Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically promote lung type 2 inflammation

Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naïve murine nasal mucosa. How IL-25 and CysLTs might cooperatively promote type 2 inflammation in the airways has not been clarified. Here, we showed that inhalation of LTC4 in combination with a subthreshold dose of IL-25 led to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, inflammatory type 2 innate lymphoid cell (ILC2) proliferation and type 2 cytokine production, dendritic cell (DC) and CD4+ T cell recruitment and goblet cell hyperplasia. While lung eosinophilia was dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells required signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of Ltc4s, the terminal enzyme required for CysLT production, reduced lung inflammation and the systemic immune response after inhalation of the mold aeroallergen Alternaria; this effect was further enhanced by concomitant blockade of IL-25. Our findings identified a potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs. Overall design: For single-cell preparations of tuft cells from the nasal mucosa, mouse snouts were processed with dispase solution, followed by mechanical separation and a second digestion with a papain containing tyrode-based solution (15, 73). Tuft cells were defined as as EpCAMhighTomato+ in ChatCreTomato and ChatCreTomatoLtc4sfl/fl mice, EpCAMhighCD45neg/lowSSClow in Ltc4sfl/fl and ChatCreLtc4sfl/fl mice. To isolate macrophages and dendritic cells, the snouts were enzymatically dissociated with a PBS-based solution containing collagenase IV, dispase and DNAse. Macrophages were defined as CD11b+CD11c+SiglecF+ and dendritic cells as SiglecF-CD11b+CD11c+MHCII+ (Fig. S5C). For RNA sequencing, at least 500 but no more than 1300 tuft cells, macrophages or dendritic cells from 1 naïve digested mouse snout were sorted into TCL buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Gibco).