Project 1: Novel mechanisms driving mucin secretion and mucus plugging in type 2-high asthma

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PROJECT 1 SUMMARY Mucus plugging is a major cause of cause of airway obstruction, morbidity, and mortality in severe asthma. Work performed in our Center has helped to establish a strong association between type 2 inflammation and mucus plugging and has revealed that direct effects of the type 2 cytokine IL-13 on airway epithelium induce changes in the composition and organization of mucus that result in plugging. The overall objective of this project is to understand novel molecular mechanisms that alter secretory cell and mucus function in asthma. In Aim 1, we will build on new preliminary data showing that the IL-13-inducible goblet cell protein, tetraspanin 8 (TSPAN8) is required for stimulated mucin release. Based upon our results and existing information about other tetraspanins, we hypothesize that TSPAN8-enriched microdomains in goblet cells play critical roles in mediating intracellular signals required for mucin secretion and in the organization and function of mucin-containing secretory granules. We will test this hypothesis using primary human bronchial epithelial cells and in a mouse model, which will allow us to determine the importance of this molecule in vivo. In Aim 2, we will investigate the impact of cathepsin cysteine proteases and their inhibitors on mucus. Cystatin SN and cystatin S are human cysteine protease inhibitors (absent in mice). We found that these cystatins are strongly induced by IL-13 and in type 2-high asthma and that CRISPR targeting either cystatin or addition of excess cathepsin to mucus prevents IL-13-induced impairments in mucociliary clearance. We hypothesize that these cystatin SN and cystatin S impair mucociliary clearance by inhibiting cathepsin-mediated degradation of mucins and activation of ENaC, a channel that is important for mucus hydration and mucociliary transport. We will test this hypothesis by using in vitro assays to study the biochemical functions of relevant cathepsins and cystatins and by assessing the physiologic role of these molecules in the primary HBEC culture model. In Aim 3, we will test whether mucus plugging in lung specimens from individuals with asthma is associated with strong local type 2 immune responses and prominent IL-13-related epithelial changes. Preliminary data from our Center and others is consistent with the hypothesis that mucus plugging occurs in the setting of a strong local type 2 immune response and strong epithelial cell induction of the mucin MUC5AC after exposure to IL-13. We will test this hypothesis by comparing IL-13 responses in epithelial cells from individuals with asthma and controls and by collaborating closely with the Clinical Subject and Biospecimen Processing and Analysis Core to obtain detailed spatial cellular and transcriptional data about immune and epithelial states associated with mucus plugs. The impact of this work will be to substantially advance our understanding of known and novel pathways responsible for formation and persistence of mucus plugs, and by doing so provide new clues to therapeutic approaches likely to benefit those with the most severe forms of asthma.