Interleukin-9 induces goblet cell hyperplasia during repair of human airway epithelia

Asthma is characterized by airway inflammation, smooth muscle hyperreactivity, and airway remodeling with excessive mucus production. The effect cytokines like interleukin (IL)-9 have on airway epithelia has been addressed using murine models of asthma, as well as transgenic and knockout mice. Though highly informative, differences exist between mouse and human airway epithelia, including cellular composition (e.g., Clara cells) and stem cell/plasticity capabilities. Therefore, to address cytokine effects on human airway epithelia, we have used a primary model system to ask whether IL-9 can alter cell fates of human airway epithelia. Here, we show that IL-9 has little effect on fully differentiated ciliated human airway epithelia. However, in the setting of airway injury repair, IL-9 results in goblet cell hyperplasia. A similar response was observed when the epithelium was exposed to IL-9 before it became fully differentiated. Moreover, exposure to IL-9 resulted in increased lysozyme and mucus production by the epithelia. Thus, a combination of IL-9 and mechanical injury can explain, in part, goblet cell hyperplasia that is evident in the lungs of individuals with asthma. These data suggest that interventions that limit airway epithelial damage, block IL-9, or modulate the repair process should result in decreased airway remodeling and prevent the chronic manifestations of this disease.     

Macrophage-intrinsic DUOX1 contributes to type 2 inflammation and mucus metaplasia during allergic airway disease

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.     

Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium

Mucus overproduction in inflammatory and obstructive airway diseases is associated with goblet cell (GC) metaplasia in airways. Although the mechanisms involved in GC metaplasia and mucus hypersecretion are not completely understood, association with oxidative stress and epidermal growth factor receptor (EGFR) signaling has been reported. To explore the mechanisms involved in oxidative stress-induced GC metaplasia, cultures of differentiated normal human bronchial epithelial cells grown at the air-liquid interface were exposed to reactive oxygen species (ROS) generated by xanthine/xanthine oxidase. EGFR activation and signaling was assessed by measuring EGF and transforming growth factor-alpha release and EGFR and (44/42)MAPK phosphorylation. The GC population was evaluated by confocal microscopy. ROS-induced EGFR activation resulted in GC proliferation and increased MUC5AC gene and protein expression. Signaling was due to pro-EGF processing by tissue kallikrein (TK), which was activated by ROS-induced hyaluronan breakdown. It was inhibited by catalase, a TK inhibitor, and EGF-blocking antibodies. Exposure to recombinant TK mimicked the ROS effects, increasing the expression of MUC5AC and lactoperoxidase. In addition, ROS induced the antiapoptotic factor Bcl-2 in a TK-dependent fashion. In conclusion, ROS-induced GC metaplasia in normal human bronchial epithelial cells is associated with HA depolymerization and EGF processing by TK followed by EGFR signaling, suggesting that increases in TK activity could contribute to GC metaplasia and mucus hypersecretion in diseases such as asthma and chronic bronchitis. The data also suggest that increases in GC population could be sustained by the associated upregulation of Bcl-2 in airway epithelial cells.     

Lipopolysaccharide administration to the allergic nose contributes to lower airway inflammation

Background Allergic rhinitis (AR) is an inflammatory reaction not confined to a single local compartment, but rather involving the whole airway system. Allergens known to induce AR are not always the sole trigger of the inflammatory reaction as infections and organic dust might also cause exacerbations of rhinitis and associated conditions. Objective To examine the effects of intranasal lipopolysaccharide (LPS) exposure, as a surrogate for upper airway bacterial infections, in patients with symptomatic AR. Methods Fourteen patients with a history of moderate to severe pollen‐induced AR were challenged intranasally with LPS. After 3–6 weeks, the same patients were challenged again, first with allergen and 24 h later with LPS. Nasal symptom scores, nasal lavage leucocyte counts and nasal airway resistance were assessed at 6–24 h after each provocation along with measurements of nitric oxide (NO) levels in the nose and lung. Results Six hours after the LPS challenge, an increased level of leucocytes could be obtained in the lavage fluid, but no symptoms were experienced and no increase in nasal resistance could be recorded. The NO production in the upper and lower airways was similar before and 6 h after the provocation. In contrast, in patients exposed to pollen before the LPS challenge, both the nasal and the pulmonary NO levels were enhanced. This was accompanied by an increase in leucocytes. Conclusion The present study demonstrates a priming effect of allergen on the nasal response to LPS as well as the presence of a systemic link between airway mucosal sites in the upper and lower airways. This suggests that exogenously derived signals, like upper airway infections, can interfere with the initiation, maintenance and progression of asthma.     

Chronic airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis during allergic airway disease induced by Aspergillus fumigatus

Clinical allergic airway disease is associated with persistent airway hyperreactivity and remodeling, but little is known about the mechanisms leading to these alterations. This paucity of information is related in part to the absence of chronic models of allergic airway disease. Herein we describe a model of persistent airway hyperreactivity, goblet cell hyperplasia, and subepithelial fibrosis that is initiated by the intratracheal introduction of Aspergillus fumigatus spores or conidia into the airways of mice previously sensitized to A. fumigatus. Similar persistent airway alterations were not observed in nonsensitized mice challenged with A. fumigatus conidia alone. A. fumigatus-sensitized mice exhibited significantly enhanced airway hyperresponsiveness to a methacholine challenge that was still present at 30 days after the conidia challenge. Eosinophils and lymphocytes were present in bronchoalveolar lavage (BAL) samples from A. fumigatus-sensitized mice at all times after conidia challenge. Compared with levels measured in A. fumigatus-sensitized mice immediately before conidia, significantly elevated interferon-gamma (IFN-gamma) and transforming growth factor (TGF-beta) levels were present in whole lung homogenates up to 7 days after the conidia challenge. At day 30 after conidia challenge, significantly elevated levels of interleukin-4 (IL-4) and IL-13 were present in the A. fumigatus-sensitized mice. Histological analysis revealed profound goblet cell hyperplasia and airway fibrosis at days 30 after conidia, and the latter finding was confirmed by hydroxyproline measurements. Thus the introduction of A. fumigatus conidia into A. fumigatus-sensitized mice results in persistent airway hyperresponsiveness, fibrosis, and goblet cell hyperplasia.     

Effect of suplatast tosilate on goblet cell metaplasia in patients with asthma

BACKGROUND: Goblet cell metaplasia is a pathologic characteristic of asthma, associated with excess mucus secretion. Interleukin (IL)-4 and IL-13 plays an important role in mucus hypersecretion. Suplatast tosilate (suplatast), an antiallergic agent, is a Th2 cytokine inhibitor that suppresses the synthesis of IL-4, IL-5, IL-13, and eosinophilic airway inflammation. OBJECTIVE: We examined the effects of suplatast on mucus production in bronchial biopsy specimens taken from asthmatic subjects. METHODS: Oral suplatast 300 mg daily, or placebo was administered for 3 months in a double-blind, parallel-group study in 25 patients with asthma. Biopsy specimens were evaluated at before and after treatment for alcian blue/period acid-Schiff (AB/PAS), MUC5AC staining in bronchial epithelium and IL-4+, IL-13+ cells as well as inflammatory cells in lamina propria. RESULTS: There were significant decreases in the percentage of AB/PAS (P < 0.01) and MUC5AC (P < 0.01) stained area in the suplatast group. These changes were accompanied by significant decreases in IL-4+ and IL-13+ cells in suplatast-treated subjects. Additionally, we have observed that the number of infiltrating eosinophils and CD4+ T cells significantly decreased. CONCLUSIONS: These findings suggest that suplatast prevents goblet cell metaplasia through modulation of Th2 cytokine production and the recruitment of eosinophils and CD4+ T cells in the asthmatic airways.     

Gob-5 contributes to goblet cell hyperplasia and modulates pulmonary tissue inflammation

Gob-5 is a member of the calcium-activated chloride channel family and has been associated with allergic response in mouse models of pulmonary inflammation. Gene expression of Gob-5 has been shown to be induced in allergic airways and has been strongly associated with mucin gene regulation and goblet cell hyperplasia. We investigated the physiologic role of Gob-5 in murine models of pulmonary inflammation using mice deficient in Gob-5. After sensitization and aerosol challenge with ovalbumin (OVA), Gob-5 knockout mice exhibit significantly increased bronchoalveolar lavage (BAL) inflammation as compared with wild-type controls. The augmented inflammation in BAL consisted predominantly of neutrophils. Examination of perivascular inflammation revealed that tissue inflammation was decreased in OVA-challenged Gob-5-/- mice. OVA-challenged Gob-5 knockout mice also had decreased goblet cell hyperplasia as well as decreased mucus production. These mice also had decreased airway hypersensitivity after cholinergic provocation with methacholine. Gob-5 knockout mice were also challenged via intranasal LPS, a TLR-4 agonist. Gob-5-/- mice responded with increased neutrophilic BAL inflammation and decreased perivascular tissue inflammation as compared with wild-type controls. There was little effect on goblet cell hyperplasia and mucus production after LPS challenge. These observations reinforce findings that associate Gob-5 with goblet cell hyperplasia and mucus production in the allergic immune response, but also implicate Gob-5 in the regulation of tissue inflammation in the innate immune response.     

Dynamic expression pattern of Sonic hedgehog in developing cochlear spiral ganglion neurons

Sonic hedgehog (Shh) signaling plays important roles in the formation of the auditory epithelium. However, little is known about the detailed expression pattern of Shh and the cell sources from which Shh is secreted. By analyzing Shh^CreEGFP/+ mice, we found that Shh was first expressed in all cochlear spiral ganglion neurons by embryonic day 13.5, after which its expression gradually decreased from base to apex. By postnatal day 0, it was not detected in any spiral ganglion neurons. Genetic cell fate mapping results also confirmed that Shh was exclusively expressed in all spiral ganglion neurons and not in surrounding glia cells. The basal‐to‐apical wave of Shh decline strongly resembles that of hair cell differentiation, supporting the idea that Shh signaling inhibits hair cell differentiation. Furthermore, this Shh^CreEGFP/+ mouse is a useful Cre line in which to delete floxed genes specifically in spiral ganglion neurons of the developing cochlea. Developmental Dynamics 239:1674–1683, 2010. © 2010 Wiley‐Liss, Inc.     

Increased surfactant protein D in rat airway goblet and Clara cells during ovalbumin-induced allergic airway inflammation

BACKGROUND: Structural remodelling of airways in asthma that follows inflammation may be affected by surfactant protein D (SP-D)-mediated effects on the immune response. OBJECTIVE: To determine potential sites of SP-D interaction with the pulmonary immune response, we examined the distribution of immunoreactive SP-D in an experimental model of allergen-induced airway inflammation using immunohistochemistry, biochemical methods and in situ hybridization. METHODS: The experimental model used subcutaneous injection of ovalbumin in adult rats, which induced an airway response to inhaled nebulized ovalbumin. Three groups of rats (ovalbumin, ovalbumin + dexamethasone and saline) were challenged thrice weekly for 3 weeks. A fourth group of seven rats (naive) were taken from the same delivery of rats as the other groups. Lungs were then lavaged to determine total cell count, eosinophil count, ovalbumin-specific IgE by enzyme-linked immunosorbent assay and SP-D by immunoblot. Tissue samples were fixed and embedded, and sections were studied for the infiltration of eosinophils and for expression of SP-D protein by histochemistry and mRNA by in situ hybridization. RESULTS: Ovalbumin induced perivascular and peribronchiolar eosinophilia which could be prevented by dexamethasone treatment. In addition, the ovalbumin-specific IgE levels in serum and bronchoalveolar lavage fluid of ovalbumin-challenged animals were enhanced. Increased amount of SP-D in lavage and tissue, particularly in type II pneumocytes, in Clara cells and, surprisingly, in hyperplastic goblet cells of inflamed lungs was found. SP-D mRNA was detected in goblet cells as well as in type II pneumocytes and Clara cells. Dexamethasone treatment did not affect level of SP-D immunoreactivity. CONCLUSION: SP-D accumulation is increased in this model of allergen-induced eosinophilia, both in upper and lower airways. The increase is unaffected by dexamethasone.     

Monoaminergic regulation of Sonic hedgehog signaling cascade expression in the adult rat hippocampus

Monoamines are implicated in the modulation of adult hippocampal neurogenesis in depression models and following chronic antidepressant treatment. Given the key role of Sonic hedgehog (Shh) in adult neurogenesis, we examined whether monoaminergic perturbations regulate the expression of Shh or its co-receptors Smoothened (Smo) and Patched (Ptc). Combined depletion of both serotonin and norepinephrine with para-chlorophenylalanine (PCPA) resulted in a significant decrease in Smo and Ptc mRNA within the dentate gyrus subfield of the hippocampus. However, selective depletion of serotonin, using the serotonergic neurotoxin 5,7-dihyrdroxytryptamine (5,7-DHT), or norepinephrine, using the noradrenergic neurotoxin DSP-4, did not alter expression of Shh and its co-receptors, Smo and Ptc. Acute treatment with the monoamine releasing agent, para-chloroamphetamine (PCA) significantly upregulated Smo mRNA within the dentate gyrus. However, acute or chronic treatment with pharmacological antidepressants that modulate monoaminergic neurotransmission did not regulate Shh cascade expression. These results indicate that robust changes in monoamine levels can regulate the expression of the Shh signaling cascade in the adult rodent brain.     

Decreased expression of Fas (CD95/APO1) associated with goblet cell metaplasia in Barrett's esophagus

Fas ligand (FasL) has been shown to induce apoptosis in cells expressing its receptor Fas. We have recently shown that Fas ligand is overexpressed in all cases of Barrett's metaplasia (BM) with dysplasia and esophageal adenocarcinomas, and in a few cases of BM negative with dysplasia. The aim of this work was to determine the status of Fas expression in BM with and without dysplasia or carcinoma. Formalin-fixed and paraffin-embedded tissue sections from esophageal biopsies and esophagectomy specimens with BM, with and without dysplasia and carcinoma, were immunostained for Fas and FasL using the immunoperoxidase technique. The percentage of positive cells in each case was evaluated and compared with the degree of dysplasia. When Fas expression was assessed in glands with goblet cell metaplasia, Fas immunoreactivity was either undetected or present in less than 10% of the cells in 85% of the cases, and only 1 (4%) of the 28 cases examined showed Fas immunoreactivity in more than 25% of the cells. When we compared Fas expression in goblet cell-containing glands with glands of gastric cardia phenotype, we found that in the 26 cases of BM with or without dysplasia Fas was completely undetectable in goblet cell-containing glands in 15 (58%) of the cases but was undetectable in only 3 (12%) of the glands with gastric cardia phenotype (P = .002). Fas is usually undetectable or is expressed at a low level in BM with or without dysplasia or carcinoma. Fas expression in goblet cell-containing glands is less frequent than in glands with gastric cardia phenotype in the same specimens. BM with dysplasia or carcinoma overexpress FasL, so decreased Fas expression may protect BM with dysplasia and carcinoma from self-destruction while allowing them to evade immune surveillance.     

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract

Background: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel’s diverticulum.Methods: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form.Results: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach.Conclusion: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.     

Reduced expression of psoriasin in human airway cystic fibrosis epithelia

Psoriasin is a low molecular weight Ca(2+)-binding protein with known antimicrobial activity. Since human airway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence, we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expression was investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells, derived from a cystic fibrosis patient. We also examined psoriasin expression following treatment with factors pertinent to the CF lung-oxidant stress and exposure to pro-inflammatory cytokines. CFBE41o- cells demonstrated much reduced psoriasin levels compared to the 16HBE14o- cells. Increased psoriasin expression was seen following treatment with IL-22 and a cytomix of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ; however, the oxidant stressor tert-butyl hydroperoxide had no apparent effect. Over-expression of human psoriasin into both cell lines resulted in increased internalization of Pseudomonas aeruginosa. In conclusion, expression of psoriasin - which has known anti-microbial activity in other systems - appears to be reduced in CFBE410- compared to 16HBE14o- cells, and its expression modified by exposure to pro-inflammatory cytokines.     

Immunohistochemical expression of Sonic hedgehog in intraductal papillary mucinous tumor of the pancreas.

Aberrant expression of Sonic hedgehog (Shh) has been reported in many human cancers including ductal carcinoma of the pancreas. The intraductal papillary mucinous tumor (IPMT) has been considered as one of the precursor lesions of invasive ductal carcinoma of the pancreas. Shh expression in pancreatic IPMT has not been reported. We investigated an immunohistochemical (IHC) expression of Shh in 55 cases of pancreatic IPMT. We analyzed the IHC expression of Shh in the following histologic grades of tumor: adenoma (AD), moderate dysplasia (MD), noninvasive carcinoma (NIC), and invasive carcinoma (IC), and with the following histologic subtype classification: intestinal, pancreatobiliary, null, and unclassifiable type. IHC Shh expression was noted in 6 (46.2%) of 13 AD, 5 (35.7%) of 14 MD, 12 (80%) of 15 NIC, and 11 (84.6%) of 13 IC. Shh expression was significantly increased in malignant IPMT (NIC+IC) compared with nonmalignant IPMT (AD+MD) (82.1% vs. 40.7%, P=0.0005). IHC Shh expression was found in 11 (68.8%) of 16 intestinal types, 13 (92.8%) of 14 pancreatobiliary types, 8 (38.1%) of 21 null types, and 2 (50%) of 4 unclassifiable types. Intestinal and pancreatobiliary subtypes showed a high expression of Shh compared with the null and unclassifiable type of IPMT. All 3 cases of node metastasis showed IHC Shh expression in tumor cells of metastatic lymph nodes. Therefore, Shh expression may have a critical role in the late stage of carcinogenesis of IPMT, and may impact metastatic progression to the lymph nodes in malignant IPMT.     

Respiratory syncytial virus-induced CCL5/RANTES contributes to exacerbation of allergic airway inflammation

Severe respiratory syncytial virus (RSV) infection has a significant impact on airway function and may induce or exacerbate the response to a subsequent allergic challenge. In a murine model combining early RSV infection with later cockroach allergen (CRA) challenge, we examined the role of RSV-induced CCL5/RANTES production on allergic airway responses. RSV infection increased CCL5 mRNA and protein levels, peaking at days 8 and 12, respectively. Administration of CCL5 antiserum during days 0-14 of the RSV infection did not significantly alter viral protein expression when compared to mice treated with control serum. In mice receiving the combined RSV-allergen challenge, lungs collected on day 22 exhibited significantly increased numbers of CD4- and CD8-positive T cells. This increase in T cell numbers was not observed in mice receiving alpha-CCL5. On day 43, peribronchial eosinophilia and leukotriene levels were increased in RSV-allergen mice. Pretreatment with CCL5 antiserum resulted in decreased recruitment of inflammatory cells to bronchoalveolar and peribronchial regions of the lungs and these reductions were associated with a reduction in both T cell recruitment into the bronchoalveolar space, leukotriene release and chemokine generation. Thus, CCL5 released during RSV infection has a significant effect on the inflammatory response to subsequent allergic airway challenges.     

Sonic hedgehog inversely regulates the expression of angiopoietin-1 and angiopoietin-2 in fibroblasts

Nerves and blood vessels have similar branching patterns and use common morphogenic molecules during development. Recent studies show that sonic hedgehog (Shh), a traditional neurogenic morphogen, is required for embryonic arterial differentiation and can induce angiogenesis. We investigated whether Shh regulates the expression of angiogenic factors. Using NIH3T3 embryonic fibroblast cells, we demonstrated that Shh increased the mRNA levels of angiopoietin-1 (Ang-1), a secreted ligand that regulates endothelial interaction with mural cells (pericytes and smooth muscle cells) and promotes blood vessel maturation. In contrast, Shh decreased mRNA levels of angiopoietin-2 (Ang-2), a negative modulator of Ang-1. By contrast, Shh did not change the expression of vascular endothelial growth factor (VEGF) mRNA, a potent endothelial mitogen. The effect of Shh appeared to be cell-type specific as the addition of Shh to neural progenitor cells or neurons did not alter Ang-1, Ang-2 or VEGF mRNA levels. The addition of cyclopamine, an inhibitor of Shh signaling, to NIH3T3 cells, suppressed the regulation of Ang-1 and Ang-2 mRNA levels in the presence of Shh. Collectively, our results suggest that Shh may contribute to blood vessel growth, maturation and stabilization in a neurovascular network by reciprocally regulating the vascular morphogens Ang-1 and Ang-2 in a cell-type-specific manner.