Gunnar C. Hansson

This study provides the first spatiotemporal map of Foxi1 ⁺ pulmonary ionocytes in mouse airways. These rare cells, which express the majority of CFTR mRNA, emerge postnatally near developing submucosal glands and concentrate in the laryngeal subglottis during adulthood. Their distribution remains unaltered in Cftr ^{ΔF508/ΔF508} mice, suggesting CFTR dysfunction does not disrupt ionocyte patterning.

Background: The gel-forming mucins MUC5B and MUC5AC constitute the main structural components of mucus in the respiratory system where MUC5AC is known to form nets. The MUC5B (rs35705950) Single Nucleotide Polymorphism (SNP) has been shown to increase the risk for Idiopathic Pulmonary Fibrosis (IPF). The MUC5AC SNP rs878913005, common in European populations, results in an Arg1201Trp substitution, resulting in more stabilized secreted mucin nets. Here we investigate the association of rs878913005 with the development of IPF and the presence of the two mucins in IPF diagnostic honeycomb cysts. Methods: We compared patient groups using whole genome sequences from the UK-Biobank for the SNPs in MUC5AC and MUC5B mucin genes. Lung tissue from IPF patients were immunostained for these mucins. Results: The MUC5AC SNP shows a significant 1.49x increased frequency in IPF patients relative to controls ( p < 0.0001) and an odds ratio of 2.09. The well-known IPF-associated SNP in the MUC5B promoter (rs35705950) has a stronger linkage to IPF when combined with the MUC5AC SNP. These two SNPs are located on the same chromosome and are in linkage disequilibrium (r ² = 0.10 in controls and up to r ² = 0.17 in IPF). Microscopic honeycomb cysts, typical for IPF, are filled with both the MUC5B and MUC5AC mucins with MUC5AC associated to the cysts’ epithelial surface. Conclusion: A genetic variant with more stable MUC5AC nets in peripheral thin airways may increase the risk of IPF.

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.

Enterotoxigenic Escherichia coli (ETEC) infections are a leading cause of diarrheal illness, responsible for an estimated 100,000 deaths annually. ETEC pathogenesis is driven by various virulence factors, including toxins, adhesins, and noncanonical factors such as the protease EatA. The first line of host defense against intestinal pathogenic bacterial infections is the protective intestinal mucus layer. Here, we demonstrate the mechanism by which EatA degrades the core mucus component MUC2, thereby facilitating access to the epithelial cell surface and promoting infection. We identify the specific cleavage site region localized at the C-terminal of MUC2. EatA’s protease activity depends on the interaction between two distinct, uniquely spaced domains in human MUC2, which defines species specificity. We confirm this using a novel transgenic mouse model exclusively expressing human MUC2, which allows us to study the role of the mucus layer in the infection by human intestinal pathogens. These findings highlight how ETEC is adapted to specifically degrade the mucus layer of its human host.

The mucosal surfaces of the body are the most vulnerable points for infection because they are lined by single or multiple layers of very active epithelial cells. The main protector of these cells is the mucus system generated by the specialized goblet cell secreting its main components, the gel-forming mucins. The organization of the mucus varies from an attached mucus that is impenetrable to bacteria in the large intestine to a nonattached, more penetrable mucus in the small intestine. The respiratory tract mucus system clears particles and microorganisms from healthy lungs but causes disease if reorganized to an attached mucus that cannot be efficiently transported. Similarly, transformation of large intestine mucus from impenetrable to penetrable causes chronic inflammation directed toward the intestinal microbiota. Mucus-producing goblet cells are regulated by and responsive to signals from immune cells, and at the same time signal back to the immune system. In this review we focus on the relationship of immune cells with intestinal goblet cells and mucus, making parallels to the respiratory tract.

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.

Enterotoxigenic Escherichia coli (ETEC) infections are a leading cause of diarrheal illness, responsible for an estimated 100,000 deaths annually. ETEC pathogenesis is driven by various virulence factors, including toxins, adhesins, and noncanonical factors such as the protease EatA. The first line of host defense against intestinal pathogenic bacterial infections is the protective intestinal mucus layer. Here, we demonstrate the mechanism by which EatA facilitates access to the epithelial cell surface by degrading the core mucus component MUC2, thereby aiding to the infection. We identify the specific cleavage site region localized at the C-terminal of MUC2. EatA’s protease activity depends on the interaction between two distinct domains, which are uniquely spaced in human MUC2, contributing to species specificity. This was confirmed using a novel chimeric mouse model solely expressing human MUC2, which allowed us to study the role of the mucus layer in the infection of human intestinal pathogens. These findings highlight how ETEC has adapted to specifically degrade the mucus layer of its human host.

Gel-forming mucins MUC5AC and MUC5B constitute the main structural component of the mucus in the respiratory system. Secreted mucins interact specifically with each other and other molecules giving mucus specific properties. We determined the cryoEM structures of the wild type D3 assembly of the human MUC5AC mucin and the structural single nucleotide polymorphisms (SNP) variants Arg996Gln and Arg1201Trp that affect intermolecular interactions. Our structures explain the MUC5AC N-terminal non-covalent oligomerization after secretion. The D3 assembly forms covalent dimers that can appear in two alternative conformations, open and closed, where the closed conformation dimers interact through an arginine-rich loop in the TIL3 domain to form tetramers. Our study provides a model to explain MUC5AC net-like structures and how the two SNPs will affect mucus organization, something that might affect lung and other diseases.

Urinary tract infections (UTIs) are highly recurrent and frequently caused by Uropathogenic Escherichia coli (UPEC) strains that can be found in patient intestines. Seeding of the urinary tract from this intestinal reservoir likely contributes to UTI recurrence (rUTI) rates. Thus, understanding the factors that promote UPEC intestinal colonization is of critical importance to designing therapeutics to reduce rUTI incidence. Although E. coli is found in high abundance in large intestine mucus, little is known about how it is able to maintain residence in this continuously secreted hydrogel. We discovered that the FimH adhesin of type 1 pili (T1P) bound throughout the secreted mucus layers of the colon and to epithelial cells in mouse and human samples. Disruption of T1P led to reduced association with colon mucus. Notably, this mutant up-regulated flagellar production and infiltrated the protective inner mucus layer of the colon. This could explain how UPEC resists being washed off by the continuously secreted mucus layers of the colon.

Mucus in the colon is crucial for intestinal homeostasis by forming a barrier that separates microbes from the epithelium. This is achieved by the structural arrangement of the major mucus proteins, such as MUC2 and FCGBP, both of which are comprised of several von Willebrand D domains (vWD) and assemblies. Numerous disulfide bonds stabilise these domains, and intermolecular bonds generate multimers of MUC2. The oligomeric nature of FCGBP is not known. Human hFCGBP contains 13 vWD domains whereas mouse mFCGBP consists of only 7. We found unpaired cysteines in the vWD1 (human and mouse) and vWD5 (mouse)/vWD11 (human) assemblies which were not involved in disulfide bonds. However, the most C‐terminal vWD domains, vWD7 (mouse)/vWD13 (human), formed disulfide‐linked dimers. The intermolecular bond between C₅₂₈₄ and C₅₄₀₃ of human hFCGBP was observed by using mass spectrometry to generate the dimer. Cryo‐EM structure analysis of recombinant mouse mFCGBP revealed a compact dimer with two symmetric intermolecular disulfide bonds between C₂₄₆₂ and C₂₅₈₁, corresponding to the dimerising cysteines in the human hFCGBP. This compact conformation involves interactions between the vWD assemblies, but although the domains involved at the interface are the same, the nature of the interactions differ. Mouse mFCGBP was also found to exist in a semi‐extended conformation. These different interactions offer insights into the dynamic nature of the FCGBP homodimer.