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Chronic inflammatory diseases of the lung are leading causes of morbidity and mortality worldwide. Many of these disorders can be attributed to abnormal immune responses to environmental stimuli and infections. As such, understanding the innate host defense pathways and their regulatory systems will be critical to developing new approaches to treatment. In this regard, there is increasing interest in the role of microRNAs (miRNAs) in the regulation of pulmonary innate host defense responses and the inflammatory sequelae in respiratory disease. In this review, we discuss recent findings that indicate an important role for miRNAs in the regulation in mouse models of various respiratory diseases and in host defense against bacterial and viral infection. We also discuss the potential utility and limitations of targeting these molecules as anti-inflammatory strategies and also as a means to improve pathogen clearance from the lung.

Asthma is a complex disease that involves both genetic factors and environmental exposures. Aberrant epigenetic modifications, such as DNA methylation, may be important in asthma development. Fetal exposure to maternal asthma during critical periods of in utero development may lead to epigenetic alterations that predispose infants to a greater risk of developing asthma themselves. We investigated alterations in the DNA methylation profile of peripheral blood from infants exposed to maternal asthma during pregnancy.Peripheral blood was collected from 12-month-old infants born to women with (n = 25) and without (n = 15) doctor diagnosed asthma during pregnancy. Genomic DNA was extracted, bisulfite converted, and hybridized to Infinium Methylation 27 arrays (Illumina), containing over27,000 CpGs from 14,495 genes. CpG loci in only autosomal genes were classified as differentially methylated at the 99% level (P 0.01, |DiffScore| 22 and delta beta0.06).There were 70 CpG loci, corresponding to 67 genes that were significantly differentially methylated. Twelve CpG loci (11 genes) showed greater than 10% comparative difference in DNA methylation, including hyper-methylated loci of FAM181A, MRI1, PIWIL1, CHFR, DEFA1, MRPL28, AURKA, and hypo-methylated loci of NALP1L5, MAP8KIP3, ACAT2, and PM20D1 in maternal asthma. Methylation of MAPK8IP3 was significantly negatively correlated with maternal blood eosinophils (r = -0.38; P = 0.022), maternal eNO (r = -0.44; P = 0.005), and maternal serum total IgE (r = -0.39, P = 0.015). Methylation of AURKA negatively correlated with maternal hemoglobin (r = -0.43; P = 0.008), infants height (r = -0.51; P 0.001) and weight (r = -0.36; P = 0.021). Methylation of PM20D1 was lower in infants born to mothers with asthma on inhaled corticosteroid treatment. Methylation of PM20D1 was lower and MRI1 was higher in infants born to atopic mothers without asthma.In an Australian study population, exposure to maternal asthma during pregnancy is associated with differential methylation profiles of infants' peripheral blood DNA, which may act as risk factors for future asthma development.

Background Severe asthma is associated with T helper (TH) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro. Objective To determine the anti-inflammatory potential of anthraquinones in-vivo. Methods BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation. Results Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of TH2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung. Conclusion Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.

Background: Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies. Methodology/Principal Findings: Neonatal (

The pathogenesis of allergic asthma in childhood remains poorly understood. Environmental factors which appear to contribute to allergic sensitisation, with development of a Th2-biased immunological response in genetically predisposed individuals, include wheezing lower respiratory viral infections in early life and exposure to airborne environmental pollutants. These may activate pattern recognition receptors and/or cause oxidant injury to airway epithelial cells (AECs). In turn, this may promote Th2 polarisation via a “final common pathway” involving interaction between AEC, dendritic cells, and CD4+ T lymphocytes. Potentially important cytokines produced by AEC include thymic stromal lymphopoietin and interleukin-25. Their role is supported by in vitro studies using human AEC, as well as by experiments in animal models. To date, however, few investigations have employed models of the induction phase of childhood asthma. Further research may help to identify interventions that could reduce the risk of allergic asthma.

Background The role of microRNAs (miRNAs) in regulating gene expression is currently an area of intense interest. Relatively little is known, however, about the role of miRNAs in inflammatory and immunologically-driven disorders. In a mouse model, we have previously shown that miRNAs are potentially important therapeutic targets in allergic asthma, because inhibition of miR-126, one of a small subset of miRNAs upregulated in the airway wall, effectively suppressed Th2-driven airway inflammation and other features of asthma. In the present study, we extended investigation of the therapeutic potential of miRNA inhibition to our well-established model of chronic asthma. Methods Female BALB/c mice were systemically sensitised with ovalbumin (OVA) and chronically challenged with low mass concentrations of aerosolised OVA for up to 6 weeks. Airway tissue was obtained by blunt dissection and RNA was isolated for miRNA profiling. On the basis of the results obtained, animals were subsequently treated with either an antagomir to miR-126 (ant-miR-126) or a scrambled control antagomir once weekly during the 6 weeks of chronic challenge, and the effects on airway inflammation and remodelling were assessed using established morphometric techniques. Results Compared to naïve mice, there was selective upregulation of a modest number of miRNAs, notably miR-126, in the airway wall tissue of chronically challenged animals. The relative increase was maximal after 2 weeks of inhalational challenge and subsequently declined to baseline levels. Compared to treatment with the scrambled control, ant-miR-126 significantly reduced recruitment of intraepithelial eosinophils, but had no effect on the chronic inflammatory response, or on changes of airway remodelling. Conclusions In this model of chronic asthma, there was an initial increase in expression of a small number of miRNAs in the airway wall, notably miR-126. However, this later declined to baseline levels, suggesting that sustained changes in miRNA may not be essential for perpetuation of chronic asthma. Moreover, inhibition of miR-126 by administration of an antagomir suppressed eosinophil recruitment into the airways but had no effect on chronic inflammation in the airway wall, or on changes of remodelling, suggesting that multiple miRNAs are likely to regulate the development of these lesions.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is elevated in the airways of subjects with asthma and has been linked to the development of allergic airway disease by promoting STAT6-dependent T helper 2 cell (T(H) 2) effector functions. To determine whether single nucleotide polymorphisms (SNPs) in the TNFSF10 gene are associated with bronchial asthma we genotyped 498 Caucasian subjects living in Southern Germany for eight SNPs in the TNFSF10 by restriction fragment length polymorphism analysis. In contrast to single SNPs, haplotypes constructed from eight SNPs were robustly associated with asthma (p=0.00012). A small haplotype approach returned four alleles consisting of two (rs3136586/ rs3136598), three (rs12488654/rs3136586/rs3136598 and rs3136586/rs3136598/rs3136604), and four SNPs (rs12488654/ rs3136586/ rs3136598/ rs3136604) that were highly associated with asthma (p=0.00005, p=0.00008, p=0.00017 and p=0.00038). Combinations of SNPs in the TNFSF10 allele were strongly associated with asthma supporting the concept that TRAIL is important in the development of hallmark features of this disease.

Background Glucocorticoids are used as mainstay therapy for asthma, but some patients remain resistant to therapy. MicroRNAs (miRNAs) are important regulators of the immune system by promoting the catabolism of their target transcripts as well as attenuating their translation. The role of miRNA in regulating allergic inflammation remains largely unknown. Blocking miRNA function may provide a new nonsteroidal anti-inflammatory approach to treatment. Objectives To (1) determine the role of specific miRNAs in the regulation of hallmark features of allergic airways inflammation and (2) compare the efficacy of antagonizing miRNA function with that of steroid treatment. Methods Mice were sensitized and then aeroallergen-challenged with house dust mite to induce allergic airways disease, and alterations in the expression of miRNAs were characterized. Next mice were treated with antagomirs that inhibited the function of specific miRNAs in the lung or treated with dexamethasone and inflammatory lesions, and airway hyperresponsiveness was measured. Results miR-145, miR-21, and let-7b have been implicated in airway smooth muscle function, inflammation, and airways epithelial cell function, respectively. Inhibition of miR-145, but not miR-21 or lethal-7b, inhibited eosinophilic inflammation, mucus hypersecretion, T H 2 cytokine production, and airway hyperresponsiveness. The anti-inflammatory effects of miR-145 antagonism were comparable to steroid treatment. Conclusion Our study highlights the importance of understanding the contribution of miRNAs to pathogenesis of human allergic disease and their potential as novel anti-inflammatory targets.

One of the immunopathological features of allergic inflammation is the infiltration of helper T type 2 (Th2) cells to the site of disease. Activation of innate pattern recognition receptors such as Toll-like receptors (TLRs) plays a critical role in helper T type 1 cell differentiation, yet their contribution to the generation of Th2 responses to clinically relevant aeroallergens remains poorly defined.To determine the requirement for TLR2, TLR4, and the Toll/IL-1 receptor domain adaptor protein MyD88 in a murine model of allergic asthma.Wild-type and factor-deficient ((-/-)) mice were sensitized intranasally to the common allergen house dust mite (HDM) and challenged 2 weeks later on four consecutive days. Measurements of allergic airway inflammation, T-cell cytokine production, and airway hyperreactivity were performed 24 hours later.Mice deficient in MyD88 were protected from the cardinal features of allergic asthma, including granulocytic inflammation, Th2 cytokine production and airway hyperreactivity. Although HDM activated NF-kappaB in TLR2- or TLR4-expressing HEK cells, only in TLR4(-/-) mice was the magnitude of allergic airway inflammation and hyperreactivity attenuated. The diminished Th2 response present in MyD88(-/-) and TLR4(-/-) mice was associated with fewer OX40 ligand-expressing myeloid dendritic cells in the draining lymph nodes during allergic sensitization. Finally, HDM-specific IL-17 production and airway neutrophilia were attenuated in MyD88(-/-) but not TLR4(-/-) mice.Together, these data suggest that Th2- and Th17-mediated inflammation generated on inhalational HDM exposure is differentially regulated by the presence of microbial products and the activation of distinct MyD88-dependent pattern recognition receptors.

Micro (mi)RNAs are small, regulatory RNA molecules that are integral components of the genetic program in the majority of cells. They are thought to regulate up to one third of all human genes and have been linked to critical processes in disease pathogenesis. The diverse role of miRNAs in disease pathogenesis suggests that the modulation of miRNA function by utilizing techniques such as the use of antagomirs, locked nucleic acids, or miRNA sponges may produce novel therapeutic approaches. In this review, the current understanding of post-transcriptional gene regulation by miRNAs is discussed and insights into the function of miRNAs in tumorigenesis, immune responses, muscle function, organogenesis, and cell-lineage decisions are reviewed.

Uniparental disomy (UPD) describes the inheritance of two homologous chromosomes from a single parent. Disease phenotypes associated with UPD and chromosomal imprinting, rather than with mutations, include Beckwith-Wiedemann syndrome (paternal UPD11p), Angelman syndrome (paternal UPD15), Prader-Willi syndrome (maternal UPD15), and transient neonatal diabetes (paternal UPD6). Here we report on the first case of paternal uniparental isodisomy of chromosome 14 with a mosaicism for a supernumerary marker chromosome 14. The patient demonstrated a small thorax with a 'coat hanger' shape of the ribs, kyphoscoliosis, hypoplasia of the maxilla and mandible, a broad nasal bridge with anteverted nares, contractures of the wrists with ulnar deviation bilaterally, diastasis recti, and marked muscle hypotonia. Vertical skin creases under the chin and stippled epiphyses of the humeri were features not previously described in patients with paternal UPD14. This case illustrates that as with the finding of an isochromosome, a supernumerary marker chromosome can be an important clue to the presence of UPD14.

BACKGROUND: Severe, steroid-insensitive (SSI) asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote disease. SSI asthma is associated with respiratory infections and non-eosinophilic endotypes of disease, including neutrophilic asthma. OBJECTIVES: To develop and use mouse models of SSI neutrophilic asthma to investigate pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase (PI3K) and histone deacetylase (HDAC)2 in order to identify new therapeutic approaches. METHODS: Novel mouse models of respiratory infection and ovalbumin-induced, SSI neutrophilic allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles of infection-induced miR-21 expression and PI3K-dependent signalling in the lung were examined using a specific miR-21 inhibitor (antagomir-21) and the pan-PI3K inhibitor LY294002. RESULTS: Infection induced a miR-21-dependent, PI3K-mediated signalling pathway that decreased nuclear HDAC2 levels and promoted steroid-insensitive neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels and steroid sensitivity. CONCLUSIONS: We have identified a novel role for a miR-21/PI3K/HDAC2 signalling axis in SSIAAD. Our data highlights miR-21 as a novel target for treating this form of asthma.

Introduction: MicroRNAs (miRNAs) are small non-coding RNA that can bind to multiple target mRNA to repress protein production. Deregulation of miRNAs have been linked with pathogenesis of multiple human diseases. In immune cells, miRNAs regulate cell development, differentiation and production of inflammatory mediators. Aim: To investigate the roles of miRNAs in regulating immune cell function and its role in respiratory diseases. Methods: Lung miRNAs that were differentially expressed following non-typeable Haemophilus Influenzae (NTHi) challenge were identified by microarray. miRNA inhibitors (antagomirs) were used to investigate the role of miRNA in regulating bacterial clearance in in mouse models of immunosuppresion and emphysema. Results: Upon NTHi infection, 15 miRNAs were up-regulated while 49 were down-regulated by > 2.5 fold in lungs. In vitro, miR-328 knockdown reduced bacterial load and increased phagocytosis by macrophages and neutrophils. Transferred of macrophages or neutrophils (deficient in miR-328) to naïve recipient mice enhanced clearance of the bacteria in vivo. Similarly, inhibition of miR-328 by antagomirs promote NTHi clearance in mouse models of immunosuppresion and emphysema. Conclusion: : Our study identify a novel role for miR-328 in the regulation of phagocytosis and provide proof of principle that miRNA pathways can be targeted in the lung and offer a potential new anti-microbial approach for the treatment of respiratory infection.

Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases., Author Summary MicroRNAs regulate pathogen recognition pathways by modulating translation. In the immune system, miRNAs have been identified as important regulators of gene expression programs, which regulate differentiation, growth and function of innate and adaptive immune cells. Using miRNA microarray, we demonstrated that lung miRNAs were differentially expressed following non-typeable Haemophilus Influenzae (NTHi) infection in mice. To study the role of a specific miRNA in macrophages, we used antagomir (chemically modified single-stranded RNA analogues, complementary to the target miRNA) to block miRNA function. Interestingly, inhibition of microRNA-328 in mouse and human macrophages increases microbicidal activity by amplifying phagocytosis and production of reactive oxygen species. Inhibition of mR-328 in the lung enhanced bacterial clearance in mouse models of immunosuppression and emphysema. Our study provides proof of principle that miRNA pathways can be targeted in the lung and offer a potential new anti-microbial approach for the treatment of respiratory infection.

S A T U R D A Y 247 Functional Analysis of Calpain-14 in Eosinophilic Esophagitis Benjamin P. Davis, MD, Leah Claire Kottyan, PhD, Emily Stucke, BA, Joseph D. Sherrill, PhD, Marc E. Rothenberg, MD, PhD; Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, Center for Autoimmune Genomics and Etiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, Cincinnati, OH, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: We recently identified a genome-wide association susceptibility locus for eosinophilic esophagitis (EoE) at 2q23 wherein CAPN14 (p52.5 3 10210) is located. CAPN14 encodes for a member of the calpain protease family. We have shown that CAPN14 was specifically expressed in the esophagus, dynamically regulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13. Herein, we aimed to determine the function of calpain-14. METHODS: We overexpressed calpain-14 in an esophageal progenitor cell line (EPC2) grown in an air-liquid interface (ALI) culture and analyzed barrier effects by transepithial electrical resistance (TEER), fluorescein isothiocyanate (FITC)-dextran flux, and hematoxylin and eosin stain. We also identified potential calpain-14 targets via western blot and immunofluorescence (IF) of the ALI culture. RESULTS: Calpain-14 over-expression induced a 2-3 fold (p50.0034) decrease in TEER and a 2-3 fold (P

The role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in immune responses mediated by T-helper 2 (T(H)2) lymphocytes is unknown. Here we characterize the development of allergic airway disease in TRAIL-deficient (Tnfsf10(-/-)) mice and in mice exposed to short interfering RNA targeting TRAIL. We show that TRAIL is abundantly expressed in the airway epithelium of allergic mice and that inhibition of signaling impairs production of the chemokine CCL20 and homing of myeloid dendritic cells and T cells expressing CCR6 and CD4 to the airways. Attenuated homing limits T(H)2 cytokine release, inflammation, airway hyperreactivity and expression of the transcriptional activator STAT6. Activation of STAT6 by interleukin-13 restores airway hyperreactivity in Tnfsf10(-/-) mice. Recombinant TRAIL induces pathognomic features of asthma and stimulates the production of CCL20 in primary human bronchial epithelium cells. TRAIL is also increased in sputum of asthmatics. The function of TRAIL in the airway epithelium identifies this molecule as a target for the treatment of asthma.

The discovery of small "noncoding" or "nonmessenger" RNA molecules that are repressors of translation (microRNAs) has provided the opportunity to specifically suppress a gene or clusters of genes. Moreover, the recent employment of synthetic analogs of these small RNA molecules termed "antagomirs" has shown that microRNAs of interest can be specifically targeted. Understanding the role of microRNAs in fundamental processes associated with complex diseases such as asthma, chronic obstructive pulmonary disease, cancer, chronic infections, and immune disorders may aid in disease diagnosis and prognosis and potentially identify new therapeutic targets.

Objective Maternal asthma is the most common chronic disease complicating pregnancy and is a risk factor for bronchiolitis in infancy. Recurrent episodes of bronchiolitis are strongly associated with the development of childhood asthma. Methods We conducted a follow-up study of infants born to women with asthma who completed a double-blind randomised controlled trial during pregnancy. In this trial, pregnant women with asthma were assigned to treatment adjustment by an algorithm using clinical symptoms (clinical group) or the fraction of exhaled nitric oxide (FeNO group) and we showed that the FeNO group had significantly lower asthma exacerbation rates in pregnancy. Results 146 infants attended the 12-month follow-up visit. Infants born to mothers from the FeNO group were significantly less likely to have recurrent episodes of bronchiolitis in the first year of life (OR 0.08, 95% CI 0.01 to 0.62; p=0.016) as compared with the clinical group. Conclusions Optimised management of asthma during pregnancy may reduce recurrent episodes of bronchiolitis in infancy, which could potentially modulate the risk to develop or the severity of emerging childhood asthma.

The effects of semi-annual and 3.5 yr mean ozone (O 3 ) concentrations on children9s forced vital capacity (FVC) and forced expiratory volume in one second (FEV 1 ) were assessed over a study period of 3.5 yrs in 2,153 schoolchildren from 15 study sites in South Western Germany and Lower Austria. Spirometric parameters were assessed twice a year, and differences between consecutive measurements divided by days were considered as a measure of lung growth. Exposure was analysed in four classes, separately for winter and summer (semi-annual mean O 3 concentrations 22–30, 30–38, 38–46, 46–54 parts per billion (ppb) in summer and 4–12, 12–20, 20–28, 28–36 ppb in winter). Regression methods for repeated measurements were used, and these revealed a significantly lower FVC (FEV 1 ) increase estimated at −19.2 (−18.5) mL·100 days −1 for semi-annual mean O 3 exposure in summer between 46 and 54 ppb compared to exposure between 22 and 30 ppb. However, in winter, the estimated difference in FVC (FEV 1 ) was 16.4 (10.9) mL·100 days −1 between the semi-annual O 3 class 28–36 ppb and the 4–12 ppb class. By means of linear regression the study found that there was no association between growth rates and mean summer O 3 for FVC and FEV 1 over a 3.5 yr period. The authors conclude that medium-term effects on schoolchildren9s lung growth are possibly present, but are in the long-term not detectable for forced vital capacity and forced expiratory volume in one second over a 3.5 yr period due to partial reversibility.

Currently most attempts at cancer immunotherapy involve the generation of CD8(+) cytotoxic T lymphocytes (CTLs) against tumor-associated antigens. Many tumors, however, have been immunoselected to evade recognition by CTLs and thus alternative approaches to cancer immunotherapy are urgently needed. Here we demonstrate that CD4(+) T cells that recognize a secreted tumor-specific antigen and exhibit a cytokine secretion profile characteristic of Th2 cells, are capable of clearing established lung and visceral metastases of a CTL-resistant melanoma. Clearance of lung metastases by the Th2 cells was found to be totally dependent on the eosinophil chemokine, eotaxin, and partially dependent on the transcription activator signal transducer and activator of transcription 6 (STAT6), with degranulating eosinophils within the tumors inducing tumor regression. In contrast, tumor-specific CD4(+) Th1 cells, that recruited macrophages into the tumors, had no effect on tumor growth. This work provides the basis for a new approach to adoptive T cell immunotherapy of cancer.

Extensive clinical investigations have implicated eosinophils in the pathogenesis of asthma. In a recent clinical trial, humanized monoclonal antibody to interleukin (IL)-5 significantly limited eosinophil migration to the lung. However, treatment did not affect the development of the late-phase response or airways hyperresponsiveness in experimental asthma. Although IL-5 is a key regulator of eosinophilia and attenuation of its actions without signs of clinical improvement raises questions about the contribution of these cells to disease, further studies are warranted to define the effects of anti-IL-5 in the processes that lead to chronic asthma. Furthermore, eosinophil accumulation into allergic tissues should not be viewed as a process that is exclusively regulated by IL-5 but one in which IL-5 greatly contributes. Indeed, data on anti-IL-5 treatments (human and animal models) are confounded by the failure of this approach to completely resolve tissue eosinophilia and the belief that IL-5 alone is the critical molecular switch for eosinophil development and migration. The contribution of these IL-5-independent pathways should be considered when assessing the role of eosinophils in disease processes.

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by ∼50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to β-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. However, limited studies suggest a more discrete immunomodulatory role for this leukocyte during these inflammatory responses. In this investigation, we highlight the potential of eosinophils to act as APCs and thus modulators of allergic responses by influencing Th2 cell function. In response to Ag provocation of the allergic lung, eosinophils rapidly trafficked to sites of Ag deposition (airways lumen) and presentation (lung-associated lymph nodes and T cell-rich paracortical zones). Eosinophils from the allergic lung expressed class II MHC peptides, T cell costimulatory molecules (CD80 and CD86), and rapidly internalized and processed Ag that was sampled from within the airway lumen. Ag-loaded eosinophils promoted the production of IL-4, IL-5, and IL-13 in cocultures with in vitro-polarized Th2 cells and induced IL-5 production in a dose-dependent manner from Ag-specific CD4+ T cells isolated from allergic mice. In addition, Ag-loaded eosinophils primed for Th2 cell-driven allergic disease of the lung when transferred to naive mice. Thus, eosinophils have the potential to not only activate Th2 cells to release disease-modulating cytokines but also to assist in priming the immune system for allergic responses. This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate allergic inflammation by amplifying Th2 cell responses.

The potent spasmogenic properties of IL-13 have identified this molecule as a potential regulator of airways hyperreactivity (AHR) in asthma. Although IL-13 is thought to primarily signal through the IL-13Rα1-IL-4Rα complex, the cellular and molecular components employed by this cytokine to induce AHR in the allergic lung have not been identified. By transferring OVA-specific CD4+ T cells that were wild type (IL-13+/+ T cells) or deficient in IL-13 (IL-13−/− T cells) to nonsensitized mice that were then challenged with OVA aerosol, we show that T cell-derived IL-13 plays a key role in regulating AHR, mucus hypersecretion, eotaxin production, and eosinophilia in the allergic lung. Moreover, IL-13+/+ T cells induce these features (except mucus production) of allergic disease independently of the IL-4Rα chain. By contrast, IL-13+/+ T cells did not induce disease in STAT6-deficient mice. This shows that IL-13 employs a novel component of the IL-13 receptor signaling system that involves STAT6, independently of the IL-4Rα chain, to modulate pathogenesis. We show that this novel pathway for IL-13 signaling is dependent on T cell activation in the lung and is critically linked to downstream effector pathways regulated by eotaxin and STAT6.

In this investigation, we have examined the integrated relationship between IL-13, IL-4, and IL-5 for the development of airways hyperreactivity (AHR) in a model of asthma in BALB/c mice. Sensitization and aeroallergen challenge of both wild-type (WT) and IL-13 gene-targeted (IL-13−/−) mice induced allergic disease that was characterized by pulmonary eosinophilia and AHR to β-methacholine. Although these responses in IL-13−/− mice were heightened compared with WT, they could be reduced to the level in nonallergic mice by the concomitant neutralization of IL-4. Mice in which both IL-4 and IL-13 were depleted displayed a marked reduction in tissue eosinophils, despite the development of a blood eosinophilia. Similar neutralization of IL-4 in WT mice only partially reduced AHR with no effect on tissue eosinophilia. In addition, neutralization of IL-5 in IL-13−/− mice, but not in WT mice, inhibited AHR, suggesting that tissue eosinophilia is linked to the mechanism underlying AHR only in the absence of IL-13. Additionally, mucus hypersecretion was attenuated in IL-13−/− mice, despite the persistence of AHR. Taken together, our data suggest both a modulatory role for IL-13 during sensitization and a proinflammatory role during aeroallergen challenge. The latter process appears redundant with respect to IL-4.

Despite intensive research, the pivotal factors that link abnormalities in permeability of the dermal barrier with inflammation and the generation of skin lesions in allergic dermatitis (AD) remain unknown. In a landmark paper published by Jin et al.1 in the Journal of Clinical Investigation, IL-21 is identified as a critical regulator of the processes that lead to sensitization and allergic inflammation of the skin. Allergic dermatitis is an inflammatory disease of the skin of unknown aetiology.2,3 AD clinically presents early in life (under 5 years of age) and is characterized by eczematous lesions, drying and thickening of the skin, and pruritus (severe itching).2 Susceptibility may be associated with a history of asthma and other atopic conditions characterized by elevated levels of IgE (for example, allergic reactions to foods, rhinitis and urticaria). The incidence of AD is rising in the Western world and the environmental factors driving increased susceptibility are thought to be associated with the global ‘atopic march’. In general, two paradigms have emerged to describe the aetiology of AD: (1) that a defect in immunological responsiveness to antigens encountered on the skin surface (stratum corneum) underpins the development of lesions, and (2) that inherited and acquired abnormalities in permeability of the barrier are the key drivers of disease initiation and progression.2–4 Like in the case of asthma, the CD4 T helper-1 and -2 paradigm has been widely used to explain the aetiology of AD. Early in life, skin lesions are associated with an abnormal Th2 response to antigens and numerous studies have implicated Th2 cytokines in pathological events. However, like in the case of asthma, the Th2 paradigm does not fully account for the heterogeneity or chronicity of disease. As AD progresses, the T-cell response may switch or is skewed to a ‘more’ Th1 phenotype, probably because of chronic infections at the site of skin lesions. Interestingly, asthma may also evolve towards a more neutrophilic and Th1 dominant phenotype that is linked to chronic respiratory infections and is largely unresponsive to steroid therapy. Indeed, although very distinct disorders, a number of parallels can be drawn between aetiology and disease progression in AD and asthma. The second paradigm suggests that inherent abnormalities in pathways that regulate skin homeostasis predispose to broad barrier failure. This alters permeability and antimicrobial functions that lead to inflammation and the development of lesions.4 Inherited barrier abnormalities in AD have been associated with a defective stratum corneum (outer layers of the skin). In particular, lossof-function mutations in filaggrin, which regulates the structural and functional integrity of the epidermal cytoskeleton, can enhance allergic sensitization.5 Although there is open debate on the aetiology of disease and critical susceptibility factors for AD, there is clear evidence that the interactions between immunological pathways and processes that maintain barrier integrity are central to the perpetuation of disease (Figure 1). Recently, Jin et al.1 identified for the first time that protein production of IL-21 and its receptor (IL-21R) was up-regulated in skin lesions of patients suffering from active AD, but not normal controls. IL-21 was expressed in mononuclear leukocytes that infiltrate the dermis, and the IL-21R was expressed by epidermal keratinocytes and along the dermal–epidermal junction. IL-21 is a pleiotropic cytokine derived from activated CD4 T cells.6 This raised the question—Is IL-21 a critical regulator of inflammation and barrier function in AD? To advance our understanding of the potential role of IL-21 in the pathogenesis of AD, a mouse model of tape stripping of saved skin was used to mimic tissue trauma induced by scratching dry, itchy skin that underlies human AD. Tissue injury induced by tape stripping resulted in the up-regulation of IL-21 and IL-21R mRNA. Epicutaneous application of the archetypical Th2 antigen ovalbumin to tape-stripped skin resulted in allergic inflammation at the site of exposure that mimicked aspects of the inflammatory response and tissue lesions in AD. In IL-21R deficient mice epicutaneous allergic sensitization was profoundly impaired. In the absence of IL-21R the expression of CCL17/ TARC (recruits skin-homing T cells), Th2 cytokines (IL-4 and IL-13), eotaxin (chemotactic signal for eosinophils) and IFNg were not increased, and isolated T cells did not proliferate in response to Ova exposure. IL-21 signalling was also shown to be essential for the development of cutaneous inflammation in a Th1 cell model of contact hypersensitivity, highlighting the pivotal role of this pathway in promoting inflammation in the skin. The impaired systemic response to epicutaneous allergic sensitization was not related to an intrinsic defect in dendritic cell (DC) function, as IL-21-deficient DCs effectively induced proliferation of Ova-specific DO11.10 transgenic Th2 and Th1 cells in culture and at steady state and the same frequency of CCR7+CD11c+ DCs were found in peripheral tissues. However, recruitment of adoptively transferred naive Ova-specific T cells to the site of Ova challenge after tape stripping was significantly impaired, and this correlated with defective DC migration to the lymphatic ducts of tape-stripped skin (in the absence of Ova treatment). The authors did not demonstrate that DC migration was decreased after Ova exposure to tapestripped skin, which would have strengthened their proposed mechanism and this could have been achieved by using FITC-labelled Professor PS Foster and Associate Professor J Mattes are at the Centre for Asthma and Respiratory Disease (CARD), School of Biomedical Sciences, Faculty of Health, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales 2300, Australia. E-mails: Paul.Foster@newcastle.edu.au or Joerg.Mattes@newcastle.edu.au Immunology and Cell Biology (2009) 87, 359–360 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09 $32.00

The relationship between nitric oxide in exhaled air, levels of sputum eosinophils, sputum eosinophil cationic protein (ECP) and urinary eosinophil protein X (EPX) excretion has not yet been investigated in corticosteroid-dependent childhood asthma. Therefore, taking 25 children with stable asthma (mean age 11.2 yrs) treated with inhaled corticosteroids and nine nonatopic healthy control children (mean age 12.8 yrs) the level of exhaled NO was measured by means of a chemiluminescence analyser before and after sputum induction. This was conducted as a slow vital capacity manoeuvre under standardized conditions with a target flow of 70 mL x s(-1) against a resistance of 100 cm H2O x L(-1) x s. Sputum induction was performed by inhalation of hypertonic saline (3, 4, and 5%) in a standardized manner and a single sample of urine was collected. Exhaled NO (p = 0.01), absolute eosinophil cell counts in sputum (p = 0.02), sputum ECP (p = 0.09) and urinary EPX excretion (p = 0.02) were higher in asthmatics compared to control children. Exhaled NO was positively correlated with sputum ECP (r(s) = 0.59, p = 0.002), urinary EPX (r(s) = 0.42, p = 0.03), and sputum eosinophils (r(s) = 0.30, p = 0.15) in the asthmatic children. These correlations appeared to be pronounced after sputum induction, where NO values had decreased (p = 0.01). None of the correlations were observed in the group of nonatopic control subjects. Additionally there were significant correlations between sputum ECP and sputum eosinophils (r(s) = 0.69, p

We have previously shown that chlamydial lung infections in early-life drive the development of chronic airway hyper-responsiveness (AHR). To elucidate the mechanisms involved, we used a murine model of chlamydial lung infection, to investigate the influence of IL-13 on infection and infection-induced AHR. Infected IL-13-/- mice had less severe infection, reduced inflammation (neutrophils, dendritic cells, macrophages and T cells), less mucus secreting cells (MSCs) in the airways and attenuated AHR. More severe infection in wild-type (WT) mice was associated with the development of alternatively activated macrophages (increased Arg-1, FIZZ-1 and Ym-1), which were suppressed in IL-13-/- mice. Surprisingly, infection of WT mice did not induce any detectable increase in IL-13 expression. However, infection decreased the level of the IL-13 decoy receptor (IL-13Rα2), which may allow constitutive IL-13 to signal. Interestingly, reconstitution of IL-13 in IL-13-/- mice restored infection-induced inflammation and MSC formation, but not AHR. Furthermore, infection-induced inflammation and mucus were Stat-6-independent, whilst infection-induced AHR was dependent upon Stat-6. Thus, early-life respiratory chlamydial infection results in decreased decoy receptor expression, which allows constitutive IL-13 to signal and promote more severe infection, which drives the development of chronic AHR that persists into adulthood.

Background Congenital thoracic malformations (CTM) are rare lung lesions that are managed with surgical resection or active surveillance. The objective of this study was to comprehensively assess large and small airway function in children with CTM who underwent lobectomy in early life. We hypothesise that sensitive measures of lung function will demonstrate residual impairments in CTM compared to healthy children. Methods Nitrogen lung clearance index (LCI), reactance and resistance (X5Hz and R5Hz), forced expiratory volume in 1 s and forced vital capacity (FEV1 and FVC) were prospectively measured in 10 children with CTM (mean age/SD: 7.6/1.3) who had undergone surgical resection in early life and in 17 healthy children (mean age/SD: 4.8/0.4). Total lung capacity (TLC) was also conducted in children older than 7 years of age with CTM (n = 8). Results Mean LCI was 8.0 (95% CI 7.5 to 8.5) in the CTM group and 7.3 (95% CI 7.0 to 7.6) in healthy children (p = 0.016). Mean X5Hz was −0.44kPa/l/s (95% CI −0.58 to −0.31) in the CTM group and −0.31kPa/l/s (95% CI −0.35 to −0.27) in healthy children (p = 0.02). Mean Z score for X5Hz was −2.11 (95% CI −3.59 to −0.63) in the CTM group and −0.11 (95% CI −0.55 to 0.33) in healthy children (p = 0.0008). Mean FEV1 was 1.21 L (95% CI 0.97 to 1.45) in the CTM group and 1.02 L (95% CI 0.90 to 1.15) in healthy children (p = 0.22). Mean % predicted FEV1 was 83% (95% CI 74 to 92) in the CTM group and 97% (95% CI 87 to 107) in healthy children (p