Journal: Respiratory Research

Article Title: Mucus threads from surface goblet cells clear particles from the airways

doi: 10.1186/s12931-021-01898-3

Figure Lengend Snippet: Mucus bundles made by submucosal glands were transported by cilia. A Schematic drawing of the tilted table with heating to 37 °C where pig distal trachea and primary bronchi were mounted to ensure air–liquid interface and transport against gravity. B Image sequence from two sequential low-resolution time-lapses lasting five minutes each. Alcian blue stained mucus bundles (arrows) on explanted trachea from a weaned WT piglet. Speed of corresponding movies (Additional files and ) increased 16×. C Bundle thickness measured on the airway surface, each data point is the mean of at least ten measurements per bundle, median with interquartile range. WT: 3 piglets, 3 time-lapses, 8 bundles. CF: 3 piglets, 4 time-lapses, 11 bundles. D Explanted WT piglet airway mucus bundles stained with Alcian blue (blue), LTL (green) and merged low-resolution image. E Confocal high-resolution image of formalin-fixed paraffin section from a newborn piglet trachea stained with antibodies against MUC5B and MUC5AC, illustrating the production of MUC5B (green) in submucosal mucous cells and mostly MUC5AC (red) in surface goblet cells. Some MUC5B was observed in surface cells. F Surface goblet cell mucus stains with the UEA1 lectin (red), whereas mucus in submucosal glands (MUC5B) stains with the lectin LTL (green). G Explanted WT piglet airway with LTL stained mucus bundles, low-resolution image. Arrowheads indicate gland openings. H Bundle thickness measured on the airway surface compared to at the gland openings. The mean of ten measurements per opening is presented as one data point. Data presented as median with interquartile range. WT: 3 piglets, 9 time-lapses, 36 bundles. CF: 2 piglets, 3 time-lapses, 8 bundles. WT surface vs. WT opening P = 0.0025 **, Kruskal–Wallis and Dunn´s multiple comparisons test

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, Paraffin Section

Journal: Respiratory Research

Article Title: Mucus threads from surface goblet cells clear particles from the airways

doi: 10.1186/s12931-021-01898-3

Figure Lengend Snippet: Mucus threads were secreted by surface goblet cells. A Image sequence from a low-resolution time-lapse where beads were collected by threads on an explanted WT piglet trachea. Arrow points to moving mucus assemblies and arrowhead to long mucus formation with increasing thickness. Speed of corresponding movie (Additional file ) increased 16×. B The lectin UEA1 (blue) stained bead-collecting assemblies (red), low-resolution image of explanted WT piglet trachea. C Low-resolution image of explanted live UEA-stained (red) WT piglet trachea. Yellow arrows indicate surface goblet cells and white arrows point to threads secreted from the goblet cells. D High-resolution Airyscan Z-stack of explanted live WT piglet trachea stained with Syto 59 (DNA, grey) and UEA1 (mucus, red). Yellow arrows indicate surface goblet cells and white arrows point to threads secreted from the goblet cells. E Scanning electron micrographs from three different WT piglet tracheas. Threads from secreting goblet cells (sGC) are indicated by green arrows. Some goblet cells (GC) were not secreting. F Scanning electron micrograph of mucus bundle originating in a submucosal gland. Note that the mucus bundle is thicker than the mucus threads in E . Sample from a CF piglet trachea. G Comparison of mucus bundle and mucus thread thickness in scanning electron micrographs. One data point represents the mean of 10 measurements per bundle or thread. Mucus threads were thinner than bundles in both WT and CF, Mann–Whitney test: WT bundles vs. WT threads P < 0.0001 ****, CF bundles vs. CF threads P = 0.0022 **. WT bundles: median 5.9 µm, 6 bundles (4 pigs), WT threads: median 0.47 µm, 13 threads (4 pigs), CF bundles: median 3.1 µm, 6 values (4 pigs), CF threads: median 0.46 µm, 6 values (4 pigs). Data presented as median with interquartile range. H The thread median thickness was 0.5 µm after fixation

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY

Journal: Respiratory Research

Article Title: Mucus threads from surface goblet cells clear particles from the airways

doi: 10.1186/s12931-021-01898-3

Figure Lengend Snippet: Mucus threads from surface goblet cells were thinner than mucus bundles. A Image sequence from a low-resolution time-lapse where threads collected beads on an explanted WT piglet trachea. Speed of corresponding movie (Additional file ) increased 16×. Mucus bundle coming out of a gland opening indicated by light blue arrowhead. Dark blue arrow: immobile bundle. Yellow, green and purple arrows indicate different moving mucus assemblies. B Low resolution image of bundles (LTL, blue arrow) and beads gathered into threads (yellow arrow) on WT piglet tracheas (arrowhead: gland opening). C Low-resolution image of explanted WT piglet trachea with bundle from a submucosal gland, LTL (green) and threads, UEA1 (red). D Airyscan high-resolution Z-stack of LTL-stained bundles (green) from submucosal glands and UEA1-stained threads (red) in a live explanted WT piglet trachea. E Thickness analysis of mucus bundles (LTL, green) and mucus threads (UEA1, red) in the image in D . Inset: zoom of the threads in the image center. F Mucus bundle thickness was calculated as a mean of 10 measurements close to the gland opening (1A, 2A) and further along the bundle (1B, 2B), as indicated in ( E ). Bundles were thicker close to the gland opening, Mann–Whitney test: 1A vs. 1B P < 0.0001 ****, 2A vs. 2B P = 0.0052 **. Thread thickness was calculated in the same way, as indicated in ( E ). Based on bundle thickness in live explants compared to fixed scanning electron micrographs, the maximum mucus thread thickness was determined to 2 µm (dashed line). Thread T3 was thicker than T4, Mann–Whitney test P < 0.0001 ****. Bundles were thicker than threads, Mann–Whitney test P = 0.0095 **. Data presented as median with interquartile range. G Scanning electron micrograph from a WT piglet trachea with mucus bundle from a submucosal gland (blue arrowhead). H Scanning electron micrograph of WT piglet trachea mucus thread (yellow arrows), GC: goblet cell, sGC: secreting goblet cell

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY

Journal: Respiratory Research

Article Title: Mucus threads from surface goblet cells clear particles from the airways

doi: 10.1186/s12931-021-01898-3

Figure Lengend Snippet: Mucus threads collected together and on mucus bundles. A Image sequence from a high-resolution video illustrating a UEA1-positive mucus assembly (green arrow) docking to an LTL-positive bundle (blue arrow) coated with UEA1 on a newborn WT piglet trachea (Additional file ). B Image sequence from a high-resolution video illustrating a UEA1-positive mucus assembly (green arrow) docking to another UEA1-positive mucus assembly (dark green arrow) on a newborn WT piglet trachea (Additional file ). C Bead-gathering mucus formations were divided into threads and mucus assemblies based on the maximum thread thickness of 2 µm determined by the difference in thickness between hydrated and fixed mucus. One thread was observed in WT and none in CF. WT: 15 values (9 pigs), CF: 6 values (3 pigs). D Thickness measurements of UEA1-stained mucus in high-resolution images from live explant tracheas from weaned pigs, newborn WT and CF piglets resulted in 3 threads and 48 mucus assemblies (6 weaned pigs), 3 threads and 54 mucus assemblies (10 newborn WT piglets), 2 threads and 37 mucus assemblies (12 newborn CF piglets), using the same criteria as in C. Mucus assemblies in weaned pigs were thicker than the corresponding assemblies in newborn CF piglets, P = 0.0418 * Kruskal–Wallis and Dunn´s multiple comparisons test. E Alcian blue stained mucus bundle velocity in weaned, newborn WT and CF tracheas measured in low-resolution time-lapses recorded on the tilted table. Each data point represents one pig. Weaned 19 pigs, newborn WT 14 pigs and newborn CF 12 pigs. Weaned bundles were faster than newborn WT bundles, P = 0.0111 *, Mann–Whitney test. F There was no difference in velocity between bead-collecting mucus assemblies in newborn WT and CF piglet trachea. WT: 15 values, 15 time-lapses, 8 pigs. CF: 12 values, 12 time-lapses, 6 pigs. Mann–Whitney test: P > 0.05. Data in ( C – F ) presented as median with interquartile range

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY