Airway epithelial barrier, mucins and inflammasome in distinct eosinophilic, neutrophilic and mixed inflammatory phenotypes of asthma

Asthma is a complex, chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Distinct inflammatory phenotypes of eosinophilic, mixed, neutrophilic and paucigranulocytic asthma are identified in patients, but most in vivo mouse models, studying asthma mechanisms, mimic only eosinophilic phenotype in humans. The detailed unbiased in vivo studies on molecular responses among different kinds of inflammation in asthma models are lacking. Therefore, we developed mouse models representing three different inflammatory phenotypes of airway inflammation, namely eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitisation. We used microarrays to determine the global gene expression in the lungs of mice with eosinophilic, mixed and neutrophilic inflammatory phenotypes to uncover underlying differences in clinical presentation and to find novel molecular targets and pathways, which might reflect different molecular mechanisms of the disease. By whole genome transcriptome profiling, we found that airway tight junction (TJ) molecules, mucins and inflammasome-related genes are differentially expressed in distinct phenotypes of allergic airway inflammation. Next, detailed analysis of several molecules from these families by quantitative RT-PCR, western blot and confocal microscopy revealed that (i) Zo-1 and Cldn18 were downregulated in all phenotypes, while Cldn4 upregulation was characteristic for neutrophilic airway inflammation; (ii) mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic asthma, and (iii) upregulation of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1 and IL-1b was characteristic for neutrophilic asthma. Finally, we showed that inflammasome/Th-17/neutrophilic axis cytokines, namely IL-1b and IL-17 impaired epithelial barrier function and increased mucins expressions in primary human bronchial epithelial cells from normal and asthmatic donors. Our findings suggest that differential expression of TJs, mucins and inflammasome-related molecules in distinct asthma phenotypes could be mechanistically linked and might further reflect the differences observed in the clinic. Different asthma phenotypes - eosinophilic, mixed, and neutrophilic, were induced in Balb/c mice via different ways of house dust mite (HDM) sensitisation and compared to the non-sensitized control group. For eosinophilic asthma mice were sensitized by three intraperitoneal (i.p.) injections of HDM extract containing 0.75 DU Der f and 0.75 DU Der p (ALK-Abelló A/S) adsorbed to 1.5 mg Al(OH)3 (Pierce, Rockford, IL, USA) in 200 µl PBS on days 0, 7 and 21. On days 26 to 28, mice were challenged intranasally (i.n.) with the similar dose of HDM extract dissolved in 50 µl PBS. For induction of the mixed or neutrophilic endotype mice were i.n. sensitized/challenged with the HDM extract (0.75 DU Der f /0.75 DU Der p or 7.5 DU Der f/7.5 DU Der p, respectively) in 50 µl PBS once a week a total of four times Intranasal application of only PBS served as a control. Whole lungs total RNA was used for transcriptomics from 3 mice in each phenotype group and control group. Lung samples were also snap frozen in liquid nitrogen for further analyses by immunofluorescence staining, Western blotting and mRNA expression. All animal procedures were performed with approval from the University of Marburg local authorities (Regierungspräsidium Gießen). >>>CEL files are not available for this series<<<