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Abstract Background The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000–5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. Methods We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. Results Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1–4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. Conclusions These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1–4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

Abstract Background The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. Methods The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. Results We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. Conclusions These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.

Summary: The gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from microbial invasion. The epithelium has one of the highest turnover rates in the body. Using stable isotope labeling, high-resolution mass spectrometry, and computational analysis, we report a comprehensive dataset of the turnover of more than 3,000 and the expression of more than 5,000 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life is shorter in the small intestine than in the colon. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological and immune-related functions between the small and large intestine. An absence of microbiota results in an approximately 1 day longer protein half-life in germ-free animals. : Arike et al. report a turnover rate of >3,000 and expression of >5,000 proteins in epithelial cells and mucus along the mice gastrointestinal tract. Protein turnover rate is faster in the small intestine than in the colon and slower in germ-free animals. Keywords: proteomics, mass spectrometry, protein turnover, bacteria, colonization, commensals, germ-free, colon, small intestine

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

Peyer's patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.

Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular over-the-counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z -eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV(1)) in vitro. Pharmacological activation of TRPV(1) in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV(1) in the brain contributes to the analgesic effect of acetaminophen. Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV(1) knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E(2) (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV(1)-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV(1) in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test. This study shows that TRPV(1) in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV(1) in the brain.