Distinct Expression Patterns of Alveolar “Alarmins” in Subtypes of Chronic Lung Allograft Dysfunction
2014; Elsevier BV; Volume: 14; Issue: 6 Linguagem: Inglês
10.1111/ajt.12718
ISSN1600-6143
AutoresTomohito Saito, Mingyao Liu, Matthew Binnie, Masaaki Sato, D. Hwang, S. Azad, Tiago Machuca, R. Zamel, Thomas K. Waddell, Marcelo Cypel, Shaf Keshavjee,
Tópico(s)Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
ResumoThe long-term success of lung transplantation is limited by chronic lung allograft dysfunction (CLAD). The purpose of this study was to investigate the alveolar alarmin profiles in CLAD subtypes, restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome (BOS). Bronchoalveolar lavage (BAL) samples were collected from 53 recipients who underwent double lung or heart-lung transplantation, including patients with RAS (n = 10), BOS (n = 18) and No CLAD (n = 25). Protein levels of alarmins such as S100A8, S100A9, S100A8/A9, S100A12, S100P, high-mobility group box 1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE) in BAL fluid were measured. RAS and BOS showed higher expressions of S100A8, S100A8/A9 and S100A12 compared with No CLAD (p < 0.0001, p < 0.0001, p < 0.0001 in RAS vs. No CLAD, p = 0.0006, p = 0.0044, p = 0.0086 in BOS vs. No CLAD, respectively). Moreover, RAS showed greater up-regulation of S100A9, S100A8/A9, S100A12, S100P and HMGB1 compared with BOS (p = 0.0094, p = 0.038, p = 0.041, p = 0.035 and p = 0.010, respectively). sRAGE did not show significant difference among the three groups (p = 0.174). Our results demonstrate distinct expression patterns of alveolar alarmins in RAS and BOS, suggesting that RAS and BOS may represent biologically different subtypes. Further refinements in biologic profiling will lead to a better understanding of CLAD. The long-term success of lung transplantation is limited by chronic lung allograft dysfunction (CLAD). The purpose of this study was to investigate the alveolar alarmin profiles in CLAD subtypes, restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome (BOS). Bronchoalveolar lavage (BAL) samples were collected from 53 recipients who underwent double lung or heart-lung transplantation, including patients with RAS (n = 10), BOS (n = 18) and No CLAD (n = 25). Protein levels of alarmins such as S100A8, S100A9, S100A8/A9, S100A12, S100P, high-mobility group box 1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE) in BAL fluid were measured. RAS and BOS showed higher expressions of S100A8, S100A8/A9 and S100A12 compared with No CLAD (p < 0.0001, p < 0.0001, p < 0.0001 in RAS vs. No CLAD, p = 0.0006, p = 0.0044, p = 0.0086 in BOS vs. No CLAD, respectively). Moreover, RAS showed greater up-regulation of S100A9, S100A8/A9, S100A12, S100P and HMGB1 compared with BOS (p = 0.0094, p = 0.038, p = 0.041, p = 0.035 and p = 0.010, respectively). sRAGE did not show significant difference among the three groups (p = 0.174). Our results demonstrate distinct expression patterns of alveolar alarmins in RAS and BOS, suggesting that RAS and BOS may represent biologically different subtypes. Further refinements in biologic profiling will lead to a better understanding of CLAD. Chronic lung allograft dysfunction (CLAD) is a major cause of morbidity and mortality in long-term survivors of lung transplantation (1Christie JD Edwards LB Kucheryavaya AY et al.The registry of the international society for heart and lung transplantation: 29th adult lung and heart-lung transplant report-2012.J Heart Lung Transplant. 2012; 31: 1073-1086Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The 5-year survival rate associated with a functioning lung allograft is ∼50%, which is considerably inferior to other solid organ transplantation (2Organ Procurement and Transplantation Network and Scientific Registry of Transplant Recipients 2010 data report.Am J Transplant. 2012; 12: 1-156Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Recently, we described a novel form of CLAD, restrictive allograft syndrome (RAS) that shows a rapid progression with pathological diagnoses of diffuse alveolar damage and pleuroparenchymal fibroelastosis, which is distinct from bronchiolitis obliterans syndrome (BOS)—the conventional form of CLAD (3Ofek E Sato M Saito T et al.Restrictive allograft syndrome post lung transplantation is characterized by pleuroparenchymal fibroelastosis.Mod Pathol. 2013; 26: 350-356Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar,4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). These clinical and pathological distinctions lead to our hypothesis that RAS and BOS may represent biologically different CLAD subtypes. Biologic profiling of CLAD phenotypes may subsequently help to understand the underlying mechanisms and to ultimately develop precisely targeted and personalized therapy. Accumulating evidence suggests that multiple immune systems may contribute to the pathogenesis of CLAD (5Todd JL Palmer SM Bronchiolitis obliterans syndrome: The final frontier for lung transplantation.Chest. 2011; 140: 502-508Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Notably, the importance of innate immunity in the CLAD development has gained significant prominence. In fact, many of the identified risk factors of CLAD, such as primary graft dysfunction, cytomegalovirus (CMV) pneumonitis, gastroesophageal-reflux and polymorphism in Toll-like receptors, would likely activate the innate immune response (5Todd JL Palmer SM Bronchiolitis obliterans syndrome: The final frontier for lung transplantation.Chest. 2011; 140: 502-508Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,6Kastelijn EA Van Moorsel CH Rijkers GT et al.Polymorphisms in innate immunity genes associated with development of bronchiolitis obliterans after lung transplantation.J Heart Lung Transplant. 2010; 29: 665-671Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The innate immunity of the lung is reliant on recognition of an array of danger signals including damage-associated molecular patterns, also referred to as "alarmins"—intracellular constitutive molecules that can turn into pro-inflammatory mediators once extracellularly released (7Guo WA Knight PR Raghavendran K The receptor for advanced glycation end products and acute lung injury/acute respiratory distress syndrome.Intensive Care Med. 2012; 38: 1588-1598Crossref PubMed Scopus (59) Google Scholar,8Chan JK Roth J Oppenheim JJ et al.Alarmins: Awaiting a clinical response.J Clin Invest. 2012; 122: 2711-2719Crossref PubMed Scopus (368) Google Scholar). Intriguingly, our preliminary proteomic study revealed that several alarmins, such as S100 family proteins, were expressed in the bronchoalveolar lavage (BAL) fluid of patients who developed RAS, but not in those of CLAD-free recipients (9Kosanam H Sato M Batruch I et al.Differential proteomic analysis of bronchoalveolar lavage fluid from lung transplant patients with and without chronic graft dysfunction.Clin Biochem. 2012; 45: 223-230Crossref PubMed Scopus (23) Google Scholar), suggesting that the alveolar release of alarmins may play a role in the RAS development. However, it remains unclear whether alveolar alarmins are uniformly associated with both RAS and BOS. The aim of this study is to identify the biological characteristics of RAS and BOS. For this purpose, we further characterized human CLAD by profiling protein expressions of alarmins such as S100 family proteins (S100A8, S100A9, S100A8/A9 heterodimer and polymer complex, S100A12 and S100P), high-mobility group box 1 (HMGB1) and their decoy receptor, soluble receptor for advanced glycation end products (sRAGE) in BAL fluid. This study was approved by the University Health Network Research Ethics Board (REB#11-0506-T). Written consent for the use of excess BAL sample was obtained from each patient in adherence to the principles set forth in the Declaration of Helsinki. Designation of the study population is summarized in Figure 1. From banked BAL specimens, samples were selected dependent on whether the postbilateral lung or heart-lung transplant recipient developed CLAD prior to BAL. Through chart review, we initially identified 45 subjects with CLAD, of which 17 cases were excluded because of concurrent proven or probable pulmonary infection at the time of BAL, compliant with the International Society for Heart and Lung Transplantation (ISHLT) consensus statement (10Husain S Mooney ML Danziger-Isakov L et al.A 2010 working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients.J Heart Lung Transplant. 2011; 30: 361-374Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In all, 28 samples from 28 patients identified with CLAD (10 with RAS and 18 with BOS) were included. Additionally, we included 25 postbilateral lung or heart-lung transplant recipients with stable lung function as "No CLAD" controls, matching the interval between lung transplantation and BAL with that of CLAD group. Standard posttransplant care was provided as previously described (11de Perrot M Chaparro C McRae K et al.Twenty-year experience of lung transplantation at a single center: Influence of recipient diagnosis on long-term survival.J Thorac Cardiovasc Surg. 2004; 127: 1493-1501Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Our lung transplant program employed cyclosporine A, azathioprine and prednisone as initial standard regimen (11de Perrot M Chaparro C McRae K et al.Twenty-year experience of lung transplantation at a single center: Influence of recipient diagnosis on long-term survival.J Thorac Cardiovasc Surg. 2004; 127: 1493-1501Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Detailed information on posttransplant follow-up, immunosuppression and treatment for CLAD are described in Supplemental Materials. BAL fluid was collected and processed as described previously (12D'Ovidio F Mura M Ridsdale R et al.The effect of reflux and bile acid aspiration on the lung allograft and its surfactant and innate immunity molecules SP-A and SP-D.Am J Transplant. 2006; 6: 1930-1938Crossref PubMed Scopus (149) Google Scholar). The quantity of recovered BAL fluid was greater than 30 mL of 100 mL-instillation in all 53 subjects except for one case with RAS. Inflammation in BAL samples was semi-quantitatively assessed by a cytopathologist and described as follows: neutrophil-predominant (neutrophils >10% of 200 counted cells in the specimen), lymphocyte-predominant (lymphocytes >10%), mixed-type (a mix of neutrophils and lymphocytes >10%) or no inflammatory findings. Acute rejection was evaluated in the concomitant transbronchial lung biopsy (TBBx) specimen by pathologists based on the ISHLT grading system (13Stewart S Fishbein MC Snell GI et al.Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection.J Heart Lung Transplant. 2007; 26: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). We measured protein expression of S100A8, S100A9, S100A8/A9 heterodimer and polymer complex, S100A12, S100P, HMGB1 and sRAGE in BAL supernatant by using specific enzyme-linked immunosorbent assay kits (S100A8, S100A9, S100A12, S100P, sRAGE, CycLex, Nagano, Japan; S100A8/A9, ALPCO, Salem, NH; HMGB1, Shino-Test, Tokyo, Japan), following manufacturers' instructions. CLAD, RAS, BOS and acute exacerbation of RAS/BOS were defined as previously described (4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,14Sato M Hwang DM Waddell TK Singer LG Keshavjee S Progression pattern of restrictive allograft syndrome after lung transplantation.J Heart Lung Transplant. 2013; 32: 23-30Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,15Estenne M Maurer JR Boehler A et al.Bronchiolitis obliterans syndrome 2001: An update of the diagnostic criteria.J Heart Lung Transplant. 2002; 21: 297-310Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). Our goal was to identify the relationship between alarmin profiles of CLAD phenotypes using posttransplant BAL samples. Analysis of variance (ANOVA), Kruskal–Wallis test and Fisher's exact test were performed to determine differences among patients with RAS, BOS and No CLAD. Mann–Whitney test was applied to compare the timing of BAL relative to the onset of RAS and BOS. p < 0.05 were reported to be significant. To report diagnostic accuracy in differentiating CLAD from No CLAD or RAS from BOS, receiver-operating characteristic (ROC) curves were constructed and the area under the ROC curve was calculated for the S100 proteins and HMGB1. GraphPad Prism version 6.02 for Windows (GraphPad Software, San Diego, CA; Microsoft, Redmond, WA) was applied. Clinical characteristics of the study population are shown in Table 1. Recipient age at transplantation was significantly different among RAS, BOS and No CLAD (p = 0.047), but paired posttests between each group did not reach statistical significance (p = 0.091 in No CLAD vs. BOS; p = 0.189 in No CLAD vs. RAS; p > 0.999 in BOS vs. RAS). However, patients with CLAD were significantly younger than those with No CLAD (p = 0.013). No significant difference in primary diagnosis, transplantation type, gender matching, CMV serology matching, posttransplant follow-up or interval between lung transplantation and BAL were found among the three study groups. RAS and BOS did not show significant difference in interval between the disease onset and BAL, incidence of acute exacerbation at BAL or forced expiratory volume in 1 s. Tacrolimus and azithromycin administration were more common in patients with RAS and BOS compared No CLAD (tacrolimus, p = 0.060 for RAS vs. No CLAD, p = 0.062 for BOS vs. No CLAD; azithromycin, p = 0.0008 in RAS vs. No CLAD, p < 0.0001 in BOS vs. No CLAD). There was no significant difference in the use of tacrolimus and azithromycin between RAS and BOS (p > 0.999 and p > 0.999).Table 1:Demographics of study populationCharacteristicsNo CLAD (n = 25)BOS (n = 18)RAS (n = 10)p-ValueRecipient age at transplant, year53 [48–64]42 [32–58]45 [37–54]0.047Primary diagnosis (%)Chronic obstructive pulmonary disease4(16.0)2(11.1)1 (10.0)0.917Idiopathic pulmonary fibrosis8 (32.0)6 (33.3)3 (30.0)Cystic fibrosis3 (12.0)5 (27.8)2 (20.0)Others10 (40.0)5 (27.8)4 (40.0)Transplant type (%)Bilateral lung transplantation22 (88.0)17(94.4)100.659Heart-lung transplantation3(12.0)1 (5.6)—Gender matching (%)Male to male10(40.0)10(55.6)4 (40.0)0.260Male to female2 (8.0)—3 (30.0)Female to female9 (36.0)5 (27.8)1 (10.0)Female to male4(16.0)3(16.7)2 (20.0)CMV serology matching, (%)Donor−/recipient−11 (44.0)6 (33.3)1 (10.0)0.182Donor−/recipient +8 (32.0)4 (22.2)4 (40.0)Donor+/recipient+5 (20.0)3(16.7)2 (20.0)Donor+/recipient−1 (4.0)5 (27.8)3 (30.0)Posttransplant follow-up, months47 [34–52]50 [39–49]39 [23–80]0.373Timing of BALPostlung transplantation, months24 [23–24]27 [21–61]36 [12–73]0.417Postdisease onset of RAS/BOS, days—165 [14–330]38 [22–95]0.292Acute exacerbation1Acute exacerbation was defined as a sudden-onset or aggravation of respiratory distress that necessitated increased oxygen supple-mentation, hospital admission or mechanical ventilation. of RAS/BOS at BAL (%)—2 (11.1)3 (30.0)0.315Pulmonary function at BALFEV1, %baseline96.7 ± 5.658.4 ± 17.2p < 0.0001 in BOS versus No CLAD.50.3 ± 14.9**p < 0.0001 in RAS versus No CLAD,<0.0001TLC, %baseline98.4 ± 3.9102.8 ± 87†p = 0.038,78.6 ± 8.5**p < 0.0001 in RAS versus No CLAD,,#p < 0.0001 in RAS versus BOS,<0.0001Immunosuppression/treatment for CLAD at BALCyclosporine A/tacrolimus15/105/13p < 0.0001 in BOS versus No CLAD.2/80.041Azathioprine/MMF or MPA7/177/82/80.320Prednisone251810—Azithromycin administration111p < 0.0001 in BOS versus No CLAD.6*p = 0.0008,<0.0001Nonparametric continuous variables are expressed as median [interquartile range]. Parametric continuous variables are expressed as mean ± standard deviation. p-Values were calculated by the Fisher's exact test for categorical variables. Kruskal–Wallis ANOVA was applied for nonparametric continuous variables, and one-way ANOVA was used for parametric continuous variables for RAS versus BOS versus No CLAD.ANOVA, analysis of variance; BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; CMV, cytomegalovirus; FEV1, forced expiratory volume in 1 s; MMF, mycophenolate mofetil; MPA, mycophenolic acid; RAS, restrictive allograft syndrome; TLC, total lung capacity.1 Acute exacerbation was defined as a sudden-onset or aggravation of respiratory distress that necessitated increased oxygen supple-mentation, hospital admission or mechanical ventilation.* p = 0.0008,** p < 0.0001 in RAS versus No CLAD,# p < 0.0001 in RAS versus BOS,† p = 0.038,†† p < 0.0001 in BOS versus No CLAD. Open table in a new tab Nonparametric continuous variables are expressed as median [interquartile range]. Parametric continuous variables are expressed as mean ± standard deviation. p-Values were calculated by the Fisher's exact test for categorical variables. Kruskal–Wallis ANOVA was applied for nonparametric continuous variables, and one-way ANOVA was used for parametric continuous variables for RAS versus BOS versus No CLAD. ANOVA, analysis of variance; BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; CMV, cytomegalovirus; FEV1, forced expiratory volume in 1 s; MMF, mycophenolate mofetil; MPA, mycophenolic acid; RAS, restrictive allograft syndrome; TLC, total lung capacity. Concurrent pathologic and microbiologic findings are summarized in Table 2. Cytopathological assessment was performed on all but one insufficient BAL specimen from a No CLAD patient. Neutrophil-predominant inflammation was frequently found in CLAD compared with No CLAD (p = 0.0047) and in RAS compared with BOS and No CLAD (p = 0.026 and p = 0.0002). There was a greater tendency for neutrophil-predominant inflammation in BOS compared with No CLAD, but it did not reach statistical significance (p = 0.054). Adequate TBBx sampling were achieved in 40.0% (4 of 10) of RAS, 66.7% (12 of 18) of BOS and 84.0% (21 of 25) of No CLAD cases, which showed significant difference between RAS and No CLAD (p = 0.043), but not between RAS and BOS or between BOS and No CLAD (p = 0.091 and p = 0.207). No significant difference was observed in the acute rejection scores and in positivity in cultures for bacteria, acid-fast bacilli or Aspergillus across the three study groups. BAL specimens were also analyzed for CMV in 11 specimens (1 RAS, 4 BOS and 6 No CLAD), all of which showed negative results.Table 2:Concurrent pathologic and microbiologic findingsNo CLAD (n = 25)BOS (n = 18)RAS (n = 10)p-ValueBAL cytology (%)Neutrophil-predominant inflammation2/24 (8.3)5/18 (27.8)8/10 (80.0)*p = 0.0002 in RAS versus No CLAD,,**p = 0.026 in RAS versus BOS.0.0003Lymphocyte-predominant inflammation3/24 (12.5)——Mixed-type inflammation—2/18 (11.1)—No abnormal findings19/24 (79.2)11/18 (61.1)2/10 (20.0)TBBx histology (%)Acute rejectionGrade A019/23 (82.6)10/15 (66.7)3/6 (50.0)0.487Grade A12/23 (8.7)2/15 (13.3)1/6(16.7)Grade AX2/23 (8.7)3/15 (20.0)2/6 (33.3)BAL microbiology (%)Positive bacterial culture2/25 (8.0)2/18 (11.1)0/100.816P. aeruginosa: 1P. aeruginosa: 1S. maltophilia: 1H. influenzae: 1Positive acid-fast bacilli culture0/250/180/10—Positive Aspergillus culture0/251/18 (5.6)0/100.528p-Values were calculated by the Fisher's exact test.BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; RAS, restrictive allograft syndrome; TBBx, transbronchial lung biopsy.* p = 0.0002 in RAS versus No CLAD,** p = 0.026 in RAS versus BOS. Open table in a new tab p-Values were calculated by the Fisher's exact test. BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; RAS, restrictive allograft syndrome; TBBx, transbronchial lung biopsy. Protein levels of the S100 proteins, HMGB1 and sRAGE in BAL fluid are presented in Figure 2 and Table S1. S100A8, S100A8/A9 and S100A12 showed higher expressions in RAS and BOS compared with No CLAD (p < 0.0001, p < 0.0001 and p < 0.0001 for S100A8, S100A8/A9 and S100A12 between RAS and No CLAD; p = 0.0006, p = 0.0044 and p = 0.0086 for S100A8, S100A8/A9 and S100A12 between BOS and No CLAD). Moreover, up-regulation of S100A8/A9 and S100A12 was significantly greater in RAS compared with BOS (p = 0.038 and p = 0.041, respectively). Furthermore, RAS showed significantly higher expression of S100A9, S100P and HMGB1 compared with BOS and No CLAD (p = 0.0094, p = 0.035 and p = 0.010 for S100A9, S100P and HMGB1 between RAS and BOS; p < 0.0001, p < 0.0001 and p = 0.0016 for S100A9, S100P and HMGB1 between RAS and No CLAD). sRAGE did not show any significant differences across the three study groups (p = 0.174). Areas under the ROC curves are summarized in Table 3. S100A8 showed high accuracy in differentiating CLAD from No CLAD (area under the curve [AUC], 0.921; 95% confidence interval [CI], 0.853–0.990). S100A9 (AUC, 0.813; 95% CI, 0.698–0.927), S100A8/A9 (AUC, 0.889; 95% CI, 0.802–0.977) and S100A12 (AUC, 0.870; 95% CI, 0.774–0.966) showed moderate accuracy in predicting CLAD versus No CLAD. As a biomarker differentiating RAS from BOS, S100A9 (AUC, 0.889; 95% CI, 0.762–1.016), S100A8/A9 (AUC, 0.889; 95% CI, 0.763–1.015), S100A12 (AUC, 0.867; 95% CI, 0.730–1.003) and HMGB1 (AUC, 0.833; 95% CI, 0.674–0.992) showed moderate accuracy.Table 3:Diagnostic accuracy for the S100 family proteins and HMGB1ProteinArea under the ROC curve (95% confidence interval)CLAD versus No CLADRAS versus BOSS100A80.921 (0.853–0.990)—S100A90.813 (0.698–0.927)0.889 (0.762–1.016)S100A8/A9heterocomplex0.889 (0.802–0.977)0.889 (0.763–1.015)S100A120.870 (0.774–0.966)0.867 (0.730–1.003)S100P0.761 (0.633–0.890)0.817 (0.651–0.982)HMGB10.661 (0.512–0.809)0.833 (0.674–0.992)BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; HMGB1, high-mobility box group 1; RAS, restrictive allograft syndrome; ROC, receiver-operating characteristic. Open table in a new tab BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; HMGB1, high-mobility box group 1; RAS, restrictive allograft syndrome; ROC, receiver-operating characteristic. It has been recognized that CLAD is not a single entity, but rather a heterogenous one (5Todd JL Palmer SM Bronchiolitis obliterans syndrome: The final frontier for lung transplantation.Chest. 2011; 140: 502-508Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,15Estenne M Maurer JR Boehler A et al.Bronchiolitis obliterans syndrome 2001: An update of the diagnostic criteria.J Heart Lung Transplant. 2002; 21: 297-310Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar,16Meyer KC Glanville AR Bronchiolitis obliterans syndrome and chronic lung allograft dysfunction: Evolving concepts and nomenclature.in: Bronchiolitis obliterans syndrome in lung transplantation. Humana Press, New York, NY2013: 1-19Crossref Scopus (2) Google Scholar). For better understanding of CLAD, several sub-phenotypes such as RAS, neutrophilic reversible allograft dysfunction, fibroproliferative BOS and early-onset BOS have been proposed (4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,16Meyer KC Glanville AR Bronchiolitis obliterans syndrome and chronic lung allograft dysfunction: Evolving concepts and nomenclature.in: Bronchiolitis obliterans syndrome in lung transplantation. Humana Press, New York, NY2013: 1-19Crossref Scopus (2) Google Scholar). Among them, RAS has been established as a widely accepted subtype of CLAD (16Meyer KC Glanville AR Bronchiolitis obliterans syndrome and chronic lung allograft dysfunction: Evolving concepts and nomenclature.in: Bronchiolitis obliterans syndrome in lung transplantation. Humana Press, New York, NY2013: 1-19Crossref Scopus (2) Google Scholar, 17Snell GI Paraskeva M Westall GP Managing bronchiolitis obliterans syndrome (BOS) and chronic lung allograft dysfunction (CLAD) in children: What does the future hold?.Paediatr Drugs. 2013; 15: 281-289Crossref PubMed Scopus (9) Google Scholar, 18Verleden SE Ruttens D Vandermeulen E et al.Bronchiolitis obliterans syndrome and restrictive allograft syndrome: Do risk factors differ?.Transplantation. 2013; 95: 1167-1172Crossref PubMed Scopus (83) Google Scholar). We initially proposed RAS as a novel form of CLAD for: (i) its restrictive physiology that does not fit the original ISHLT definition of BOS (15Estenne M Maurer JR Boehler A et al.Bronchiolitis obliterans syndrome 2001: An update of the diagnostic criteria.J Heart Lung Transplant. 2002; 21: 297-310Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar), (ii) its prevalence of 25–35% of all CLAD (4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,18Verleden SE Ruttens D Vandermeulen E et al.Bronchiolitis obliterans syndrome and restrictive allograft syndrome: Do risk factors differ?.Transplantation. 2013; 95: 1167-1172Crossref PubMed Scopus (83) Google Scholar), and (iii) its clinical and pathological distinctions from BOS (3Ofek E Sato M Saito T et al.Restrictive allograft syndrome post lung transplantation is characterized by pleuroparenchymal fibroelastosis.Mod Pathol. 2013; 26: 350-356Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar,4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,14Sato M Hwang DM Waddell TK Singer LG Keshavjee S Progression pattern of restrictive allograft syndrome after lung transplantation.J Heart Lung Transplant. 2013; 32: 23-30Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,18Verleden SE Ruttens D Vandermeulen E et al.Bronchiolitis obliterans syndrome and restrictive allograft syndrome: Do risk factors differ?.Transplantation. 2013; 95: 1167-1172Crossref PubMed Scopus (83) Google Scholar). The heterogeneity of CLAD might be attributed to multiple immunopathological mechanisms underlying the pathogenesis (5Todd JL Palmer SM Bronchiolitis obliterans syndrome: The final frontier for lung transplantation.Chest. 2011; 140: 502-508Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar); therefore, biologic profiling of RAS and BOS in turn may help to better understand CLAD, with the hope that these discoveries will lead to the establishment of precisely targeted preventive and therapeutic strategies. Neutrophil-predominant inflammation was common in BAL samples of CLAD compared with No CLAD (Table 2). Although our semi-quantitative criteria for evaluating inflammation may limit generalizability and comparability, our results might further support previous findings describing the association between BAL neutrophilia and CLAD (18Verleden SE Ruttens D Vandermeulen E et al.Bronchiolitis obliterans syndrome and restrictive allograft syndrome: Do risk factors differ?.Transplantation. 2013; 95: 1167-1172Crossref PubMed Scopus (83) Google Scholar,19Kennedy VE Todd JL Palmer SM Bronchoalveolar lavage as a tool to predict, diagnose and understand bronchiolitis obliterans syndrome.Am J Transplant. 2013; 13: 552-561Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Intriguingly, neutrophil-predominant inflammation in BAL cytology was more common in RAS than in BOS. Since we excluded CLAD with pulmonary infection, combined with the result that all BAL from RAS patients showed negative culture results, the increase of neutrophils in BAL sample might be likely associated with RAS rather than possible concomitant infection. Activated neutrophils may contribute to the development of CLAD, especially RAS, by degrading extracellular matrix, depleting antioxidant defense and promoting fibroblast proliferation (19Kennedy VE Todd JL Palmer SM Bronchoalveolar lavage as a tool to predict, diagnose and understand bronchiolitis obliterans syndrome.Am J Transplant. 2013; 13: 552-561Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). RAS and BOS seem to share similar up-regulation of S100A8, S100A8/A9 and S100A12 in BAL fluid (Figure 2 and Table S1). S100 proteins are a family of 10–12 kD calcium-binding proteins. S100A8, S100A9, S100A8/A9 and S100A12 can be released from activated neutrophils, monocytes/macrophages and necrotic cells (8Chan JK Roth J Oppenheim JJ et al.Alarmins: Awaiting a clinical response.J Clin Invest. 2012; 122: 2711-2719Crossref PubMed Scopus (368) Google Scholar,20Wittkowski H Sturrock A van Zoelen MA et al.Neutrophil-derived S100A12 in acute lung injury and respiratory distress syndrome.Crit Care Med. 2007; 35: 1369-1375Crossref PubMed Scopus (97) Google Scholar). Extracellular S100 proteins ultimately lead to innate immune responses such as leukocyte recruitment and endothelial cell activation (20Wittkowski H Sturrock A van Zoelen MA et al.Neutrophil-derived S100A12 in acute lung injury and respiratory distress syndrome.Crit Care Med. 2007; 35: 1369-1375Crossref PubMed Scopus (97) Google Scholar). Thus, S100A8, S100A8/A9 and S100A12 may contribute to the development of both RAS and BOS by activating innate immune-dependent mechanisms. Considering the finding that RAS showed further up-regulation of alveolar S100A8/A9 and S100A12 compared with BOS, S100A8/A9 and S100A12 might be more associated with RAS development. Strikingly, up-regulation of alveolar S100A9, S100P and HMGB1 were observed exclusively in RAS (Figure 2 and Table S1). Reportedly, alveolar S100A9 may be associated with the pathogenesis of idiopathic pulmonary fibrosis (21Korthagen NM Nagtegaal MM van Moorsel CH Kazemier KM van den Bosch JM Grutters JC MRP14 is elevated in the bronchoalveolar lavage fluid of fibrosing interstitial lung diseases.Clin Exp Immunol. 2010; 161: 342-347Crossref PubMed Scopus (33) Google Scholar). Thus, S100A9 may contribute to pulmonary parenchymal fibrosis in RAS. Although the role of S100P in the context of pulmonary pathophysiology is not yet well understood, its potent function in mediating cell proliferation, metastasis and invasion may contribute to RAS development (22Jiang H Hu H Tong X Jiang Q Zhu H Zhang S Calcium-binding protein S100P and cancer: Mechanisms and clinical relevance.J Cancer Res Clin Oncol. 2012; 138: 1-9Crossref PubMed Scopus (67) Google Scholar). HMGB1 is a ubiquitous 30-kD DNA-binding nuclear protein that can be released from activated monocytes/macrophages, natural killer cells, mature myeloid dendritic cells and necrotic or apoptotic cells (8Chan JK Roth J Oppenheim JJ et al.Alarmins: Awaiting a clinical response.J Clin Invest. 2012; 122: 2711-2719Crossref PubMed Scopus (368) Google Scholar,23Li G Liang X Lotze MT HMGB1: The central cytokine for all lymphoid cells.Front Immunol. 2013; 4: 68Crossref PubMed Scopus (118) Google Scholar). Extracellular HMGB1 promotes innate immune and autoimmune responses via Toll-like receptors or RAGE (5Todd JL Palmer SM Bronchiolitis obliterans syndrome: The final frontier for lung transplantation.Chest. 2011; 140: 502-508Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,19Kennedy VE Todd JL Palmer SM Bronchoalveolar lavage as a tool to predict, diagnose and understand bronchiolitis obliterans syndrome.Am J Transplant. 2013; 13: 552-561Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,23Li G Liang X Lotze MT HMGB1: The central cytokine for all lymphoid cells.Front Immunol. 2013; 4: 68Crossref PubMed Scopus (118) Google Scholar, 24Nakagiri T Inoue M Morii E et al.Local IL-17 production and a decrease in peripheral blood regulatory T cells in an animal model of bronchiolitis obliterans.Transplantation. 2010; 89: 1312-1319Crossref PubMed Scopus (30) Google Scholar, 25Kruger B Yin N Zhang N et al.Islet-expressed TLR2 and TLR4 sense injury and mediate early graft failure after transplantation.Eur J Immunol. 2010; 40: 2914-2924Crossref PubMed Scopus (45) Google Scholar), which may contribute to RAS development. More importantly, HMGB1, S100A12 and their receptor RAGE have been demonstrated to be associated with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) (20Wittkowski H Sturrock A van Zoelen MA et al.Neutrophil-derived S100A12 in acute lung injury and respiratory distress syndrome.Crit Care Med. 2007; 35: 1369-1375Crossref PubMed Scopus (97) Google Scholar,26Ueno H Matsuda T Hashimoto S et al.Contributions of high mobility group box protein in experimental and clinical acute lung injury.Am J Respir Crit Care Med. 2004; 170: 1310-1316Crossref PubMed Scopus (333) Google Scholar). Interestingly, RAS shares radiological and pathological features with ALI/ARDS (4Sato M Waddell TK Wagnetz U et al.Restrictive allograft syndrome (RAS): A novel form of chronic lung allograft dysfunction.J Heart Lung Transplant. 2011; 30: 735-742Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,14Sato M Hwang DM Waddell TK Singer LG Keshavjee S Progression pattern of restrictive allograft syndrome after lung transplantation.J Heart Lung Transplant. 2013; 32: 23-30Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Specifically, diffuse alveolar damage that is the pathologic counterpart of ALI/ARDS is commonly observed in RAS (3Ofek E Sato M Saito T et al.Restrictive allograft syndrome post lung transplantation is characterized by pleuroparenchymal fibroelastosis.Mod Pathol. 2013; 26: 350-356Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Thus, RAS and ALI/ARDS may share common biological pathways such as the RAGE-RAGE ligand axis involving HMGB1 and S100A12. In contrast to S100s and HMGB1, alveolar sRAGE did not show significant differences among RAS, BOS and No CLAD (Figure 2 and Table S1). sRAGE is a 48-kD C-terminally truncated RAGE and could be released via proteolytic cleavage of full-length membrane-bound RAGE expressed by alveolar type I epithelial cells during tissue injury (i.e. ALI/ARDS) (27Uchida T Shirasawa M Ware LB et al.Receptor for advanced glycation end-products is a marker of type I cell injury in acute lung injury.Am J Respir Crit Care Med. 2006; 173: 1008-1015Crossref PubMed Scopus (362) Google Scholar). One possible explanation for the lack of increased detection of sRAGE in our study might be the proteolysis of sRAGE. For example, eosinophil cationic protein, implicated in the breakdown of sRAGE, is reportedly up-regulated in the BAL fluid of lung transplant recipients (28Van Crombruggen K Holtappels G De Ruyck N Derycke L Tomassen P Bachert C RAGE processing in chronic airway conditions: Involvement of Staphylococcus aureus and ECP.J Allergy Clin Immunol. 2012; 129 (e8): 1515-1521Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,29Bargagli E Madioni C Prasse A et al.Eosinophilic cationic protein in bronchoalveolar lavage fluid of lung transplant patients.Clin Chem Lab Med. 2008; 46: 563-564Crossref PubMed Scopus (3) Google Scholar). Another possible explanation might be binding of up-regulated RAGE ligands (i.e. S100s and HMGB1) to sRAGE. More importantly, sRAGE could act as a decoy receptor for RAGE ligands due to its deficient transmembrane domain (7Guo WA Knight PR Raghavendran K The receptor for advanced glycation end products and acute lung injury/acute respiratory distress syndrome.Intensive Care Med. 2012; 38: 1588-1598Crossref PubMed Scopus (59) Google Scholar). In fact, recombinant sRAGE was shown to ameliorate endotoxin-induced ALI (30Zhang H Tasaka S Shiraishi Y et al.Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury.Am J Respir Crit Care Med. 2008; 178: 356-362Crossref PubMed Scopus (115) Google Scholar). Therefore, neutralizing RAGE-RAGE ligand axis by the application of sRAGE could be explored as a potential therapeutic option for CLAD, especially RAS, although further investigation is necessary to determine its effect and optimal delivery. Specifically, S100A9 and S100A8/A9 appear to be useful to differentiate RAS from BOS, while S100A8 and S100A8/A9 appear to distinguish CLAD from No CLAD (Table 3). We recognize that since the data reported here were generated via a cross-sectional study, longitudinal observation for the potential biomarkers is necessary to reveal temporal dynamics as well as the predictive values. Limitations of this study are as follows. First, the presented results are based on a single-center study with a relatively small sample size. We acknowledge limited generalizability and the possibility of type II error. Further study is essential to validate our results, specifically the clinical utility of S100s and HMGB1 as potential markers for differentiating RAS from BOS. Second, we could not collect quantitative data regarding the recovery volume of BAL samples. Since the recovery volume of CLAD was reported to be inferior to that of stable patients (a median of 35 mL vs. 46 mL for 100 mL instillation) (31Vanaudenaerde BM Wuyts WA Geudens N et al.Broncho-alveolar lavage fluid recovery correlates with airway neutrophilia in lung transplant patients.Respir Med. 2008; 102: 339-347Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), the protein expression data here should be carefully interpreted with consideration for a possible dilution effect. However, the dilution effect seemed not likely to directly explain the elevation of alveolar alarmins in RAS, which ranged from 5 to 10 times higher than in BOS and from 5 to 30 times higher than in No CLAD. Third, we could not match immunosuppressive regimen and azithromycin administration of patients with No CLAD with those of RAS and BOS. This could be due to our protocol for initial immunosuppressive regimen and treatment for CLAD (11de Perrot M Chaparro C McRae K et al.Twenty-year experience of lung transplantation at a single center: Influence of recipient diagnosis on long-term survival.J Thorac Cardiovasc Surg. 2004; 127: 1493-1501Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), which could result in a switch from cyclosporine A to tacrolimus and/or azithromycin administration in patients with CLAD. There might be concerns that the use of tacrolimus and azithromycin may confound interpretation of the presented results. However, at least existing evidence to date does not seem to strongly support the positive correlation between the use of tacrolimus/azithromycin and expression of S100 proteins or HMGB1; azithromycin treatment reportedly reduces the plasma concentration of S100A8/A9 in patients with cystic fibrosis (32Ratjen F Saiman L Mayer-Hamblett N et al.Effect of azithromycin on systemic markers of inflammation in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa.Chest. 2012; 142: 1259-1266Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and tacrolimus/cyclosporine suppress HMGB1 release from conditioned rat astrocytes (33Gabryel B Bielecka A Bernacki J Labuzek K Herman ZS Immunosuppressant cytoprotection correlates with HMGB1 suppression in primary astrocyte cultures exposed to combined oxygen-glucose deprivation.Pharmacol Rep. 2011; 63: 392-402Crossref PubMed Scopus (16) Google Scholar). Fourth, we could not perform adequate TBBx sampling in all patients, partly due to inadequate sampling or unstable respiratory status during bronchoscopy. Although all the cases with adequate TBBx sampling showed minimal acute rejection in our study, further investigation is necessary to reveal underlying histologic condition in relation to expression of alveolar alarmins. In conclusion, our results identified distinct expression patterns of alveolar alarmins in RAS and BOS, supporting the contention that RAS and BOS may represent biologically different subtypes of CLAD. Our ultimate goal is to predict CLAD and to delineate its subtypes more accurately. Further efforts in delineating the underlying pathological mechanisms and refinements in biologic profiling will lead to a better understanding of CLAD, as well as the development of specifically targeted personalized therapies for patients with CLAD. The authors would like to express our gratitude to Jerome Valero's assistance for editing this manuscript. The authors thank Paul Chartrand, Eleanor Balmaceda and Sarah E. Gilpin for facilitating this project. The authors also thank Dr. Naoki Minato and Dr. Yukihito Saito for their mentorship. This research was supported by the Canadian Institutes of Health Research (grant#190953), Canadian Cystic Fibrosis Foundation (grant#2387), Ontario Ministry of Research and Innovation (grant#GL2-01-019) and MC's Canada Research Chair in Lung Transplantation. TS is supported by Research Fellowships of the Japan Society for Promotion of Science for Young Scientists. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. Additional Supporting Information may be found in the online version of this article. Download .docx (.02 MB) Help with docx files Supplemental Materials. Download .pdf (.1 MB) Help with pdf files Table S1: Protein expressions in bronchoalveolar lavage fluid.
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