Plethysmography Phenotype QTL in Mice Before and After Allergen Sensitization and Challenge.
ABSTRACT: Allergic asthma is common airway disease that is characterized in part by enhanced airway constriction in response to nonspecific stimuli. Genome-wide association studies have identified multiple loci associated with asthma risk in humans, but these studies have not accounted for gene-environment interactions, which are thought to be important factors in asthma. To identify quantitative trait loci (QTL) that regulate responses to a common human allergen, we applied a house dust mite mouse (HDM) model of allergic airway disease (AAD) to 146 incipient lines of the Collaborative Cross (CC) and the CC founder strains. We employed a longitudinal study design in which mice were phenotyped for response to the bronchoconstrictor methacholine both before and after HDM sensitization and challenge using whole body plethysmography (WBP). There was significant variation in methacholine responsiveness due to both strain and HDM treatment, as reflected by changes in the WBP parameter enhanced pause. We also found that distinct QTL regulate baseline [chromosome (Chr) 18] and post-HDM (Chr 19) methacholine responsiveness and that post-HDM airway responsiveness was correlated with other features of AAD. Finally, using invasive measurements of airway mechanics, we tested whether the Chr 19 QTL affects lung resistance per se using C57BL/6J mice and a consomic strain but found that QTL haplotype did not affect lung resistance. We conclude that aspects of baseline and allergen-induced methacholine responsiveness are associated with genetic variation, and that robust detection of airway resistance QTL in genetically diverse mice will be facilitated by direct measurement of airway mechanics.
Project description:BACKGROUND: There is increasing evidence that the small airways contribute significantly to the pathophysiology of asthma. However, due to the difficulty in accessing distal lung regions in clinical settings, functional changes in the peripheral airways are often overlooked in studies of asthmatic patients. The aim of the current study was to characterize progressive changes in small airway function in sheep repeatedly challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: Four spatially separate lung segments were utilized for HDM challenges. The right apical, right medial, right caudal and left caudal lung segments received 0, 8, 16 and 24 weekly challenges with HDM respectively. A wedged-bronchoscope technique was used to assess changes in peripheral resistance (R(p)) at rest, and in response to specific and non-specific stimuli throughout the trial. Allergen induced inflammatory cell infiltration into bronchoalveolar lavage and increases in R(p) in response to HDM and methacholine were localized to treated lung segments, with no changes observed in adjacent lung segments. The acute response to HDM was variable between sheep, and was significantly correlated to airway responsiveness to methacholine (r(s) = 0.095, P<0.01). There was no correlation between resting R(p) and the number of weeks of HDM exposure. Nor was there a correlation between the magnitude of early-phase airway response and the number of HDM-challenges. CONCLUSIONS: Our findings indicate that airway responses to allergic and non-allergic stimuli are localized to specific treated areas of the lung. Furthermore, while there was a decline in peripheral airway function with HDM exposure, this decrease was not correlated with the length of allergen challenge.
Project description:The endoplasmic reticulum (ER) stress response participates in many chronic inflammatory and autoimmune diseases. In the current study, we sought to examine the contribution of ER stress transducers in the pathogenesis of three principal facets of allergic asthma: inflammation, airway fibrosis, and airways hyperresponsiveness.House Dust Mite (HDM) was used as an allergen for in vitro and in vivo challenge of primary human and murine airway epithelial cells. ER stress transducers were modulated using specific small interfering RNAs (siRNAs) in vivo. Inflammation, airway remodeling, and hyperresponsiveness were measured by total bronchoalveolar lavage (BAL) cell counts, determination of collagen, and methacholine responsiveness in mice, respectively.Challenge of human bronchiolar and nasal epithelial cells with HDM extract induced the ER stress transducer, activating transcription factor 6 ? (ATF6?) as well as protein disulfide isomerase, ERp57, in association with activation of caspase-3. SiRNA-mediated knockdown of ATF6? and ERp57 during HDM administration in mice resulted in a decrease in components of HDM-induced ER stress, disulfide mediated oligomerization of Bak, and activation of caspase-3. Furthermore, siRNA-mediated knockdown of ATF6? and ERp57 led to decreased inflammation, airway hyperresponsiveness and airway fibrosis.Collectively, our work indicates that HDM induces ER stress in airway epithelial cells and that ATF6? and ERp57 play a significant role in the development of cardinal features of allergic airways disease. Inhibition of ER stress responses may provide a potential therapeutic avenue in chronic asthma and sub-epithelial fibrosis associated with loss of lung function.
Project description:We previously identified dipeptidylpeptidase 10 (DPP10) on chromosome 2 as a human asthma susceptibility gene, through positional cloning. Initial association results were confirmed in many subsequent association studies but the functional role of DPP10 in asthma remains unclear. Using the MRC Harwell N-ethyl-N-nitrosourea (ENU) DNA archive, we identified a point mutation in Dpp10 that caused an amino acid change from valine to aspartic acid in the β-propeller region of the protein. Mice carrying this point mutation were recovered and a congenic line was established (Dpp10145D ). Macroscopic examination and lung histology revealed no significant differences between wild-type and Dpp10145D/145D mice. However, after house dust mite (HDM) treatment, Dpp10 mutant mice showed significantly increased airway resistance in response to 100 mg/ml methacholine. Total serum IgE levels and bronchoalveolar lavage (BAL) eosinophil counts were significantly higher in homozygotes than in control mice after HDM treatment. DPP10 protein is present in airway epithelial cells and altered expression is observed in both tissue from asthmatic patients and in mice following HDM challenge. Moreover, knockdown of DPP10 in human airway epithelial cells results in altered cytokine responses. These results show that a Dpp10 point mutation leads to increased airway responsiveness following allergen challenge and provide biological evidence to support previous findings from human genetic studies. This article has an associated First Person interview with the first author of the paper.
Project description:BACKGROUND:Current data concerning maternal paracetamol intake during pregnancy, or intake during infancy and risk of wheezing or asthma in childhood is inconclusive based on epidemiological studies. We have investigated whether there is a causal link between maternal paracetamol intake during pregnancy and lactation and the development of house dust mite (HDM) induced allergic airways disease (AAD) in offspring using a neonatal mouse model. METHODS:Pregnant mice were administered paracetamol or saline by oral gavage from the day of mating throughout pregnancy and/or lactation. Subsequently, their pups were exposed to intranasal HDM or saline from day 3 of life for up to 6 weeks. Assessments of airway hyper-responsiveness, inflammation and remodelling were made at weaning (3 weeks) and 6 weeks of age. RESULTS:Maternal paracetamol exposure either during pregnancy and/or lactation did not affect development of AAD in offspring at weaning or at 6 weeks. There were no effects of maternal paracetamol at any time point on airway remodelling or IgE levels. CONCLUSIONS:Maternal paracetamol did not enhance HDM induced AAD in offspring. Our mechanistic data do not support the hypothesis that prenatal paracetamol exposure increases the risk of childhood asthma.
Project description:<label>BACKGROUND</label>Asthma is a chronic inflammatory disease characterized by airways hyper-responsiveness (AHR), reversible airway obstruction, and airway inflammation and remodeling. We previously showed that Syk modulates methacholine-induced airways contractility in naïve mice and in mice with allergic airways inflammation. We hypothesize that Syk plays a role in the pathogenesis of AHR; this was evaluated in a chronic 8-week mouse model of house dust mite (HDM)-induced allergic airways inflammation.<label>METHODS</label>We used the Sykflox/flox//rosa26CreERT2 conditional Syk knock-out mice to assess the role of Syk prior to HDM exposure, and treated HDM-sensitized mice with the Syk inhibitor, GSK143, to evaluate its role in established allergic airways inflammation. Respiratory mechanics and methacholine (MCh)-responsiveness were assessed using the flexiVent® system. Lungs underwent bronchoalveolar lavage to isolate inflammatory cells or were frozen for determination of gene expression in tissues.<label>RESULTS</label>MCh-induced AHR was observed following HDM sensitization in the Syk-intact (Sykflox/flox) and vehicle-treated BALB/c mice. MCh responsiveness was reduced to control levels in HDM-sensitized Sykdel/del mice and in BALB/c and Sykflox/flox mice treated with GSK143. Both Sykdel/del and GSK143-treated mice mounted appropriate immune responses to HDM, with HDM-specific IgE levels that were comparable to Sykflox/flox and vehicle-treated BALB/c mice. HDM-induced increases in bronchoalveolar lavage cell counts were attenuated in both Sykdel/del and GSK143-treated mice, due primarily to decreased neutrophil recruitment. Gene expression analysis of lung tissues revealed that HDM-induced expression of IL-17 and CXCL-1 was significantly attenuated in both Sykdel/del and GSK143-treated mice.<label>CONCLUSION</label>Syk inhibitors may play a role in the management of neutrophilic asthma.
Project description:Associations between vitamin D status and childhood asthma are increasingly reported, but direct causation and mechanisms underlying an effect remain unknown. We investigated the effect of early-life vitamin D deficiency on the development of murine neonatal allergic airways disease (AAD).In utero and early-life vitamin D deficiency was achieved using a vitamin D-deficient diet for female mice during the third trimester of pregnancy and lactation. Offspring were weaned onto a vitamin D-deficient or vitamin D-replete diet, and exposure to intranasal house dust mite (HDM) or saline was commenced from day 3 of life for up to 6 weeks, when airway hyper-responsiveness (AHR), airway inflammation and remodelling were assessed.Neonatal mice that had in utero and early-life vitamin D deficiency had significantly increased pulmonary CD3(+) CD4(+) T1ST2(+) cells and reduced CD4(+) IL-10(+) cells. This effect was enhanced following HDM exposure. AHR in HDM-exposed mice was unaffected by vitamin D status. Introduction of vitamin D into the diet at weaning resulted in a significant reduction in serum IgE levels, reduced pulmonary eosinophilia and peri-bronchiolar collagen deposition.Peri-natal vitamin D deficiency alone has immunomodulatory effects including Th2 skewing and reduced IL-10-secreting T regulatory cells, exaggerated with additional allergen exposure. Vitamin D deficiency in early life does not affect AHR, but contributes to disease severity with worse eosinophilic inflammation and airway remodelling. Importantly, supplementation with vitamin D improves both of these pathological abnormalities.
Project description:Allergic asthma is a complex disease characterized in part by granulocytic inflammation of the airways. In addition to eosinophils, neutrophils (PMN) are also present, particularly in cases of severe asthma. We sought to identify the genetic determinants of neutrophilic inflammation in a mouse model of house dust mite (HDM)-induced asthma. We applied an HDM model of allergic asthma to the eight founder strains of the Collaborative Cross (CC) and 151 incipient lines of the CC (preCC). Lung lavage fluid was analyzed for PMN count and the concentration of CXCL1, a hallmark PMN chemokine. PMN and CXCL1 were strongly correlated in preCC mice. We used quantitative trait locus (QTL) mapping to identify three variants affecting PMN, one of which colocalized with a QTL for CXCL1 on chromosome (Chr) 7. We used lung eQTL data to implicate a variant in the gene Zfp30 in the CXCL1/PMN response. This genetic variant regulates both CXCL1 and PMN by altering Zfp30 expression, and we model the relationships between the QTL and these three endophenotypes. We show that Zfp30 is expressed in airway epithelia in the normal mouse lung and that altering Zfp30 expression in vitro affects CXCL1 responses to an immune stimulus. Our results provide strong evidence that Zfp30 is a novel regulator of neutrophilic airway inflammation.
Project description:Animal models of allergic airways inflammation are useful tools in studying the pathogenesis of asthma and potential therapeutic interventions. The different allergic airways inflammation models available to date employ varying doses, frequency, duration and types of allergen, which lead to the development of different features of asthma; showing varying degrees of airways inflammation and hyper-responsiveness (AHR) and airways remodeling. Models that also exhibit airway remodeling, a key feature of asthma, in addition to AHR and airway inflammation typically require 5-12 weeks to develop. In this report, we describe a 4-week mouse model of house dust mite (HDM)-induced allergic airways inflammation, and compare the phenotypic features of two different doses of HDM exposures (10?µg and 25?µg) for 5 days/week with a well-characterized 8-week chronic HDM model. We found that 4 weeks of intranasal HDM (25?µg in 35?µl saline; 5 days/week) resulted in AHR, airway inflammation and airway remodeling that were comparable to the 8-week model. We conclude that this new 4-week HDM model is another useful tool in studies of human asthma that offers advantages of shorter duration for development and decreased costs when compared to other models that require longer durations of exposure (5-12 weeks) to develop.
Project description:BACKGROUND:Allergic asthma is characterized by chronic inflammation and remodelling of the airways, associated with dysregulated type 2 immune responses and allergen-specific IgE. T follicular helper cells (TFH ) are crucial in T-dependent B-cell responses and have been implicated in allergic airway disease (AAD). TFH , unlike other CD4+ T cells, are uniquely reliant on continuous ICOS signalling to maintain their phenotype after T-cell priming; therefore, disrupting this signal can impair TFH responses. However, the contribution of TFH to disease during chronic aero-allergen exposure and the therapeutic potential of targeting these cells have not been evaluated. METHODS:To establish AAD, female BALB/c mice were repeatedly exposed to house dust mite or Alternaria alternata three times a week for up to 5 weeks. To examine the impact of TFH on AAD, mice were allergen exposed for 5 weeks and co-administered anti-ICOS Ligand-targeted antibodies, three times a week for the last 2 weeks. RESULTS:TFH were first observed in the lung-draining lymph nodes and with further exposure were also found locally within the lungs. TFH accumulated with sustained allergen exposure, alongside germinal centre (GC) B cells. Blockade of ICOS signalling after AAD establishment successfully depleted TFH but did not affect the differentiation of other CD4+ T-cell subsets. This reduced GC responses, allergen-specific IgE, inflammation, pulmonary IL-13 and airway hyper-responsiveness. CONCLUSIONS:TFH are crucial in the regulation of AAD and the ICOS/ICOS-L pathway could represent a novel therapeutic target in allergic asthma.
Project description:Elevated levels of combustion-derived particulate matter (CDPM) are a risk factor for the development of lung diseases such as asthma. Studies have shown that CDPM exacerbates asthma, inducing acute lung dysfunction and inflammation; however, the impact of CDPM exposure on early immunological responses to allergens remains unclear. To determine the effects of early-life CDPM exposure on allergic asthma development in infants, we exposed infant mice to CDPM and then induced a mouse model of asthma using house dust mite (HDM) allergen. Mice exposed to CDPM+HDM failed to develop a typical asthma phenotype including airway hyper-responsiveness, T-helper type 2 (Th2) inflammation, Muc5ac expression, eosinophilia, and HDM-specific immunoglobulin (Ig) compared with HDM-exposed mice. Although HDM-specific IgE was attenuated, total IgE was twofold higher in CDPM+HDM mice compared with HDM mice. We further demonstrate that CDPM exposure during early life induced an immunosuppressive environment in the lung, concurrent with increases in tolerogenic dendritic cells and regulatory T cells, resulting in the suppression of Th2 responses. Despite having early immunosuppression, these mice develop severe allergic inflammation when challenged with allergen as adults. These findings demonstrate a mechanism whereby CDPM exposure modulates adaptive immunity, inducing specific antigen tolerance while amplifying total IgE, and leading to a predisposition to develop asthma upon rechallenge later in life.