Phenotypic and genetic aspects of epithelial barrier function in asthmatic patients

The bronchial epithelium is continuously exposed to a multitude of noxious challenges in inhaled air. Cellular contact with most damaging agents is reduced by the action of the mucociliary apparatus and by formation of a physical barrier that controls passage of ions and macromolecules. In conjunction with these defensive barrier functions, immunomodulatory cross-talk between the bronchial epithelium and tissue-resident immune cells controls the tissue microenvironment and barrier homeostasis. This is achieved by expression of an array of sensors that detect a wide variety of viral, bacterial, and nonmicrobial (toxins and irritants) agents, resulting in production of many different soluble and cell-surface molecules that signal to cells of the immune system. The ability of the bronchial epithelium to control the balance of inhibitory and activating signals is essential for orchestrating appropriate inflammatory and immune responses and for temporally modulating these responses to limit tissue injury and control the resolution of inflammation during tissue repair. In asthmatic patients abnormalities in many aspects of epithelial barrier function have been identified. We postulate that such abnormalities play a causal role in immune dysregulation in the airways by translating gene-environment interactions that underpin disease pathogenesis and exacerbation.

many different soluble and cell-surface molecules, collectively termed the "epimmunome" 51 histocompatibility (MHC) gene products) that recruit and activate cells such as macrophages 159 and neutrophils involved in inflammation and the induction of adaptive immunity. Together 160 these responses enable many infections to be controlled by the immune system with limited 161 damage to host tissues, however it is important to note that both innate and adaptive immune 162 signaling events are involved in mediating tissue damage 52 . For example, macrophages, 163 neutrophils and eosinophils release a range of molecules, including cytotoxic cytokines, 164 cationic proteins, lipid mediators, metalloproteinases and reactive oxygen species that induce 165 tissue damage or malfunction. Therefore, the ability of the epithelium to control the balance 166 of inhibitory and activating signals is essential not only for initiating an appropriate immune 167 response to environmental challenges, if required ( Figure 2), but also for temporally 168 orchestrating these responses to limit tissue injury and control the resolution of inflammatory  Epithelial cells express CD200 which binds to the inhibitory immune receptor, CD200R, 182 expressed at high levels on lung macrophages. This not only maintains a strong threshold for 9 response in the context of inhaled, but non-pathogenic antigens 56

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Targeted studies of the bronchial epithelium have demonstrated a range of abnormalities at 218 many levels of barrier function and innate immunity ( Figure 3). However, unbiased 219 transcriptomic approaches are now enabling in-depth analysis of epithelial gene expression 220 profiles 8;9 to provide evidence of molecular mechanisms that may eventually define specific 221 epithelial endotypes of asthma. We will first summarise key abnormalities identified in the 222 epithelial barrier in asthma and then put these into the context of the newer clusters that have 223 been identified and how these relate to genetic susceptibilities.

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The mucociliary apparatus is modified in asthma as evidenced by an increase the number of 225 goblet cells with increased mucin gene expression, an increase in MUC5AC protein relative 226 to MUC5B and a reduction in ciliated cell number 71-73 . In addition, decreased ciliary beat 227 frequency, dyskinesia, and ciliary disorientation have been reported in severe asthma 74 .

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Together, mucous hypersecretion and ciliary dysfunction in asthma may result in stimulation 229 of neural receptors that result in cough 75 and mucous plugging which, over time, can lead to 230 severe airflow obstruction.

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The increase in MUC5AC relative to MUC5B seen in asthma has been postulated to affect 232 mucous clearance, reduce eosinophil apoptosis 76 and/or contribute to abnormal innate 233 immune responses 57 . Reprogramming of epithelial differentiation towards a hypersecretory 234 phenotype has been linked to increased expression of the epidermal growth factor receptor 72 , 235 and to the activity of Th2 cytokines including IL-13 and IL-9 77;78 . Consistent with this, the 236 'Th2 high' asthmatics have significantly increased airway mucin gene expression 79 . Th2   retaining signaling capacity. Analyses of epithelial brush RNA suggest that Δ exon 3,4 is 320 strongly associated with airway Type 2 inflammation, whereas full-length IL33 is not 124 .

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These results suggest that therapeutic IL-33 inhibitors will need to block all biologically 322 active isoforms.

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TSLP is an interleukin 7-like cytokine that can trigger dendritic cell-mediated Th2  Assuming that these phenotypes are stable, rather than fluctuations due to disease activity,

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The focus on epithelial repair genes in asthma has been limited to date, but promoter variants 504 in TGFB1 and TGFB2 that increase TGFβ expression are associated with asthma 195;196 and 505 airflow obstruction 197 . It is also interesting to note that genes like HHIP (hedgehog 506 interacting protein) and PTCH1 (patched homolog 1), that may play a role in epithelial repair 507 have been identified through genetic association with reduced lung function 198 , suggesting 508 that impaired repair may drive ECM deposition and tissue remodelling.

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Most of the asthma-associated SNPs identified by GWAS are not coding-change variants.