Roberto Adachi

Rationale: Finely tuned production and secretion of the polymeric mucins MUC5AC and MUCB are required for lung health, but knowledge of many details between their translation and their packaging into secretory granules is lacking. Objective: To analyze the structure and function of the Golgi apparatus, a key site of mucin glycosylation, folding, polymerization and packaging, in airway epithelial secretory cells. Methods: Lung tissue was obtained from mice stimulated or not with IL-13 to upregulate mucin production, and from normal human lungs. Golgi elements in mouse and human tissue were imaged by high-resolution immunofluorescence microscopy and electron microscopy. Tissue from mice with deletion of both polymeric mucins was also examined. Measurements and Main Results: By immunofluorescence microscopy, both mouse and human airway secretory cells contained ∼100 dispersed puncta labeled by markers of medial and trans Golgi cisternae and the trans-Golgi network (TGN), but only a few perinuclear puncta were labeled by markers of cis-Golgi cisternae. By electron microscopy, secretory cells contained both a perinuclear Golgi ribbon and numerous dispersed Golgi stacks, termed satellites. In mucous metaplastic cells, satellites were concentrated among immature mucin granules. Increasing mucin production by cytokine stimulation did not increase the number of TGN puncta, nor did preventing polymeric mucin production by gene deletion reduce TGN puncta. Conclusions: Mucin-producing airway secretory cells express an unusual Golgi structure consisting of a conventional perinuclear ribbon as well as dispersed satellites. While the Golgi satellites are likely an adaptation for mucin production and packaging, their presence is specified developmentally, independent of mucin production.

Rationale: Finely tuned production and secretion of the polymeric mucins MUC5AC and MUCB are required for lung health, but knowledge of many details between their translation and their packaging into secretory granules is lacking. Objectives: To analyze the structure and function of the Golgi apparatus, a key site of mucin glycosylation, folding, polymerization and packaging, in airway epithelial secretory cells. Methods: Lung tissue was obtained from mice stimulated or not with IL-13 to upregulate mucin production, and from normal human lungs. Golgi elements in mouse and human tissue were imaged by high-resolution immunofluorescence microscopy and electron microscopy. Tissue from mice with deletion of both polymeric mucins was also examined. Measurements and Main Results: By immunofluorescence microscopy, both mouse and human airway secretory cells contained ∼100 dispersed puncta labeled by markers of medial and trans Golgi cisternae and the trans-Golgi network (TGN), but only a few perinuclear puncta were labeled by markers of cis-Golgi cisternae. By electron microscopy, secretory cells contained both a perinuclear Golgi ribbon and numerous dispersed Golgi stacks, termed satellites. In mucous metaplastic cells, satellites were concentrated among immature mucin granules. Increasing mucin production by cytokine stimulation did not increase the number of TGN puncta, nor did preventing polymeric mucin production by gene deletion reduce TGN puncta. Conclusions: Mucin-producing airway secretory cells express an unusual Golgi structure consisting of a conventional perinuclear Golgi ribbon as well as dispersed satellites. While the Golgi satellites are likely an adaptation for mucin production and packaging, their presence is specified developmentally, independent of mucin production.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal–distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1β and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell–deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

Background: Granulation tissue is a common complication of airway stenting, but no published methods can quantify the volume and type of tissue that develops. Objective: To use design-based stereology to quantify changes in tissue volume and type associated with airway stenting. Methods: We compared drug-eluting stents (DES) filled with gendine to standard silicone stents in pigs in an assessor-blinded randomized trial. Tracheal stents were placed via rigid bronchoscopy. After 1 month, animals were euthanized and necropsies were performed. Antimicrobial effects of the DES were assessed in trachea tissue samples, on the DES surface, and with residual gel from the DES reservoir. Tracheal thickness was measured using orthogonal intercepts. Design-based stereology was used to quantify the volume density of tissues using a point-counting method. The volume of each tissue was normalized to cartilage volume, which is unaffected by stenting. Results: Pigs were randomized to DES (n = 36) or control stents (n = 9). The drug was successfully eluted from the DES, and the stent surface showed antibacterial activity. DES and controls did not differ in tissue microbiology, tracheal thickness, or granulation tissue volume. Compared to nonstented controls, stented airways demonstrated a 110% increase in soft-tissue volume (p = 0.005). Submucosal connective tissue (118%; p < 0.0001), epithelium (70%; p < 0.0001), submucosal glands (47%; p = 0.001), and smooth muscle (41%; p < 0.0001) increased in volume. Conclusion: Stenting doubles the volume of soft tissue in the trachea. Design-based stereology can quantify the tissue changes associated with airway stenting.