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Background: Results from recent clinical studies suggest potential efficacy of immune training (IT)-based approaches for protection against severe lower respiratory tract infections in infants, but underlying mechanisms are unclear., Objective: We used systems-level analyses to elucidate IT mechanisms in infants in a clinical trial setting., Methods: Pre- and posttreatment peripheral blood mononuclear cells from a placebo-controlled trial in which winter treatment with the IT agent OM85 reduced infant respiratory infection frequency and/or duration were stimulated for 24 hours with the virus/bacteria mimics polyinosinic:polycytidylic acid/lipopolysaccharide. Transcriptomic profiling via RNA sequencing, pathway and upstream regulator analyses, and systems-level gene coexpression network analyses were used sequentially to elucidate and compare responses in treatment and placebo groups., Results: In contrast to subtle changes in antivirus-associated polyinosinic:polycytidylic acid response profiles, the bacterial lipopolysaccharide-triggered gene coexpression network responses exhibited OM85 treatment-associated upregulation of IFN signaling. This was accompanied by network rewiring resulting in increased coordination of TLR4 expression with IFN pathway-associated genes (especially master regulator IRF7); segregation of TNF and IFN-γ (which potentially synergize to exaggerate inflammatory sequelae) into separate expression modules; and reduced size/complexity of the main proinflammatory network module (containing, eg, IL-1,IL-6, and CCL3). Finally, we observed a reduced capacity for lipopolysaccharide-induced inflammatory cytokine (eg, IL-6 and TNF) production in the OM85 group., Conclusion: These changes are consistent with treatment-induced enhancement of bacterial pathogen detection/clearance capabilities concomitant with enhanced capacity to regulate ensuing inflammatory response intensity and duration. We posit that IT agents exemplified by OM85 potentially protect against severe lower respiratory tract infections in infants principally by effects on innate immune responses targeting the bacterial components of the mixed respiratory viral/bacterial infections that are characteristic of this age group., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Multiple regulatory mechanisms have been identified employing conventional hypothesis-driven approaches as contributing to allergen-specific immunotherapy outcomes, but understanding of how these integrate to maintain immunological homeostasis is incomplete., Objective: To explore the potential for unbiased systems-level gene co-expression network analysis to advance understanding of immunotherapy mechanisms., Methods: We profiled genome-wide allergen-induced Th-cell responses prospectively during 24 months subcutaneous immunotherapy (SCIT) in 25 rhinitis, documenting changes in immunoinflammatory pathways and associated co-expression networks and their relationships to symptom scores out to 36 months., Results: Prior to immunotherapy, mite-induced Th-cell response networks involved multiple discrete co-expression modules including those related to Th2-, type1 IFN-, inflammation- and FOXP3/IL2-associated signalling. A signature comprising 109 genes correlated with symptom scores, and these mapped to cytokine signalling/T-cell activation-associated pathways, with upstream drivers including hallmark Th1/Th2- and inflammation-associated genes. Reanalysis after 3.5 months SCIT updosing detected minimal changes to pathway/upstream regulator profiles despite 32.5% symptom reduction; however, network analysis revealed underlying merging of FOXP3/IL2-with inflammation-and Th2-associated modules. By 12 months SCIT, symptoms had reduced by 41% without further significant changes to pathway/upstream regulator or network profiles. Continuing SCIT to 24 months stabilized symptoms at 47% of baseline, accompanied by upregulation of the type1 IFN-associated network module and its merging into the Th2/FOXP3/IL2/inflammation module., Conclusions: Subcutaneous immunotherapy stimulates progressive integration of mite-induced Th cell-associated Th2-, FOXP3/IL2-, inflammation- and finally type1 IFN-signalling subnetworks, forming a single highly integrated co-expression network module, maximizing potential for stable homeostatic control of allergen-induced Th2 responses via cross-regulation. Th2-antagonistic type1 IFN signalling may play a key role in stabilizing clinical effects of SCIT., (© 2020 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Respiratory viral infections, particularly those caused by rhinovirus, exacerbate chronic respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the primary site of rhinovirus replication and responsible of initiating the host immune response to infection. Numerous studies have reported that the anti-viral innate immune response (including type I and type III interferon) in asthma is less effective or deficient leading to the conclusion that epithelial innate immunity is a key determinant of disease severity during a rhinovirus induced exacerbation. However, deficient rhinovirus-induced epithelial interferon production in asthma has not always been observed. We hypothesized that disparate in vitro airway epithelial infection models using high multiplicity of infection (MOI) and lacking genome-wide, time course analyses have obscured the role of epithelial innate anti-viral immunity in asthma and COPD. To address this, we developed a low MOI rhinovirus model of differentiated primary epithelial cells obtained from healthy, asthma and COPD donors. Using genome-wide gene expression following infection, we demonstrated that gene expression patterns are similar across patient groups, but that the kinetics of induction are delayed in cells obtained from asthma and COPD donors. Rhinovirus-induced innate immune responses were defined by interferons (type-I, II, and III), interferon response factors (IRF1, IRF3, and IRF7), TLR signaling and NF-κB and STAT1 activation. Induced gene expression was evident at 24 h and peaked at 48 h post-infection in cells from healthy subjects. In contrast, in cells from donors with asthma or COPD induction was maximal at or beyond 72-96 h post-infection. Thus, we propose that propensity for viral exacerbations of asthma and COPD relate to delayed (rather than deficient) expression of epithelial cell innate anti-viral immune genes which in turns leads to a delayed and ultimately more inflammatory host immune response., (Copyright © 2020 Veerati, Troy, Reid, Li, Nichol, Kaur, Maltby, Wark, Knight, Bosco, Grainge and Bartlett.)

RNA-Seq is increasingly used for the diagnosis of patients, targeting of therapies and for single cell transcriptomics. These applications require cost effective, fast and reliable ways of capturing and analyzing gene expression data. Here we compared Lexogen's QuantSeq which captures only the 3' end of RNA transcripts and Illumina's TruSeq, using both Tophat2 and Salmon for gene quantification. We also compared these results to microarray. This analysis was performed on peripheral blood mononuclear cells stimulated with Poly (I:C), a viral mimic that induces innate antiviral responses. This provides a well-established model to determine if RNA-Seq and QuantSeq identify the same biological signatures. Gene expression levels in QuantSeq and RNA-Seq were strongly correlated (Spearman's rho ~0.8), Salmon and Tophat2 (Spearman's rho > 0.9). There was high consistency in protein coding genes, non-concordant genes had a high proportion of shorter, non-coding features. RNA-Seq identified more differentially expressed genes than QuantSeq, both methods outperformed microarray. The same key biological signals emerged in each of these approaches. We conclude that QuantSeq, coupled with a fast quantification method such as Salmon, should provide a viable alternative to traditional RNA-Seq in many applications and may be of particular value in the study of the 3'UTR region of mRNA.

Asthma exacerbations are triggered by rhinovirus infections. We employed a systems biology approach to delineate upper-airway gene network patterns underlying asthma exacerbation phenotypes in children. Cluster analysis unveiled distinct IRF7 hi versus IRF7 lo molecular phenotypes, the former exhibiting robust upregulation of Th1/type I IFN responses and the latter an alternative signature marked by upregulation of cytokine and growth factor signaling and downregulation of IFN-γ. The two phenotypes also produced distinct clinical phenotypes. For IRF7 lo children, symptom duration prior to hospital presentation was more than twice as long from initial symptoms ( p = 0.011) and nearly three times as long for cough ( p < 0.001), the odds ratio of admission to hospital was increased more than 4-fold ( p = 0.018), and time to recurrence was shorter ( p = 0.015). In summary, our findings demonstrate that asthma exacerbations in children can be divided into IRF7 hi versus IRF7 lo phenotypes with associated differences in clinical phenotypes., (Copyright © 2019 by The American Association of Immunologists, Inc.)

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 (HDM S ) 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 HDM S -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.

Though human rhinoviruses (HRVs) are usually innocuous viruses, they can trigger serious consequences in certain individuals, especially in the setting of impaired interferon (IFN) synthesis. Plasmacytoid dendritic cells (pDCs) are key IFN producing cells, though we know little about the role of pDC in HRV-induced immune responses. Herein, we used gene expression microarrays to examine HRV-activated peripheral blood mononuclear cells (PBMCs) from healthy people, in combination with pDC depletion, to assess whether observed gene expression patterns were pDC dependent. As expected, pDC depletion led to a major reduction in IFN-α release. This was associated with profound differences in gene expression between intact PBMC and pDC-depleted PBMC, and major changes in upstream regulators: 70-80% of the HRV activated genes appeared to be pDC dependent. Real-time PCR confirmed key changes in gene expression, in which the following selected genes were shown to be highly pDC dependent: the transcription factor IRF7 , both IL-27 chains ( IL-27p28 and EBI3 ), the alpha chain of the IL-15 receptor ( IL-15RA ) and the IFN-related gene IFI27 . HRV-induced IL-6, IFN-γ, and IL-27 protein synthesis were also highly pDC dependent. Supplementing pDC-depleted cultures with recombinant IL-15, IFN-γ, IL-27, or IL-6 was able to restore the IFN-α response, thereby compensating for the absence of pDC. Though pDC comprise only a minority population of migratory leukocytes, our findings highlight the profound extent to which these cells contribute to the immune response to HRV.

Infection-associated inflammatory stress during pregnancy is the most common cause of fetal growth restriction and/or miscarriage. Treatment strategies for protection of at-risk mothers are limited to a narrow range of vaccines, which do not cover the bulk of the common pathogens most frequently encountered. Using mouse models, we demonstrate that oral treatment during pregnancy with a microbial-derived immunomodulator (OM85), currently used clinically for attenuation of infection-associated airway inflammatory symptoms in infants-adults, markedly reduces risk for fetal loss/growth restriction resulting from maternal challenge with bacterial lipopolysaccharide or influenza. Focusing on LPS exposure, we demonstrate that the key molecular indices of maternal inflammatory stress, notably high levels of RANTES, MIP-1α, CCL2, KC, and G-CSF (granulocyte colony-stimulating factor) in gestational tissues/serum, are abrogated by OM85 pretreatment. Systems-level analyses conducted in parallel using RNASeq revealed that OM85 pretreatment selectively tunes LPS-induced activation in maternal gestational tissues for attenuated expression of TNF, IL1, and IFNG-driven proinflammatory networks, without constraining Type1-IFN-associated networks central to first-line antimicrobial defense. This study suggests that broad-spectrum protection-of-pregnancy against infection-associated inflammatory stress, without compromising capacity for efficient pathogen eradication, represents an achievable therapeutic goal.

Respiratory viral infections are a leading cause of disease and mortality. The severity of these illnesses can vary markedly from mild or asymptomatic upper airway infections to severe wheezing, bronchiolitis or pneumonia. In this article, we review the viral sensing pathways and organizing principles that govern the innate immune response to infection. Then, we reconstruct the molecular networks that differentiate symptomatic from asymptomatic respiratory viral infections, and identify the underlying molecular drivers of these networks. Finally, we discuss unique aspects of the biology and pathogenesis of infections with respiratory syncytial virus, rhinovirus and influenza, drawing on insights from genomics.

Vitamin D deficiency is associated with asthma risk. Vitamin D deficiency may enhance the inflammatory response, and we have previously shown that airway remodeling and airway hyperresponsiveness is increased in vitamin D-deficient mice. In this study, we hypothesize that vitamin D deficiency would exacerbate house dust mite (HDM)-induced inflammation and alterations in lung structure and function. A BALB/c mouse model of vitamin D deficiency was established by dietary manipulation. Responsiveness to methacholine, airway smooth muscle (ASM) mass, mucus cell metaplasia, lung and airway inflammation, and cytokines in bronchoalveolar lavage (BAL) fluid were assessed. Gene expression patterns in mouse lung samples were profiled by RNA-Seq. HDM exposure increased inflammation and inflammatory cytokines in BAL, baseline airway resistance, tissue elastance, and ASM mass. Vitamin D deficiency enhanced the HDM-induced influx of lymphocytes into BAL, ameliorated the HDM-induced increase in ASM mass, and protected against the HDM-induced increase in baseline airway resistance. RNA-Seq identified nine genes that were differentially regulated by vitamin D deficiency in the lungs of HDM-treated mice. Immunohistochemical staining confirmed that protein expression of midline 1 (MID1) and adrenomedullin was differentially regulated such that they promoted inflammation, while hypoxia-inducible lipid droplet-associated, which is associated with ASM remodeling, was downregulated. Protein expression studies in human bronchial epithelial cells also showed that addition of vitamin D decreased MID1 expression. Differential regulation of these genes by vitamin D deficiency could determine lung inflammation and pathophysiology and suggest that the effect of vitamin D deficiency on HDM-induced allergic airways disease is complex., (Copyright © 2016 the American Physiological Society.)

Background: Asthma is strongly associated with allergic sensitization, but the mechanisms that determine why only a subset of atopics develop asthma are not well understood. The aim of this study was to test the hypothesis that variations in allergen-driven CD4 T cell responses are associated with susceptibility to expression of asthma symptoms., Methods: The study population consisted of house dust mite (HDM) sensitized atopics with current asthma (n = 22), HDM-sensitized atopics without current asthma (n = 26), and HDM-nonsensitized controls (n = 24). Peripheral blood mononuclear cells from these groups were cultured in the presence or absence of HDM extract for 24 h. CD4 T cells were then isolated by immunomagnetic separation, and gene expression patterns were profiled on microarrays., Results: Differential network analysis of HDM-induced CD4 T cell responses in sensitized atopics with or without asthma unveiled a cohort of asthma-associated genes that escaped detection by more conventional data analysis techniques. These asthma-associated genes were enriched for targets of STAT6 signaling, and they were nested within a larger coexpression module comprising 406 genes. Upstream regulator analysis suggested that this module was driven primarily by IL-2, IL-4, and TNF signaling; reconstruction of the wiring diagram of the module revealed a series of hub genes involved in inflammation (IL-1B, NFkB, STAT1, STAT3), apoptosis (BCL2, MYC), and regulatory T cells (IL-2Ra, FoxP3). Finally, we identified several negative regulators of asthmatic CD4 T cell responses to allergens (e.g. IL-10, type I interferons, microRNAs, drugs, metabolites), and these represent logical candidates for therapeutic intervention., Conclusion: Differential network analysis of allergen-induced CD4 T cell responses can unmask covert disease-associated genes and pin point novel therapeutic targets.

Rationale: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function., Aim of the Study: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function., Materials and Methods: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests., Results: HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection., Conclusion: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.