated in 26% of total patients with asthma, it was not significantly correlated with asthma severity. Two modules (grey60 and thistle2) were identified that were inversely related to disease severity. Grey60 was enriched with genes involved in epithelial growth and repair, and thistle2 was enriched with genes involved in neuronal development and function. Overall, the data show that severe asthma and related traits are associated with up-regulation of gene networks involved in mitosis and Th2 inflammation and down-regulation of networks involved in epithelial growth and repair and neuronal development and function. To identify genes that were strongly associated with clinical traits and central components of the modules, the authors selected

Basophil levels in PBMC population during childhood acute wheeze/asthma are associated with future exacerbations

Our data suggest that a basophil level above 0.18% of the PBMC population during an acute respiratory exacerbation is associated with an increased risk for future exacerbations in children with asthma and/or wheeze.

Atopy-dependent and independent immune responses in the heightened severity of atopics to respiratory viral infections: Rat model studies

Allergic (Th2high immunophenotype) asthmatics have a heightened susceptibility to common respiratory viral infections such as human rhinovirus. Evidence suggests that the innate interferon response is deficient in asthmatic/atopic individuals, while other studies show no differences in antiviral response pathways. Unsensitized and OVA-sensitized/challenged Th2high (BN rats) and Th2low immunophenotype (PVG rats) animals were inoculated intranasally with attenuated mengovirus (vMC0). Sensitized animals were exposed/unexposed during the acute viral response phase. Cellular and transcriptomic profiling was performed on bronchoalveolar lavage cells. In unsensitized PVG rats, vMC0 elicits a prototypical antiviral response (neutrophilic airways inflammation, upregulation of Th1/type I interferon-related pathways). In contrast, response to infection in the Th2high BN rats was associated with a radically altered intrinsic host response to respiratory viral infection, characterized by macrophage influx/Th2-associated pathways. In sensitized animals, response to virus infection alone was not altered compared to unsensitized animals. However, allergen exposure of sensitized animals during viral infection unleashes a notably exaggerated airways inflammatory response profile orders of magnitude higher in BN versus PVG rats despite similar viral loads. The co-exposure responses in the Th2high BN incorporated type I interferon/Th1, alternative macrophage activation/Th2 and Th17 signatures. Similar factors may underlie the hyper-susceptibility to infection-associated airways inflammation characteristic of the human Th2high immunophenotype.