Elysia M. Hollams

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.

The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.

Summary The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

mRNA Stability and the Control of Gene Expression: Implications for Human Disease

Regulation of gene expression is essential for the homeostasis of an organism, playing a pivotal role in cellular proliferation, differentiation, and response to specific stimuli. Multiple studies over the last two decades have demonstrated that the modulation of mRNA stability plays an important role in regulating gene expression. The stability of a given mRNA transcript is determined by the presence of sequences within an mRNA known as cis-elements, which can be bound by trans-acting RNA-binding proteins to inhibit or enhance mRNA decay. These cis-trans interactions are subject to a control by a wide variety of factors including hypoxia, hormones, and cytokines. In this review, we describe mRNA biosynthesis and degradation, and detail the cis-elements and RNA-binding proteins known to affect mRNA turnover. We present recent examples in which dysregulation of mRNA stability has been associated with human diseases including cancer, inflammatory disease, and Alzheimers disease.

Vitamin D and atopy and asthma phenotypes in children: a longitudinal cohort study

Vitamin D has been linked in some studies with atopy- and asthma-associated phenotypes in children with established disease, but its role in disease inception at the community level is less clear. The aim of the present study was to investigate associations between vitamin D status and biological signatures indicative of allergy and asthma development in children aged 6 and 14 years in Perth, WA, Australia (latitude 32° S). Serum vitamin D was assayed in 989 6-yr-olds and 1,380 14-yr-olds from an unselected community birth cohort; 689 subjects were assessed at both ages. Vitamin D levels were assessed as a risk modifier for respiratory and allergic outcomes at both ages, using previously ascertained phenotypic data. The predictive value of vitamin D levels at age 6 yrs for development of clinical phenotypes at age 14 yrs was also examined. Serum vitamin D levels in children of both ages were negatively associated with concurrent allergic phenotypes; sex stratification revealed that this association was restricted mainly to males. Furthermore, vitamin D levels at age 6 yrs were significant predictors of subsequent atopy/asthma-associated phenotypes at age 14 yrs. In an unselected community setting, children (particularly males) with inadequate vitamin D are at increased risk of developing atopy, and subsequently bronchial hyperresponsiveness (BHR) and asthma. In a large unselected cohort, males with inadequate vitamin D at 6 and 14 yrs of age had increased atopy and BHR. Low vitamin D at age 6 yrs was a predictor of atopy and asthma at 14 yrs of age.

Toward improved prediction of risk for atopy and asthma among preschoolers: A prospective cohort study

BACKGROUND 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. OBJECTIVE We sought to identify clinical and laboratory biomarkers in 2-year-olds that are predictive of the risk for persistent atopy and wheeze at age 5 years. METHODS We prospectively studied 198 atopic family history-positive children to age 5 years. Clinical and laboratory assessments related to asthma history and atopy status were undertaken annually; episodes of acute respiratory illness were assessed and classified throughout and graded by severity. RESULTS Aeroallergen-specific IgE titers cycled continuously within the low range in nonatopic subjects. Atopic subjects displayed similar cycling in infancy but eventually locked into a stable pattern of upwardly trending antibody production and T(H)2-polarized cellular immunity. The latter was associated with stable expression of IL-4 receptor in allergen-specific T(H)2 memory responses, which was absent from responses during infancy. Risk for persistent wheeze was strongly linked to early sensitization and in turn to early infection. Integration of these data by means of logistic regression revealed that attaining mite-specific IgE titers of greater than 0.20 kU/L by age 2 years was associated with a 12.7% risk of persistent wheeze, increasing progressively to an 87.2% risk with increasing numbers of severe lower respiratory tract illnesses experienced. CONCLUSION The risk for development of persistent wheeze in children can be quantified by means of integration of measures related to early sensitization and early infections. Follow-up studies along similar lines in larger unselected populations to refine this approach are warranted.

Presymptomatic differences in Toll-like receptor function in infants who have allergy.

BACKGROUND Microbial exposure might play a key role in allergy development, but little is known about the role of Toll-like receptors (TLRs). OBJECTIVE This study explored the association between neonatal TLR microbial recognition/function, allergy risk (maternal allergy), and prospective allergy development. METHODS Cord blood mononuclear cells (n = 111) were cultured either alone or with optimal concentrations of TLR ligands: lipoteichoic acid (TLR2), polyinosinicpolycytidylic acid (TLR3), LPS with IFN-gamma (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), or CpG (TLR9). Cytokine responses were assessed in relation to allergy risk (maternal allergy) and allergy outcomes (sensitization, food allergy, and atopic dermatitis) at 12 months of age. RESULTS Maternal allergy (n = 59) was associated with significantly higher neonatal IL-12 and IFN-gamma responses to TLR2, TLR3, and TLR4 activation, whereas TNF-alpha and IL-6 responses to TLR2, TLR4, and TLR5 activation were significantly higher in newborns who subsequently had allergic disease (n = 32). Notably, consistent with previous reports, newborns who had disease had lower T(H)1 IFN-gamma response to mitogens (PHA). CONCLUSION Allergic disease was associated with increased (rather than decreased) perinatal TLR responses. Further studies are needed to determine how these responses track in the postnatal period and whether this relative hyperresponsiveness is a product of intrauterine influences, including maternal atopy, functional genetic polymorphisms, or both.

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.

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.

Elucidation of asthma phenotypes in atopic teenagers through parallel immunophenotypic and clinical profiling

BACKGROUND Current treatment strategies for asthma in teenagers derive primarily from information on chronic disease in adults. More detailed understanding of risk factors related to teenage asthma might aid in the development of improved preventive and treatment strategies for this age group. OBJECTIVE We sought to identify biomarkers associated with asthma phenotypes in teenagers, particularly atopic asthma, and to identify markers that aid in discriminating between atopic subjects at high versus low risk of asthma. METHODS We studied 1380 unselected 14-year-olds and collected data on clinical history, allergic sensitization, and respiratory and immunoinflammatory function. The latter comprised measurements of circulating inflammatory markers and in vitro innate and adaptive immune functions, including house dust mite T-cell responses. We integrated the data into regression models to identify variables most strongly associated with asthma risk and severity among atopic subjects. RESULTS Eight hundred twenty-seven subjects were atopic, 140 subjects were asthmatic, and 81% of asthmatic subjects were also atopic. We identified asthma risk variables related to atopy intensity, including specific IgE and eosinophil levels, plus an additional series external to the T(H)2 cascade but that modified risk only in atopic subjects, including IFN-gamma, IL-10, and IL-12 responses and neutrophil numbers in blood. Moreover, bronchial hyperresponsiveness was associated strongly with atopic but not nonatopic asthma, and the bronchial hyperresponsiveness risk profile was itself dominated by atopy-associated variables. CONCLUSIONS Asthma in teenagers is predominantly driven by atopy acting in concert with a second tier of T(H)2-independent immunoinflammatory mechanisms, which contribute to pathogenesis only against the background of pre-existing inhalant allergy.

Persistent Effects of Maternal Smoking during Pregnancy on Lung Function and Asthma in Adolescents

RATIONALE The extent to which maternal smoking in pregnancy (MSP) has persisting effects on respiratory health remains uncertain and the mechanisms involved are not fully understood. Alterations in immune function have been proposed as a mechanism contributing to respiratory disease. OBJECTIVES To determine whether MSP increases risk of respiratory disorders in adolescence and, if so, whether this occurs by decreased lung function, altered immune function, and/or enhanced atopy. METHODS Data on spirometry, bronchial responsiveness, respiratory symptoms, total and allergen-specific IgE and IgG4, immune function, and inflammatory markers were obtained from 1,129 participants in the 14-year follow-up of the Western Australian Pregnancy (Raine) Cohort and related to MSP using regression analyses. MEASUREMENTS AND MAIN RESULTS MSP was reported for 21.0% (237 of 1,129) of participants, with 92 (8.1%) reporting current smoking. MSP was associated with some altered immune measures at age 14. MSP was strongly related to reduced lung function in current nonsmokers (forced expiratory flow midexpiratory phase [FEF25-75%], P = 0.016; FEV1/FVC, P = 0.009) and increased risk for current asthma (odds ratio [OR], 1.84; 95% confidence interval [CI], 1.16-2.92; P = 0.01), current wheeze (OR, 1.77; 95% CI, 1.14-2.75; P = 0.011), and exercise-induced wheeze (OR, 2.29; 95% CI, 1.37-3.85; P = 0.002), but not for bronchial hyperresponsiveness or atopy. Adjustment for immune measures and/or lung function in multivariate models did not greatly alter these associations and the increased risks for asthma and wheeze were not modified by sex, atopy, or maternal history of asthma or atopy. CONCLUSIONS MSP increases risk of asthma and wheezing in adolescence; mechanisms go beyond reducing lung function and exclude altering immune function or enhancing atopy.