Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.mucus | cystic fibrosis | lung | asthma | COPD
While pathological and clinical data suggest that small airways are involved in early cystic fibrosis (CF) lung disease development, little is known about how the lack of cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to disease pathogenesis in these small airways. Large and small airway epithelia are exposed to different airflow velocities, temperatures, humidity, and CO2 concentrations. The cellular composition of these two regions is different, and small airways lack submucosal glands. To better understand the ion transport properties and impacts of lack of CFTR function on host defense function in small airways, we adapted a novel protocol to isolate small airway epithelial cells from CF and non-CF pigs and established an organotypic culture model. Compared with non-CF large airways, non-CF small airway epithelia cultures had higher Cl− and bicarbonate (HCO3−) short-circuit currents and higher airway surface liquid (ASL) pH under 5% CO2 conditions. CF small airway epithelia were characterized by minimal Cl− and HCO3− transport and decreased ASL pH, and had impaired bacterial killing compared with non-CF small airways. In addition, CF small airway epithelia had a higher ASL viscosity than non-CF small airways. Thus, the activity of CFTR is higher in the small airways, where it plays a role in alkalinization of ASL, enhancement of antimicrobial activity, and lowering of mucus viscosity. These data provide insight to explain why the small airways are a susceptible site for the bacterial colonization.
Without CFTR-mediated HCO3secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this timecourse matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNFα and IL-17, two inflammatory cytokines that are elevated in CF airways. TNFα+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl -/HCO3exchanger. Moreover, when CF epithelia were exposed to TNFα+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cellautonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.
The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base (HCO3¯) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing HCO3¯ secretion. TNFα+IL-17 increased expression and function of two apical HCO3¯ transporters, CFTR anion channels and pendrin Cl-/HCO3- exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was co-expressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate HCO3- secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.
Key points• The Na + -bicarbonate cotransporter NBCe1 regulates cell and tissue pH, as well as ion movement across cell layers in organs such as kidney, gut, and pancreas.• We previously showed that the signalling molecule PIP 2 stimulates the cloned A variant of NBCe1 in a patch of biological membrane. • In the current study, we characterize the effect of injecting PIP 2 into intact oocytes expressing an NBCe1 variant (A, B, or C).• PIP 2 stimulates the B and C variants, but not the A variant, through hydrolysis to IP 3 .Stimulation requires an intracellular Ca 2+ store and kinase activity.• The results will contribute to our understanding of multiple HCO 3 − -dependent transporters with different modes of regulation, as well as how molecules that stimulate specific membrane receptors lead to changes in cell/tissue pH, and perhaps how pathologies such as stroke and ischaemia that lead to energy deficiency cause tissue acidosis. Abstract Electrogenic Na+ -bicarbonate cotransporter NBCe1 variants contribute to pH i regulation, and promote ion reabsorption or secretion by many epithelia. Most Na + -coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH-and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP 2 on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP 2 (10 μM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO 3 − -induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP 2 hydrolysis and endoplasmic reticulum (ER) Ca 2+ release. Stimulation by PIP 2 injection was mimicked by injecting IP 3 , but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca 2+ with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca 2+ channels (SOCCs) to trigger a Ca 2+ influx mimicked the PIP 2 /IP 3 stimulation of the B and C variants. Activating the endogenous G q protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH i -recovery rate from a CO 2 -induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP 3 -induced stimulation of NBCe1-C. In summary, injecting PIP 2 stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP 3 /Ca 2+ that involves a staurosporine-sensitive kinase. In ...
Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30–50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.
SignificancePrevious studies localized the hedgehog (HH) signaling system to primary cilia. We discovered that motile cilia on airway epithelia also contain HH signaling proteins, indicating that like primary cilia, these motile cilia have an important sensory function. However, in contrast to the function of HH signaling in most primary cilia, sonic hedgehog (SHH) elicits noncanonical signaling, reducing cellular levels of cAMP. These findings suggest that airway SHH may quiet airway defenses. Involvement of SHH in lung disease and positioning of motile cilia where they sample SHH and other ligands in the airway lumen suggest that noncanonical HH signaling might modulate airway responses to the environment in health and disease.
Glutamate dysregulation occurs in multiple sclerosis (MS), but whether excitotoxic mechanisms in mature oligodendrocytes contribute to demyelination and axonal injury is unexplored. Although current treatments modulate the immune system, long-term disability ensues, highlighting the need for neuroprotection. Glutamate is elevated before T2-visible white matter lesions appear in MS. We previously reported that myelin-reactive T cells provoke microglia to release glutamate from the system xc− transporter promoting myelin degradation in experimental autoimmune encephalomyelitis (EAE). Here, we explore the target for glutamate in mature oligodendrocytes. Most glutamate-stimulated calcium influx into oligodendrocyte somas is AMPA receptor (AMPAR)–mediated, and genetic deletion of AMPAR subunit GluA4 decreased intracellular calcium responses. Inducible deletion of GluA4 on mature oligodendrocytes attenuated EAE and loss of myelinated axons was selectively reduced compared to unmyelinated axons. These data link AMPAR signaling in mature oligodendrocytes to the pathophysiology of myelinated axons, demonstrating glutamate regulation as a potential neuroprotective strategy in MS.
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