Fig. 6: Upregulation of CD86 on dendritic cells (DCs) is dependent on ATP released from tuft cells.

A–F FACS analysis of CD86 expression on sorted pulmonary DCs (CD45⁺Ly6G^-F4/80^-CD11c⁺) co-cultured with sorted Trpm5-DREADD tracheal epithelium (EpCAM⁺CD45^-) (n = 7 mice). Clozapine-n-oxide (CNO) (100 µM) was used to stimulate Trpm5-DREADD tracheal epithelium in presence or absence of ATP hydrolyzing enzyme apyrase (5 U/ml) or nicotinic receptor antagonist mecamylamine (10 µM) and muscarinic receptor antagonist atropine (1 µM) in the co-culture system. The percentage of CD86-expressing DCs was significantly higher when DCs were co-cultured with CNO-stimulated Trpm5-DREADD tracheal epithelium compared to CNO treated DCs (n = 10 mice) or CNO treated Trpm5^+/+ tracheal epithelium. Treatment of CNO-stimulated Trpm5-DREADD tracheal epithelium with apyrase but not mecamylamine/atropine reduced the percentages of CD86⁺ DCs (n = 4 mice). One-way ANOVA. G Co-culture of pulmonary DCs from Panx1^−/− mice with CNO-stimulated Trpm5-DREADD tracheal tuft cells significantly increased the percentages of CD86 expressing DCs compared to CNO treated Panx1^−/− DCs (n = 4 mice). Two-tailed Mann-Whitney test. H The median fluorescence intensity (MFI) of MHCII in pulmonary DCs was significantly increased after being co-cultured with CNO-stimulated Trpm5-DREADD tracheal epithelium (DC: n = 10 mice, DC + TE: n = 8 mice). This was reduced when CNO-stimulated Trpm5-DREADD tracheal epithelium was treated with apyrase (n = 3 mice), or mecamylamine and atropine (n = 4 mice). One-way ANOVA. I Blood agar plate of phagocytosed P. aeruginosa NH57388A CFUs after treatment of DCs with supernatant (SN) of Trpm5^+/+ tracheae stimulated with 1 mM denatonium, with supernatant of Trpm5^+/+ tracheae stimulated with denatonium in the presence 5 U/ml apyrase and with supernatants of denatonium-treated Trpm5^−/− tracheae and untreated control conditions. J Quantification of (A) (n = 5 mice). Data are shown as a box-and-whisker plot displaying the minimum and maximum values, with the median represented by a horizontal line. One-way ANOVA. K Schematic drawing. Activation of tuft cells with denatonium or CNO leads to an ATP-dependent increased in the number and activation of DCs. (* p < 0.05, ** p < 0.01). Source data are provided in the Source Data file. Data in panels (F, G and H) are presented as mean ± SEM.