Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection.

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B.

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance.

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

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-1ß 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.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration.

QUESTION: Cystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (i.e. acidic airway surface liquid (ASL) pH, low bicarbonate (HCO3 -) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown. MATERIALS/METHODS: We characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties. RESULTS: Loss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO3 -, normalised mucus recovery to modulator-treated cell levels. CONCLUSION: These results indicate that airway dehydration, not acidic pH and/or low [HCO3 -], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Mucins and CFTR: Their Close Relationship.

Mucociliary clearance is a critical defense mechanism for the lungs governed by regionally coordinated epithelial cellular activities, including mucin secretion, cilia beating, and transepithelial ion transport. Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, is characterized by failed mucociliary clearance due to abnormal mucus biophysical properties. In recent years, with the development of highly effective modulator therapies, the quality of life of a significant number of people living with CF has greatly improved; however, further understanding the cellular biology relevant to CFTR and airway mucus biochemical interactions are necessary to develop novel therapies aimed at restoring CFTR gene expression in the lungs. In this article, we discuss recent advances of transcriptome analysis at single-cell levels that revealed a heretofore unanticipated close relationship between secretory MUC5AC and MUC5B mucins and CFTR in the lungs. In addition, we review recent findings on airway mucus biochemical and biophysical properties, focusing on how mucin secretion and CFTR-mediated ion transport are integrated to maintain airway mucus homeostasis in health and how CFTR dysfunction and restoration of function affect mucus properties.

Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus.

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

Differential responses to e-cig generated aerosols from humectants and different forms of nicotine in epithelial cells from nonsmokers and smokers.

In the United States, millions of adults use electronic cigarettes (e-cigs), and a majority of these users are former or current cigarette smokers. It is unclear, whether prior smoking status affects biological responses induced by e-cigs. In this study, differentiated human nasal epithelial cells (hNECs) from nonsmokers and smokers at air-liquid interface were acutely exposed to the e-cig generated aerosols of humectants, propylene glycol (PG), and glycerol (GLY). Mucin levels were examined in the apical washes, and cytokine levels were assessed in the basolateral supernatants 24 h postexposure. The aerosol from the GLY exposure increased mucin 5, subtype AC (MUC5AC) levels in the apical wash of hNECs from nonsmokers, but not smokers. However, the aerosol from GLY induced pro-inflammatory responses in hNECs from smokers. We also exposed hNECs from nonsmokers and smokers to e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt. The PG:GLY with freebase nicotine exposure increased MUC5AC and mucin 5, subtype B (MUC5B) levels in hNECs from nonsmokers, but the nicotine salt exposure did not. The PG:GLY with nicotine salt exposure increased pro-inflammatory cytokines in hNECs from smokers, which was not seen with the freebase nicotine exposure. Taken together, these data indicate that the e-cig generated aerosols from the humectants, mostly GLY, and the type of nicotine used cause differential effects in airway epithelial cells from nonsmokers and smokers. As e-cig use is increasing, it is important to understand that the biological effects of e-cig use are likely dependent on prior cigarette smoke exposure.

Airway Mucus Hyperconcentration in Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

Localization of Secretory Mucins MUC5AC and MUC5B in Normal/Healthy Human Airways.

RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 nonsmoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/colocalization were assessed by RNA in situ hybridization and immunohistochemistry in five lungs with histologically healthy airways. Droplet digital PCR and cell cultures were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. MEASUREMENTS AND MAIN RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA in situ hybridization revealed MUC5AC and MUC5B were colocalized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-copositive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium and glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP-positive secretory cells in superficial airway epithelia.

An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases.

RATIONALE: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. OBJECTIVES: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. METHODS: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. MEASUREMENTS AND MAIN RESULTS: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in ßENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. CONCLUSIONS: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.

Endotracheal tube mucus as a source of airway mucus for rheological study.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs.

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ~20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an "expanded" rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Mucus aberrant properties in CF: Insights from cells and animal models.

Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, is characterized by mucus accumulation in the lungs, the intestinal tract, and the pancreatic ducts. Mucins are high-molecular-weight glycoproteins that govern the biochemical and biophysical properties of mucus. In the CF lung, increased mucus viscoelasticity is associated with decreased mucociliary clearance and defects in host defense mechanisms. The link between defective ion channel and abnormal mucus properties has been investigated in studies involving cell and animal models. In this review article, we discuss recent progress toward understanding the different regions and cells that express CFTR in the airways and how mucus is produced and cleared from the lungs. In addition, we reflect on animal models that provided insights into the organization and the role of the mucin network and how mucus and antimicrobial activities act in concert to protect the lungs from invading pathogens.