Rhiannon B. Werder

Gene-based analysis of regulatory variants identifies 4 putative novel asthma risk genes related to nucleotide synthesis and signaling

Background: Hundreds of genetic variants are thought to contribute to variation in asthma risk by modulating gene expression. Methods that increase the power of genome‐wide association studies (GWASs) to identify risk‐associated variants are needed. Objective: We sought to develop a method that aggregates the evidence for association with disease risk across expression quantitative trait loci (eQTLs) of a gene and use this approach to identify asthma risk genes. Methods: We developed a gene‐based test and software package called EUGENE that (1) is applicable to GWAS summary statistics; (2) considers both cis‐ and trans‐eQTLs; (3) incorporates eQTLs identified in different tissues; and (4) uses simulations to account for multiple testing. We applied this approach to 2 published asthma GWASs (combined n = 46,044) and used mouse studies to provide initial functional insights into 2 genes with novel genetic associations. Results: We tested the association between asthma and 17,190 genes that were found to have cis‐ and/or trans‐eQTLs across 16 published eQTL studies. At an empirical FDR of 5%, 48 genes were associated with asthma risk. Of these, for 37, the association was driven by eQTLs located in established risk loci for allergic disease, including 6 genes not previously implicated in disease cause (eg, LIMS1, TINF2, and SAFB). The remaining 11 significant genes represent potential novel genetic associations with asthma. The association with 4 of these replicated in an independent GWAS: B4GALT3, USMG5, P2RY13, and P2RY14, which are genes involved in nucleotide synthesis or nucleotide‐dependent cell activation. In mouse studies, P2ry13 and P2ry14—purinergic receptors activated by adenosine 5‐diphosphate and UDP‐sugars, respectively—were upregulated after allergen challenge, notably in airway epithelial cells, eosinophils, and neutrophils. Intranasal exposure with receptor agonists induced the release of IL‐33 and subsequent eosinophil infiltration into the lungs. Conclusion: We identified novel associations between asthma and eQTLs for 4 genes related to nucleotide synthesis/signaling and demonstrated the power of gene‐based analyses of GWASs.

The influence of the microbiome on early-life severe viral lower respiratory infections and asthma-Food for thought?

Severe viral lower respiratory infections are a major cause of infant morbidity. In developing countries, respiratory syncytial virus (RSV)-bronchiolitis induces significant mortality, whereas in developed nations the disease represents a major risk factor for subsequent asthma. Susceptibility to severe RSV-bronchiolitis is governed by gene–environmental interactions that affect the host response to RSV infection. Emerging evidence suggests that the excessive inflammatory response and ensuing immunopathology, typically as a consequence of insufficient immunoregulation, leads to long-term changes in immune cells and structural cells that render the host susceptible to subsequent environmental incursions. Thus, the initial host response to RSV may represent a tipping point in the balance between long-term respiratory health or chronic disease (e.g., asthma). The composition and diversity of the microbiota, which in humans stabilizes in the first year of life, critically affects the development and function of the immune system. Hence, perturbations to the maternal and/or infant microbiota are likely to have a profound impact on the host response to RSV and susceptibility to childhood asthma. Here, we review recent insights describing the effects of the microbiota on immune system homeostasis and respiratory disease and discuss the environmental factors that promote microbial dysbiosis in infancy. Ultimately, this knowledge will be harnessed for the prevention and treatment of severe viral bronchiolitis as a strategy to prevent the onset and development of asthma.

RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma

Asthma is a chronic inflammatory disease. Although many patients with asthma develop type-2 dominated eosinophilic inflammation, a number of individuals develop paucigranulocytic asthma, which occurs in the absence of eosinophilia or neutrophilia. The aetiology of paucigranulocytic asthma is unknown. However, both respiratory syncytial virus (RSV) infection and mutations in the receptor for advanced glycation endproducts (RAGE) are risk factors for asthma development. Here, we show that RAGE deficiency impairs anti-viral immunity during an early-life infection with pneumonia virus of mice (PVM; a murine analogue of RSV). The elevated viral load was associated with the release of high mobility group box-1 (HMGB1) which triggered airway smooth muscle remodelling in early-life. Re-infection with PVM in later-life induced many of the cardinal features of asthma in the absence of eosinophilic or neutrophilic inflammation. Anti-HMGB1 mitigated both early-life viral disease and asthma-like features, highlighting HMGB1 as a possible novel therapeutic target. DOI: http://dx.doi.org/10.7554/eLife.21199.001

Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation

Memory Th2 cell responses underlie the development and perpetuation of allergic diseases. Because these states result from immune dysregulation, established Th2 cell responses represent a significant challenge for conventional immunotherapies. New approaches that overcome the detrimental effects of immune dysregulation are required. We tested whether memory Th2 cell responses were silenced using a therapeutic approach where allergen expression in DCs is transferred to sensitized recipients using BM cells as a vector for therapeutic gene transfer. Development of allergen-specific Th2 responses and allergen-induced airway inflammation was blocked by expression of allergen in DCs. Adoptive transfer studies showed that Th2 responses were inactivated by a combination of deletion and induction of T cell unresponsiveness. Transfer of BM encoding allergen expression targeted to DCs terminated, in an allergen-specific manner, Th2 responses in sensitized recipients. Importantly, when preexisting airway inflammation was present, there was effective silencing of Th2 cell responses, airway inflammation was alleviated, and airway hyperreactivity was reversed. The effectiveness of DC-targeted allergen expression to terminate established Th2 responses in sensitized animals indicates that exploiting cell-intrinsic T cell tolerance pathways could lead to development of highly effective immunotherapies.

Chronic IL-33 expression predisposes to virus-induced asthma exacerbations by increasing type 2 inflammation and dampening antiviral immunity

Background Rhinovirus infection triggers acute asthma exacerbations. IL‐33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. Objective We sought to determine whether anti–IL‐33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and in vitro human primary airway epithelial cells (AECs). Methods Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti–IL‐33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti–IL‐33 after rhinovirus infection. Results Anti–IL‐33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL‐33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti–IL‐33 or dexamethasone diminished exacerbation severity, and anti–IL‐33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN‐&lgr; levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti–IL‐33 decreased rhinovirus replication and increased IFN‐&lgr; levels at the gene and protein levels. Conclusion Anti–IL‐33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus‐induced acute exacerbation; however, only anti–IL‐33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus‐infected human AECs, suggesting that anti–IL‐33 therapy has the additional benefit of enhancing host defense.

Coinfection with Blood-Stage Plasmodium Promotes Systemic Type I Interferon Production during Pneumovirus Infection but Impairs Inflammation and Viral Control in the Lung

ABSTRACT Acute lower respiratory tract infections (ALRTI) are the leading cause of global childhood mortality, with human respiratory syncytial virus (hRSV) being a major cause of viral ALRTI in young children worldwide. In sub-Saharan Africa, many young children experience severe illnesses due to hRSV or Plasmodium infection. Although the incidence of malaria in this region has decreased in recent years, there remains a significant opportunity for coinfection. Recent data show that febrile young children infected with Plasmodium are often concurrently infected with respiratory viral pathogens but are less likely to suffer from pneumonia than are non-Plasmodium-infected children. Here, we hypothesized that blood-stage Plasmodium infection modulates pulmonary inflammatory responses to a viral pathogen but does not aid its control in the lung. To test this, we established a novel coinfection model in which mice were simultaneously infected with pneumovirus of mice (PVM) (to model hRSV) and blood-stage Plasmodium chabaudi chabaudi AS (PcAS) parasites. We found that PcAS infection was unaffected by coinfection with PVM. In contrast, PVM-associated weight loss, pulmonary cytokine responses, and immune cell recruitment to the airways were substantially reduced by coinfection with PcAS. Importantly, PcAS coinfection facilitated greater viral dissemination throughout the lung. Although Plasmodium coinfection induced low levels of systemic interleukin-10 (IL-10), this regulatory cytokine played no role in the modulation of lung inflammation or viral dissemination. Instead, we found that Plasmodium coinfection drove an early systemic beta interferon (IFN-β) response. Therefore, we propose that blood-stage Plasmodium coinfection may exacerbate viral dissemination and impair inflammation in the lung by dysregulating type I IFN-dependent responses to respiratory viruses.

Plasmacytoid dendritic cells protect from viral bronchiolitis and asthma through semaphorin 4a-mediated T reg expansion

Respiratory syncytial virus–bronchiolitis is a major independent risk factor for subsequent asthma, but the causal mechanisms remain obscure. We identified that transient plasmacytoid dendritic cell (pDC) depletion during primary Pneumovirus infection alone predisposed to severe bronchiolitis in early life and subsequent asthma in later life after reinfection. pDC depletion ablated interferon production and increased viral load; however, the heightened immunopathology and susceptibility to subsequent asthma stemmed from a failure to expand functional neuropilin-1+ regulatory T (T reg) cells in the absence of pDC-derived semaphorin 4a (Sema4a). In adult mice, pDC depletion predisposed to severe bronchiolitis only after antibiotic treatment. Consistent with a protective role for the microbiome, treatment of pDC-depleted neonates with the microbial-derived metabolite propionate promoted Sema4a-dependent T reg cell expansion, ameliorating both diseases. In children with viral bronchiolitis, nasal propionate levels were decreased and correlated with an IL-6high/IL-10low microenvironment. We highlight a common but age-related Sema4a-mediated pathway by which pDCs and microbial colonization induce T reg cell expansion to protect against severe bronchiolitis and subsequent asthma.

The Absence of Interferon-β Promotor Stimulator-1 (IPS-1) Predisposes to Bronchiolitis and Asthma-like Pathology in Response to Pneumoviral Infection in Mice

Respiratory syncytial virus (RSV)-bronchiolitis is a major cause of infant morbidity and mortality and a risk factor for subsequent asthma. We showed previously that toll-like receptor (TLR)7 in plasmacytoid dendritic cells (pDCs) is critical for protection against bronchiolitis and asthma in mice infected with pneumonia virus of mice (PVM), the mouse homolog of RSV. This lack of redundancy was unexpected as interferon-β promotor stimulator-1 (IPS-1) signalling, downstream of RIG-I-like receptor (RLR) and not TLR7 activation, contributes to host defence in hRSV-inoculated adult mice. To further clarify the role of IPS-1 signalling, we inoculated IPS-1−/− and WT mice with PVM in early-life, and again in later-life, to model the association between bronchiolitis and asthma. IPS-1 deficiency predisposed to severe PVM bronchiolitis, characterised by neutrophilic inflammation and necroptotic airway epithelial cell death, high mobility group box 1 (HMGB1) and IL-33 release, and downstream type-2 inflammation. Secondary infection induced an eosinophilic asthma-like pathophysiology in IPS-1−/− but not WT mice. Mechanistically, we identified that IPS-1 is necessary for pDC recruitment, IFN-α production and viral control. Our findings suggest that TLR7 and RLR signalling work collaboratively to optimally control the host response to pneumovirus infection thereby protecting against viral bronchiolitis and subsequent asthma.

PGD2/DP2 receptor activation promotes severe viral bronchiolitis by suppressing IFN-λ production

RSV-induced prostaglandin D2 release contributes to disease severity by impairing IFN-λ production. RSV gives innate immunity the runaround Asthma can be exacerbated by pathogens such as respiratory syncytial virus (RSV); prostaglandin D2 (PGD2) is also important in asthma and is being investigated as a therapeutic target. Werder et al. used multiple models to examine how RSV infection may perturb immune responses and influence asthma pathogenesis. Samples from infants with bronchiolitis or primary pediatric epithelial cells infected with RSV had elevated PGD2. Modulating PGD2 signaling in a mouse model of severe bronchiolitis improved antiviral immunity and dampened asthmatic symptoms later in life. This protection was not due to preventing type 2 immunity but instead a restoration of IFN-λ production. Their findings shed light on this host-pathogen interaction and suggest new therapeutic avenues. Prostaglandin D2 (PGD2) signals through PGD2 receptor 2 (DP2, also known as CRTH2) on type 2 effector cells to promote asthma pathogenesis; however, little is known about its role during respiratory syncytial virus (RSV) bronchiolitis, a major risk factor for asthma development. We show that RSV infection up-regulated hematopoietic prostaglandin D synthase expression and increased PGD2 release by cultured human primary airway epithelial cells (AECs). Moreover, PGD2 production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis compared to healthy controls. In a neonatal mouse model of severe viral bronchiolitis, DP2 antagonism decreased viral load, immunopathology, and morbidity and ablated the predisposition for subsequent asthma onset in later life. This protective response was abolished upon dual DP1/DP2 antagonism and replicated with a specific DP1 agonist. Rather than mediating an effect via type 2 inflammation, the beneficial effects of DP2 blockade or DP1 agonism were associated with increased interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] expression and were lost upon IL-28A neutralization. In RSV-infected AEC cultures, DP1 activation up-regulated IFN-λ production, which, in turn, increased IFN-stimulated gene expression, accelerating viral clearance. Our findings suggest that DP2 antagonists or DP1 agonists may be useful antivirals for the treatment of viral bronchiolitis and possibly as primary preventatives for asthma.

Aeroallergen-induced IL-33 predisposes to respiratory virus-induced asthma by dampening type I and III interferon production

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.