Tillie L. Hackett

Lung eQTLs to Help Reveal the Molecular Underpinnings of Asthma

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55×10−151). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.

Airway epithelial regulation of pulmonary immune homeostasis and inflammation.

Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. Further, there is now strong evidence that epithelium dysfunction is involved in the development of inflammatory disorders of the lung. Here we review the characteristic immune responses that are orchestrated by the epithelium in response to diverse triggers such as pollutants, cigarette smoke, bacterial peptides, and viruses. We focus in part on the role of epithelium-derived interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), as well as CC family chemokines as critical regulators of the immune response. We cite examples of the function of the epithelium in host defense and the role of epithelium dysfunction in the development of inflammatory diseases.

E-cadherin: gatekeeper of airway mucosa and allergic sensitization

The airway epithelium plays a role in immune regulation during environmental challenge, which is intertwined with its barrier function and capacity to limit submucosal access of environmental factors. In asthma, mucosal barrier function is often compromised, with disrupted expression of the adhesion molecule E-cadherin. Recent progress suggests that E-cadherin contributes to the structural and immunological function of airway epithelium, through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators that can modulate the immune response. Here, we discuss this novel role for E-cadherin in mediating the crucial immunological decision between maintenance of tolerance versus induction of innate and adaptive immunity.

The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation

Background House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation. Objectives The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo. Methods Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model. Results The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity. Conclusions This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

A gene expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK

BackgroundChronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time.MethodsIn order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans.ResultsWe identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFβ) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFβ pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFβ-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin β1. Furthermore, addition of GHK or TGFβ restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.ConclusionsThese results demonstrate that gene-expression changes associated with regional emphysema severity within an individuals lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFβ and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression.

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

ABSTRACT Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.

TGFβ1 induces αvβ3 integrin expression in human lung fibroblasts via a β3 integrin, c-Src and p38MAPK dependent pathway

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

Mechanical properties of asthmatic airway smooth muscle

Airway smooth muscle (ASM) is the major effector of excessive airway narrowing in asthma. Changes in some of the mechanical properties of ASM could contribute to excessive narrowing and have not been systematically studied in human ASM from nonasthmatic and asthmatic subjects. Human ASM strips (eight asthmatic and six nonasthmatic) were studied at in situ length and force was normalised to maximal force induced by electric field stimulation (EFS). Measurements included: passive and active force versus length before and after length adaptation, the force–velocity relationship, maximal shortening and force recovery after length oscillation. Force was converted to stress by dividing by cross-sectional area of muscle. The only functional differences were that the asthmatic tissue was stiffer at longer lengths (p<0.05) and oscillatory strain reduced isometric force in response to EFS by 19% as opposed to 36% in nonasthmatics (p<0.01). The mechanical properties of human ASM from asthmatic and nonasthmatic subjects are comparable except for increased passive stiffness and attenuated decline in force generation after an oscillatory perturbation. These data may relate to reduced bronchodilation induced by a deep inspiration in asthmatic subjects.

Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-β1.

Objective Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-β1 into the extracellular milieu. Methods/Results Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-β1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-β1 release. Our data confirmed that GrB liberated TGF-β1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-β1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. Conclusion In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.