The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

The disulfide catalyst QSOX1 maintains the colon mucosal barrier by regulating Golgi glycosyltransferases.

Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.

Elucidation of the physical factors that control activated transport of penetrants in chemically complex glass-forming liquids.

Understanding the activated transport of penetrant or tracer atoms and molecules in condensed phases is a challenging problem in chemistry, materials science, physics, and biophysics. Many angstrom- and nanometer-scale features enter due to the highly variable shape, size, interaction, and conformational flexibility of the penetrant and matrix species, leading to a dramatic diversity of penetrant dynamics. Based on a minimalist model of a spherical penetrant in equilibrated dense matrices of hard spheres, a recent microscopic theory that relates hopping transport to local structure has predicted a novel correlation between penetrant diffusivity and the matrix thermodynamic dimensionless compressibility, S0(T) (which also quantifies the amplitude of long wavelength density fluctuations), as a consequence of a fundamental statistical mechanical relationship between structure and thermodynamics. Moreover, the penetrant activation barrier is predicted to have a factorized/multiplicative form, scaling as the product of an inverse power law of S0(T) and a linear/logarithmic function of the penetrant-to-matrix size ratio. This implies an enormous reduction in chemical complexity that is verified based solely on experimental data for diverse classes of chemically complex penetrants dissolved in molecular and polymeric liquids over a wide range of temperatures down to the kinetic glass transition. The predicted corollary that the penetrant diffusion constant decreases exponentially with inverse temperature raised to an exponent determined solely by how S0(T) decreases with cooling is also verified experimentally. Our findings are relevant to fundamental questions in glassy dynamics, self-averaging of angstrom-scale chemical features, and applications such as membrane separations, barrier coatings, drug delivery, and self-healing.

Odd-even effect on the thermal conductivity of liquid crystalline epoxy resins.

Rapid developments in high-performance computing and high-power electronics are driving needs for highly thermal conductive polymers and their composites for encapsulants and interface materials. However, polymers typically have low thermal conductivities of ∼0.2 W/(m K). We studied the thermal conductivity of a series of epoxy resins cured by one diamine hardener and seven diepoxide monomers with different precise ethylene linker lengths (x = 2-8). We found pronounced odd-even effects of the ethylene linker length on the liquid crystalline order, mass density, and thermal conductivity. Epoxy resins with even x have liquid crystalline structure with the highest density of 1.44 g/cm3 and highest thermal conductivity of 1.0 W/(m K). Epoxy resins with odd x are amorphous with the lowest density of 1.10 g/cm3 and lowest thermal conductivity of 0.17 W/(m K). These findings indicate that controlling precise linker length in dense networks is a powerful route to molecular design of thermally conductive polymers.

Emerging cell and molecular targets for treating mucus hypersecretion in asthma.

Mucus provides a protective barrier that is crucial for host defense in the lungs. However, excessive or abnormal mucus can have pathophysiological consequences in many pulmonary diseases, including asthma. Patients with asthma are treated with agents that relax airway smooth muscle and reduce airway inflammation, but responses are often inadequate. In part, this is due to the inability of existing therapeutic agents to directly target mucus. Accordingly, there is a critical need to better understand how mucus hypersecretion and airway plugging are affected by the epithelial cells that synthesize, secrete, and transport mucus components. This review highlights recent advances in the biology of mucin glycoproteins with a specific focus on MUC5AC and MUC5B, the chief macromolecular components of airway mucus. An improved mechanistic understanding of key steps in mucin production and secretion will help reveal novel potential therapeutic strategies.

Idiopathic Pulmonary Fibrosis: A Genetic Disease That Involves Mucociliary Dysfunction of the Peripheral Airways.

Idiopathic pulmonary fibrosis (IPF) is an incurable complex genetic disorder that is associated with sequence changes in 7 genes (MUC5B, TERT, TERC, RTEL1, PARN, SFTPC, and SFTPA2) and with variants in at least 11 novel loci. We have previously found that 1) a common gain-of-function promoter variant in MUC5B rs35705950 is the strongest risk factor (genetic and otherwise), accounting for 30-35% of the risk of developing IPF, a disease that was previously considered idiopathic; 2) the MUC5B promoter variant can potentially be used to identify individuals with preclinical pulmonary fibrosis and is predictive of radiologic progression of preclinical pulmonary fibrosis; and 3) MUC5B may be involved in the pathogenesis of pulmonary fibrosis with MUC5B message and protein expressed in bronchiolo-alveolar epithelia of IPF and the characteristic IPF honeycomb cysts. Based on these considerations, we hypothesize that excessive production of MUC5B either enhances injury due to reduced mucociliary clearance or impedes repair consequent to disruption of normal regenerative mechanisms in the distal lung. In aggregate, these novel considerations should have broad impact, resulting in specific etiologic targets, early detection of disease, and novel biologic pathways for use in the design of future intervention, prevention, and mechanistic studies of IPF.

Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Muc5b plays a role in the development of inflammation and fibrosis in hypersensitivity pneumonitis induced by Saccharopolyspora rectivirgula.

Previously we have shown that a gain-of-function MUC5B promoter variant (rs35705950) is the strongest risk factor for the development of idiopathic pulmonary fibrosis. We have also found that Muc5b overexpression reduces mucociliary clearance in mice, potentially leading to recurrent injury to the bronchoalveolar epithelia. Hypersensitivity pneumonitis (HP) is induced by inhalation of numerous causative antigens that may be affected by mucociliary clearance. We conducted this study to determine the role of Muc5b in a mouse model of HP induced by Saccharopolyspora rectivirgula (SR) antigen. We used Muc5b-deficient and wild-type (WT) mice to determine whether Muc5b plays a role in inflammation and fibrosis at 3 and 6 wk in an SR model of HP. We measured cell concentrations and MUC5B expression in whole lung lavage (WLL) and quantified fibrosis using hydroxyproline assay and second harmonic generation. Muc5b expression in WLL fluid was significantly increased in SR-exposed WT mice compared with saline controls. WT mice challenged with SR developed more inflammation and lung fibrosis at 6 wk compared with 3 wk postexposure. Moreover, we found that 6 wk following challenge with SR, Muc5b-deficient mice had less lung inflammation and less lung fibrosis than Muc5b WT mice. Furthermore, Muc5b-deficient mice had significantly lower concentrations of TGF-ß1 in the WLL compared with Muc5b WT mice at 6 wk of exposure. Muc5b appears to play a role in fibrosis in the animal model of HP and this may have implications for HP in humans.

Characterizing mucociliary clearance in young children with cystic fibrosis.

BACKGROUND: This research characterized mucociliary clearance (MCC) in young children with cystic fibrosis (CF). METHODS: Fourteen children (5-7 years old) with CF underwent: two baseline MCC measurements (Visits 1 and 2); one MCC measurement approximately 1 year later (Visit 3); and measurements of lung clearance index (LCI), a measure of ventilation inhomogeneity. RESULTS: Median (range) percent MCC through 60 min (MCC60) was similar on Visits 1 and 2 with 11.0 (0.9-33.7) and 12.8 (2.7-26.8), respectively (p = 0.95), and reproducible (Spearman Rho = 0.69; p = 0.007). Mucociliary clearance did not change significantly over 1 year with median percent MCC60 on Visit 3 [12.8 (3.7-17.6)] similar to Visit 2 (p = 0.58). Lower percent MCC60 on Visit 3 was significantly associated with higher LCI scores on Visit 3 (N = 14; Spearman Rho = -0.56; p = 0.04). CONCLUSIONS: Tests of MCC were reproducible and reliable over a 2-week period and stable over a 1-year period in 5-7-year-old children with CF. Lower MCC values were associated with increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF. IMPACT: This is the first study to characterize mucociliary clearance (MCC) in children with cystic fibrosis (CF) who were 5-7 years old. Measurements of mucociliary clearance were reproducible and reliable over a 2-week period and stable over a 1-year period. Variability in MCC between children was associated with differences in ventilation homogeneity, such that children with lower MCC values had increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF.

Impact of dynamic covalent chemistry and precise linker length on crystallization kinetics and morphology in ethylene vitrimers.

Vitrimers, dynamic polymer networks with topology conserving exchange reactions, have emerged as a promising platform for sustainable and reprocessable materials. While prior work has documented how dynamic bonds impact stress relaxation and viscosity, their role on crystallization has not been systematically explored. Precise ethylene vitrimers with 8, 10, or 12 methylene units between boronic ester junctions were investigated to understand the impact of bond exchange on crystallization kinetics and morphology. Compared to linear polyethylene which has been heavily investigated for decades, a long induction period for crystallization is seen in the vitrimers ultimately taking weeks in the densest networks. An increase in melting temperatures (Tm) of 25-30 K is observed with isothermal crystallization over 30 days. Both C10 and C12 networks initially form hexagonal crystals, while the C10 network transforms to orthorhombic over the 30 day window as observed with wide angle X-ray scattering (WAXS) and optical microscopy (OM). After 150 days of isothermal crystallization, the three linker lengths led to double diamond (C8), orthorhombic (C10), and hexagonal (C12) crystals indicating the importance of precision on final morphology. Control experiments on a precise, permanent network implicate dynamic bonds as the cause of long-time rearrangements of the crystals, which is critical to understand for applications of semi-crystalline vitrimers. The dynamic bonds also allow the networks to dissolve in water and alcohol-based solvents to monomers, followed by repolymerization while preserving the mechanical properties and melting temperatures.

Static and Dynamic Gradient Based Directional Transportation of Neutral Molecules in Swollen Polymer Films.

Materials which selectively transport molecules offer powerful opportunities for concentrating and separating chemical agents. Here, utilizing static and dynamic chemical gradients, transport of molecules within swollen crosslinked polymers is demonstrated. Using an ≈200 µm static hydroxyl to hexyl gradient, the neutral ambipolar nerve agent surrogate diethyl (cyanomethyl)phosphonate (DECP) is directionally transported and concentrated 60-fold within 4 hours. To accelerate transport kinetics, a dynamic gradient (a "travelling wave") is utilized. Here, the non-polar dye pyrene was transported. The dynamic gradient is generated by an ion exchange process triggered by the localized introduction of an aqueous NaCl solution, which converts the gel from hydrophobic to hydrophilic. As the hydrophilic region expands, associated water enters the gel, and pyrene is pushed ahead of the expansion front. The dynamic gradient provides about 10-fold faster transport kinetics than the static gradient.

Effect of precise linker length, bond density, and broad temperature window on the rheological properties of ethylene vitrimers.

Dynamic networks which undergo topology conserving exchange reactions, sometimes called vitrimers, show properties intermediate to thermosets and thermoplastics. The dynamic nature of the networks results in complex rheological properties and has attracted much attention in the past decade for self-healing, malleable and recyclable polymers. Here, we investigate a series of precise, high crosslink density telechelic ethylene vitrimers as a function of temperature and crosslink density. The networks show a rubbery plateau at high frequencies and a terminal flow regime at lower frequencies. With increasing crosslink density, the rubbery plateau modulus shows a monotonic increase and the terminal flow shifts to lower frequencies. The plateau modulus at high frequency increases as a function of temperature, as expected for a conserved network topology. When plotted against inverse temperature, the zero shear viscosities show a characteristic Arrhenius behavior, and the activation energy monotonically increases with crosslink density. Crossover frequency and shift factors (from time temperature superposition) also show Arrhenius behavior with activation energies in good agreement with those determined from zero shear viscosity. A positive deviation from this Arrhenius trend is observed beginning as high as 100 K above the glass transition temperature for C6 and C8 networks. Further investigations of such networks are critical for the development of sustainable and recyclable replacements for commercial plastics.

Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin.

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b-/-, Muc5ac-/-, and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In Muc5ac-/- mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b-/- mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.

Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Catalyst-Free Dynamic Networks for Recyclable, Self-Healing Solid Polymer Electrolytes.

Polymer networks with dynamic covalent cross-links act as solids but can flow at high temperatures. They have been widely explored as reprocessable and self-healing materials, but their use as solid electrolytes is limited. Here we report poly(ethylene oxide)-based networks with varying amounts of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) to understand the impact of a salt on the ion transport and network dynamics. We observed that the conductivity of our dynamic networks reached a maximum of 3.5 × 10-4 S/cm at an optimal LiTFSI concentration. Rheological measurements showed that the amount of LiTFSI significantly affects the mechanical properties, as the shear modulus varies between 1 and 10 MPa and the stress relaxation by 2 orders of magnitude. Additionally, we found that these networks can efficiently dissolve back to pure monomers and heal to recover their conductivity after damage, showing the potential of dynamic networks as sustainable solid electrolytes.

Role of mucins in lung homeostasis: regulated expression and biosynthesis in health and disease.

In humans and mice, the first line of innate defense against inhaled pathogens and particles in the respiratory tract is airway mucus. The primary solid components of the mucus layer are the mucins MUC5AC and MUC5B, polymeric glycoproteins whose changes in abundance and structure can dramatically affect airway defense. Accordingly, MUC5AC/Muc5ac and MUC5B/Muc5b are tightly regulated at a transcriptional level by tissue-specific transcription factors in homeostasis and in response to injurious and inflammatory triggers. In addition to modulated levels of mucin gene transcription, translational and post-translational biosynthetic processes also exert significant influence upon mucin function. Mucins are massive macromolecules with numerous functional domains that contribute to their structural composition and biophysical properties. Single MUC5AC and MUC5B apoproteins have molecular masses of >400 kDa, and von Willebrand factor D-like as well as other cysteine-rich domain segments contribute to mucin polymerization and flexibility, thus increasing apoprotein length and complexity. Additional domains serve as sites for O-glycosylation, which increase further mucin mass several-fold. Glycosylation is a defining process for mucins that is specific with respect to additions of glycans to mucin apoprotein backbones, and glycan additions influence the physical properties of the mucins via structural modifications as well as charge interactions. Ultimately, through their tight regulation and complex assembly, airway mucins follow the biological rule of 'form fits function' in that their structural organization influences their role in lung homeostatic mechanisms.