Stephanie T. Yerkovich

An immunoepidemiological approach to asthma: identification of in-vitro T-cell response patterns associated with different wheezing phenotypes in children

BACKGROUND Increasing evidence suggests that patterns of T-cell immunity to inhalant allergens in genetically diverse human populations are more heterogeneous than previously assumed, and that covert differences in expression patterns might underlie variations in airway disease phenotypes. We tested this proposition in a community sample of children. METHODS We analysed data from 172 individuals who had been recruited antenatally to a longitudinal birth cohort study. Of the 194 birth cohort participants, data from the 147 probands (age range 8.6-13.5 years) who consented to blood collection were included along with data from 25 consenting siblings (mean age 11 years [range 7.4-17.4]). We ascertained clinical phenotypes related to asthma and allergy. We measured T-cell responses to allergens and mitogens, together with blood eosinophils and IgE/IgG antibodies, and assessed associations between these indices and clinical phenotypes. FINDINGS Atopy was associated with allergen-specific T-helper (Th)2 responses dominated by interleukin 4, interleukin 5, interleukin 9, interleukin 13, whereas interleukin 10, tumour necrosis factor alpha, and interferon gamma responses were common to both atopics and non-atopics. The wheal size from skin prick with allergen was positively associated with in-vitro interleukin 5 and interferon gamma responses, and negatively associated with interleukin 10. Asthma, especially in atopics, was strongly associated with eosinophilia/interleukin 5, and bronchial hyper-responsiveness (BHR) was associated with eosinophilia plus polyclonal interferon gamma production. BHR in non-atopics was associated with elevated allergen-specific and polyclonal interleukin 10 production. INTERPRETATION Parallel immunological and clinical profiling of children identified distinctive immune response patterns related to asthma and wheeze compared with BHR, in atopics non-atopics. Immunological hyper-responsiveness, including within the Th1 cytokine compartment, is identified as a hallmark of BHR. RELEVANCE TO PRACTICE These findings highlight the heterogeneity of immune response patterns in asthmatic children, including those with seemingly homogeneous Th2-driven atopic asthma. Further elucidation of the covert relationships between wheezing phenotypes and underlying immunophenotypes in this age group will potentially lead to more effective treatments for what is an unexpectedly heterogeneous collection of disease subtypes.

Postnatal Development of Monocyte Cytokine Responses to Bacterial Lipopolysaccharide

Early childhood is a period of heightened susceptibility to infection due to immaturity of the immune system, and the nature of these developmental deficiencies is only partially understood. In this study, we focused on the ontogeny of the innate immune system by investigating the capacity of mononuclear cells to secrete a wide spectrum of inflammatory cytokines in response to interferon (IFN)-γ priming and lipopolysaccharide (LPS) stimulation, namely IL-6, IL-10, IL-12, IL-18, IL-23, tumor necrosis factor (TNF)-α, and myxovirus resistance protein A, induced by type-I IFN, at several time points between birth (cord blood) and adulthood. Competence to produce all these cytokines followed a similar developmental pattern, with slow postnatal up-regulation from the response observed in cord blood. Unexpectedly, IL-6, IL-10, TNF-α, and IFN-γ showed slow postnatal up-regulation but also elevated cord blood responses equal to or greater than the adult level. This was transient and not observed at 2 mo of age, and was not related to predelivery stress of the newborns. Variations in Toll-like receptor (TLR)4 function may account for these age related differences in cytokine responses, as TLR4 expression on neonatal monocytes post LPS stimulation was elevated and sustained relative to infants and adults.

A phase 1b study of placenta-derived mesenchymal stromal cells in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a degenerative disease characterized by fibrosis following failed epithelial repair. Mesenchymal stromal cells (MSC), a key component of the stem cell niche in bone marrow and possibly other organs including lung, have been shown to enhance epithelial repair and are effective in preclinical models of inflammation‐induced pulmonary fibrosis, but may be profibrotic in some circumstances.

Th2-associated local reactions to the acellular diphtheria-tetanus-pertussis vaccine in 4- to 6-year-old children.

ABSTRACT Acellular vaccines against diphtheria-tetanus-pertussis (acellular pertussis) (DTaP) are being progressively introduced into vaccination programs worldwide, with the aim of reducing T-helper 1 (Th1)-associated reactogenicity associated with the cellular diphtheria-tetanus-pertussis (whole-cell pertussis) (DTwP) vaccine. The DTaP vaccine has an improved safety profile in infants, but little information is available concerning the nature of the ensuing immunological memory in older children and how this may affect the reactogenicity of DTaP booster doses. We have addressed this question in the present study by assessing polyclonal and vaccine antigen-specific humoral and cellular immune responses to boosting with DTaP in 4- to 6-year-old children primed during infancy with DTaP (n = 30) or DTwP (n = 16) and by correlating these parameters, in particular cytokine responses, with expression of local side effects at the injection site. Large local reactions (≥50-mm diameter) 24 to 72 h after receiving the DTaP booster occurred in 43% of exclusively DTaP-primed children, in contrast to 6% of children primed with DTwP. These reactions were associated with vigorous T helper 2 (Th2)-polarized memory responses to vaccine antigen exemplified by interleukin 5 (IL-5), IL-6, and IL-13 production and log-scale boosting of tetanus-specific immunoglobulin E and occurred most frequently among children who are intrinsically “high Th2 responders” as detected by in vitro responsiveness to polyclonal mitogen. Our findings suggest that priming during infancy with DTaP promotes stable, boostable Th2-polarized immunity against vaccine antigens, which in a significant subset of children is subsequently associated with local reactions at the booster site. The time course of these reactions suggests that the underlying mechanism involves reactivation of Th2-polarized cellular immune memory.

Reduced soluble receptor for advanced glycation end-products in COPD

The soluble receptor for advanced glycation end-products (sRAGE) has anti-inflammatory properties, and deficiency of circulating sRAGE is associated with various human diseases. Whether sRAGE concentrations are reduced in chronic obstructive pulmonary disease (COPD) has not been determined. The aim of this study was to determine plasma levels of sRAGE in COPD patients and establish whether sRAGE varies in relation to forced expiratory volume in 1 s (FEV1) and other inflammatory markers. 61 COPD patients and 42 healthy controls were recruited. Plasma sRAGE, C-reactive protein (CRP) and serum amyloid A (SAA) were measured in patients with stable COPD. A subgroup had measurements during acute exacerbations of COPD (AECOPD). sRAGE was significantly lower in stable COPD than in healthy controls (p<0.001), while CRP (p<0.001) and SAA (p = 0.015) were higher in stable COPD than in healthy controls. Multiple linear regression confirmed that COPD was negatively associated with sRAGE (p<0.001). Plasma sRAGE was positively correlated with FEV1 (r2 = 0.530, p<0.001), while CRP and SAA were inversely proportional to FEV1. Multiple linear regression analysis showed that only sRAGE was a strong predictor of FEV1. AECOPD were associated with even lower sRAGE levels that increased with convalescence. Circulating sRAGE is lower in COPD and shows a strong correlation to the degree of airflow limitation.

CpG methylation patterns in the IFN promoter in naive T cells: Variations during Th1 and Th2 differentiation and between atopics and non-atopics

Interferon‐γ (IFNγ) gene expression is tightly regulated in early life, and exaggerated negative control of IFNγ 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 IFNγ promoter in neonates, a mechanism which in mice leads to strong suppression of IFNγ gene transcription. In the present study, the methylation status of six CpG sites in the proximal promoter of the human IFNγ 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 IFNγ 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, IFNγ promoter methylation was reduced in CD8+ T cells from atopic children in the age range in which hyperproduction of IFNγ as recently been identified as a common feature of the atopic phenotype. The findings demonstrate the potency of IFNγ promoter methylation as a mechanism for control of human IFNγ gene expression, particularly during early life. Differential regulation of IFNγ promoter methylation in T cells may be an important contributory factor in atopy development in childhood, and this possibility warrants further detailed investigation.

Reestablishment of Recipient-associated Microbiota in the Lung Allograft Is Linked to Reduced Risk of Bronchiolitis Obliterans Syndrome

RATIONALE Bronchiolitis obliterans syndrome (BOS) is the primary limiting factor for long-term survival after lung transplantation, and has previously been associated with microbial infections. OBJECTIVES To cross-sectionally and longitudinally characterize microbial communities in allografts from transplant recipients with and without BOS using a culture-independent method based on high-throughput sequencing. METHODS Allografts were sampled by bronchoalveolar lavage, and microbial communities were profiled using 16S rRNA gene amplicon pyrosequencing. Community profiles were compared using the weighted Unifrac metric and the relationship between microbial populations, BOS, and other covariates was explored using PERMANOVA and logistic regression. MEASUREMENTS AND MAIN RESULTS Microbial communities in transplant patients fell into two main groups: those dominated by Pseudomonas or those dominated by Streptococcus and Veillonella, which seem to be mutually exclusive lung microbiomes. Aspergillus culture was also negatively correlated with the Pseudomonas-dominated group. The reestablishment of dominant populations present in patients pretransplant, notably Pseudomonas in individuals with cystic fibrosis, was negatively correlated with BOS. CONCLUSIONS Recolonization of the allograft by Pseudomonas in individuals with cystic fibrosis is not associated with BOS. In general, reestablishment of pretransplant lung populations in the allograft seems to have a protective effect against BOS, whereas de novo acquisition of microbial populations often belonging to the same genera may increase the risk of BOS.

Toll-like receptor 7 function is reduced in adolescents with asthma

Anti-viral innate immune responses may be impaired in asthma, although the mechanisms are not well understood. Toll-like receptors (TLRs) 7 and 3 are particularly relevant for initiating responses to common respiratory viruses, as they recognise single-stranded viral RNA and double-stranded viral RNA, respectively. The aim of the present study was to investigate TLR7 and TLR3 function in 14-yr-old adolescents with asthma. Blood mononuclear cells obtained from 17 atopic asthmatics, 29 atopic, non-asthmatics and 21 healthy, non-atopic individuals, were stimulated with the TLR7 agonist imiquimod and the TLR3 agonist poly I:C. Expression of anti-viral molecules was measured by real-time PCR. Concentrations of interferon-γ-inducible cytokine protein (IP)-10 and interleukin (IL)-6 were measured by ELISA. TLR7-induced myxovirus resistance protein A and 2′5′ oligoadenylate synthetase mRNA expression and protein levels of IP-10 were significantly lower in asthma subjects compared with healthy subjects (p = 0.041, p = 0.003 and p = 0.001 respectively). There was a significant negative correlation between total serum immunoglobulin E and IP-10 following TLR7 stimulation. However, TLR3-induced responses did not vary with asthma or atopy. IL-10 mRNA and IL-6 protein synthesis were similar in asthmatic and control subjects. In conclusion, TLR7 function is reduced in adolescents with asthma and this may contribute to susceptibility to respiratory viral infections.

Mesenchymal stem cells and the lung

Mesenchymal stem cells (MSC) are a population of tissue‐resident adult progenitor cells that were originally identified in bone marrow, but have now been identified in many organs including the lung. Although their precise role in organ function remains incompletely defined, mounting evidence suggests that they are an important component of the parenchymal progenitor cell niche and orchestrate organ homeostasis and repair following injury. In this review, what is known about MSC biology will be outlined with particular emphasis on lung biology, and the therapeutic potential of MSC‐based cell therapy will also be highlighted.

Allergen-enhanced thrombomodulin (blood dendritic cell antigen 3, CD141) expression on dendritic cells is associated with a TH2-skewed immune response.

BACKGROUND 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. OBJECTIVE To identify novel genes expressed by house dust mite (HDM) allergen-exposed DCs. METHODS We screened for allergen-induced gene expression by microarray, and validated differentially expressed genes at the mRNA and protein levels. RESULTS Thrombomodulin (CD141, blood dendritic cell antigen 3) expression by microarray was higher on HDM-stimulated DCs from atopic (relative to nonatopic) individuals. These findings were confirmed at both the mRNA and protein levels in an independent group. Purified thrombomodulin(+) DCs induced a strongly T(H)2-polarized cytokine response by allergen-specific T cells compared with DCs lacking thrombomodulin. In vivo, thrombomodulin(+) circulating DCs were significantly more frequent in subjects with HDM allergy and asthma, compared with control subjects. Furthermore, thrombomodulin expression in blood leukocytes was higher in children with acute asthma than at convalescence 6 weeks later. CONCLUSION Thrombomodulin expression on DCs may be involved in the pathogenesis of atopy and asthma.