EDITORIALS
Measuring Mucociliary Transport and Mucus Properties in Multiple Regions of Airway Epithelial Surfaces Helps Clarify Cystic Fibrosis Defects
364
tracking (PT) revealed increased recovery times and reduced particle movement in CF mucus, indicating elevated viscosity (Figure 1B).
A Airway lumen Airway lumen MCT
Rostral PCL
ASL
CBF epithelial cells lamina propria
CF vs. control
B
PCL ASL Piglet CBF Tracheas & HBE cultures MCT PT FRAP
↓ ↓
MCT vs. PCL
C MCT (microns/sec)
Cystic fibrosis (CF) lung disease occurs when mutations disable CF transmembrane conductance regulator (CFTR), an ion channel mediating Cl2 and HCO32 transport across epithelia. Unlike some gastrointestinal organs that are abnormal at birth, the lungs of humans and CF animal models appear to be healthy, although modest structural differences emerge under scrutiny (1). This near-normal appearance belies fundamental differences in ion transport that are seen in humans and animal models: CFTRdependent, anion-mediated fluid secretion from surface epithelia and glands is reduced (2, 3). These transport defects cause CF mucus properties to differ: mucus is reduced in volume (3), has higher solids and viscosity (4), is more acidic (5), and is less able to kill bacteria (6). In addition, mucus clearance via ciliary action and cough, which may be the most important innate defense mechanism of the airways (7), also appears to be slowed. To repeat, all of these defects appear to arise from reduced anion-mediated fluid secretion, with reduced HCO32 secretion playing a critical role (8–10). The primary defects in CF mucus quickly lead to bacterial lung infections, which become chronic under present standards of care. Chronic infections drastically alter the milieu of CF airways, giving rise to purulent sputum that is dominated by bacterial loads that can reach 5.9 3 1012 cfu/ml (11) as well as very high leukocyte numbers, producing a vile witch’s brew of products from dead, injured, and inflamed cells. The analysis of this material has preoccupied many CF workers for decades, and this focus is arguably one reason why progress in mapping out the links between the loss of CFTR function and lung disease has been retarded. Surprisingly, in spite of the widespread conviction that mucus clearance is disrupted in people with CF, quantitative evidence that this is a primary rather than an acquired defect has been controversial, given the difficulties of working with human infants, the rapid onset of infections, and the drastic changes in sputum properties that quickly ensue. This knowledge gap is now being addressed by studying airway disease in a growing set of CF animal models. The article by Birket and colleagues in this issue of the Journal (pp. 421–432) exemplifies this approach by applying the powerful new method of microoptical coherence tomography (mOCT) to visualize details of the airway surface and mucus transport in CF and control ex vivo piglet tracheas and cultured human bronchial epithelial cells (HBEs); these studies were reinforced by studies of adult pig tracheas treated with inhibitors of Cl2 and HCO32 transport (12). Figure 1A diagrams four airway features that were quantified with mOCT: (1) the depth of periciliary liquid (PCL) = maximal cilia height, (2) depth of airway surface liquid (ASL) = PCL 1 mucus layer, (3) ciliary beat frequency (CBF), and (4) mucociliary transport (MCT). All of these measures were reduced in the CF piglet tracheas and HBE cultures. These results are consistent with the idea that less fluid is being secreted by the CF cells. Also, fluorescence recovery from photobleaching and particle
15 10 Control
CF
5 0 0
5
10
15
PCL height (microns)
D
Anion inhibition
Adult pig tracheas
PCL* MCT PT
↓
Figure 1. (A) Four anatomical features of airway surface were measured. (B) In two preparations, all four features were reduced in cystic fibrosis (CF); two measures of mucus viscosity were increased. (C) When small periciliary layer (PCL) and mucociliary transport (MCT) were coordinately measured in small regions, CF showed an inverse relationship. (D) Inhibition of HCO32 or Cl2 secretion caused equivalent reductions in PCL, MCT, and particle movement within the mucus, but regional correlations for PCL and MCT after HCO32 inhibition showed a pattern like CF with slower MCT at higher PCL (indicated by *). ASL = airway surface liquid; CBF = ciliary beat frequency; FRAP = fluorescence recovery after photobleaching; HBE = human bronchial epithelia cells; PT = particle tracking.
American Journal of Respiratory and Critical Care Medicine Volume 190 Number 4 | August 15 2014
EDITORIALS When the average values for each trachea were supplemented with correlated measures of MCT and PCL depth in localized regions of the trachea (a new metric made possible by mOCT), a striking reversal was seen between CF and control pigs in the relation between these values. Normally, increased fluid on the airway surface would be expected to increase MCT. That expectation was confirmed in control piglets, but the opposite was observed in the CF piglets (Figure 1C). Why should this be? One possibility is that the mucus viscosity was increased in spite of increased volume because it lacked HCO32, consistent with the hypothesis advanced by Paul Quinton that proper expansion of mucin molecules requires HCO32 (10). To explore the relations between anion secretion and MCT, adult pig tracheas were studied with and without inhibitors of HCO32 or Cl2 secretion. In these experiments, only PCL depth, MCT, and PT measures of viscosity were reported. PT showed large and nearly equivalent increases in viscosities after either HCO32 or Cl2 was inhibited. Also, inhibition of either anion produced similar large reductions in MCT. Average PCL depths were decreased by z9 and 18% by inhibition of HCO32 or Cl2, respectively (the 9% reduction was not significant). Thus, these results do not discriminate between the anions. However, once again, mOCT sampling of multiple small regions of the adult tracheas showed a negative correlation between PCL depth and MCT at those specific regions for 4,49-dinitrostilbene-2,29-disulfonic acid–inhibited tracheas, whereas there was no correlation between PCL depth and MCT in controls or bumetanide-treated tracheas. Although the negative correlation was weak, it gains interest because it is unexpected and bolsters what was observed in CF piglets. This work is impressive for its range of methods, the use of three different preparations, and especially for the innovative correlative measurements at multiple airway regions using mOCT. Given the innovative approaches, it is natural that new questions arise. For example, it would have been interesting to see how ASL depth and CBF correlated with MCT after inhibition of Cl2 and HCO32, but these data were not reported. In Figure 6 of Birket and colleagues (12) the intriguing observation is given in passing that bumetanide virtually abolished ASL, which would have been predicted to greatly slow MCT. Indeed, just this result was recently reported for ex vivo ferret tracheas in which MCT had been stimulated with forskolin (13). The importance of the novel approach of measuring PCL, ASL, CBF, and MCT in discrete, localized regions of tracheas is evident, because it provided the only data to support the hypothesis that altered mucus properties and not reduced mucus volume alone contributed to slowing of MCT. Because so much rests on the microsampling method, future work will require more careful consideration of sampling methodology, statistical treatments of multiple measures, and data presentation. For example, it was noted in passing that the mucus layer was thicker near glands, raising the possibility
Editorials
that proximity to glands rather than mucus layer depth is a factor of importance. n Author disclosures are available with the text of this article at www.atsjournals.org. Jeffrey J. Wine, Ph.D. Cystic Fibrosis Research Laboratory Stanford University Stanford, California
References 1. Meyerholz DK, Stoltz DA, Namati E, Ramachandran S, Pezzulo AA, Smith AR, Rector MV, Suter MJ, Kao S, McLennan G, et al. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Respir Crit Care Med 2010; 182:1251–1261. 2. Chen JH, Stoltz DA, Karp PH, Ernst SE, Pezzulo AA, Moninger TO, Rector MV, Reznikov LR, Launspach JL, Chaloner K, et al. Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia. Cell 2010;143: 911–923. 3. Joo NS, Cho HJ, Khansaheb M, Wine JJ. Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs. J Clin Invest 2010;120:3161–3166. 4. Jayaraman S, Joo NS, Reitz B, Wine JJ, Verkman AS. Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na(1)] and pH but elevated viscosity. Proc Natl Acad Sci USA 2001;98:8119–8123. 5. Song Y, Salinas D, Nielson DW, Verkman AS. Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis. Am J Physiol Cell Physiol 2006;290:C741–C749. 6. Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MH, Ramachandran S, Moninger TO, Karp PH, Wohlford-Lenane CL, Haagsman HP, van Eijk M, et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 2012;487:109–113. 7. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002; 109:571–577. 8. Yang N, Garcia MA, Quinton PM. Normal mucus formation requires cAMP-dependent HCO3- secretion and Ca21-mediated mucin exocytosis. J Physiol 2013;591:4581–4593. 9. Quinton PM. Birth of mucus. Am J Physiol Lung Cell Mol Physiol 2010; 298:L13–L14. 10. Quinton PM. Role of epithelial HCO3⁻ transport in mucin secretion: lessons from cystic fibrosis. Am J Physiol Cell Physiol 2010;299: C1222–C1233. 11. Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TW, Carroll MP, Hoffman LR, Jones G, Allen CE, Patel N, et al. Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation? J Cyst Fibros 2011;10:357–365. 12. Birket SE, Chu KK, Liu L, Houser GH, Diephuis BJ, Wilsterman EJ, Dierksen G, Mazur M, Shastry S, Li Y, et al. A functional anatomic defect of the cystic fibrosis airway. Am J Respir Crit Care Med 2014;190:421–432. 13. Jeong JH, Joo NS, Hwang PH, Wine JJ. Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea. Am J Physiol Lung Cell Mol Physiol 2014;307:L83–L93.
Copyright © 2014 by the American Thoracic Society
365
Measuring Mucociliary Transport and Mucus Properties in Multiple Regions of Airway Epithelial Surfaces Helps Clarify Cystic Fibrosis Defects
364
tracking (PT) revealed increased recovery times and reduced particle movement in CF mucus, indicating elevated viscosity (Figure 1B).
A Airway lumen Airway lumen MCT
Rostral PCL
ASL
CBF epithelial cells lamina propria
CF vs. control
B
PCL ASL Piglet CBF Tracheas & HBE cultures MCT PT FRAP
↓ ↓
MCT vs. PCL
C MCT (microns/sec)
Cystic fibrosis (CF) lung disease occurs when mutations disable CF transmembrane conductance regulator (CFTR), an ion channel mediating Cl2 and HCO32 transport across epithelia. Unlike some gastrointestinal organs that are abnormal at birth, the lungs of humans and CF animal models appear to be healthy, although modest structural differences emerge under scrutiny (1). This near-normal appearance belies fundamental differences in ion transport that are seen in humans and animal models: CFTRdependent, anion-mediated fluid secretion from surface epithelia and glands is reduced (2, 3). These transport defects cause CF mucus properties to differ: mucus is reduced in volume (3), has higher solids and viscosity (4), is more acidic (5), and is less able to kill bacteria (6). In addition, mucus clearance via ciliary action and cough, which may be the most important innate defense mechanism of the airways (7), also appears to be slowed. To repeat, all of these defects appear to arise from reduced anion-mediated fluid secretion, with reduced HCO32 secretion playing a critical role (8–10). The primary defects in CF mucus quickly lead to bacterial lung infections, which become chronic under present standards of care. Chronic infections drastically alter the milieu of CF airways, giving rise to purulent sputum that is dominated by bacterial loads that can reach 5.9 3 1012 cfu/ml (11) as well as very high leukocyte numbers, producing a vile witch’s brew of products from dead, injured, and inflamed cells. The analysis of this material has preoccupied many CF workers for decades, and this focus is arguably one reason why progress in mapping out the links between the loss of CFTR function and lung disease has been retarded. Surprisingly, in spite of the widespread conviction that mucus clearance is disrupted in people with CF, quantitative evidence that this is a primary rather than an acquired defect has been controversial, given the difficulties of working with human infants, the rapid onset of infections, and the drastic changes in sputum properties that quickly ensue. This knowledge gap is now being addressed by studying airway disease in a growing set of CF animal models. The article by Birket and colleagues in this issue of the Journal (pp. 421–432) exemplifies this approach by applying the powerful new method of microoptical coherence tomography (mOCT) to visualize details of the airway surface and mucus transport in CF and control ex vivo piglet tracheas and cultured human bronchial epithelial cells (HBEs); these studies were reinforced by studies of adult pig tracheas treated with inhibitors of Cl2 and HCO32 transport (12). Figure 1A diagrams four airway features that were quantified with mOCT: (1) the depth of periciliary liquid (PCL) = maximal cilia height, (2) depth of airway surface liquid (ASL) = PCL 1 mucus layer, (3) ciliary beat frequency (CBF), and (4) mucociliary transport (MCT). All of these measures were reduced in the CF piglet tracheas and HBE cultures. These results are consistent with the idea that less fluid is being secreted by the CF cells. Also, fluorescence recovery from photobleaching and particle
15 10 Control
CF
5 0 0
5
10
15
PCL height (microns)
D
Anion inhibition
Adult pig tracheas
PCL* MCT PT
↓
Figure 1. (A) Four anatomical features of airway surface were measured. (B) In two preparations, all four features were reduced in cystic fibrosis (CF); two measures of mucus viscosity were increased. (C) When small periciliary layer (PCL) and mucociliary transport (MCT) were coordinately measured in small regions, CF showed an inverse relationship. (D) Inhibition of HCO32 or Cl2 secretion caused equivalent reductions in PCL, MCT, and particle movement within the mucus, but regional correlations for PCL and MCT after HCO32 inhibition showed a pattern like CF with slower MCT at higher PCL (indicated by *). ASL = airway surface liquid; CBF = ciliary beat frequency; FRAP = fluorescence recovery after photobleaching; HBE = human bronchial epithelia cells; PT = particle tracking.
American Journal of Respiratory and Critical Care Medicine Volume 190 Number 4 | August 15 2014
EDITORIALS When the average values for each trachea were supplemented with correlated measures of MCT and PCL depth in localized regions of the trachea (a new metric made possible by mOCT), a striking reversal was seen between CF and control pigs in the relation between these values. Normally, increased fluid on the airway surface would be expected to increase MCT. That expectation was confirmed in control piglets, but the opposite was observed in the CF piglets (Figure 1C). Why should this be? One possibility is that the mucus viscosity was increased in spite of increased volume because it lacked HCO32, consistent with the hypothesis advanced by Paul Quinton that proper expansion of mucin molecules requires HCO32 (10). To explore the relations between anion secretion and MCT, adult pig tracheas were studied with and without inhibitors of HCO32 or Cl2 secretion. In these experiments, only PCL depth, MCT, and PT measures of viscosity were reported. PT showed large and nearly equivalent increases in viscosities after either HCO32 or Cl2 was inhibited. Also, inhibition of either anion produced similar large reductions in MCT. Average PCL depths were decreased by z9 and 18% by inhibition of HCO32 or Cl2, respectively (the 9% reduction was not significant). Thus, these results do not discriminate between the anions. However, once again, mOCT sampling of multiple small regions of the adult tracheas showed a negative correlation between PCL depth and MCT at those specific regions for 4,49-dinitrostilbene-2,29-disulfonic acid–inhibited tracheas, whereas there was no correlation between PCL depth and MCT in controls or bumetanide-treated tracheas. Although the negative correlation was weak, it gains interest because it is unexpected and bolsters what was observed in CF piglets. This work is impressive for its range of methods, the use of three different preparations, and especially for the innovative correlative measurements at multiple airway regions using mOCT. Given the innovative approaches, it is natural that new questions arise. For example, it would have been interesting to see how ASL depth and CBF correlated with MCT after inhibition of Cl2 and HCO32, but these data were not reported. In Figure 6 of Birket and colleagues (12) the intriguing observation is given in passing that bumetanide virtually abolished ASL, which would have been predicted to greatly slow MCT. Indeed, just this result was recently reported for ex vivo ferret tracheas in which MCT had been stimulated with forskolin (13). The importance of the novel approach of measuring PCL, ASL, CBF, and MCT in discrete, localized regions of tracheas is evident, because it provided the only data to support the hypothesis that altered mucus properties and not reduced mucus volume alone contributed to slowing of MCT. Because so much rests on the microsampling method, future work will require more careful consideration of sampling methodology, statistical treatments of multiple measures, and data presentation. For example, it was noted in passing that the mucus layer was thicker near glands, raising the possibility
Editorials
that proximity to glands rather than mucus layer depth is a factor of importance. n Author disclosures are available with the text of this article at www.atsjournals.org. Jeffrey J. Wine, Ph.D. Cystic Fibrosis Research Laboratory Stanford University Stanford, California
References 1. Meyerholz DK, Stoltz DA, Namati E, Ramachandran S, Pezzulo AA, Smith AR, Rector MV, Suter MJ, Kao S, McLennan G, et al. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Respir Crit Care Med 2010; 182:1251–1261. 2. Chen JH, Stoltz DA, Karp PH, Ernst SE, Pezzulo AA, Moninger TO, Rector MV, Reznikov LR, Launspach JL, Chaloner K, et al. Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia. Cell 2010;143: 911–923. 3. Joo NS, Cho HJ, Khansaheb M, Wine JJ. Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs. J Clin Invest 2010;120:3161–3166. 4. Jayaraman S, Joo NS, Reitz B, Wine JJ, Verkman AS. Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na(1)] and pH but elevated viscosity. Proc Natl Acad Sci USA 2001;98:8119–8123. 5. Song Y, Salinas D, Nielson DW, Verkman AS. Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis. Am J Physiol Cell Physiol 2006;290:C741–C749. 6. Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MH, Ramachandran S, Moninger TO, Karp PH, Wohlford-Lenane CL, Haagsman HP, van Eijk M, et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 2012;487:109–113. 7. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002; 109:571–577. 8. Yang N, Garcia MA, Quinton PM. Normal mucus formation requires cAMP-dependent HCO3- secretion and Ca21-mediated mucin exocytosis. J Physiol 2013;591:4581–4593. 9. Quinton PM. Birth of mucus. Am J Physiol Lung Cell Mol Physiol 2010; 298:L13–L14. 10. Quinton PM. Role of epithelial HCO3⁻ transport in mucin secretion: lessons from cystic fibrosis. Am J Physiol Cell Physiol 2010;299: C1222–C1233. 11. Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TW, Carroll MP, Hoffman LR, Jones G, Allen CE, Patel N, et al. Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation? J Cyst Fibros 2011;10:357–365. 12. Birket SE, Chu KK, Liu L, Houser GH, Diephuis BJ, Wilsterman EJ, Dierksen G, Mazur M, Shastry S, Li Y, et al. A functional anatomic defect of the cystic fibrosis airway. Am J Respir Crit Care Med 2014;190:421–432. 13. Jeong JH, Joo NS, Hwang PH, Wine JJ. Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea. Am J Physiol Lung Cell Mol Physiol 2014;307:L83–L93.
Copyright © 2014 by the American Thoracic Society
365
