Fig 2: Extracellular succinate stimulates SUCNR1-dependent SMG contraction.A) Representative reflected light image of SMGs in newborn pig trachea after cartilage and smooth muscle layers were removed.B) Representative images of SMGs (EPCAM+, red) before and after succinate treatment. Closed lines outline the selected SMG regions for area measurement.C) Summary of SMG area treated with succinate or methyl succinate during live imaging of SMGs. 3 glands each group.D, E) Quantification of succinate-induced SMG contraction. Relative SMG area 3 minutes after a treatment. Panel D, SMGs were treated with 10 mM methyl succinate or succinate. Panel E, Airway surface epithelia were removed. SMGs were pre-incubated with or without SUCNR1 antagonist NF-56-EJ40 (5 µM) and induced with succinate. Data points are the mean of 3 SMGs from each pig; p values are shown in graph; p > 0.05 is considered as not significant (n.s), Student’s t test.F, G) Succinate prevents SMG swelling due to mucus secretion triggered by substance P. Panel F, representative images of SMGs that were pre-incubated with or without NF-56-EJ40 and treated with substance P and/or succinate. Panel G, quantification of SMG swelling response to substance P and/or succinate treatment. P values are shown in graph, n = 5 pigs, paired Student’s t test.H) Quantification of acinar cell shrinkage under succinate and/or substance P stimulation. P = 0.0006, n = 5 pigs, one way ANOVA. See also Figure S5.

Fig 3: PNECs are localized in porcine and human SMGs.A) PNEC marker gene expression in single-cell RNA sequencing (scRNA-seq) data from porcine SMGs (Yu et al., 2022). Ser, serous cell; Myoep, myoepithelial cell; Muc, mucous cell.B, C) Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) of PNEC markers (ASCL1, INSM1, KRT20) in SMGs (EPCAM+).D) Quantification of PNECs in individual SMGs. Upper panel, immunostaining of PNECs (KRT20+, green) in whole mount SMGs (EPCAM+, red). Lower panel, summary of total cells and PNECs in individual SMGs. Each data point represents number of cells or PNECs in a SMG. P value was calculated between WT and CF based on individual SMGs; p > 0.05 is considered as not significant (n.s.), unpaired Student’s t test; n = 3–4 pigs.E) SMG PNEC reconstruction. Enlarged images show apical and basolateral portions (arrows) of a PNEC connecting to the acinar lumen (dashed outline) and other SMG cells.F) Representative transmission electron microscopic images of rare cells (PNECs) with dense core vesicles (arrowheads) in SMGs. Sec, secretory cell of SMGs; BL, Basal lamina; Fib, fibroblast.G) Immunohistochemistry of gastrin-releasing peptide (GRP, human PNEC marker) in human airways (AW) and SMGs.See also Figure S1 and Figure S2.

Fig 4: PQQ treatment restores intercellular mitochondrial transport by MSC-Ob to airway epithelial cells and enhances their therapeutic potential.A Schema showing the development of HDM-induced airway allergic inflammation model. B Representative images show Mito-GFP (green) donation by MSCs to bronchial epithelial cells stained for CCSP (RED). Mito-GFP tagged MSCs were transplanted into the mice lungs via intra-tracheal infusion, and tissue sections were prepared after 24 h of infusion. C Images from B were subjected to image analysis to determine the signal of mito-GFP in CCSP positive epithelial cells and represented as integrated density. D Similarly, mitochondrial donation by MSCs was calculated using flow cytometry analysis. Single cells were prepared from lung tissue and stained for EpCAM to mark epithelial cells, which were gated to calculate the GFP signal. E, F Similarly, cells were stained with mitoSOX red and TMRE, respectively, to determine the mtROS and mitochondrial membrane potential in EpCAM stained epithelial cells. G Lung tissue was obtained from different groups of mice to prepare total lung protein (TLP). The ATP levels were measured in the TLP immediately after preparation. H Representative TUNEL images from lung tissue sections prepared from animals after 48 h of MSC transplantation. I No. of TUNEL positive cells were calculated by counting 100 bronchial epithelial cell nuclei per section, representing cell death. Mean ± SEM. ****P < 0.001; ***P < 0.005; **P < 0.01; *P < 0.05; ns (non-significant). Scale bars: 50 µm.

Fig 5: Expression levels of AQP5 in submandibular gland tissue of WT and Negr1 KO mice(A and B) Quantitative PCR analysis of AQP5 mRNA in submandibular gland cells from WT and Negr1 KO mice.(A) Real-time change in fluorescence with PCR cycle number in a representative quantitative PCR experiment. The result was determined in technical triplicates and data are plotted as lines.(B) Bar graph indicating the Aqp5 mRNA expression of WT (n = 4) and Negr1 KO mice (n = 4) was normalized to a reference gene (Gapdh).(C–E) AQP5 surface expression of submandibular gland cells in WT and Negr1 KO mice was measured by flow cytometry.(C) Representative density plots are shown: Left gating of epithelial cell adhesion molecule (EpCAM)(+) cells, right gating of AQP5(+) cells. Percentages reported in the flow plots indicate the percentage of EpCAM-labeled AQP5(+) cells in SMG isolated from WT or Negr1KO mice.(D) Representative histogram of EpCAM-labeled AQP5(+) cells in SMG isolated from WT or Negr1 KO mice. The black line from the black box of WT and red line from the red box of KO in panel C.(E) Quantification of AQP5(+) cells of EpCAM(+) cells in WT (n = 3) and Negr1 KO mice (n = 3) by flow cytometry. Data are represented as mean ± SD.