Am J Respir Cell Mol Biol. 2016 Apr;54(4):562-73. doi: 10.1165/rcmb.2015-0210OC.

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins.

 

Lynn TM1, Molloy EL1, Masterson JC2, Glynn SF3, Costello RW3, Avdalovic MV4, Schelegle ES4, Miller LA4, Hyde DM4, O’Dea S1.

  • 1 Biology Department, Maynooth University, County Kildare, Ireland.
  • 2 Department of Pediatrics, University of Colorado Denver, Aurora, Colorado.
  • 3 Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland.
  • 4 California National Primate Research Center, University of California, Davis, School of Veterinary Medicine, Davis, California.

 

Abstract

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P < 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P < 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways.

KEYWORDS: BMP; airways; asthma; homeostasis; repair

PMID: 26414797

 

Supplement:

The architecture of the lungs and the epithelial cells types that line the airways change in the descending respiratory tract to reflect specialised functions therein. The trachea and bronchi are lined with ciliated and secretory cells in the form of a pseudostratified epithelium which aid the clearance of inhaled pathogens. At the terminal end of the bronchioles, a single layer of airway epithelial cells (AEC) type I and II line the alveolar sacs. These cells facilitate the movement of gases between the airways and the blood 1. The establishment of the complex architecture of the lung and these distinct cell niches occurs during lung formation in the womb and is tightly controlled by a complex interplay between different signalling molecules, growth factors and morphogens such as Fibroblast Growth Factors (FGF), Wnt and Bone Morphogenetic Proteins (BMP).

Our group is specifically interested in the expression of BMP signalling in the airways. BMPs are a highly conserved group of signalling molecules and are members of the transforming growth factor (TGF) superfamily2. We know from seminal studies in the field that BMP-4 is expressed at varying concentrations along the developing airways and is responsible for co-ordinating correct branching morphogenesis and epithelial cell differentiation throughout the respiratory axis3,4. These studies highlighted a link between regionalised expression of BMP4 and specialised airway epithelial function. Despite the importance of localised expression of BMP4 during lung formation during development, the concept of BMP expression gradients in adult airways remains poorly explored.

The airways are constantly challenged by irritants, pathogens and other disease-causing agents. These can cause injury to the airways inducing a number of local cellular responses such as the recruitment of inflammatory mediators and the release of inflammatory cytokines as the lungs attempt to remove the pathogen and restore normal airway function. The signalling pathways involved in lung morphogenesis, including BMPs, are believed to be reactivated during this process and are involved in orchestrating the cell responses. Previous work in our lab showed that BMP signalling is activated during repair in rodent models of airway injury using nitronaphthalene exposure. The results showed that an increase in BMP signalling in airway epithelial cells (AECs) coincides with airway inflammation5. Furthermore, using primary AECs in a model of airway injury, we showed that BMP signalling is involved in promoting epithelial cell migration during airway restitution and co-ordinates the movement of epithelial cells to repair the injured site by inducing epithelial-mesenchymal-transition (EMT) 6. These studies highlighted the role of BMP signalling in airway injury, inflammation and restitution. BMP signalling is also activated in lung stem cell niches following injury, highlighting the importance of the BMP pathway in epithelial cell differentiation and proliferation during repair7.

In the present study we explored the concept of BMP expression gradients in adult airways. We hypothesized that a BMP signalling gradient exists in healthy adult airways and this gradient is altered during injury, repair and in chronic inflammatory disease. Furthermore, we were interested in the potential interplay between BMP signalling and the local inflammatory response.

In the first part of this work we used rhesus macaque monkeys as a model of healthy airways. We examined BMP signalling expression in the descending left cranial bronchus of 6 and 12 month old monkeys by immunofluorescence. The level of expression was then quantified using stereology.  As outlined in the paper, membrane localised BMPRIa and nuclear pSMAD 1/5/8 was present in both groups of healthy monkeys. In addition, a gradient in pSMAD 1/5/8 expression was found along the airways of healthy 12 month old rhesus macaques (n=3, p<0.005). These results indicated that BMP signalling is active in healthy adult airways.

We then examined BMP signalling in an established model of allergic airway disease in rhesus macaques, a model that displays the hallmarks of human asthma.  Monkeys were exposed to cyclic doses of house dust mite allergen (HDMA) and ozone which causes an increased levels of allergen-specific antibodies in their blood and higher numbers of inflammatory cells (eosinophils) in washings taken from the airways8. In addition, the monkeys displayed rapid shallow breathing and a cough during aerosol challenge. Furthermore, they displayed thickened basement membrane zone in the airways with increased eosinophil accumulation in the conducting airways and displayed higher numbers of mucus cells in the airways 8,9.

As outlined in the paper, exposure to HDMA and ozone caused significant downregulation of nuclear pSMAD 1/5/8 in the epithelium of the upper airways. When the animals were provided with a recovery period in filtered air, nuclear PCNA, pSMAD 1/5/8 and membrane-localised BMPRIa expression were all significantly increased in the epithelium of conducting airways (p<0.005). These results support the hypothesise that the repeated exposure model causing allergic airway disease alters the basic growth factor patterns that co-ordinate correct respiratory tract formation and repair and epithelial cell distribution8.

In addition to reduced pSMAD 1/5/8, nuclear localisation of BMPRIa was evident in the upper airway epithelium of the asthma group. Alongside extensive airway remodelling and reduced airway functionality, infiltrating inflammatory cells (eosinophils) were previously shown to be significantly elevated in the conducting airways and distal bronchioles of these monkeys 8. Many studies have shown that eosinophils and specifically, their secreted proteins, play a central role in airway remodelling, contributing to airway hyper-responsiveness in asthma and inflammatory airway diseases. The signalling pathways governing these events remain poorly understood 10–12. We hypothesised a link between these inflammatory cells and the altered BMPRIa expression evident in our results.

To examine this further we applied eosinophilic proteins to cultured mouse AECs and studied the trafficking of BMPRIa between the membrane and the nucleus, as outlined in the paper. We found that the eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) caused transient nuclear translocation of membrane-bound BMPRIa.

Based on these findings we suggest that the reduced level of BMP signalling present in the asthmatic monkeys is due to eosinophil-induced nuclear translocation of membrane-bound BMPRIa, as outlined in Figure 1. Subsequent inhibition of downstream BMP signalling pathways may interfere with appropriate epithelial repair processes mediated by BMP and its signalling partners13. Abnormal signalling processes could contribute to a chronic disease state promoting incorrect epithelial turnover, an inflammatory phenotype and epithelial remodelling, all of which are well-established hallmarks of asthma. While BMP receptor processing and trafficking between the nucleus and the membrane are essential for correct signal transduction, incorrect receptor trafficking has been shown to contribute to disease, as evidenced by BMPRII mutations in pulmonary hypertension14,15.

The importance of this paper are two-fold. Firstly, we demonstrate that BMP signalling is active in the healthy airways of juvenile non-human primate airways and that a signalling gradient exists in the airways. This indicates an important role for BMP signalling in maintaining adult airway homeostasis in addition to its role during lung morphogenesis. In addition, we have shown that the BMP signalling pathway is altered during chronic inflammatory disease. This could be contributing to the diseased phenotype of the airways. By subsequently treating AECs with eosinophil-derived proteins, we have highlighted a potential novel mechanistic relationship between eosinophils and BMP receptor trafficking in asthma.

 

 

fig1

Figure 1: Hypothesis of eosinophil-induced transient trafficking of BMPRIa

Healthy airway epithelial cells express BMPRIa at the membrane and normal homeostatic BMP signalling occurs via downstream activation of pSMAD 1/5/8. Following airway insult and the induction of an inflammatory response in the airway epithelium, infiltrating eosinophils are recruited to the site which illicit their response by releasing cationic proteins. Either by binding BMPRIa directly or inducing BMP ligands, eosinophilic cationic proteins potentially induce BMPRIa nuclear-translocation and the BMP signalling pathway is disrupted. Reduced BMP signalling occurs which in turn affects cell proliferation and epithelium remodelling events. Removal of the pathogen facilitates the restoration of membrane bound BMPRIa signalling and activation of normal BMP signalling events. This encourages active epithelial repair and restoration of epithelial integrity.

 

 

References:

  1. Chang, M. M., Shih, L. & Wu, R. Pulmonary Epithelium : Cell Types. (2008).
  2. Miyazono, K., Kamiya, Y. & Morikawa, M. Bone morphogenetic protein receptors and signal transduction. J. Biochem. 147, 35–51 (2010).
  3. Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. & Hogan, B. L. Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122, 1693–702 (1996).
  4. Weaver, M., Yingling, J. M., Dunn, N. R., Bellusci, S. & Hogan, B. L. Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 126, 4005–15 (1999).
  5. Masterson, J. C. et al. Bone morphogenetic protein signalling in airway epithelial cells during regeneration. Cell. Signal. 23, 398–406 (2011).
  6. McCormack, N., Molloy, E. L. & ODea, S. Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium. Respir. Res. 14, 1 (2013).
  7. Sountoulidis, A. et al. Activation of the canonical bone morphogenetic protein (BMP) pathway during lung morphogenesis and adult lung tissue repair. PLoS One 7, e41460 (2012).
  8. Schelegle, E. S. et al. Repeated episodes of ozone inhalation attenuates airway injury/repair and release of substance P, but not adaptation. Toxicol. Appl. Pharmacol. 186, 127–142 (2003).
  9. Van Winkle, L. S. et al. Epithelial cell distribution and abundance in rhesus monkey airways during postnatal lung growth and development. J. Appl. Physiol. 97, 2355–63; discussion 2354 (2004).
  10. Humbles, A. A. et al. A critical role for eosinophils in allergic airways remodeling. Science 305, 1776–9 (2004).
  11. Costello, Glynn, S., Walsh, M. & RW. Eosinophil Mediated Airway Remodelling and the Bone Morphogenetic Protein (BMP) Pathway in Asthma. Am. Thorac. Soc. Int. Conf. Meet. Abstr. (2009). at <http://www.atsjournals.org/doi/abs/10.1164/ajrccm-conference.2009.179.1_MeetingAbstracts.A6325>
  12. Gleich, G. J. & Adolphson, C. Bronchial hyperreactivity and eosinophil granule proteins. Agents Actions. Suppl. 43, 223–30 (1993).
  13. Desai, T. J. & Cardoso, W. V. Growth factors in lung development and disease: friends or foe? Respir. Res. 3, 2 (2002).
  14. Lane, K. B. et al. Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat. Genet. 26, 81–4 (2000).
  15. Gleason, R. J., Akintobi, A. M., Grant, B. D. & Padgett, R. W. BMP signaling requires retromer-dependent recycling of the type I receptor. Proc. Natl. Acad. Sci. U. S. A. 111, 2578–83 (2014).

 

 

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