Project description: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.

Project description:Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling refractory to corticosteroid treatment. CD4+ T cells and their canonical effector molecules, like type 2 cytokines, play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting these cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4+ T cell subsets and their effector molecules may drive airway inflammation and remodeling. To address this issue in a hypothesis-free manner, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics' airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma. This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, IL-17A, CSF2, LIGHT) that could fuel persistent airway inflammation and remodeling. Furthermore, we found that CD4+ T cells isolated ex vivo from airways of severe asthmatics displayed signatures linked to corticosteroid resistance (FKBP5, DDIT4), as well as reduced expression of transcripts encoding for molecules that dampen TCR signaling and T cell effector functions (CREM, DUSP1, TNFAIP3). Overall, our single-cell transcriptomic analysis highlights the pathogenic properties and clinical associations of airway CD4+ T cell subsets in severe asthmatics, features that can potentially trigger their unrestrained activation, and that these findings were more pronounced in males. These pathways may therefore be crucial for driving disease severity in adult male severe asthmatics.

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Systemic inflammation is associated with differential gene expression and airway neutrophilia in asthma

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