Anthony Bosco

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• Zhai, J., Voraphani, N., Imboden, M., Keidel, D., Liu, C., Stern, D. A., Venker, C., Petersen, H., Bosco, A., Sherrill, D. L., Morgan, W. J., Tesfaigzi, Y., Probst-Hensch, N. M., Martinez, F. D., Halonen, M., & Guerra, S. (2024). Circulating biomarkers of airflow limitation across the life span. The Journal of allergy and clinical immunology.
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  Airflow limitation is a hallmark of COPD, which can develop through different lung function trajectories across the life span. There is a need for longitudinal studies aimed at identifying circulating biomarkers of airflow limitation across different stages of life.
• Bosco, A. (2023). Emerging role for interferons in respiratory viral infections and childhood asthma. Frontiers in immunology, 14, 1109001.
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  Respiratory syncytial virus (RSV) and Rhinovirus (RV) infections are major triggers of severe lower respiratory illnesses (sLRI) in infants and children and are strongly associated with the subsequent development of asthma. Decades of research has focused on the role of type I interferons in antiviral immunity and ensuing airway diseases, however, recent findings have highlighted several novel aspects of the interferon response that merit further investigation. In this perspective, we discuss emerging roles of type I interferons in the pathogenesis of sLRI in children. We propose that variations in interferon response patterns exist as discrete endotypes, which operate locally in the airways and systemically through a lung-blood-bone marrow axis. We discuss new insights into the role of interferons in immune training, bacterial lysate immunotherapy, and allergen-specific immunotherapy. Interferons play complex and diverse roles in the pathogenesis of sLRI and later asthma, providing new directions for mechanistic studies and drug development.
• Jones, A. C., Leffler, J., Laing, I. A., Bizzintino, J., Khoo, S. K., LeSouef, P. N., Sly, P. D., Holt, P. G., Strickland, D. H., & Bosco, A. (2023). LPS binding protein and activation signatures are upregulated during asthma exacerbations in children. Respiratory research, 24(1), 184.
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  Asthma exacerbations in children are associated with respiratory viral infection and atopy, resulting in systemic immune activation and infiltration of immune cells into the airways. The gene networks driving the immune activation and subsequent migration of immune cells into the airways remains incompletely understood. Cellular and molecular profiling of PBMC was employed on paired samples obtained from atopic asthmatic children (n = 19) during acute virus-associated exacerbations and later during convalescence. Systems level analyses were employed to identify coexpression networks and infer the drivers of these networks, and validation was subsequently obtained via independent samples from asthmatic children. During exacerbations, PBMC exhibited significant changes in immune cell abundance and upregulation of complex interlinked networks of coexpressed genes. These were associated with priming of innate immunity, inflammatory and remodelling functions. We identified activation signatures downstream of bacterial LPS, glucocorticoids and TGFB1. We also confirmed that LPS binding protein was upregulated at the protein-level in plasma. Multiple gene networks known to be involved positively or negatively in asthma pathogenesis, are upregulated in circulating PBMC during acute exacerbations, supporting the hypothesis that systemic pre-programming of potentially pathogenic as well as protective functions of circulating immune cells preceeds migration into the airways. Enhanced sensitivity to LPS is likely to modulate the severity of acute asthma exacerbations through exposure to environmental LPS.
• Orozco Morales, M. L., Rinaldi, C. A., de Jong, E., Lansley, S. M., Lee, Y. C., Zemek, R. M., Bosco, A., Lake, R. A., & Lesterhuis, W. J. (2023). Geldanamycin treatment does not result in anti-cancer activity in a preclinical model of orthotopic mesothelioma. PloS one, 18(5), e0274364.
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  Mesothelioma is characterised by its aggressive invasive behaviour, affecting the surrounding tissues of the pleura or peritoneum. We compared an invasive pleural model with a non-invasive subcutaneous model of mesothelioma and performed transcriptomic analyses on the tumour samples. Invasive pleural tumours were characterised by a transcriptomic signature enriched for genes associated with MEF2C and MYOCD signaling, muscle differentiation and myogenesis. Further analysis using the CMap and LINCS databases identified geldanamycin as a potential antagonist of this signature, so we evaluated its potential in vitro and in vivo. Nanomolar concentrations of geldanamycin significantly reduced cell growth, invasion, and migration in vitro. However, administration of geldanamycin in vivo did not result in significant anti-cancer activity. Our findings show that myogenesis and muscle differentiation pathways are upregulated in pleural mesothelioma which may be related to the invasive behaviour. However, geldanamycin as a single agent does not appear to be a viable treatment for mesothelioma.
• Read, J. F., Serralha, M., Armitage, J. D., Iqbal, M. M., Cruickshank, M. N., Saxena, A., Strickland, D. H., Waithman, J., Holt, P. G., & Bosco, A. (2023). Single cell transcriptomics reveals cell type specific features of developmentally regulated responses to lipopolysaccharide between birth and 5 years. Frontiers in immunology, 14, 1275937.
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  Human perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution.
• Tu, X., Gomez, H. M., Kim, R. Y., Brown, A. C., de Jong, E., Galvao, I., Faiz, A., Bosco, A., Horvat, J. C., Hansbro, P., & Donovan, C. (2023). Airway and parenchyma transcriptomics in a house dust mite model of experimental asthma. Respiratory research, 24(1), 32.
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  Lung transcriptomics studies in asthma have provided valuable information in the whole lung context, however, deciphering the individual contributions of the airway and parenchyma in disease pathogenesis may expedite the development of novel targeted treatment strategies. In this study, we performed transcriptomics on the airway and parenchyma using a house dust mite (HDM)-induced model of experimental asthma that replicates key features of the human disease. HDM exposure increased the expression of 3,255 genes, of which 212 were uniquely increased in the airways, 856 uniquely increased in the parenchyma, and 2187 commonly increased in both compartments. Further interrogation of these genes using a combination of network and transcription factor enrichment analyses identified several transcription factors that regulate airway and/or parenchymal gene expression, including transcription factor EC (TFEC), transcription factor PU.1 (SPI1), H2.0-like homeobox (HLX), metal response element binding transcription factor-1 (MTF1) and E74-like factor 4 (ets domain transcription factor, ELF4) involved in controlling innate immune responses. We next assessed the effects of inhibiting lung SPI1 responses using commercially available DB1976 and DB2313 on key disease outcomes. We found that both compounds had no protective effects on airway inflammation, however DB2313 (8 mg/kg) decreased mucus secreting cell number, and both DB2313 (1 mg/kg) and DB1976 (2.5 mg/kg and 1 mg/kg) reduced small airway collagen deposition. Significantly, both compounds decreased airway hyperresponsiveness. This study demonstrates that SPI1 is important in HDM-induced experimental asthma and that its pharmacological inhibition reduces HDM-induced airway collagen deposition and hyperresponsiveness.
• Ashley, S. E., Jones, A. C., Anderson, D., Holt, P. G., Bosco, A., & Tang, M. L. (2022). Remission of peanut allergy is associated with rewiring of allergen-driven T helper 2-related gene networks. Allergy, 77(10), 3015-3027.
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  The immunological changes underpinning acquisition of remission (also called sustained unresponsiveness) following food immunotherapy remain poorly defined. Limited access to effective therapies and biosamples from treatment responders has prevented progress. Probiotic peanut oral immunotherapy is highly effective at inducing remission, providing an opportunity to investigate immune changes.
• Lee, K. H., Bosco, A., O'Sullivan, M., Song, Y., Metcalfe, J., Yu, K., Mullins, B. J., Loh, R., & Zhang, G. (2022). Identifying gene network patterns and associated cellular immune responses in children with or without nut allergy. The World Allergy Organization journal, 15(2), 100631.
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  Although evidence suggests that the immune system plays a key role in the pathophysiology of nut allergy, the precise immunological mechanisms of nut allergy have not been systematically investigated. The aim of the present study was to identify gene network patterns and associated cellular immune responses in children with or without nut allergy.
• Orozco Morales, M. L., Rinaldi, C. A., de Jong, E., Lansley, S. M., Gummer, J. P., Olasz, B., Nambiar, S., Hope, D. E., Casey, T. H., Lee, Y. C., Leslie, C., Nealon, G., Shackleford, D. M., Powell, A. K., Grimaldi, M., Balaguer, P., Zemek, R. M., Bosco, A., Piggott, M. J., , Vrielink, A., et al. (2022). PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective. iScience, 25(1), 103571.
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  Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity . However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma.
• Read, J. F., Serralha, M., Mok, D., Holt, B. J., Cruickshank, M., Karpievitch, Y. V., Broadhurst, D. I., Sly, P. D., Strickland, D. H., Reinke, S. N., Holt, P. G., & Bosco, A. (2022). Lipopolysaccharide-induced interferon response networks at birth are predictive of severe viral lower respiratory infections in the first year of life. Frontiers in immunology, 13, 876654.
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  Appropriate innate immune function is essential to limit pathogenesis and severity of severe lower respiratory infections (sLRI) during infancy, a leading cause of hospitalization and risk factor for subsequent asthma in this age group. Employing a systems biology approach to analysis of multi-omic profiles generated from a high-risk cohort (n=50), we found that the intensity of activation of an LPS-induced interferon gene network at birth was predictive of sLRI risk in infancy (AUC=0.724). Connectivity patterns within this network were stronger among susceptible individuals, and a systems biology approach identified IRF1 as a putative master regulator of this response. These findings were specific to the LPS-induced interferon response and were not observed following activation of viral nucleic acid sensing pathways. Comparison of responses at birth versus age 5 demonstrated that LPS-induced interferon responses but not responses triggered by viral nucleic acid sensing pathways may be subject to strong developmental regulation. These data suggest that the risk of sLRI in early life is in part already determined at birth, and additionally that the developmental status of LPS-induced interferon responses may be a key determinant of susceptibility. Our findings provide a rationale for the identification of at-risk infants for early intervention aimed at sLRI prevention and identifies targets which may be relevant for drug development.
• Tilsed, C. M., Casey, T. H., de Jong, E., Bosco, A., Zemek, R. M., Salmons, J., Wan, G., Millward, M. J., Nowak, A. K., Lake, R. A., & Lesterhuis, W. J. (2022). Retinoic Acid Induces an IFN-Driven Inflammatory Tumour Microenvironment, Sensitizing to Immune Checkpoint Therapy. Frontiers in oncology, 12, 849793.
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  With immune checkpoint therapy (ICT) having reshaped the treatment of many cancers, the next frontier is to identify and develop novel combination therapies to improve efficacy. Previously, we and others identified beneficial immunological effects of the vitamin A derivative tretinoin on anti-tumour immunity. Although it is known that tretinoin preferentially depletes myeloid derived suppressor cells in blood, little is known about the effects of tretinoin on the tumour microenvironment, hampering the rational design of clinical trials using tretinoin in combination with ICT. Here, we aimed to identify how tretinoin changed the tumour microenvironment in mouse tumour models, using flow cytometry and RNAseq, and we sought to use that information to establish optimal dosing and scheduling of tretinoin in combination with several ICT antibodies in multiple cancer models. We found that tretinoin rapidly induced an interferon dominated inflammatory tumour microenvironment, characterised by increased CD8+ T cell infiltration. This phenotype completely overlapped with the phenotype that was induced by ICT itself, and we confirmed that the combination further amplified this inflammatory milieu. The addition of tretinoin significantly improved the efficacy of anti-CTLA4/anti-PD-L1 combination therapy, and staggered scheduling was more efficacious than concomitant scheduling, in a dose-dependent manner. The positive effects of tretinoin could be extended to ICT antibodies targeting OX40, GITR and CTLA4 monotherapy in multiple cancer models. These data show that tretinoin induces an interferon driven, CD8+ T cell tumour microenvironment that is responsive to ICT.
• Troy, N. M., Strickland, D., Serralha, M., de Jong, E., Jones, A. C., Read, J., Galbraith, S., Islam, Z., Kaur, P., Mincham, K. T., Holt, B. J., Sly, P. D., Bosco, A., & Holt, P. G. (2022). Protection against severe infant lower respiratory tract infections by immune training: Mechanistic studies. The Journal of allergy and clinical immunology, 150(1), 93-103.
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  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.
• Tu, X., Kim, R. Y., Brown, A. C., de Jong, E., Jones-Freeman, B., Ali, M. K., Gomez, H. M., Budden, K. F., Starkey, M. R., Cameron, G. J., Loering, S., Nguyen, D. H., Nair, P. M., Haw, T. J., Alemao, C. A., Faiz, A., Tay, H. L., Wark, P. A., Knight, D. A., , Foster, P. S., et al. (2022). Airway and parenchymal transcriptomics in a novel model of asthma and COPD overlap. The Journal of allergy and clinical immunology, 150(4), 817-829.e6.
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  Asthma and chronic obstructive pulmonary disease (COPD) are common chronic respiratory diseases, and some patients have overlapping disease features, termed asthma-COPD overlap (ACO). Patients characterized with ACO have increased disease severity; however, the mechanisms driving this have not been widely studied.
• Zemek, R. M., Chin, W. L., Fear, V. S., Wylie, B., Casey, T. H., Forbes, C., Tilsed, C. M., Boon, L., Guo, B. B., Bosco, A., Forrest, A. R., Millward, M. J., Nowak, A. K., Lake, R. A., Lassmann, T., & Lesterhuis, W. J. (2022). Temporally restricted activation of IFNβ signaling underlies response to immune checkpoint therapy in mice. Nature communications, 13(1), 4895.
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  The biological determinants of the response to immune checkpoint blockade (ICB) in cancer remain incompletely understood. Little is known about dynamic biological events that underpin therapeutic efficacy due to the inability to frequently sample tumours in patients. Here, we map the transcriptional profiles of 144 responding and non-responding tumours within two mouse models at four time points during ICB. We find that responding tumours display on/fast-off kinetics of type-I-interferon (IFN) signaling. Phenocopying of this kinetics using time-dependent sequential dosing of recombinant IFNs and neutralizing antibodies markedly improves ICB efficacy, but only when IFNβ is targeted, not IFNα. We identify Ly6C/CD11b inflammatory monocytes as the primary source of IFNβ and find that active type-I-IFN signaling in tumour-infiltrating inflammatory monocytes is associated with T cell expansion in patients treated with ICB. Together, our results suggest that on/fast-off modulation of IFNβ signaling is critical to the therapeutic response to ICB, which can be exploited to drive clinical outcomes towards response.
• Armitage, J. D., Newnes, H. V., McDonnell, A., Bosco, A., & Waithman, J. (2021). Fine-Tuning the Tumour Microenvironment: Current Perspectives on the Mechanisms of Tumour Immunosuppression. Cells, 10(1).
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  Immunotherapy has revolutionised the treatment of cancers by harnessing the power of the immune system to eradicate malignant tissue. However, it is well recognised that some cancers are highly resistant to these therapies, which is in part attributed to the immunosuppressive landscape of the tumour microenvironment (TME). The contexture of the TME is highly heterogeneous and contains a complex architecture of immune, stromal, vascular and tumour cells in addition to acellular components such as the extracellular matrix. While understanding the dynamics of the TME has been instrumental in predicting durable responses to immunotherapy and developing new treatment strategies, recent evidence challenges the fundamental paradigms of how tumours can effectively subvert immunosurveillance. Here, we discuss the various immunosuppressive features of the TME and how fine-tuning these mechanisms, rather than ablating them completely, may result in a more comprehensive and balanced anti-tumour response.
• Audsley, K. M., Wagner, T., Ta, C., Newnes, H. V., Buzzai, A. C., Barnes, S. A., Wylie, B., Armitage, J., Kaisho, T., Bosco, A., McDonnell, A., Cruickshank, M., Fear, V. S., Foley, B., & Waithman, J. (2021). IFNβ Is a Potent Adjuvant for Cancer Vaccination Strategies. Frontiers in immunology, 12, 735133.
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  Cancer vaccination drives the generation of anti-tumor T cell immunity and can be enhanced by the inclusion of effective immune adjuvants such as type I interferons (IFNs). Whilst type I IFNs have been shown to promote cross-priming of T cells, the role of individual subtypes remains unclear. Here we systematically compared the capacity of distinct type I IFN subtypes to enhance T cell responses to a whole-cell vaccination strategy in a pre-clinical murine model. We show that vaccination in combination with IFNβ induces significantly greater expansion of tumor-specific CD8 T cells than the other type I IFN subtypes tested. Optimal expansion was dependent on the presence of XCR1 dendritic cells, CD4 T cells, and CD40/CD40L signaling. Therapeutically, vaccination with IFNβ delayed tumor progression when compared to vaccination without IFN. When vaccinated in combination with anti-PD-L1 checkpoint blockade therapy (CPB), the inclusion of IFNβ associated with more mice experiencing complete regression and a trend in increased overall survival. This work demonstrates the potent adjuvant activity of IFNβ, highlighting its potential to enhance cancer vaccination strategies alone and in combination with CPB.
• Coleman, L. A., Khoo, S. K., Franks, K., Prastanti, F., Le Souëf, P., Karpievitch, Y. V., Laing, I. A., & Bosco, A. (2021). Personal Network Inference Unveils Heterogeneous Immune Response Patterns to Viral Infection in Children with Acute Wheezing. Journal of personalized medicine, 11(12).
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  Human rhinovirus (RV)-induced exacerbations of asthma and wheeze are a major cause of emergency room presentations and hospital admissions among children. Previous studies have shown that immune response patterns during these exacerbations are heterogeneous and are characterized by the presence or absence of robust interferon responses. Molecular phenotypes of asthma are usually identified by cluster analysis of gene expression levels. This approach however is limited, since genes do not exist in isolation, but rather work together in networks. Here, we employed personal network inference to characterize exacerbation response patterns and unveil molecular phenotypes based on variations in network structure. We found that personal gene network patterns were dominated by two major network structures, consisting of interferon-response versus FCER1G-associated networks. Cluster analysis of these structures divided children into subgroups, differing in the prevalence of atopy but not RV species. These network structures were also observed in an independent cohort of children with virus-induced asthma exacerbations sampled over a time course, where we showed that the FCER1G-associated networks were mainly observed at late time points (days four-six) during the acute illness. The ratio of interferon- and FCER1G-associated gene network responses was able to predict recurrence, with low interferon being associated with increased risk of readmission. These findings demonstrate the applicability of personal network inference for biomarker discovery and therapeutic target identification in the context of acute asthma which focuses on variations in network structure.
• McGrath, F. M., Francis, A., Fatovich, D. M., Macdonald, S. P., Arendts, G., Bosco, A., Woo, A., & Bosio, E. (2021). Small nucleolar RNA networks are up-regulated during human anaphylaxis. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 51(10), 1310-1321.
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  Anaphylaxis is a severe, potentially life-threatening allergic reaction driven primarily by the activation of mast cells. We still fail to understand factors underlying reaction severity. Furthermore, there is currently no reliable diagnostic test to confirm anaphylaxis in the emergency department (ED).
• Newnes, H. V., Armitage, J. D., Audsley, K. M., Bosco, A., & Waithman, J. (2021). Directing the Future Breakthroughs in Immunotherapy: The Importance of a Holistic Approach to the Tumour Microenvironment. Cancers, 13(23).
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  Immunotherapy has revolutionised the treatment of cancers by exploiting the immune system to eliminate tumour cells. Despite the impressive response in a proportion of patients, clinical benefit has been limited thus far. A significant focus to date has been the identification of specific markers associated with response to immunotherapy. Unfortunately, the heterogeneity between patients and cancer types means identifying markers of response to therapy is inherently complex. There is a growing appreciation for the role of the tumour microenvironment (TME) in directing response to immunotherapy. The TME is highly heterogeneous and contains immune, stromal, vascular and tumour cells that all communicate and interact with one another to form solid tumours. This review analyses major cell populations present within the TME with a focus on their diverse and often contradictory roles in cancer and how this informs our understanding of immunotherapy. Furthermore, we discuss the role of integrated omics in providing a comprehensive view of the TME and demonstrate the potential of leveraging multi-omics to decipher the underlying mechanisms of anti-tumour immunity for the development of novel immunotherapeutic strategies.
• Ralph, A. P., Webb, R., Moreland, N. J., McGregor, R., Bosco, A., Broadhurst, D., Lassmann, T., Barnett, T. C., Benothman, R., Yan, J., Remenyi, B., Bennett, J., Wilson, N., Mayo, M., Pearson, G., Kollmann, T., & Carapetis, J. R. (2021). Searching for a technology-driven acute rheumatic fever test: the START study protocol. BMJ open, 11(9), e053720.
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  The absence of a diagnostic test for acute rheumatic fever (ARF) is a major impediment in managing this serious childhood condition. ARF is an autoimmune condition triggered by infection with group A . It is the precursor to rheumatic heart disease (RHD), a leading cause of health inequity and premature mortality for Indigenous peoples of Australia, New Zealand and internationally. METHODS AND ANALYSIS: 'Searching for a Technology-Driven Acute Rheumatic Fever Test' (START) is a biomarker discovery study that aims to detect and test a biomarker signature that distinguishes ARF cases from non-ARF, and use systems biology and serology to better understand ARF pathogenesis. Eligible participants with ARF diagnosed by an expert clinical panel according to the 2015 Revised Jones Criteria, aged 5-30 years, will be recruited from three hospitals in Australia and New Zealand. Age, sex and ethnicity-matched individuals who are healthy or have non-ARF acute diagnoses or RHD, will be recruited as controls. In the discovery cohort, blood samples collected at baseline, and during convalescence in a subset, will be interrogated by comprehensive profiling to generate possible diagnostic biomarker signatures. A biomarker validation cohort will subsequently be used to test promising combinations of biomarkers. By defining the first biomarker signatures able to discriminate between ARF and other clinical conditions, the START study has the potential to transform the approach to ARF diagnosis and RHD prevention.
• de Jong, E., & Bosco, A. (2021). Unlocking immune-mediated disease mechanisms with transcriptomics. Biochemical Society transactions, 49(2), 705-714.
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  The transcriptome represents the entire set of RNA transcripts expressed in a cell, reflecting both the underlying genetic and epigenetic landscape and environmental influences, providing a comprehensive view of functional cellular states at any given time. Recent technological advances now enable the study of the transcriptome at the resolution of individual cells, providing exciting opportunities to characterise cellular and molecular events that underpin immune-medicated diseases. Here, we draw on recent examples from the literature to highlight the application of advanced bioinformatics tools to extract mechanistic insight and disease biology from bulk and single-cell transcriptomic profiles. Key considerations for the use of available analysis techniques are presented throughout.
• de Jong, E., Lauzon-Joset, J. F., Leffler, J., Serralha, M., Larcombe, A. N., Christophersen, C. T., Holt, P. G., Strickland, D. H., & Bosco, A. (2021). IRF7-Associated Immunophenotypes Have Dichotomous Responses to Virus/Allergen Coexposure and OM-85-Induced Reprogramming. Frontiers in immunology, 12, 699633.
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  High risk for virus-induced asthma exacerbations in children is associated with an IRF7lo immunophenotype, but the underlying mechanisms are unclear. Here, we applied a Systems Biology approach to an animal model comprising rat strains manifesting high (BN) low susceptibility (PVG) to experimental asthma, induced by virus/allergen coexposure, to elucidate the mechanism(s)-of-action of the high-risk asthma immunophenotype. We also investigated potential risk mitigation pretreatment with the immune training agent OM-85. Virus/allergen coexposure in low-risk PVG rats resulted in rapid and transient airways inflammation alongside IRF7 gene network formation. In contrast, responses in high-risk BN rats were characterized by severe airways eosinophilia and exaggerated proinflammatory responses that failed to resolve, and complete absence of IRF7 gene networks. OM-85 had more profound effects in high-risk BN rats, inducing immune-related gene expression changes in lung at baseline and reducing exaggerated airway inflammatory responses to virus/allergen coexposure. In low-risk PVG rats, OM-85 boosted IRF7 gene networks in the lung but did not alter baseline gene expression or cellular influx. Distinct IRF7-associated asthma risk immunophenotypes have dichotomous responses to virus/allergen coexposure and respond differentially to OM-85 pretreatment. Extrapolating to humans, our findings suggest that the beneficial effects OM-85 pretreatment may preferentially target those in high-risk subgroups.
• Anderson, D., Jones, A. C., Gaido, C. M., Carter, K. W., Laing, I. A., Bosco, A., Thomas, W. R., & Hales, B. J. (2020). Differential Gene Expression of Lymphocytes Stimulated with Rhinovirus A and C in Children with Asthma. American journal of respiratory and critical care medicine, 202(2), 202-209.
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  Individuals with asthma have heightened antibody responses to rhinoviruses (RVs), although those specific for RV-C are lower than responses specific for RV-A, suggesting poor immunity to this species. To ascertain and compare T-cell memory responses induced by RV-A and RV-C in children with and without asthma. Peripheral blood mononuclear cells from 17 children with asthma and 19 control subjects without asthma were stimulated with peptide formulations to induce representative species-specific responses to RV-A and RV-C. Molecular profiling (RNA sequencing) was used to identify enriched pathways and upstream regulators. Responses to RV-A showed higher expression of IFNG and STAT1 compared with RV-C, and significant expression of CXCL9, 10, and 11 was not found for RV-C. There was no reciprocal increase of T-helper cell type 2 (Th2) cytokine genes or the Th2 chemokine genes CCL11, CCL17, and CCL22. RV-C induced higher expression of CCL24 (eotaxin-2) than RV-A in the responses of children with and without asthma. Upstream regulator analysis showed both RV-A and, although to a lesser extent, RV-C induced predominant Th1 and inflammatory cytokine expression. The responses of children with asthma compared with those without asthma were lower for both RV-A and RV-C while retaining the pattern of gene expression and upstream regulators characteristic of each species. All groups showed activation of the IL-17A pathway. RV-C induced memory cells with a lower IFN-γ-type response than RV-A without T-helper cell type 2 (Th2) upregulation. Children with asthma had lower recall responses than those without asthma while largely retaining the same gene activation profile for each species. RV-A and RV-C, therefore, induce qualitatively different T-cell responses.
• Bosco, A., Bartlett, N. W., Grainge, C., Knight, D. A., Wark, P. A., Maltby, S., Kaur, P., Nichol, K., Li, N. F., Reid, A. T., Troy, N. M., & Veerati, P. C. (2020). Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD. Frontiers in Immunology. doi:10.3389/fimmu.2020.00974
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  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.
• Bosco, A., Veerati, P. C., Loo, S., Reid, A. T., Grainge, C., Parsons, D., Roscioli, E., Farrow, N., Landwehr, K. R., McLean, S. A., Schofield, C., Laucirica, D. R., Poh, M. W., Millington, N., McGee, K., Hunter, M., Larcombe, A. N., Schoep, T., Forsyth, I., , Heaney, M., et al. (2020). Assessing the unified airway hypothesis in children via transcriptional profiling of the airway epithelium. The Journal of Allergy and Clinical Immunology. doi:10.1016/j.jaci.2020.02.018
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  Background Emerging evidence suggests that disease vulnerability is expressed throughout the airways, the so-called unified airway hypothesis, but the evidence to support this is predominantly indirect. Objectives We sought to establish the transcriptomic profiles of the upper and lower airways and determine their level of similarity irrespective of airway symptoms (wheeze) and allergy. Methods We performed RNA sequencing on upper and lower airway epithelial cells from 63 children with or without wheeze and accompanying atopy, using differential gene expression and gene coexpression analyses to determine transcriptional similarity. Results We observed approximately 91% homology in the expressed genes between the 2 sites. When coexpressed genes were grouped into modules relating to biological functions, all were found to be conserved between the 2 regions, resulting in a consensus network containing 16 modules associated with ribosomal function, metabolism, gene expression, mitochondrial activity, and antiviral responses through IFN activity. Although symptom-associated gene expression changes were more prominent in the lower airway, they were reflected in nasal epithelium and included IL-1 receptor like 1, prostaglandin-endoperoxide synthase 1, CCL26, and periostin. Through network analysis we identified a cluster of coexpressed genes associated with atopic wheeze in the lower airway, which could equally distinguish atopic and nonatopic phenotypes in upper airway samples. Conclusions We show that the upper and lower airways are significantly conserved in their transcriptional composition, and that variations associated with disease are present in both nasal and tracheal epithelium. Findings from this study supporting a unified airway imply that clinical insight regarding the lower airway in health and disease can be gained from studying the nasal epithelium.
• Guo, J., Song, Y., Saiganesh, A., Hales, B. J., Bosco, A., Le Souëf, P. N., & Zhang, G. (2020). Toll-like receptor signalling has inverted U-shaped response over time with the Western environment. Allergy, 75(10), 2665-2667.
• Jones, A. C., Anderson, D., Troy, N. M., Mallon, D., Hartmann, R., Serralha, M., Holt, B., Bosco, A., & Holt, P. G. (2020). Rewiring of gene networks underlying mite allergen-induced CD4 + Th-cell responses during immunotherapy. Allergy, 75(9), 2330-2341.
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  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.
• Kicic, A., de Jong, E., Ling, K. M., Nichol, K., Anderson, D., Wark, P. A., Knight, D. A., Bosco, A., Stick, S. M., , W., & , A. (2020). Assessing the unified airway hypothesis in children via transcriptional profiling of the airway epithelium. The Journal of allergy and clinical immunology, 145(6), 1562-1573.
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  Emerging evidence suggests that disease vulnerability is expressed throughout the airways, the so-called unified airway hypothesis, but the evidence to support this is predominantly indirect.
• Mincham, K. T., Jones, A. C., Bodinier, M., Scott, N. M., Lauzon-Joset, J. F., Stumbles, P. A., Bosco, A., Holt, P. G., & Strickland, D. H. (2020). Transplacental Innate Immune Training via Maternal Microbial Exposure: Role of XBP1-ERN1 Axis in Dendritic Cell Precursor Programming. Frontiers in immunology, 11, 601494.
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  We recently reported that offspring of mice treated during pregnancy with the microbial-derived immunomodulator OM-85 manifest striking resistance to allergic airways inflammation, and localized the potential treatment target to fetal conventional dendritic cell (cDC) progenitors. Here, we profile maternal OM-85 treatment-associated transcriptomic signatures in fetal bone marrow, and identify a series of immunometabolic pathways which provide essential metabolites for accelerated myelopoiesis. Additionally, the cDC progenitor compartment displayed treatment-associated activation of the XBP1-ERN1 signalling axis which has been shown to be crucial for tissue survival of cDC, particularly within the lungs. Our forerunner studies indicate uniquely rapid turnover of airway mucosal cDCs at baseline, with further large-scale upregulation of population dynamics during aeroallergen and/or pathogen challenge. We suggest that enhanced capacity for XBP1-ERN1-dependent cDC survival within the airway mucosal tissue microenvironment may be a crucial element of OM-85-mediated transplacental innate immune training which results in postnatal resistance to airway inflammatory disease.
• Read, J. F., & Bosco, A. (2020). Decoding Susceptibility to Respiratory Viral Infections and Asthma Inception in Children. International journal of molecular sciences, 21(17).
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  Human Respiratory Syncytial Virus and Human Rhinovirus are the most frequent cause of respiratory tract infections in infants and children and are major triggers of acute viral bronchiolitis, wheezing and asthma exacerbations. Here, we will discuss the application of the powerful tools of systems biology to decode the molecular mechanisms that determine risk for infection and subsequent asthma. An important conceptual advance is the understanding that the innate immune system is governed by a Bow-tie architecture, where diverse input signals converge onto a few core pathways (e.g., IRF7), which in turn generate diverse outputs that orchestrate effector and regulatory functions. Molecular profiling studies in children with severe exacerbations of asthma/wheeze have identified two major immunological phenotypes. The IRF7hi phenotype is characterised by robust upregulation of antiviral response networks, and the IRF7lo phenotype is characterised by upregulation of markers of TGFβ signalling and type 2 inflammation. Similar phenotypes have been identified in infants and children with severe viral bronchiolitis. Notably, genome-wide association studies supported by experimental validation have identified key pathways that increase susceptibility to HRV infection (ORMDL3 and CHDR3) and modulate TGFβ signalling (GSDMB, TGFBR1, and SMAD3). Moreover, functional deficiencies in the activation of type I and III interferon responses are already evident at birth in children at risk of developing febrile lower respiratory tract infections and persistent asthma/wheeze, suggesting that the trajectory to asthma begins at birth or in utero. Finally, exposure to microbes and their products reprograms innate immunity and provides protection from the development of allergies and asthma in children, and therefore microbial products are logical candidates for the primary prevention of asthma.
• Veerati, P. C., Troy, N. M., Reid, A. T., Li, N. F., Nichol, K. S., Kaur, P., Maltby, S., Wark, P. A., Knight, D. A., Bosco, A., Grainge, C. L., & Bartlett, N. W. (2020). Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD. Frontiers in immunology, 11, 974.
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  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 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.
• Zemek, R. M., Fear, V. S., Forbes, C., de Jong, E., Casey, T. H., Boon, L., Lassmann, T., Bosco, A., Millward, M. J., Nowak, A. K., Lake, R. A., & Lesterhuis, W. J. (2020). Bilateral murine tumor models for characterizing the response to immune checkpoint blockade. Nature protocols, 15(5), 1628-1648.
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  The therapeutic response to immune checkpoint blockade (ICB) is highly variable, not only between different cancers but also between patients with the same cancer type. The biological mechanisms underlying these differences in response are incompletely understood. Identifying correlates in patient tumor samples is challenging because of genetic and environmental variability. Murine studies usually compare different tumor models or treatments, introducing potential confounding variables. This protocol describes bilateral murine tumor models, derived from syngeneic cancer cell lines, that display a symmetrical yet dichotomous response to ICB. These models enable detailed analysis of whole tumors in a highly homogeneous background, combined with knowledge of the therapeutic outcome within a few weeks, and could potentially be used for mechanistic studies using other (immuno-)therapies. We discuss key considerations and describe how to use two cell lines as fully optimized models. We discuss experimental details, including proper inoculation technique to achieve symmetry and one-sided surgical tumor removal, which takes only 5 min per mouse. Furthermore, we outline the preparation of bulk tissue or single-cell suspensions for downstream analyses such as bulk RNA-seq, immunohistochemistry, single-cell RNA-seq and flow cytometry.
• Corley, S. M., Troy, N. M., Bosco, A., & Wilkins, M. R. (2019). QuantSeq. 3' Sequencing combined with Salmon provides a fast, reliable approach for high throughput RNA expression analysis. Scientific reports, 9(1), 18895.
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  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.
• Jones, A. C., Anderson, D., Galbraith, S., Fantino, E., Cardenas, D. G., Read, J. F., Serralha, M., Holt, B. J., Strickland, D. H., Sly, P. D., Bosco, A., & Holt, P. G. (2019). Immunoinflammatory responses to febrile lower respiratory infections in infants display uniquely complex/intense transcriptomic profiles. The Journal of allergy and clinical immunology, 144(5), 1411-1413.
• Jones, A. C., Anderson, D., Galbraith, S., Fantino, E., Gutierrez Cardenas, D., Read, J. F., Serralha, M., Holt, B. J., Strickland, D. H., Sly, P. D., Bosco, A., & Holt, P. G. (2019). Personalized Transcriptomics Reveals Heterogeneous Immunophenotypes in Children with Viral Bronchiolitis. American journal of respiratory and critical care medicine, 199(12), 1537-1549.
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  A subset of infants are hypersusceptible to severe/acute viral bronchiolitis (AVB), for reasons incompletely understood. To characterize the cellular/molecular mechanisms underlying infant AVB in circulating cells/local airway tissues. Peripheral blood mononuclear cells and nasal scrapings were obtained from infants (
• Khoo, S. K., Read, J., Franks, K., Zhang, G., Bizzintino, J., Coleman, L., McCrae, C., Öberg, L., Troy, N. M., Prastanti, F., Everard, J., Oo, S., Borland, M. L., Maciewicz, R. A., Le Souëf, P. N., Laing, I. A., & Bosco, A. (2019). Upper Airway Cell Transcriptomics Identify a Major New Immunological Phenotype with Strong Clinical Correlates in Young Children with Acute Wheezing. Journal of immunology (Baltimore, Md. : 1950), 202(6), 1845-1858.
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  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 versus IRF7 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 children, symptom duration prior to hospital presentation was more than twice as long from initial symptoms ( = 0.011) and nearly three times as long for cough ( < 0.001), the odds ratio of admission to hospital was increased more than 4-fold ( = 0.018), and time to recurrence was shorter ( = 0.015). In summary, our findings demonstrate that asthma exacerbations in children can be divided into IRF7 versus IRF7 phenotypes with associated differences in clinical phenotypes.
• Leffler, J., Read, J. F., Jones, A. C., Mok, D., Hollams, E. M., Laing, I. A., Le Souef, P. N., Sly, P. D., Kusel, M. M., de Klerk, N. H., Bosco, A., Holt, P. G., & Strickland, D. H. (2019). Progressive increase of FcεRI expression across several PBMC subsets is associated with atopy and atopic asthma within school-aged children. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 30(6), 646-653.
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  Antigen-specific IgE binds the Fcε receptor I (FcεRI) expressed on several types of immune cells, including dendritic cells (DCs). Activation of FcεRI on DCs in atopics has been shown to modulate immune responses that potentially contribute to asthma development. However, the extent to which DC subsets differ in FcεRI expression between atopic children with or without asthma is currently not clear. This study aimed to analyse the expression of FcεRI on peripheral blood mononuclear cells (PBMCs) from atopic children with and without asthma, and non-atopic/non-asthmatic age-matched healthy controls.
• Mehr, S., Lee, E., Hsu, P., Anderson, D., de Jong, E., Bosco, A., & Campbell, D. E. (2019). Innate immune activation occurs in acute food protein-induced enterocolitis syndrome reactions. The Journal of allergy and clinical immunology, 144(2), 600-602.e2.
• Zemek, R. M., De Jong, E., Chin, W. L., Schuster, I. S., Fear, V. S., Casey, T. H., Forbes, C., Dart, S. J., Leslie, C., Zaitouny, A., Small, M., Boon, L., Forrest, A. R., Muiri, D. O., Degli-Esposti, M. A., Millward, M. J., Nowak, A. K., Lassmann, T., Bosco, A., , Lake, R. A., et al. (2019). Sensitization to immune checkpoint blockade through activation of a STAT1/NK axis in the tumor microenvironment. Science translational medicine, 11(501).
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  Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line-derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti-IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.
• Hurgobin, B., de Jong, E., & Bosco, A. (2018). Insights into respiratory disease through bioinformatics. Respirology (Carlton, Vic.), 23(12), 1117-1126.
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  Respiratory diseases such as asthma, chronic obstructive pulmonary disease and lung cancer represent a critical area for medical research as millions of people are affected globally. The development of new strategies for treatment and/or prevention, and the identification of biomarkers for patient stratification and early detection of disease inception are essential to reducing the impact of lung diseases. The successful translation of research into clinical practice requires a detailed understanding of the underlying biology. In this regard, the advent of next-generation sequencing and mass spectrometry has led to the generation of an unprecedented amount of data spanning multiple layers of biological regulation (genome, epigenome, transcriptome, proteome, metabolome and microbiome). Dealing with this wealth of data requires sophisticated bioinformatics and statistical tools. Here, we review the basic concepts in bioinformatics and genomic data analysis and illustrate the application of these tools to further our understanding of lung diseases. We also highlight the potential for data integration of multi-omic profiles and computational drug repurposing to define disease subphenotypes and match them to targeted therapies, paving the way for personalized medicine.
• Jones, A. C., Troy, N. M., White, E., Hollams, E. M., Gout, A. M., Ling, K. M., Kicic, A., Stick, S. M., Sly, P. D., Holt, P. G., Hall, G. L., & Bosco, A. (2018). Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics. Scientific reports, 8(1), 1511.
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  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) 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-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.
• Lauzon-Joset, J. F., Jones, A. C., Mincham, K. T., Thomas, J. A., Rosenthal, L. A., Bosco, A., Holt, P. G., & Strickland, D. H. (2018). Atopy-Dependent and Independent Immune Responses in the Heightened Severity of Atopics to Respiratory Viral Infections: Rat Model Studies. Frontiers in immunology, 9, 1805.
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  Allergic (Th2 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 Th2 (BN rats) and Th2 immunophenotype (PVG rats) animals were inoculated intranasally with attenuated mengovirus (vMC). 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, vMC elicits a prototypical antiviral response (neutrophilic airways inflammation, upregulation of Th1/type I interferon-related pathways). In contrast, response to infection in the Th2 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 Th2 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 Th2 immunophenotype.
• Leffler, J., Jones, A. C., Hollams, E. M., Prastanti, F., Le Souëf, P. N., Holt, P. G., Bosco, A., Laing, I. A., & Strickland, D. H. (2018). Basophil counts in PBMC populations during childhood acute wheeze/asthma are associated with future exacerbations. The Journal of allergy and clinical immunology, 142(5), 1639-1641.e5.
• Wylie, B., Read, J., Buzzai, A. C., Wagner, T., Troy, N., Syn, G., Stone, S. R., Foley, B., Bosco, A., Cruickshank, M. N., & Waithman, J. (2018). CD8XCR1 Dendritic Cells Express High Levels of Toll-Like Receptor 5 and a Unique Complement of Endocytic Receptors. Frontiers in immunology, 9, 2990.
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  Conventional dendritic cells (cDC) resident in the lymphoid organs of mice have been classically divided into CD8 and CD8 subsets. It is well-established that CD8 dendritic cells (DCs) and their migratory counterparts in the periphery comprise the cross-presenting cDC1 subset. In contrast, CD8 DCs are grouped together in the heterogeneous cDC2 subset. CD8 DCs are relatively poor cross-presenters and drive more prominent CD4 T cell responses against exogenous antigens. The discovery of the X-C motif chemokine receptor 1 (XCR1) as a specific marker of cross-presenting DCs, has led to the identification of a divergent subset of CD8 DCs that lacks the ability to cross-present. Here, we report that these poorly characterized CD8XCR1 DCs have a gene expression profile that is consistent with both plasmacytoid DCs (pDCs) and cDC2. Our data demonstrate that CD8XCR1 DCs possess a unique pattern of endocytic receptors and a restricted toll-like receptor (TLR) profile that is particularly enriched for TLR5, giving them a unique position within the DC immunosurveillance network.
• de Jong, E., & Bosco, A. (2018). Dissecting Asthma Transcriptomics: Does Site Matter?. American journal of respiratory cell and molecular biology, 58(2), 144-146.
• DeVries, A., Wlasiuk, G., Miller, S. J., Bosco, A., Stern, D. A., Lohman, I. C., Rothers, J., Jones, A. C., Nicodemus-Johnson, J., Vasquez, M. M., Curtin, J. A., Simpson, A., Custovic, A., Jackson, D. J., Gern, J. E., Lemanske, R. F., Guerra, S., Wright, A. L., Ober, C., , Halonen, M., et al. (2017). Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers. The Journal of allergy and clinical immunology, 140(2), 534-542.
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  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.
• Gardeux, V., Berghout, J., Achour, I., Schissler, A. G., Li, Q., Kenost, C., Li, J., Shang, Y., Bosco, A., Saner, D., Halonen, M. J., Jackson, D. J., Li, H., Martinez, F. D., & Lussier, Y. A. (2017). A genome-by-environment interaction classifier for precision medicine: personal transcriptome response to rhinovirus identifies children prone to asthma exacerbations. Journal of the American Medical Informatics Association : JAMIA, 24(6), 1116-1126.
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  To introduce a disease prognosis framework enabled by a robust classification scheme derived from patient-specific transcriptomic response to stimulation.
• Jones, A. C., & Bosco, A. (2017). Using Network Analysis to Understand Severe Asthma Phenotypes. American journal of respiratory and critical care medicine, 195(11), 1409-1411.
• Lesterhuis, W. J., Bosco, A., Millward, M. J., Small, M., Nowak, A. K., & Lake, R. A. (2017). Dynamic versus static biomarkers in cancer immune checkpoint blockade: unravelling complexity. Nature reviews. Drug discovery, 16(4), 264-272.
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  Recently, there has been a coordinated effort from academic institutions and the pharmaceutical industry to identify biomarkers that can predict responses to immune checkpoint blockade in cancer. Several biomarkers have been identified; however, none has reliably predicted response in a sufficiently rigorous manner for routine use. Here, we argue that the therapeutic response to immune checkpoint blockade is a critical state transition of a complex system. Such systems are highly sensitive to initial conditions, and critical transitions are notoriously difficult to predict far in advance. Nevertheless, warning signals can be detected closer to the tipping point. Advances in mathematics and network biology are starting to make it possible to identify such warning signals. We propose that these dynamic biomarkers could prove to be useful in distinguishing responding from non-responding patients, as well as facilitate the identification of new therapeutic targets for combination therapy.
• Tarique, A. A., Sly, P. D., Holt, P. G., Bosco, A., Ware, R. S., Logan, J., Bell, S. C., Wainwright, C. E., & Fantino, E. (2017). CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 16(4), 475-482.
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  The role of the macrophages in cystic fibrosis (CF) lung disease has been poorly studied. We hypothesized that alternatively activated M2 macrophages are abnormal in CF lung disease.
• Xi, Y., Troy, N. M., Anderson, D., Pena, O. M., Lynch, J. P., Phipps, S., Bosco, A., & Upham, J. W. (2017). Critical Role of Plasmacytoid Dendritic Cells in Regulating Gene Expression and Innate Immune Responses to Human Rhinovirus-16. Frontiers in immunology, 8, 1351.
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  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 , both chains ( and ), the alpha chain of the IL-15 receptor () and the IFN-related gene . 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.
• Bosco, A., Wiehler, S., & Proud, D. (2016). Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection. BMC genomics, 17, 76.
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  Human rhinoviruses (HRV) cause the majority of colds and trigger exacerbations of chronic lower airway diseases. Airway epithelial cells are the primary site for HRV infection and replication, and the initiation of host inflammatory responses. At present, the molecular mechanisms that underpin HRV responses in airway epithelial cells are incompletely understood. The aim of this study was to employ microarray profiling, upstream regulator analysis, and siRNA mediated gene silencing to further our understanding of the role of interferon regulatory factor 7 (IRF7) in this response.
• Coleman, L., Laing, I. A., & Bosco, A. (2016). Rhinovirus-induced asthma exacerbations and risk populations. Current opinion in allergy and clinical immunology, 16(2), 179-85.
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  This article discusses recent findings into the mechanisms that determine how viruses trigger asthma exacerbations.
• Eguíluz-Gracia, I., Bosco, A., Dollner, R., Melum, G. R., Lexberg, M. H., Jones, A. C., Dheyauldeen, S. A., Holt, P. G., Bækkevold, E. S., & Jahnsen, F. L. (2016). Rapid recruitment of CD14(+) monocytes in experimentally induced allergic rhinitis in human subjects. The Journal of allergy and clinical immunology, 137(6), 1872-1881.e12.
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  Activated TH2 cells and eosinophils are hallmarks of the allergic inflammation seen in patients with allergic rhinitis (AR). However, which cells activate and attract T cells and eosinophils to the inflammatory lesion has not been determined.
• Foong, R. E., Bosco, A., Troy, N. M., Gorman, S., Hart, P. H., Kicic, A., & Zosky, G. R. (2016). Identification of genes differentially regulated by vitamin D deficiency that alter lung pathophysiology and inflammation in allergic airways disease. American journal of physiology. Lung cellular and molecular physiology, 311(3), L653-63.
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  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.
• Holt, P. G., Strickland, D., Bosco, A., Belgrave, D., Hales, B., Simpson, A., Hollams, E., Holt, B., Kusel, M., Ahlstedt, S., Sly, P. D., & Custovic, A. (2016). Distinguishing benign from pathologic TH2 immunity in atopic children. The Journal of allergy and clinical immunology, 137(2), 379-87.
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  Although most children with asthma and rhinitis are sensitized to aeroallergens, only a minority of sensitized children are symptomatic, implying the underlying operation of efficient anti-inflammatory control mechanisms.
• Looi, K., Troy, N. M., Garratt, L. W., Iosifidis, T., Bosco, A., Buckley, A. G., Ling, K. M., Martinovich, K. M., Kicic-Starcevich, E., Shaw, N. C., Sutanto, E. N., Zosky, G. R., Rigby, P. J., Larcombe, A. N., Knight, D. A., Kicic, A., & Stick, S. M. (2016). Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability. Experimental lung research, 42(7), 380-395.
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  No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function.
• Troy, N. M., & Bosco, A. (2016). Respiratory viral infections and host responses; insights from genomics. Respiratory research, 17(1), 156.
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  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.
• Troy, N. M., Hollams, E. M., Holt, P. G., & Bosco, A. (2016). Differential gene network analysis for the identification of asthma-associated therapeutic targets in allergen-specific T-helper memory responses. BMC medical genomics, 9, 9.
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  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.
• Foong, R. E., Bosco, A., Jones, A. C., Gout, A., Gorman, S., Hart, P. H., & Zosky, G. R. (2015). The effects of in utero vitamin D deficiency on airway smooth muscle mass and lung function. American journal of respiratory cell and molecular biology, 53(5), 664-75.
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  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.
• Gardeux, V., Bosco, A., Li, J., Halonen, M. J., Jackson, D., Martinez, F. D., & Lussier, Y. A. (2015). Towards a PBMC "virogram assay" for precision medicine: Concordance between ex vivo and in vivo viral infection transcriptomes. Journal of biomedical informatics, 55, 94-103.
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  Understanding individual patient host-response to viruses is key to designing optimal personalized therapy. Unsurprisingly, in vivo human experimentation to understand individualized dynamic response of the transcriptome to viruses are rarely studied because of the obvious limitations stemming from ethical considerations of the clinical risk.
• Lesterhuis, W. J., Rinaldi, C., Jones, A., Rozali, E. N., Dick, I. M., Khong, A., Boon, L., Robinson, B. W., Nowak, A. K., Bosco, A., & Lake, R. A. (2015). Network analysis of immunotherapy-induced regressing tumours identifies novel synergistic drug combinations. Scientific reports, 5, 12298.
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  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.
• Agdestein, A., Jones, A., Flatberg, A., Johansen, T. B., Heffernan, I. A., Djønne, B., Bosco, A., & Olsen, I. (2014). Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection. BMC genomics, 15, 58.
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  Mycobacterium 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.
• Lesterhuis, W. J., Bosco, A., & Lake, R. A. (2014). Comment on "drug discovery: turning the titanic". Science translational medicine, 6(229), 229le2.
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  The pathobiology-based approach to research and development has been the dominant paradigm for successful drug discovery over the last decades. We propose that the molecular and cellular events that govern a resolving, rather than an evolving, disease may reveal new druggable pathways.
• Stone, S. F., Bosco, A., Jones, A., Cotterell, C. L., van Eeden, P. E., Arendts, G., Fatovich, D. M., & Brown, S. G. (2014). Genomic responses during acute human anaphylaxis are characterized by upregulation of innate inflammatory gene networks. PloS one, 9(7), e101409.
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  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.
• Ramsey, K. A., Bosco, A., McKenna, K. L., Carter, K. W., Elliot, J. G., Berry, L. J., Sly, P. D., Larcombe, A. N., & Zosky, G. R. (2013). In utero exposure to arsenic alters lung development and genes related to immune and mucociliary function in mice. Environmental health perspectives, 121(2), 244-50.
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  Exposure to arsenic via drinking water is a global environmental health problem. In utero exposure to arsenic via drinking water increases the risk of lower respiratory tract infections during infancy and mortality from bronchiectasis in early adulthood.
• Bosco, A., Ehteshami, S., Panyala, S., & Martinez, F. D. (2012). Interferon regulatory factor 7 is a major hub connecting interferon-mediated responses in virus-induced asthma exacerbations in vivo. The Journal of allergy and clinical immunology, 129(1), 88-94.
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  Exacerbations are responsible for a substantial burden of morbidity and health care use in children with asthma. Most asthma exacerbations are triggered by viral infections; however, the underlying mechanisms have not been systematically investigated.
• Rate, A., Bosco, A., McKenna, K. L., Holt, P. G., & Upham, J. W. (2012). Airway epithelial cells condition dendritic cells to express multiple immune surveillance genes. PloS one, 7(9), e44941.
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  Increasing evidence suggests that crosstalk between airway epithelial cells (AEC) and adjacent dendritic cells (DC) tightly regulates airway mucosal DC function in steady state. AEC are known to express multiple immuno-modulatory factors, though detailed information on how this influences human DC function remains incomplete. We recently demonstrated using an in vitro coculture model that AEC alter differentiation of monocytes into DC in a manner that inhibits expression of potentially damaging Th2 effector function. In the current study, we have extended these findings to examine other aspects of DC function. Using micro-array technology we show that multiple genes important for immune surveillance are significantly over expressed in purified AEC-conditioned DC, compared to control DC. These findings were confirmed by quantitative real time PCR or flow cytometry in an independent sample set. In particular, AEC-conditioned DC showed selective upregulation of chemokines that recruit Th1 cells, but minimal change in chemokines linked to Th2 cell recruitment. AEC-conditioned DC were also characterized by enhanced expression of complement family genes (C1QB, C2, CD59 and SERPING1), Fcγ receptor genes (FCGR1A, FCGR2A, FCGR2B and FCGR2C), signaling lymphocytic activation molecule family member 1 (SLAM), programmed death ligands 1 and 2, CD54 and CD200R1, relative to control DC. These findings suggest that AEC conditioning facilitates the capacity of DC to react to danger signals, to enhance leukocyte recruitment, especially of Th1 effector cells, and to interact with other immune cell populations while minimizing the risks of excessive inflammation leading to tissue damage.
• White, O. J., McKenna, K. L., Bosco, A., H J van den Biggelaar, A., Richmond, P., & Holt, P. G. (2012). A genomics-based approach to assessment of vaccine safety and immunogenicity in children. Vaccine, 30(10), 1865-74.
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  Immune responses to vaccines in infants and young children are typically Th2-biased, giving rise to concerns regarding potential atopy-like side effects, and antagonism of Th1-associated sterilising immunity. Conventional immunological methodology has limited capacity to effectively address these problems because of the inherent complexity of the immune responses involved. In the present study, we sought to develop an unbiased systems biology approach to elucidate superficially similar Th2-associated responses to paediatric vaccines and allergens, and to differentiate between them via gene coexpression network analysis. We demonstrate below that in immune responses to the diptheria/acellular pertussis/tetanus and pneumococcal polysaccharide conjugate vaccines, potentially antagonistic Th1-/IFN-associated and Th2-associated gene networks coexist in an apparent state of dynamic equilibrium, whereas in Th2-dominant allergen-specific responses of atopics the Th1 and IFN networks are respectively disrupted and downregulated. Capacity to detect and interpret these covert differences between responses to vaccines and allergens relies on the use of sophisticated algorithms that underpin coexpression network analysis, which identify genes that function co-ordinately in complex pathways. This methodology has significant potential to identify covert interactions between inflammatory pathways triggered by vaccination, and as such may be a useful tool in prediction of vaccine safety/efficacy.
• van den Biggelaar, A. H., Pomat, W., Bosco, A., Phuanukoonnon, S., Devitt, C. J., Nadal-Sims, M. A., Siba, P. M., Richmond, P. C., Lehmann, D., & Holt, P. G. (2011). Pneumococcal conjugate vaccination at birth in a high-risk setting: no evidence for neonatal T-cell tolerance. Vaccine, 29(33), 5414-20.
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  Concerns about the risk of inducing immune deviation-associated "neonatal tolerance" as described in mice have restricted the widespread adoption of neonatal vaccination. The aim of this study was to demonstrate the immunological feasibility of neonatal pneumococcal conjugate vaccination (PCV) which could potentially protect high-risk infants in resource poor countries against severe pneumococcal disease and mortality in the early critical period of life. Papua New Guinean infants were randomized to be vaccinated with the 7-valent PCV (7vPCV) at birth, 1 and 2 months (neonatal group, n=104) or at 1, 2 and 3 months of age (infant group, n=105), or to not receive 7vPCV at all (control group, n=109). Analysis of vaccine responses at 3 and 9 months of age demonstrated persistently higher type-1 (IFN-γ) and type-2 (IL-5 and IL-13) T-cell responses to the protein carrier CRM(197) and IgG antibody titres to 7vPCV serotypes in children vaccinated with 7vPCV according to either schedule as compared to unvaccinated children. In a comprehensive immuno-phenotypic analysis at 9 months of age, no differences in the quantity or quality of vaccine-specific T cell memory responses were found between neonatal vaccinations versus children given their first PCV dose at one month. Hospitalization rates in the first month of life did not differ between children vaccinated with PCV at birth or not. These findings demonstrate that neonatal 7vPCV vaccination is safe and not associated with immunological tolerance. Neonatal immunisation schedules should therefore be considered in high-risk areas where this may result in improved vaccine coverage and the earliest possible protection against pneumococcal disease and death.
• Holt, P. G., Rowe, J., Kusel, M., Parsons, F., Hollams, E. M., Bosco, A., McKenna, K., Subrata, L., de Klerk, N., Serralha, M., Holt, B. J., Zhang, G., Loh, R., Ahlstedt, S., & Sly, P. D. (2010). Toward improved prediction of risk for atopy and asthma among preschoolers: a prospective cohort study. The Journal of allergy and clinical immunology, 125(3), 653-9, 659.e1-659.e7.
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  Atopy and asthma are commonly initiated during early life, and there is increasing interest in the development of preventive treatments for at-risk children. However, effective methods for assessing the level of risk in individual children are lacking.
• Bosco, A., McKenna, K. L., Firth, M. J., Sly, P. D., & Holt, P. G. (2009). A network modeling approach to analysis of the Th2 memory responses underlying human atopic disease. Journal of immunology (Baltimore, Md. : 1950), 182(10), 6011-21.
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  Complex cellular functions within immunoinflammatory cascades are conducted by networks of interacting genes. In this study, we employed a network modeling approach to dissect and interpret global gene expression patterns in allergen-induced Th cell responses that underpin human atopic disease. We demonstrate that a subnet of interconnected genes enriched for Th2 and regulatory T cell-associated signatures plus many novel genes is hardwired into the atopic response and is a hallmark of atopy at the systems level. We show that activation of this subnet is stabilized via hyperconnected "hub" genes, the selective disruption of which can collapse the entire network in a comprehensive fashion. Finally, we investigated gene expression in different Th cell subsets and show that regulatory T cell- and Th2-associated signatures partition at different stages of Th memory cell differentiation. Moreover, we demonstrate the parallel presence of a core element of the Th2-associated gene signature in bystander naive cells, which can be reproduced by rIL-4. These findings indicate that network analysis provides significant additional insight into atopic mechanisms beyond that achievable with conventional microarray analyses, predicting functional interactions between novel genes and previously recognized members of the allergic cascade. This approach provides novel opportunities for design of therapeutic strategies that target entire networks of genes rather than individual effector molecules.
• Holt, P. G., Strickland, D. H., Bosco, A., & Jahnsen, F. L. (2009). Pathogenic mechanisms of allergic inflammation: atopic asthma as a paradigm. Advances in immunology, 104, 51-113.
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  Prospective studies tracking birth cohorts over periods of years indicate that the seeds for atopic asthma in adulthood are sewn during early life. The key events involve programming of functional phenotypes within the immune and respiratory systems which determine long-term responsiveness to ubiquitous environmental stimuli, particularly respiratory viruses and aeroallergens. A crucial component of asthma pathogenesis is early sensitization to aeroallergens stemming from a failure of mucosal tolerance mechanisms during the preschool years, which is associated with delayed postnatal maturation of a range of adaptive and innate immune functions. These maturational defects also increase risk for severe respiratory infections, and the combination of sensitization and infections maximizes risk for early development of the persistent asthma phenotype. Interactions between immunoinflammatory pathways stimulated by these agents also sustain the disease in later life as major triggers of asthma exacerbations. Recent studies on the nature of these interactions suggest the operation of an infection-associated lung:bone marrow axis involving upregulation of FcERlalpha on myeloid precursor populations prior to their migration to the airways, thus amplifying local inflammation via IgE-mediated recruitment of bystander atopic effector mechanisms. The key participants in the disease process are airway mucosal dendritic cells and adjacent epithelial cells, and transiting CD4(+) effector and regulatory T-cell populations, and increasingly detailed characterization of their roles at different stages of pathogenesis is opening up novel possibilities for therapeutic control of asthma. Of particular interest is the application of genomics-based approaches to drug target identification in cell populations of interest, exemplified by recent findings discussed below relating to the gene network(s) triggered by activation of Th2-memory cells from atopics.
• Rate, A., Upham, J. W., Bosco, A., McKenna, K. L., & Holt, P. G. (2009). Airway epithelial cells regulate the functional phenotype of locally differentiating dendritic cells: implications for the pathogenesis of infectious and allergic airway disease. Journal of immunology (Baltimore, Md. : 1950), 182(1), 72-83.
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  Atopic asthma pathogenesis is driven by the combined effects of airway inflammation generated during responses to viral infections and aeroallergens, and both these pathways are regulated by dendritic cells (DC) that differentiate locally from monocytic precursors. These DCs normally exhibit a sentinel phenotype characterized by active Ag sampling but attenuated presentation capability, which limits the intensity of local expression of adaptive immunity. How this tight control of airway DC functions is normally maintained, and why it breaks down in some atopics leading to immunopathological changes in airway tissues, is unknown. We postulated that signals from adjacent airway epithelial cells (AEC) contribute to regulation of local differentiation of DC. We tested this in a coculture model containing both cell types in a GM-CSF-IL-4-enriched cytokine milieu characteristic of the atopic asthmatic airway mucosa. We demonstrate that contact with AEC during DC differentiation up-regulates expression of the function-associated markers MHC class II, CD40, CD80, TLR3, and TLR4 on DCs with concomitant up-regulation of Ag uptake/processing. Moreover, the AEC-conditioned DCs displayed increased LPS responsiveness evidenced by higher production of IL-12, IL-6, IL-10, and TNF-alpha. The Th2 memory-activating properties of AEC-conditioned DCs were also selectively attenuated. Data from microarray and blocking experiments implicate AEC-derived type 1 IFNs and IL-6 in modulation of DC differentiation. Collectively, these findings suggest that resting AECs modulate local DC differentiation to optimize antimicrobial defenses in the airways and in the process down-modulate capacity for expression of potentially damaging Th2 immunity.
• Subrata, L. S., Bizzintino, J., Mamessier, E., Bosco, A., McKenna, K. L., Wikström, M. E., Goldblatt, J., Sly, P. D., Hales, B. J., Thomas, W. R., Laing, I. A., LeSouëf, P. N., & Holt, P. G. (2009). Interactions between innate antiviral and atopic immunoinflammatory pathways precipitate and sustain asthma exacerbations in children. Journal of immunology (Baltimore, Md. : 1950), 183(4), 2793-800.
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  Severe asthma exacerbations in children requiring hospitalization are typically associated with viral infection and occur almost exclusively among atopics, but the significance of these comorbidities is unknown. We hypothesized that underlying interactions between immunoinflammatory pathways related to responses to aeroallergen and virus are involved, and that evidence of these interactions is detectable in circulating cells during exacerbations. To address this hypothesis we used a genomics-based approach involving profiling of PBMC subpopulations collected during exacerbation vs convalescence by microarray and flow cytometry. We demonstrate that circulating T cells manifest the postactivated "exhausted" phenotype during exacerbations, whereas monocyte/dendritic cell populations display up-regulated CCR2 expression accompanied by phenotypic changes that have strong potential for enhancing local inflammation after their recruitment to the atopic lung. Notably, up-regulation of FcepsilonR1, which is known to markedly amplify capacity for allergen uptake/presentation to Th2 effector cells via IgE-mediated allergen capture, and secondarily programming of IL-4/IL-13-dependent IL-13R(+) alternatively activated macrophages that have been demonstrated in experimental settings to be a potent source of autocrine IL-13 production. We additionally show that this disease-associated activation profile can be reproduced in vitro by cytokine exposure of atopic monocytes, and furthermore that IFN-alpha can exert both positive and negative roles in the process. Our findings suggest that respiratory viral infection in atopic children may initiate an atopy-dependent cascade that amplifies and sustains airway inflammation initiated by innate antiviral immunity via harnessing underlying atopy-associated mechanisms. These interactions may account for the unique susceptibility of atopics to severe viral-induced asthma exacerbations.
• Yerkovich, S. T., Roponen, M., Smith, M. E., McKenna, K., Bosco, A., Subrata, L. S., Mamessier, E., Wikström, M. E., Le Souef, P., Sly, P. D., Holt, P. G., & Upham, J. W. (2009). Allergen-enhanced thrombomodulin (blood dendritic cell antigen 3, CD141) expression on dendritic cells is associated with a TH2-skewed immune response. The Journal of allergy and clinical immunology, 123(1), 209-216.e4.
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  Dendritic cells (DCs) are important in allergic diseases such as asthma, although little is known regarding the mechanisms by which DCs induce T(H)2-polarized responses in atopic individuals. It has been suggested that intrinsic properties of allergens can directly stimulate T(H)2 polarizing functions of DCs, but little is known of the underlying mechanisms.
• Zhang, G., Rowe, J., Kusel, M., Bosco, A., McKenna, K., de Klerk, N., Sly, P. D., & Holt, P. G. (2009). Interleukin-10/interleukin-5 responses at birth predict risk for respiratory infections in children with atopic family history. American journal of respiratory and critical care medicine, 179(3), 205-11.
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  Respiratory infections in early life are associated with risk for wheezing bronchiolitis, especially in children at high risk of atopy. The underlying mechanisms are unknown, but are suspected to involve imbalance(s) in host defense responses against pathogens stemming from functional immaturity of the immune system in this age group.
• Bosco, A., McKenna, K. L., Devitt, C. J., Firth, M. J., Sly, P. D., & Holt, P. G. (2006). Identification of novel Th2-associated genes in T memory responses to allergens. Journal of immunology (Baltimore, Md. : 1950), 176(8), 4766-77.
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  Atopic diseases are associated with hyperexpression of Th2 cytokines by allergen-specific T memory cells. However, clinical trials with recently developed Th2 inhibitors in atopics have proven disappointing, suggesting underlying complexities in atopy pathogenesis which are not satisfactorily explained via the classical Th1/Th2 paradigm. One likely possibility is that additional Th2-associated genes which are central to disease pathogenesis remain unidentified. The aim of the present study was to identify such novel Th2-associated genes in recall responses to the inhalant allergen house dust mite. In contrast to earlier human microarray studies in atopy which focused on mitogen-activated T cell lines and clones, we concentrated on PBMC-derived primary T cells stimulated under more physiological conditions of low dose allergen exposure. We screened initially for allergen-induced gene activation by microarray, and validated novel genes in independent panels of subjects by quantitative RT-PCR. Kinetic analysis of allergen responses in PBMC revealed an early wave of novel atopy-associated genes involved in signaling which were coexpressed with IL-4 and IL-4R, followed by a later wave of genes encoding the classical Th2 effector cytokines. We further demonstrate that these novel activation-associated Th2 genes up-regulate in response to another atopy-associated physiological stimulus bacterial superantigen, but remain quiescent in nonphysiological responses in primary T cells or cell lines driven by potent mitogens, which may account for their failure to be detected in earlier microarray studies.
• White, G. P., Hollams, E. M., Yerkovich, S. T., Bosco, A., Holt, B. J., Bassami, M. R., Kusel, M., Sly, P. D., & Holt, P. G. (2006). CpG methylation patterns in the IFNgamma promoter in naive T cells: variations during Th1 and Th2 differentiation and between atopics and non-atopics. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 17(8), 557-64.
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  Interferon-gamma (IFNgamma) gene expression is tightly regulated in early life, and exaggerated negative control of IFNgamma production in CD4(+) T cells has been associated with risk for subsequent development of atopy. Recent studies have demonstrated hypermethylation of CpG sites in the IFNgamma promoter in neonates, a mechanism which in mice leads to strong suppression of IFNgamma gene transcription. In the present study, the methylation status of six CpG sites in the proximal promoter of the human IFNgamma gene was determined by bisulphite sequencing. Cell populations studied were Th1 or Th2 polarized cell lines derived from neonatal and adult CD4(+)/CD45RA(+) T cells, CD4(+) and CD8(+) naive T cells from cord blood of children followed to outcome age 2 for assessment of atopy status, and CD4(+) and CD8(+) naive T cells from 6 yr old and adult atopics and controls. We demonstrate that in vitro differentiation of CD4(+) T cells down the Th1 pathway (but not the Th2 pathway) is accompanied by progressive demethylation of CpG sites in the IFNgamma promoter, which is most marked in neonatal cells. Atopy development by age 2 was not associated with variations in methylation patterns in cord blood T cells. However, IFNgamma promoter methylation was reduced in CD8(+) T cells from atopic children in the age range in which hyperproduction of IFNgamma as recently been identified as a common feature of the atopic phenotype. The findings demonstrate the potency of IFNgamma promoter methylation as a mechanism for control of human IFNgamma gene expression, particularly during early life. Differential regulation of IFNgamma promoter methylation in T cells may be an important contributory factor in atopy development in childhood, and this possibility warrants further detailed investigation.
• Hales, B. J., Bosco, A., Mills, K. L., Hazell, L. A., Loh, R., Holt, P. G., & Thomas, W. R. (2004). Isoforms of the major peanut allergen Ara h 2: IgE binding in children with peanut allergy. International archives of allergy and immunology, 135(2), 101-7.
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  The major peanut allergen Ara h 2 consists of two isoforms, namely Ara h 2.0101 and Ara h 2.0201. The recently identified Ara h 2.0201 isoform contains an extra 12 amino acids including an extra copy of the reported immunodominant epitope DPYSPS. This study aimed to evaluate the IgE binding of the two Ara h 2 isoforms.

Others

• Kicic-Starcevich, E., Hancock, D. G., Iosifidis, T., Agudelo-Romero, P., Caparros-Martin, J. A., Silva, D., Turkovic, L., Le Souef, P. N., Bosco, A., Martino, D. J., Kicic, A., Prescott, S. L., & Stick, S. M. (2023). Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) birth cohort: study protocol. medRxiv : the preprint server for health sciences.
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  Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, exposures, and respiratory exposures in the first year of life.