scholarly journals Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells

2012 ◽  
Vol 446 (3) ◽  
pp. 383-394 ◽  
Author(s):  
Kyubo Kim ◽  
Youlia M. Petrova ◽  
Brenton L. Scott ◽  
Rupesh Nigam ◽  
Anurag Agrawal ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Essential Gene ◽  
Allergic Inflammation ◽  
Airway Epithelial Cells ◽  
Secretory Cells ◽  
Airway Epithelial ◽  
Response Element ◽  
Protein Levels ◽  
Mucous Metaplasia ◽  
Specificity Protein

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.

Differential expression of chitinases identify subsets of murine airway epithelial cells in allergic inflammation

2006 ◽  
Vol 291 (3) ◽  
pp. L502-L511 ◽  
Author(s):  
Robert J. Homer ◽  
Zhou Zhu ◽  
Lauren Cohn ◽  
Chun Gun Lee ◽  
Wendy I. White ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Allergic Inflammation ◽  
Secretory Protein ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Parasitic Nematodes ◽  
Secretory Cells ◽  
Airway Epithelial ◽  
Fungal Cell ◽  
Clara Cell Secretory Protein

The mammalian chitinase family includes members both with and without enzymatic activity against chitin, a product of fungal cell walls, exoskeletons of crustaceans and insects, and the microfilarial sheaths of parasitic nematodes. Two members of that family, Ym1 and acidic mammalian chitinase (AMCase), are strongly upregulated in pulmonary T helper (Th) 2 inflammation but not in Th1 inflammation. The sites of expression of these products are incompletely known. We show here that, in two different models of Th2 inflammation, Ym1 and AMCase are mutually exclusively expressed in proximal vs. distal airway epithelium, respectively, whereas both are expressed in alveolar macrophages. This regional difference along the airway corresponds to the previously noted distinction between mucus positive proximal cells and mucus negative distal cells under the same conditions. Among distal cells, AMCase colocalizes with epithelial cells expressing the Clara cell marker Clara cell secretory protein. These AMCase-expressing cells retain expression of FOXA2, a transcription factor whose downregulation in association with IL-13 signaling has previously been associated with production of mucus in proximal airway epithelial cells. These results provide evidence that secretory cells of proximal and distal airways undergo fundamentally different gene expression programs in response to allergic inflammation. Furthermore, AMCase provides the first positive molecular marker of distal Clara cell secretory protein-expressing cells under these conditions.

scholarly journals TGF-β2 reduces nitric oxide synthase mRNA through a ROCK-dependent pathway in airway epithelial cells

2011 ◽  
Vol 301 (3) ◽  
pp. L361-L367 ◽  
Author(s):  
Jingjing Jiang ◽  
Steven C. George
Keyword(s):  
Epithelial Cells ◽  
Gas Phase ◽  
Airway Epithelial Cells ◽  
No Synthase ◽  
Arginase Activity ◽  
Inos Mrna ◽  
Airway Epithelial ◽  
Protein Levels ◽  
No Release ◽  
Dependent Pathway

Exhaled NO (eNO) is a potential noninvasive biomarker of inflammation in asthma. The significant intersubject variability of eNO within clinically similar patients has contributed to its limited clinical application. Arginase and NO synthase (NOS) utilize the same substrate (l-arginine) and contribute to the fibrotic and inflammatory features of asthma, respectively. Interestingly, TGF-β2 can increase the expression of arginase, stimulates fibrosis, and is overexpressed in asthma. We hypothesized that TGF-β2-enhanced arginase activity would decrease gas phase NO release from lung epithelial cells by limiting l-arginine availability for NOS. Our results show that TGF-β2 (5 ng/ml) significantly enhances total arginase activity up to two- to threefold in both primary small airway epithelial cells (SAECs) and the A549 cell line. Preincubation with TGF-β2 prior to cytokine (IL-1β, TNF-α, and IFN-γ, 10 ng/ml each) stimulation decreases gas phase NO release to baseline levels (from 1.66 ± 0.52 to 0.30 ± 0.12 pl·s−1·cm−2 and from 0.27 ± 0.03 pl·s−1·cm−2 to near zero in SAEC and A549 cells, respectively). Addition of arginase inhibitor ( Nω-hydroxy-nor-l-arginine) or small interfering RNA only partly reverses the reduction. In contrast, Rho-kinase (ROCK) pathway inhibitor (Y-27632) completely recovers the cytokine-induced NO flux in the present of TGF-β2. Inducible NO synthase (iNOS) mRNA and protein levels change in a similar trend as NO release from the cells. We conclude that TGF-β2 impacts cytokine-induced NO production in airway epithelial cells by reducing iNOS mRNA and protein levels through a ROCK-dependent pathway.

scholarly journals ACE2 Expression is elevated in Airway Epithelial Cells from aged and male donors but reduced in asthma

2020 ◽  
Author(s):  
Peter Wark ◽  
Prabuddha Pathinyake ◽  
Gerard Kaiko ◽  
Kristy Nichol ◽  
Ayesha Ali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Serine Proteases ◽  
Airway Epithelial Cells ◽  
Lower Airway ◽  
Chronic Obstructive ◽  
Airway Epithelial ◽  
Obstructive Pulmonary Disease ◽  
Protein Levels ◽  
Asthma Patients

Rationale: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter airway epithelial cells (AECs). Objective: To determine what factors are associated with ACE2 expression particularly in patients with asthma and chronic obstructive pulmonary disease (COPD). Methods: We obtained upper and lower AECs from 145 people from two independent cohorts, aged 2-89, Newcastle (n=115), and from Perth (n= 30) Australia. The Newcastle cohort was enriched with people with asthma (n=37) and COPD (n=38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry were assessed by quantitative PCR, protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AECs. Results: Increased gene expression of ACE2 was associated with older age (p=0.02) and male sex (p=0.03), but not pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma (p=0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in asthma (p=0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface was increased (p=0.02). ACE2 protein levels were lower in endobronchial biopsies from asthma patients. Conclusions: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

scholarly journals Obesity impairs therapeutic efficacy of mesenchymal stem cells by inhibiting cardiolipin-dependent mitophagy and intercellular mitochondrial transfer in mouse models of airway allergic inflammation

2021 ◽  
Author(s):  
Shakti Sagar ◽  
Md. Imam Faizan ◽  
Nisha Chaudhary ◽  
Atish Gheware ◽  
Khushboo Sharma ◽  
...  
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Human Subjects ◽  
Allergic Inflammation ◽  
Allergic Airway Inflammation ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Mitochondrial Transfer ◽  
Metabolic Conditions

Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on MSCs in general, and IMT in particular, has largely remained unexplored. This study has identified a molecular pathway that primarily governs the metabolic function and IMT of MSCs. We found underlying mitochondrial dysfunction, impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mice (MSC-Ob). Mechanistically, MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue metabolic deficits and cell death in stress-induced epithelial cells. In a small molecule screen, we found pyrroloquinoline quinone (PQQ) as a regulator of mitophagy and IMT. Long-term culture of MSC-Ob with PQQ (MSC-ObPQQ) restored cardiolipin content and sequestration of mitochondria to autophagosomes with concomitant activation of mitophagy. Upon co-culture, MSC-ObPQQ rescued cell death in stress-induced epithelial cells by enhancing IMT. The beneficial effect of PQQ was also evident in MSCs derived from human subjects in an in vitro model. In two independent mice models, the transplantation of MSC-ObPQQ restored IMT to airway epithelial cells, improved their mitochondrial metabolism and attenuated features of allergic airway inflammation (AAI). However, unmodulated MSC-Ob failed to do so. In summary, we uncover the molecular mechanism leading to the therapeutic decline of obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention.

scholarly journals Syndecan-1 Amplifies Ovalbumin-Induced Airway Remodeling by Strengthening TGFβ1/Smad3 Action

2021 ◽  
Vol 12 ◽  
Author(s):  
Dong Zhang ◽  
Xin-rui Qiao ◽  
Wen-Jing Cui ◽  
Jin-tao Zhang ◽  
Yun Pan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Remodeling ◽  
Airway Epithelial Cells ◽  
Collagen I ◽  
Chronic Asthma ◽  
Airway Epithelial ◽  
Protein Levels ◽  
Rna Targeting

Syndecan-1 (SDC-1) is a transmembrane proteoglycan of heparin sulfate that can regulate various cell signal transduction pathways in the airway epithelial cells and fibroblasts. Airway epithelial cells and human bronchial fibroblasts are crucial in airway remodeling. However, the importance of SDC-1 in the remodeling of asthmatic airways has not been confirmed yet. The present study was the first to uncover SDC-1 overexpression in the airways of humans and mice with chronic asthma. This study also validated that an increase in SDC-1 expression was correlated with TGFβ1/Smad3-mediated airway remodeling in vivo and in vitro. A small interfering RNA targeting SDC-1 (SDC-1 siRNA) and homo-SDC-1 in pcDNA3.1 (pc-SDC-1) was designed to assess the effects of SDC-1 on TGFβ1/Smad3-mediated collagen I expression in Beas-2B (airway epithelial cells) and HLF-1 (fibroblasts) cells. Downregulation of the SDC-1 expression by SDC-1 siRNA remarkably attenuated TGFβ1-induced p-Smad3 levels and collagen I expression in Beas-2B and HLF-1 cells. In addition, SDC-1 overexpression with pc-SDC-1 enhanced TGFβ1-induced p-Smad3 level and collagen I expression in Beas-2B and HLF-1 cells. Furthermore, the levels of p-Smad3 and collagen I induced by TGFβ1 were slightly increased after the addition of the recombinant human SDC-1 protein to Beas-2B and HLF-1 cells. These findings in vitro were also confirmed in a mouse model. A short hairpin RNA targeting SDC-1 (SDC-1 shRNA) to interfere with SDC-1 expression considerably reduced the levels of p-Smad3 and remodeling protein (α-SMA, collagen I) in the airways induced by ovalbumin (OVA). Similarly, OVA-induced p-Smad3 and remodeling protein levels in airways increased after mice inhalation with the recombinant mouse SDC-1 protein. These results suggested that SDC-1 of airway epithelial cells and fibroblasts plays a key role in the development of airway remodeling in OVA-induced chronic asthma.

IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury

1998 ◽  
Vol 275 (3) ◽  
pp. L524-L532 ◽  
Author(s):  
Mary Mann-Jong Chang ◽  
Reen Wu ◽  
Charles G. Plopper ◽  
Dallas M. Hyde
Keyword(s):  
Epithelial Cells ◽  
Neutralizing Antibody ◽  
Airway Epithelial Cells ◽  
Time Profile ◽  
Ozone Exposure ◽  
Neutrophil Chemotaxis ◽  
Airway Epithelial ◽  
Cell Secretion ◽  
Protein Levels

A rhesus monkey interleukin (IL)-8 cDNA clone with >94% homology to the human IL-8 gene was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. In situ hybridization and immunohistochemistry showed increased IL-8 mRNA and protein levels in epithelial cells at 1 h but not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine in acute ozone-induced airway inflammation in primates.

scholarly journals MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure

2019 ◽  
Vol 316 (2) ◽  
pp. L334-L347 ◽  
Author(s):  
John D. Dickinson ◽  
Jenea M. Sweeter ◽  
Elizabeth B. Staab ◽  
Amy J. Nelson ◽  
Kristina L. Bailey ◽  
...  
Keyword(s):  
Mucous Cell ◽  
Airway Epithelial Cells ◽  
Dust Exposure ◽  
Secretory Cells ◽  
Airway Epithelial ◽  
Organic Dust ◽  
Protein Levels ◽  
Activation Conditions ◽  
Deficient Mice

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

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