Immunohistochemical demonstration of airway epithelial cell markers of guinea pig

The guinea pig (Cavea porcellus) is a mammalian non-rodent species in the Caviidae family. The sensitivity of the respiratory system and the susceptibility to infectious diseases allows the guinea pig to be a useful model for both infectious and non-infectious lung diseases such as asthma and tuberculosis. In this report, we demonstrated for the first time, the major cell types and composition in the guinea pig airway epithelium, using cell type-specific markers by immunohistochemical staining using the commercial available immunological reagents that cross-react with guinea pig. Our results revealed the availability of antibodies cross-reacting with airway epithelial cell types of basal, non-ciliated columnar, ciliated, Clara, goblet and alveolar type II cells, as well as those cells expressing Mucin 5AC, Mucin 2, Aquaporin 4 and Calcitonin Gene Related Peptide. The distribution of these various cell types were quantified in the guinea pig airway by immunohistochemical staining and were comparable with morphometric studies using an electron microscopy assay. Moreover, this study also demonstrated that goblet cells are the main secretory cell type in the guinea pig's airway, distinguishing this species from rats and mice. These results provide useful information for the understanding of airway epithelial cell biology and mechanisms of epithelial–immune integration in guinea pig models.     

Age differences in cholinergic airway responsiveness in relation with muscarinic receptor subtypes

Children seem more susceptible to increased airway reactivity than adults. Such an age-dependent discrepancy in airway reactivity may involve different airway smooth muscle functions. Therefore, we compared the in vivo and in vitro responsiveness of airway smooth muscles between two age groups of animals. Rats of 6 and 21 weeks old were challenged in vivo with acetylcholine (ACh) infused intravenously and airway resistance (R(aw)) was measured. Tracheal muscle was also isolated and the isometric force developed to ACh or KCl was measured. Furthermore, the level of genes encoding muscarinic receptor subtypes (M(1-3)) and acetylcholinesterase (AChE) expressed in the tracheal muscle was determined by RT-PCR. In results, the basal R(aw) was similar in the two age groups. The R(aw) at each ACh dose was significantly greater in young rats than older rats (p<0.05, n=22-27). Tracheal muscles from young rats were more sensitive to ACh than older rats (p<0.05, n=20-21), while receptor-independent muscle contraction to KCl was greater in older rats (p<0.05, n=10-19). Genes encoding AChE, M(2) and M(3) muscarinic receptors were more highly expressed in the tracheal muscles from young than older rats (p<0.05, n=4-6). In conclusion, airway smooth muscle in young rat is more sensitive to cholinergic stimulation in vivo and in vitro compared to older rats, which may be due to a higher expression of M(2) and M(3) muscarinic receptors in airway smooth muscle.     

Effects of airway surface liquid pH on host defense in cystic fibrosis

Cystic fibrosis is a lethal genetic disorder characterized by viscous mucus and bacterial colonization of the airways. Airway surface liquid represents a first line of pulmonary defense. Studies in humans and animal models of cystic fibrosis indicate that the pH of airway surface liquid is reduced in the absence of cystic fibrosis transmembrane conductance regulator function. Many aspects of the innate host defense system of the airways are pH sensitive, including antimicrobial peptide/protein activity, the rheological properties of secreted mucins, mucociliary clearance, and the activity of proteases. This review will focus on how changes in airway surface liquid pH may contribute to the host defense defect in cystic fibrosis soon after birth. Understanding how changes in pH impact mucosal immunity may lead to new therapies that can modify the airway surface liquid environment, improve airway defenses, and alter the disease course.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Delay of airway epithelial wound repair in COPD is associated with airflow obstruction severity

Background

Airway epithelium integrity is essential to maintain its role of mechanical and functional barrier. Recurrent epithelial injuries require a complex mechanism of repair to restore its integrity. In chronic obstructive pulmonary disease (COPD), an abnormal airway epithelial repair may participate in airway remodeling. The objective was to determine if airway epithelial wound repair of airway epithelium is abnormal in COPD.

Methods

Patients scheduled for lung resection were prospectively recruited. Demographic, clinical data and pulmonary function tests results were recorded. Emphysema was visually scored and histological remodeling features were noted. Primary bronchial epithelial cells (BEC) were extracted and cultured for wound closure assay. We determined the mean speed of wound closure (MSWC) and cell proliferation index, matrix metalloprotease (MMP)-2, MMP-9 and cytokines levels in supernatants of BEC 18 hours after cell wounding. In a subset of patients, bronchiolar epithelial cells were also cultured for wound closure assay for MSWC analyze.

Results

13 COPD and 7 non COPD patients were included. The severity of airflow obstruction and the severity of emphysema were associated with a lower MSWC in BEC (p = 0.01, 95% CI [0.15-0.80]; p = 0.04, 95% CI [−0.77;-0.03] respectively). Cell proliferation index was decreased in COPD patients (19 ± 6% in COPD vs 27 ± 3% in non COPD, p = 0.04). The severity of COPD was associated with a lower level of MMP-2 (7.8 ± 2 10⁵ AU in COPD GOLD D vs 12.8 ± 0.13 10⁵ AU in COPD GOLD A, p = 0.04) and a lower level of IL-4 (p = 0.03, 95% CI [0.09;0.87]). Moreover, higher levels of IL-4 and IL-2 were associated with a higher MSWC (p = 0.01, 95% CI [0.17;0.89] and p = 0.02, 95% CI [0.09;0.87] respectively). Clinical characteristics and smoking history were not associated with MSWC, cell proliferation index or MMP and cytokines levels. Finally, we showed an association of the MSWC of bronchial and corresponding bronchiolar epithelial cells obtained from the same patients (p = 0.02, 95% CI [0.12;0.89]).

Conclusion

Our results showed an abnormal bronchial epithelial wound closure process in severe COPD. Further studies are needed to elucidate the contribution and the regulation of this mechanism in the complex pathophysiology of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0151-9) contains supplementary material, which is available to authorized users.     

Gene expression in whole lung and pulmonary macrophages reflects the dynamic pathology associated with airway surface dehydration

Background

Defects in airway mucosal defense, including decreased mucus clearance, contribute to the pathogenesis of human chronic obstructive pulmonary diseases. Scnn1b-Tg mice, which exhibit chronic airway surface dehydration from birth, can be used as a model to study the pathogenesis of muco-obstructive lung disease across developmental stages. To identify molecular signatures associated with obstructive lung disease in this model, gene expression analyses were performed on whole lung and purified lung macrophages collected from Scnn1b-Tg and wild-type (WT) littermates at four pathologically relevant time points. Macrophage gene expression at 6 weeks was evaluated in mice from a germ-free environment to understand the contribution of microbes to disease development.

Results

Development- and disease-specific shifts in gene expression related to Scnn1b over-expression were revealed in longitudinal analyses. While the total number of transgene-related differentially expressed genes producing robust signals was relatively small in whole lung (n = 84), Gene Set Enrichment Analysis (GSEA) revealed significantly perturbed biological pathways and interactions between normal lung development and disease initiation/progression. Purified lung macrophages from Scnn1b-Tg mice exhibited numerous robust and dynamic gene expression changes. The expression levels of Classically-activated (M1) macrophage signatures were significantly altered at post-natal day (PND) 3 when Scnn1b-Tg mice lung exhibit spontaneous bacterial infections, while alternatively-activated (M2) macrophage signatures were more prominent by PND 42, producing a mixed M1-M2 activation profile. While differentially-regulated, inflammation-related genes were consistently identified in both tissues in Scnn1b-Tg mice, there was little overlap between tissues or across time, highlighting time- and tissue-specific responses. Macrophages purified from adult germ-free Scnn1b-Tg mice exhibited signatures remarkably similar to non-germ-free counterparts, indicating that the late-phase macrophage activation profile was not microbe-dependent.

Conclusions

Whole lung and pulmonary macrophages respond independently and dynamically to local stresses associated with airway mucus stasis. Disease-specific responses interact with normal developmental processes, influencing the final state of disease in this model. The robust signatures observed in Scnn1b-Tg lung macrophages highlight their critical role in disease pathogenesis. These studies emphasize the importance of region-, cell-type-, and time-dependent analyses to fully dissect the natural history of disease and the consequences of disease on normal lung development.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-726) contains supplementary material, which is available to authorized users.     

Tryptase activates TGFbeta in human airway smooth muscle cells via direct proteolysis

Transforming growth factor β (TGFβ) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFβ bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFβ from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFβ activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFβ activation without affecting levels of total TGFβ. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH₂. Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFβ via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.     

Lycopene suppresses ovalbumin-induced airway inflammation in a murine model of asthma

In this study, we attempt to determine whether lycopene regulates inflammatory mediators in the ovalbumin (OVA)-induced murine asthma model. To address this, mice were sensitized and challenged with OVA, and then treated with lycopene before the last OVA challenge. Administration of lycopene significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Administration of lycopene also resulted in a significant inhibition of the infiltration of inflammatory immunocytes into the bronchoalveolar lavage, and attenuated the gelatinolytic activity of matrix metalloproteinase-9 and the expression of eosinophil peroxidase. Additionally, lycopene reduced the increased levels of GATA-3 mRNA level and IL-4 expression in OVA-challenged mice. However, it increased T-bet mRNA level and IFN-γ expression in lycopene-challenged mice. These findings provide new insight into the immunopharmacological role of lycopene in terms of its effects in a murine model of asthma.     

The proximal airway of the bat Tadarida brasiliensis: a minimum entropy production design

The bronchial tree of most mammalian lungs is a good example of an efficient distribution system whose geometry and dimensions of branched structures are important factors in determining the efficiency of respiration. Small and flying endothermic animals have high-energy requirements, requiring morphological and physiological adaptations to reduce energy loss. Here we show that Tadarida brasiliensis, a nocturnal small bat whose energy requirements are exacerbated by this small size and by their frequent exposure to high altitude, has a different morphology in the proximal airway, sustained by a wider trachea and better scaling factors, than other non-flying mammals. This design allows a great decrease of the volume specific resistance of the proximal airway and in consequence a very low entropy production during breathing, approximately 1/18 of that expected for a non-flying mammals of similar body size.