Preclinical studies underlined the role of CCSP in the response of lung epithelium to ischemia and reperfusion. In vitro, CCSP protein increased epithelial cell proliferation while protecting against cell death by oxidative stress.17 In vivo, CCSP deprivation has been associated with an increased susceptibility to oxidative stress.18 The first clinical study exploring the role of CCSP after LT focused on bronchiolitis obliterans syndrome (BOS), the most frequent pattern of CLAD characterized by a fibrosing process of the small airways causing irreversible airway obstruction. After 22 LT recipients over 2 years, Nord et al19 found that levels of CCSP in serum and BAL were lowered in BOS patients, suggesting that recipient serum CCSP concentration could be an early marker for BOS. Ten years later, Diamond et al20 focused on PGD on a prospective cohort of 104 LT recipients, and determined levels of plasma CCSP at 3 time points: pretransplant, 6 hours posttransplant, and 24 hours posttransplant. Interestingly, elevated CCSP levels at 6 hours posttransplant were associated with increased odds of PGD in univariate and multivariate analysis. Concomitantly, Gilpin et al21 studied the kinetics of CCSP(+) bone marrow cells in the first days after LT in 30 recipients, and found a significant increase in these progenitors at 24 hours, with a decrease by 48 hours. More recently, Shah et al22 studied the impact of preoperative recipient levels of 5 biomarkers (CCSP, sRAGE, ICAM-1, IL-8, and protein C) on the occurrence of severe PGD in 714 patients and found preoperative recipient levels of CCSP to be associated with PGD in non-IPF subjects (OR for highest quartile of CCSP, 2.87; 95% CI, 1.37-6.00, P = 0.005) but not in subjects with IPF (OR, 1.38; 95% CI, 0.43-4.45; P = 0.59). All together, these data suggest that lung IR leads to increased levels of CCSP that correlate with altered alveolar epithelial permeability, whereas increased CCSP(+) peripheral blood mononuclear cell mobilization after LT may have beneficial effects on lung oxygenation and recovery time, pointing CCSP and CCSP(+) peripheral blood mononuclear cell as a key determinant of early graft function. Unfortunately, these studies did not take into account the genetic variability of donor and recipients.
The gene encoding CCSP is indeed subject to a frequent polymorphism that may alter its expression at baseline or its response to external injuries. In humans, the gene encoding CCSP has 3 short exons and 2 introns for a total length of 4.1 kb. It is located on chromosome 11q12.3-13.1 in the vicinity of other genes associated with inflammatory and immune processes.23,24 Studies of the noncoding region of the exon 1 of CCSP gene have identified a number of binding sites for transcription factors.16 This region is also subject to an SNP located 38-bp downstream of the transcription initiation site, defined as dbSNP rs3741240, and characterized by an adenine/guanine substitution.25,26 This CCSP G38A polymorphism is found in 34% of the population and associated with 25% reduced transcription levels as compared with the G allele.11,12 Interestingly, we report similar proportions of CCSP G38A polymorphism in donors and recipients, but associated decrease in serum CCSP levels were found in donors but not in recipients. These results should be interpreted cautiously. It could suggest that in healthy individuals, such as lung donors, CCSP polymorphism is associated with decreased serum CCSP levels, but that in patients with end-stage lung disease such as lung recipients, CCSP polymorphism is associated with sustained serum CCSP levels—that is, that CCSP G38A polymorphism could be associated with a resistance to cell signals associated with end-stage respiratory disease. In the literature, CCSP G38A polymorphism has been studied mostly in sarcoidosis and asthma. The association between the presence of an A allele and the development of asthma in the general population is still questioned,13,27 whereas serum CCSP levels have been independently related to small airway hyperresponsiveness in asymptomatic individuals,28 COPD patients,6 and asthmatic patients.29 The fact that CCSP polymorphism has not been formally associated with the development of asthma, but serum CCSP levels has been associated with small airway hyperresponsiveness in various situations, could be explained by confounding factors. Individual exposure to cigarette smoke, air pollution, and professional toxics could interact with individual genetic susceptibility to impact the serum concentration of CCSP and the development of symptoms.16
This concept of gene-environment interaction could be of tremendous importance when considering lung grafts subjected to IR. In our study, the AG genotype in the donor was associated with a decreased risk of severe PGD both in univariate and multivariate analyses. Tentative explanation might include the resistance of the A allele to p53, as recently suggested.16 Lung IR has been associated with increased levels of p53 in the airway epithelium,3 that correlates with increased apoptosis of airway epithelial cells.30 Interestingly, Knabe et al16 recently reported that p53 binds to the promoter of CCSP and causes a decrease in CCSP gene expression in vitro. In humans, this binding site is located at the position of the G38A polymorphism site, thus explaining the resistance of CCSP G38A to p53-mediated CCSP downregulation in vitro. Specifically, BEAS-2B cells were transfected by either the CCSP 38G or 38A construct, in the presence/absence of cigarette smoke extract and modulating transcription factor p53. Baseline CCSP transcription levels were similar between the wild and variant constructs. Cigarette smoke extract decreased more profoundly the CCSP transcription level of 38A-transfected cells, but p53 decreased the CCSP transcription level more intensely with the wild than with the variant construct.16 Such experiments have not been performed after IR injury. Our hypothesis is that in lung grafts with the G allele of CCSP polymorphism, IR causes an increase in p53 associated with an increase in apoptosis and a decrease in the production of CCSP in lung epithelial cells, thus constituting a vicious circle that may alter epithelial function and lead to PGD. Conversely, in lung grafts with the A allele of CCSP G38A polymorphism, IR-induced p53 increase has no effect on CCSP expression and CCSP levels (Figure 4), and is thus associated with sustained lung epithelial function. This p53 resistance of G38A binding site may therefore explain the decrease frequency of PGD in grafts harboring the CCSP G38A genotype. Unfortunately, no blood sample has been collected immediately after transplant in the COLT study. As a consequence, we have not been able to determine the levels of CCSP posttransplant and to study the impact of donor CCSP polymorphism on the evolution of CCSP levels immediately after transplant. Even though it has been indirectly corroborated by recent publications,3,16,30 our hypothesis should therefore be confirmed both experimentally and clinically.
Our study has some limitations, including its retrospective design, the absence of information on genetic background, the limited number of patients, borderline P value, lack of long-term analysis after the 1-year timepoint, and lack of validation cohort. These limitations might be balanced by the multicentric and prospective design of COLT, the determination of PGD status in each participating center, and the centralized analysis of CCSP concentrations and CCSP polymorphism blinded from the clinical characteristics and studied outcome. However, the borderline P value of the multivariate analysis and the lack of validation cohort still constitute major limitations of this study.
The authors would like to thank Aurore Foureau for administrative assistance, the Nantes University Hospital Biologic Resources Centre for DNA extraction (BRIF : BB-0033-00040), the members of the COLT consortium for their involvement into the study, and the patients and families for their participation to the COLT study.
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10. Wutzler S, Backhaus L, Henrich D, et al. Clara cell protein 16: a biomarker for detecting secondary respiratory complications in patients with multiple injuries. J Trauma Acute Care Surg
11. Kim YS, Kang D, Kwon DY, et al. Uteroglobin gene polymorphisms affect the progression of immunoglobulin A nephropathy by modulating the level of uteroglobin expression. Pharmacogenetics
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Members of the COLT consortium.
Cohort Of Lung Transplantation-COLT (associating surgeons; anesthetists-intensivists; physicians, research staff). Bordeaux: J. Jougon, J.-F. Velly; H. Rozé; E. Blanchard, C. Dromer; Grenoble : E. Arnaud-Crozat, O. Chavanon, S. Guigard, R. Hacini, C. Martin, A. Pirvu, P. Porcu; P. Albaladejo, C. Allègre, A. Bataillard, D. Bedague, E. Briot, M. Casez-Brasseur, D. Colas, G. Dessertaine, M. Durand, G. Francony, A. Hebrard, M.R. Marino, D. Protar, D. Rehm, S. Robin, M. Rossi-Blancher; C. Augier, P. Bedouch, A. Boignard, H. Bouvaist, E. Brambilla, A. Briault, B. Camara, J. Claustre, S. Chanoine, M. Dubuc, S. Quétant, J. Maurizi, P. Pavèse, C. Pison, C. Saint-Raymond, N. Wion; C. Chérion; Lyon : R. Grima, O. Jegaden, J.-M. Maury, F. Tronc; C. Flamens, S. Paulus; J.-F. Mornex, F. Philit, A. Senechal, J.-C. Glérant, S. Turquier; D. Gamondes; L. Chalabresse, F. Thivolet-Bejui; C Barnel, C. Dubois, A. Tiberghien; Paris, Hôpital Européen Georges Pompidou : F. Le Pimpec-Barthes, A. Bel, P. Mordant, P. Achouh; V. Boussaud; R. Guillemain, D. Méléard, M.O. Bricourt, B. Cholley; V. Pezella; Marseille: G. Brioude, X.B. D’Journo, C. Doddoli. P. Thomas, D. Trousse; S. Dizier, M. Leone, L. Papazian; F. Bregeon, B. Coltey, N. Dufeu, H. Dutau, S. Garcia, JY. Gaubert, C. Gomez, S. Laroumagne, G. Mouton, A. Nieves, − Ch. Picard, M. Reynaud-Gaubert, JM. Rolain, E. Sampol, V. Secq; Nantes : P. Lacoste, C. Perigaud, J.C. Roussel, T. Senage, A Mugniot; I. Danner, A Haloun A. Magnan, A Tissot, S Abbes, C Bry, FX Blanc; T. Lepoivre, K. Botturi-Cavaillès, S. Brouard, R. Danger, J. Loy, M. Bernard, E. Godard, P.-J. Royer, E. Durand, K. Henrio, M. Durand, C. Brosseau, A.Foureau; Le Plessis Robinson, Hôpital Marie Lannelongue : Ph. Dartevelle, D. Fabre, E. Fadel, O. Mercier, S. Mussot; F. Stephan, P. Viard; J. Cerrina, P. Dorfmuller, S. Feuillet M. Ghigna, Ph. Hervé, F. Le Roy Ladurie, J. Le Pavec, V. Thomas de Montpreville; L. Lamrani; Paris, Hôpital Bichat : Y. Castier, P. Mordant, P. Cerceau, P. Augustin, S. Jean-Baptiste, S. Boudinet, P. Montravers; O. Brugière, G. Dauriat, G. Jébrak, H. Mal, A. Marceau, A.-C. Métivier, G. Thabut, E. Lhuillier, C. Dupin, V. Bunel; Strasbourg : P. Falcoz, G. Massard, N. Santelmo; A. Olland, J. Reeb, J. Seitlinger, G. Ajob, O. Collange O. Helms, J. Hentz, A. Roche; T. Degot, A. Dory, S. Hirschi, S. Ohlmann-Caillard, L. Kessler, R. Kessler, A. Schuller;B. Renaud-Picard, M. Porzio, Sarah Idris-Khodja, Julien Stauder; Suresnes, Hôpital Foch : P. Bonnette, A. Chapelier, P. Puyo, E. Sage; J. Bresson, V. Caille, C. Cerf, J. Devaquet, V. Dumans-Nizard, ML. Felten, M. Fischler, AG. Si Larbi, M. Leguen, L. Ley, N. Liu, G. Trebbia; S. De Miranda, B. Douvry, F. Gonin, D. Grenet, A.M. Hamid, H. Neveu, F. Parquin, C. Picard, A. Roux, M. Stern; F. Bouillioud, P. Cahen, M. Colombat, C. Dautricourt, M. Delahousse, B. D’Urso, J. Gravisse, A. Guth, S. Hillaire, P. Honderlick, M. Lequintrec, E. Longchampt, F. Mellot, A. Scherrer, L. Temagoult, L. Tricot; M. Vasse, C. Veyrie, L. Zemoura; Toulouse : M Dahan, M Murris, H Benahoua, J Berjaud, A Le Borgne Krams, L Crognier, L Brouchet, O Mathe, A Didier.