D21-11 The Role of Mast Cells in Tslp Mediated Airway Hyperresponsiveness Revealed by Single Cell RNA Sequencing

Abstract Rationale Asthma is a chronic inflammatory disease of the airways, and airway hyperresponsiveness (AHR) is one of its principal pathophysiologic features. However, the detailed mechanisms underlying AHR are not yet fully understood.Thymic stromal lymphopoietin (TSLP) has recently been recognized as a crucial molecule, particularly in relation to AHR. Mast cells are also conventionally recognized as important effector cells in asthma. In our previous single-cell RNA sequencing study, mast-cell derived genes were markedly decreased in TSLP receptor deficient mice compared with control mice. The objective of this study is to elucidate the interaction between TSLP and mast cells in a mouse model of asthma. Methods Female BALB/c mice were exposed to phosphate-buffered saline, house dust mite (HDM) or HDM + TSLP for 3 weeks. AHR, bronchoalveolar lavage fluid (BALF) cell counts, and cytokine and chemokine levels in lung homogenates were measured by multiplex assays or ELISA. Historical analysis by H.E. staining and PAS staining were performed to quantify inflammation and goblet cell metaplasia. For mast-cell analyses, mast cell protease (Mcpt) transcripts were quantified by real-time PCR, mast cell counts were evaluated by flow cytometry, and histopathological changes were assessed by immunohistochemistry of KIT or Tryptase staining in control, HDM, and HDM+TSLP groups. To explore the mechanisms, single-cell RNA sequencing of lung-immune cells in control, HDM, or HDM+TSLP was performed to focus on mast cell-derived genes. Finally, Cpa3-Cre mice, known as mast cells depletion mice, were used to determine whether these responses were mast cell-dependent and attenuated. Results Compared with the control, HDM groups showed increased AHR accompanied by eosinophilic airway inflammation, and cytokines (IL-4, IL-5, IL-13) in lung were significantly elevated and those were further increased in HDM+TSLP groups. Mcpt2 and Mcpt5 gene expressions were also increased in parallel with higher mast-cell numbers in lung tissue. Histological findings showed accumulation of inflammatory cells and goblet-cell metaplasia with accumulation of mast cells in the HDM+TSLP group. In single cell RNA sequencing, mast cell derived gene were significantly altered by TSLP stimulation. In Cpa3-Cre mice, AHR induced by HDM+TSLP was attenuated, while eosinophilic airway inflammation persisted, compared with the HDM group. Conclusions TSLP exacerbates AHR in association with increased mast-cell abundance and function. This abstract is funded by: None