LKB1 deficiency upregulates RELM-α to drive airway goblet cell metaplasia

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

PI3Kδ inhibition prevents IL33, ILC2s and inflammatory eosinophils in persistent airway inflammation

Background Phosphoinositide-3-kinase-delta (PI3Kδ) inhibition is a promising therapeutic approach for inflammatory conditions due to its role in leucocyte proliferation, migration and activation. However, the effect of PI3Kδ inhibition on group 2 innate lymphoid cells (ILC2s) and inflammatory eosinophils remains unknown. Using a murine model exhibiting persistent airway inflammation we sought to understand the effect of PI3Kδ inhibition, montelukast and anti-IL5 antibody treatment on IL33 expression, group-2-innate lymphoid cells, inflammatory eosinophils, and goblet cell metaplasia. Results Mice were sensitised to house dust mite and after allowing inflammation to resolve, were re-challenged with house dust mite to re-initiate airway inflammation. ILC2s were found to persist in the airways following house dust mite sensitisation and after re-challenge their numbers increased further along with accumulation of inflammatory eosinophils. In contrast to montelukast or anti-IL5 antibody treatment, PI3Kδ inhibition ablated IL33 expression and prevented group-2-innate lymphoid cell accumulation. Only PI3Kδ inhibition and IL5 neutralization reduced the infiltration of inflammatory eosinophils. Moreover, PI3Kδ inhibition reduced goblet cell metaplasia. Conclusions Hence, we show that PI3Kδ inhibition dampens allergic inflammatory responses by ablating key cell types and cytokines involved in T-helper-2-driven inflammatory responses.

Inhibition of β-Catenin/CREB Binding Protein Signaling Attenuates House Dust Mite-Induced Goblet Cell Metaplasia in Mice

Excessive mucus production is a major feature of allergic asthma. Disruption of epithelial junctions by allergens such as house dust mite (HDM) results in the activation of β-catenin signaling, which has been reported to stimulate goblet cell differentiation. β-catenin interacts with various co-activators including CREB binding protein (CBP) and p300, thereby regulating the expression of genes involved in cell proliferation and differentiation, respectively. We specifically investigated the role of the β-catenin/CBP signaling pathway in goblet cell metaplasia in a HDM-induced allergic airway disease model in mice using ICG-001, a small molecule inhibitor that blocks the binding of CBP to β-catenin. Female 6- 8-week-old BALB/c mice were sensitized to HDM/saline on days 0, 1, and 2, followed by intranasal challenge with HDM/saline with or without subcutaneous ICG-001/vehicle treatment from days 14 to 17, and samples harvested 24 h after the last challenge/treatment. Differential inflammatory cells in bronchoalveolar lavage (BAL) fluid were enumerated. Alcian blue (AB)/Periodic acid–Schiff (PAS) staining was used to identify goblet cells/mucus production, and airway hyperresponsiveness (AHR) was assessed using invasive plethysmography. Exposure to HDM induced airway inflammation, goblet cell metaplasia and increased AHR, with increased airway resistance in response to the non-specific spasmogen methacholine. Inhibition of the β-catenin/CBP pathway using treatment with ICG-001 significantly attenuated the HDM-induced goblet cell metaplasia and infiltration of macrophages, but had no effect on eosinophils, neutrophils, lymphocytes or AHR. Increased β-catenin/CBP signaling may promote HDM-induced goblet cell metaplasia in mice.

Esophageal granular cell tumor and eosinophils: a multicenter experience

Background Esophageal granular cell tumor (eGCT) is rare, and the recent literature suggests a link between eosinophilic esophagitis (EoE) and eGCT. The aim of our study was to determine if EoE or other disorders associated with eosinophilia are consistently associated with eGCT. Methods We retrospectively searched pathology databases of three academic institutions from 1999 to 2018 for eGCTs. The archived slides and medical records were reviewed. Results From 294,855 esophagogastroduodenoscopy procedures, 45 patients (17 males and 28 females) with eGCTs were identified. The patients (30–73 years in age, median 50) had eGCT 0.2–2.0 cm in size (average 0.71). Thirteen had a history of gastroesophageal reflux disease, 5 had Barrett esophagus/goblet cell metaplasia and 1 had EoE. Thirty-four eGCTs had intralesional eosinophils (14 with peak > 10 eosinophils/400x hpf); of these, 21 also had eosinophils in lamina propria (9 with peak > 10 eosinophils/hpf). eGCT with atypical features (including nuclear enlargement and prominent nucleoli) were more likely to have increased eosinophils in non-epithelial compartments than those without atypia. Pleomorphism and spindled cells were seen in 3 eGCT cases (mean peak intralesional eosinophils: 43 per hpf); 2 of these had goblet cell metaplasia. We found no association between EoE and eGCT, p = 0.5966, (95% C.I. 0.0276, 6.5389, Fisher’s exact test). Instead, most patients had gastroesophageal reflux disease or Barrett esophagus. Conclusion Eosinophilia, common in eGCT and adjacent stroma, likely drives atypical/reactive histologic features, but a pathogenic relationship between eosinophil rich inflammatory conditions and eGCT has not yet been established.

Less airway inflammation and goblet cell metaplasia in an IL-33-induced asthma model of leptin-deficient obese mice

Background Obesity-associated asthma is a phenotype of severe asthma. Late-onset, non-eosinophilic and female-dominant phenotype is highly symptomatic and difficult to treat. Leptin, an adipokine, exerts an immunomodulatory effect. IL-33 associated with innate immunity induces type 2 inflammation and is present in adipose tissue. The purpose of this study was to elucidate the pathogenesis of obesity-associated asthma by focusing on the interaction between leptin and IL-33. Methods In leptin-deficient obese (ob/ob) and wild-type mice, IL-33 was instilled intranasally on three consecutive days. In part of the mice, leptin was injected intraperitoneally prior to IL-33 treatment. The mice were challenged with methacholine, and airway hyperresponsiveness (AHR) was assessed by resistance (Rrs) and elastance (Ers) of the respiratory system using the forced oscillation technique. Cell differentiation, IL-5, IL-13, eotaxin, keratinocyte-derived chemokine (KC) in bronchoalveolar lavage fluid (BALF) and histology of the lung were analyzed. For the in vitro study, NCI-H292 cells were stimulated with IL-33 in the presence or absence of leptin. Mucin-5AC (MUC5AC) levels were measured using an enzyme-linked immunosorbent assay. Results Ob/ob mice showed greater Rrs and Ers than wild-type mice. IL-33 with leptin, but not IL-33 alone, enhanced Ers rather than Rrs challenged with methacholine in ob/ob mice, whereas it enhanced Rrs alone in wild-type mice. IL-33-induced eosinophil numbers, cytokine levels in BALF, eosinophilic infiltration around the bronchi, and goblet cell metaplasia were less in ob/ob mice than in wild-type mice. However, leptin pretreatment attenuated these changes in ob/ob mice. MUC5AC levels were increased by co-stimulation with IL-33 and leptin in vitro. Conclusions Ob/ob mice show innate AHR. IL-33 with leptin, but not IL-33 alone, induces airway inflammation and goblet cell metaplasia and enhances AHR involving peripheral airway closure. This is presumably accelerated by mucus in ob/ob mice. These results may explain some aspects of the pathogenesis of obesity-associated asthma.

CLCA1 Regulates Airway Mucus Production and Ion Secretion Through TMEM16A

TMEM16A, a Ca2+-activated chloride channel (CaCC), and its regulator, CLCA1, are associated with inflammatory airway disease and goblet cell metaplasia. CLCA1 is a secreted protein with protease activity that was demonstrated to enhance membrane expression of TMEM16A. Expression of CLCA1 is particularly enhanced in goblet cell metaplasia and is associated with various lung diseases. However, mice lacking expression of CLCA1 showed the same degree of mucous cell metaplasia and airway hyperreactivity as asthmatic wild-type mice. To gain more insight into the role of CLCA1, we applied secreted N-CLCA1, produced in vitro, to mice in vivo using intratracheal instillation. We observed no obvious upregulation of TMEM16A membrane expression by CLCA1 and no differences in ATP-induced short circuit currents (Iscs). However, intraluminal mucus accumulation was observed by treatment with N-CLCA1 that was not seen in control animals. The effects of N-CLCA1 were augmented in ovalbumin-sensitized mice. Mucus production induced by N-CLCA1 in polarized BCi-NS1 human airway epithelial cells was dependent on TMEM16A expression. IL-13 upregulated expression of CLCA1 and enhanced mucus production, however, without enhancing purinergic activation of Isc. In contrast to polarized airway epithelial cells and mouse airways, which express very low levels of TMEM16A, nonpolarized airway cells express large amounts of TMEM16A protein and show strong CaCC. The present data show an only limited contribution of TMEM16A to airway ion secretion but suggest a significant role of both CLCA1 and TMEM16A for airway mucus secretion.

Esophageal Granular Cell Tumor and Eosinophils: A Multicenter Experience

Background: Esophageal granular cell tumor (eGCT) is rare, and the recent literature suggests a link between eosinophilic esophagitis (EoE) and eGCT. The aim of our study was to determine if EoE or other disorders associated with eosinophilia are consistently associated with eGCT. Methods: We retrospectively searched pathology databases of three academic institutions from 1999 to 2018 for eGCTs. The archived slides and medical records were reviewed.Results: From 294,855 esophagogastroduodenoscopy procedures, 45 patients (17 males and 28 females) with eGCTs were identified. The patients were 30-73 years in age (median 50) had eGCT (0.2 – 2.0 cm, average 0.71). Thirteen had a history of gastroesophageal reflux disease, 5 had Barrett esophagus/goblet cell metaplasia and 1 had EoE. Thirty-four eGCTs had intralesional eosinophils (14 with peak >10 eosinophils/400x hpf); of these, 21 also had eosinophils in lamina propria (9 with peak >10 eosinophils/hpf). eGCT with atypical features (including nuclear enlargement and prominent nucleoli) were more likely to have increased eosinophils in non-epithelial compartments than those without atypia. Pleomorphism and spindled cells were seen in 3 eGCT cases (mean peak intralesional eosinophils: 43 per hpf); 2 of these had goblet cell metaplasia. We found no association between EoE and eGCT, p= 0.5966, (95% C.I. 0.0276, 6.5389, Fisher’s exact test). Instead, most patients had gastroesophageal reflux disease or Barrett esophagus.Conclusion: Eosinophilia, common in eGCT and adjacent stroma, likely drives atypical/reactive histologic features, but a pathogenic relationship between eosinophil rich inflammatory conditions and eGCT has not yet been established.

PI3Kδ Inhibition Prevents IL33, ILC2s and Inflammatory Eosinophils in Persistent Airway Inflammation

Background: Phosphoinositide-3-kinase-delta (PI3Kδ) inhibition is a promising therapeutic approach for inflammatory conditions due to its role in leucocyte proliferation, migration and activation. However, the effect of PI3Kδ inhibition on group-2-innate lymphoid cells (ILC2s) and inflammatory eosinophils remains unknown. Using a murine model exhibiting persistent airway inflammation we sought to understand the effect of PI3Kδ inhibition, montelukast and anti-IL5 antibody treatment on IL33 expression, group-2-innate lymphoid cells, inflammatory eosinophils, and goblet cell metaplasia. Results: Mice were sensitised to house dust mite and after allowing inflammation to resolve, were re-challenged with house dust mite to re-initiate airway inflammation. ILC2s were found to persist in the airways following house dust mite sensitisation and after re-challenge their numbers increased further along with accumulation of inflammatory eosinophils. In contrast to montelukast or anti-IL5 antibody treatment, PI3Kδ inhibition ablated IL33 expression and prevented group-2-innate lymphoid cell accumulation. Only PI3Kδ inhibition and IL5 neutralization reduced the infiltration of inflammatory eosinophils. Moreover, PI3Kδ inhibition reduced goblet cell metaplasia. Conclusions: Hence, we show that PI3Kδ inhibition dampens allergic inflammatory responses by ablating key cell types and cytokines involved in T-helper-2-driven inflammatory responses.

Less Airway Inflammation and Goblet Cell Metaplasia in an IL-33-Induced Asthma Model of Leptin-Deficient Obese Mice

BackgroundAsthma with obesity is a phenotype of severe asthma. Leptin exerts an immunomodulatory effect and its level is increased in obesity. IL-33 is associated with innate immunity and induces type 2 inflammation, and is present in adipose tissue. However, the role of IL-33 and leptin in obesity-associated asthma is not fully understood. We examined the effect of IL-33 on eosinophilic inflammation, goblet cell metaplasia, and airway responsiveness in leptin-deficient obese (ob/ob) and wild-type mice, and examined the effect of exogenous leptin pretreatment. MethodsIn ob/ob and wild-type mice, IL-33 was instilled intranasally on three consecutive days. In part of the animals, leptin was injected intraperitoneally prior to IL-33 treatment. The mice were challenged with methacholine and resistance of the respiratory system (Rrs) was measured using the forced oscillation technique. Cell differentiation, IL-5, IL-13, eotaxin, KC in bronchoalveolar lavage fluid (BALF), and histology of the lung were analyzed. For the in vitro study, NCI-H292 cells were stimulated with IL-33 in the presence or absence of leptin, and MUC5AC levels were measured by ELISA. ResultsOb/ob mice showed greater baseline Rrs than wild-type mice. IL-33 and IL-33 with leptin did not enhance Rrs challenged with methacholine compared to non-treatment in ob/ob mice, whereas IL-33 with leptin enhanced Rrs in wild-type mice. Ob/ob mice showed less IL-33-induced eosinophil numbers, IL-5, IL-13, eotaxin, and KC levels in BALF and eosinophilic infiltration around bronchi and goblet cell metaplasia than wild-type mice, but leptin pretreatment attenuated these changes in ob/ob mice. MUC5AC levels were increased by co-stimulation with IL-33 and leptin in vitro . ConclusionsLeptin plays an important role in IL-33-induced inflammation and goblet cell metaplasia in the airway, but obesity per se increases airway hyperresponsiveness independent of inflammation. These results explain some aspects of the pathogenesis of obesity-related asthma.