Welcome to the club: Opening the door for club cell secretory protein as biomarker in lung transplantation

Airway epithelial injury is considered critical for the development of small airways fibrosis present in posttransplant chronic lung allograft dysfunction (CLAD). Club cells (formerly known as Clara cells) are nonciliated, secretory cells of the airway epithelium, mainly situated in the terminal and respiratory bronchioles, identified by expression of club cell secretory protein (CCSP) (also known as CC-16, CC-10, uteroglobin, or secretoglobin family 1A member 1 [SCGB1A1]). In physiological conditions, club cells, which constitute up to 44% of all proliferating cells in the small airways, function as epithelial progenitors for regeneration and repair of lung injury.1Rokicki W Rokicki M Wojtacha J Dżeljijli A. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases.Kardiochir Torakochir Pol. 2016; 13: 26-30https://doi.org/10.5114/kitp.2016.58961Crossref PubMed Scopus (67) Google Scholar,2Zuo WL Shenoy SA Li S et al.Ontogeny and biology of human small airway epithelial club cells.Am J Respir Crit Care Med. 2018; 198: 1375-1388https://doi.org/10.1164/rccm.201710-2107OCCrossref PubMed Scopus (57) Google Scholar Moreover, club cells are thought to play a vital role in lung host defenses, by regulating pulmonary homeostasis and modulating the pulmonary immune system via CCSP, which displays broad anti-inflammatory, antioxidative, and immunoregulatory functions. For instance, CCSP inhibits phospholipase A2, expressed by alveolar macrophages and airway epithelial cells, which is the primary enzyme responsible for inflammatory arachidonic acid release.3Yoshikawa S Miyahara T Reynolds SD et al.Clara cell secretory protein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice.J Appl Physiol (1985). 2005; 98: 1264-1271https://doi.org/10.1152/japplphysiol.01150.2004Crossref PubMed Scopus (43) Google Scholar CCSP also inhibits the release of oxidants from activated neutrophils and enhances their phagocytic ability.4Katavolos P Ackerley CA Clark ME Bienzle D. Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils.Vet Immunol Immunopathol. 2011; 139: 1-9https://doi.org/10.1016/j.vetimm.2010.07.021Crossref PubMed Scopus (21) Google Scholar Club cells are the principal site of localization of the cytochrome P-450 monooxygenase system in the human airway tract, and engage in xenobiotic metabolism of inhaled compounds, such as chemicals (i.e., sulfur mustard, naphthalene, chlorine, and diacetyl), particulate matter (i.e., cigarette smoke) and toxins.5Enlo-Scott Z Bäckström E Mudway I Forbes B. Drug metabolism in the lungs: opportunities for optimising inhaled medicines.Expert Opin Drug Metab Toxicol. 2021; 17: 611-625https://doi.org/10.1080/17425255.2021.1908262Crossref PubMed Scopus (17) Google Scholar,6Teitz-Tennenbaum S Viglianti SP Jomma A et al.Sustained club cell injury in mice induces histopathologic features of deployment-related constrictive bronchiolitis.Am J Pathol. 2022; 192: 410-425https://doi.org/10.1016/j.ajpath.2021.11.012Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Club cell metabolism, injury, loss and/or alterations in CCSP levels have all been associated with pathophysiologic small airways remodeling, and the presence of obliterative or constrictive bronchiolitis, in inhalation exposures,6Teitz-Tennenbaum S Viglianti SP Jomma A et al.Sustained club cell injury in mice induces histopathologic features of deployment-related constrictive bronchiolitis.Am J Pathol. 2022; 192: 410-425https://doi.org/10.1016/j.ajpath.2021.11.012Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar chronic obstructive pulmonary disease,7Laucho-Contreras ME Polverino F Tesfaigzi Y Pilon A Celli BR Owen CA. Club cell protein 16 (CC16) augmentation: a potential disease-modifying approach for chronic obstructive pulmonary disease (COPD).Expert Opin Ther Targets. 2016; 20: 869-883https://doi.org/10.1517/14728222.2016.1139084Crossref PubMed Scopus (54) Google Scholar asthma,8Wang M Tang K Gao P et al.Club cell 10-kDa protein (CC10) as a surrogate for identifying type 2 asthma phenotypes.J Asthma. 2023; 60: 203-211https://doi.org/10.1080/02770903.2022.2040531Crossref PubMed Scopus (2) Google Scholar and cystic fibrosis.9Zhai J Emond MJ Spangenberg A et al.Club cell secretory protein and lung function in children with cystic fibrosis.J Cyst Fibros. 2022; 21: 811-820https://doi.org/10.1016/j.jcf.2022.03.007Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar A key role for club cells has also been demonstrated in the onset of pulmonary fibrosis.10Park SY Hong JY Lee SY et al.Club cell-specific role of programmed cell death 5 in pulmonary fibrosis.Nat Commun. 2021; 12: 2923https://doi.org/10.1038/s41467-021-23277-8Crossref PubMed Scopus (8) Google Scholar,11Fukumoto J Soundararajan R Leung J et al.The role of club cell phenoconversion and migration in idiopathic pulmonary fibrosis.Aging (Albany NY). 2016; 8: 3091-3109https://doi.org/10.18632/aging.101115Crossref PubMed Scopus (19) Google Scholar Interestingly, in the context of lung transplantation, club cells/CCSP may suppress alloreactivity. Indeed, in a murine orthotopic lung transplant model it was shown that club cell loss alone was sufficient to trigger obliterative bronchiolitis pathology and to promote adaptive immunity, with expansion of Th1 and Th17 effector T cells.12Liu Z Liao F Scozzi D et al.An obligatory role for club cells in preventing obliterative bronchiolitis in lung transplants.JCI Insight. 2019; 5e124732https://doi.org/10.1172/jci.insight.124732Crossref Scopus (19) Google Scholar Also, CCSP inhibits leukocyte to endothelial cell adherence by binding to integrin α4β1, also known as adhesion molecule "very late antigen 4" (VLA-4).13Johnson MDL Younis US Menghani SV et al.CC16 Binding to α4β1 integrin protects against Mycoplasma pneumoniae infection.Am J Respir Crit Care Med. 2021; 203: 1410-1418https://doi.org/10.1164/rccm.202006-2576OCCrossref PubMed Scopus (14) Google Scholar VLA-4 is present on all leukocytes except neutrophils, and plays a key role in lymphocyte differentiation, extravasation, and T-cell receptor co-stimulation.14Bertoni A Alabiso O Galetto AS Baldanzi G. Integrins in T cell physiology.Int J Mol Sci. 2018; 19: 485https://doi.org/10.3390/ijms19020485Crossref PubMed Scopus (44) Google Scholar CCSP is easily detectable in serum following diffusion into the bloodstream and is therefore thought to be involved in modulation of local T-cell responses, migration, and accumulation in the lungs. Furthermore, reduced CCSP levels were recently associated with exosome release from natural killer cells and immune responses to donor HLA and lung self-antigens in chronic lung allograft rejection.15Itabashi Y Ravichandran R Bansal S et al.Decline in club cell secretory proteins, exosomes induction and immune responses to lung self-antigens, kα1 tubulin and collagen V, leading to chronic rejection after human lung transplantation.Transplantation. 2021; 105: 1337-1346https://doi.org/10.1097/TP.0000000000003428Crossref PubMed Scopus (12) Google Scholar,16Ravichandran R Itabashi Y Liu W et al.A decline in club cell secretory proteins in lung transplantation is associated with release of natural killer cells exosomes leading to chronic rejection.J Heart Lung Transplant. 2021; 40: 1517-1528https://doi.org/10.1016/j.healun.2021.08.016Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Mounting clinical evidence corroborates a putative role of club cells/CCSP in CLAD pathophysiology, as lung recipients with bronchiolitis obliterans syndrome (BOS) demonstrated selective loss of CCSP-expressing cells in the airways (decreased CCSP transcripts and protein expression in lung tissue), and reduced CCSP levels in bronchoalveolar lavage fluid (BALF).17Kelly FL Kennedy VE Jain R et al.Epithelial Clara cell injury occurs in bronchiolitis obliterans syndrome after human lung transplantation.Am J Transplant. 2012; 12: 3076-3084https://doi.org/10.1111/j.1600-6143.2012.04201.xAbstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Also, early posttransplant BALF and/or serum CCSP levels were lower in patients who later developed BOS after lung transplantation18Nord M Schubert K Cassel TN Andersson O Riise GC. Decreased serum and bronchoalveolar lavage levels of Clara cell secretory protein (CC16) is associated with bronchiolitis obliterans syndrome and airway neutrophilia in lung transplant recipients.Transplantation. 2002; 73: 1264-1269https://doi.org/10.1097/00007890-200204270-00013Crossref PubMed Scopus (55) Google Scholar or after allogeneic stem cell transplantation.19Mattsson J Remberger M Andersson O Sundberg B Nord M. Decreased serum levels of Clara cell secretory protein (CC16) are associated with bronchiolitis obliterans and may permit early diagnosis in patients after allogeneic stem-cell transplantation.Transplantation. 2005; 79: 1411-1416https://doi.org/10.1097/01.tp.0000158354.39635.abCrossref PubMed Scopus (0) Google Scholar Conversely, an increase in CCSP-expressing cells 1 to 3 months posttransplant was detected in patients who remained free from BOS, whereas cell numbers remained unchanged in those who later developed CLAD.20Bourdin A Mifsud NA Chanez B et al.Donor Clara cell secretory protein polymorphism is a risk factor for bronchiolitis obliterans syndrome after lung transplantation.Transplantation. 2012; 94: 652-658https://doi.org/10.1097/TP.0b013e31825ffca6Crossref PubMed Scopus (27) Google Scholar Interestingly, donor CCSP A38G gene polymorphism has been associated with decreased donor blood CCSP levels before lung transplantation,21Hin A Kannengiesser C Roussel A et al.Donor club cell secretory protein G38A polymorphism is associated with a decreased risk of primary graft dysfunction in the French cohort in lung transplantation.Transplantation. 2018; 102: 1382-1390https://doi.org/10.1097/TP.0000000000002143Crossref PubMed Scopus (5) Google Scholar and with decreased BALF CCSP levels early after lung transplantation,20Bourdin A Mifsud NA Chanez B et al.Donor Clara cell secretory protein polymorphism is a risk factor for bronchiolitis obliterans syndrome after lung transplantation.Transplantation. 2012; 94: 652-658https://doi.org/10.1097/TP.0b013e31825ffca6Crossref PubMed Scopus (27) Google Scholar putting those recipients at risk for later BOS. However, until now, evidence from large multicenter studies on CCSP in lung transplant recipients was missing. In the current issue, Todd et al investigate the relationship between BALF CCSP levels and early posttransplant allograft injury; and determine if reductions in early posttransplant BALF CCSP levels are indicative for later CLAD.22Todd JL Weber JM Kelly FL et al.Early posttransplant reductions in club cell secretory protein associate with future risk for chronic allograft dysfunction in lung recipients: results from a multicenter study.J Heart Lung Transplant. 2023; (To be completed by Editorial office)Abstract Full Text Full Text PDF Scopus (1) Google Scholar For this, they evaluated 1606 BALF samples, paired with a biopsy gradable for acute perivascular cellular rejection, serially collected over the first posttransplant year from 392 adult lung recipients at 5 centers from the Clinical Trials in Organ Transplantation (CTOT)-20 (NCT02631720) study, a prospective observational study of newly transplanted adult lung recipients in North America. BALF CCSP levels were normalized to BALF total protein content and determined using a commercial ELISA assay. The presence of BALF microorganisms was assessed by routine culture or polymerase chain reaction, according to each center's standard operating procedures. Histologic acute cellular rejection (AR) and lymphocytic bronchiolitis (LB) grading of concurrent biopsies was performed according to consensus guidelines23Stewart 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: 1229-1242https://doi.org/10.1016/j.healun.2007.10.017Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar; and assessment of nonrejection pathology, such as acute lung injury (ALI) and organizing pneumonia (OP), was harmonized by means of a pathologists’ working group to share microscopic descriptors and diagnostic features. Next, protein-normalized BALF CCSP concentrations were correlated with allograft histology or infection events, using generalized estimating equations to fit linear models to account for potential correlation from using multiple samples from the same patient. Analyses were performed both unadjusted and adjusted for donor age, time posttransplant and transplant type (single vs double lung transplant). Finally, using a time-dependent binary covariate for reduced normalized BALF CCSP levels in the first year posttransplant, cox proportional hazards model assessed the risk for later development of probable or definite CLAD.24Verleden GM Glanville AR Lease ED et al.Chronic lung allograft dysfunction: definition, diagnostic criteria, and approaches to treatment: a consensus report from the Pulmonary Council of the ISHLT.J Heart Lung Transplant. 2019; 38: 493-503https://doi.org/10.1016/j.healun.2019.03.009Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar Interestingly, compared to "healthy" BALF samples, CCSP concentrations were significantly reduced in "anomalous" samples, i.e., BALF with concurrent AR/LB (19% lower), AR/LB with another histologic/microbiological finding (37%), OP/ALI (33%), or OP/ALI with another histologic/microbiological finding (48%). This association between lower CCSP and AR was strengthened when considering severe rejection (A2-grade) only (p=0.006). On the other hand, no differences in BALF CCSP concentrations were seen between infection-only vs healthy samples. This suggests that rejection-mediated airway epithelial injury, rather than airway infection, lowers CCSP levels. Reduced CCSP levels may thus be a proxy for selective loss of (CCSP-producing) club cells in the airways, and since club cells/CCSP have been suggested to play a role in the pathobiology of CLAD, CCSP may thus be an interesting biomarker to assess the risk for future CLAD development. Indeed, Todd et al. next demonstrated that any occurrence of a BALF CCSP level below the median (8.63 (4.1-16.7) ng/µg) during the first posttransplant year was associated with an almost 2-fold increased risk for later probable CLAD, independent of other factors previously linked to CLAD (adjusted hazard ratio 1.95; p = 0.035), with a median time of 474 (235-720) days between reduced CCSP and CLAD onset. These findings are interesting and may open the door for the use of CCSP as a biomarker for monitoring of posttransplant complications, and for early posttransplant risk stratification. However, by looking at the data, several questions arise. For instance, overall CCSP levels were almost 30% lower in single (n = 96, with 391 BALF samples) vs double lung transplant recipients (n = 296, with 1215 BALF samples), which is difficult to explain from a physiologic perspective since BALF was selectively obtained by wedged bronchoscopy sampling from the lung allograft, which approach prevents "spill over" from the contralateral lung, thus not explaining the higher CCSP levels found in double lung recipients. Also, it is neither clear how CCSP levels evolve over time in a patient with a single AR/LB event vs in those with repeated AR/LB, nor is the evolution of CCSP levels following treatment of rejection. Moreover, no distinction was made between AR vs LB events, for severity of LB (B1 vs B2 -grade), nor between minimal-mild (A1/2) vs moderate-severe (A3/4-grade) AR samples, because of limited sample numbers (i.e., A-grade biopsies: 15.8% A1 and 5.9% ≥ A2; B-grade biopsies: 3.3% ≥ B1). Furthermore, the observed absence of a relation of CCSP levels with infection is surprising, since certain infections appear to be more harmful to the graft, i.e., are more likely to induce epithelial injury, OP/ALI, or have been associated with CLAD (i.e., cytomegalovirus, Pseudomonas aeruginosa). Yet the authors did not look into the type of microbial infection and CCSP levels, despite that CMV infection within the first post00operative year was the strongest independent risk factor for CLAD, more than CCSP levels, in adjusted multivariable risk analysis (i.e., adjusted HR 2.62 (1.60-4.28, p < 0.001) for CMV vs 1.95 (1.05-3.63, p = 0.035) for reduced CCSP levels). Also, differences in CCSP regarding CLAD phenotypes (i.e., BOS, Restrictive Allograft Syndrome/RAS, Mixed) were not evaluated, because of the modest number of CLAD events in this cohort which precludes appropriate powered analysis according to the authors. Nevertheless, after a median follow-up of 3 years, a total of 100 patients developed probable CLAD (prevalence 26.2%) and 76 developed definite CLAD (19.9%), which numbers should allow to assess possible differences in CCSP between CLAD phenotypes. Since RAS patients display more extensive pulmonary remodeling compared to BOS, which is predominantly an airway-centered disorder, pathologic differences in CLAD phenotypes could affect BALF CCSP levels prior to CLAD onset and/or during subsequent CLAD progression. Additional (unpublished) sub-analysis by the authors of the univariable hazard ratio (HR) for the association between reduced BALF CCSP levels and probable CLAD according to CLAD phenotypes, however, demonstrated an apparently higher HR in BOS compared to RAS: BOS n = 42 or 45.2%, HR 2.90 (1.15-7.31) vs RAS n = 51 or 54.8%, HR 1.97 (0.83-4.70), which is surprising and deserves further study. Finally, BALF samples were collected within the first postoperative year, with over 50% of samples obtained within the first 140 days, thus mainly reflecting (very) early posttransplant allograft injury, which questions then how these CCSP levels relate to particular donor demographics (i.e., donor age and smoking history) and perioperative risk factors for graft injury (i.e., ischemic time). Indeed, BAL CCSP levels were significantly lower in older (≥55 years) compared to younger donor lungs (<55 years), and BALF CCSP levels decreased on average 6% per 5-year increase in donor age in the current cohort (unpublished). Therefore, donor age needed to be included as adjustment variable in the correlation analysis by the authors. Again, this finding of lower CCSP levels with increasing donor/allograft age requires further study, given that senescence-related loss of (CCSP-producing) club cells was previously shown to cause progressive lung remodeling and airway-centric fibrosis, similar to the lung remodeling and fibrosis characteristic of CLAD.25Naikawadi RP Green G Jones KD et al.Airway epithelial telomere dysfunction drives remodeling similar to chronic lung allograft dysfunction.Am J Respir Cell Mol Biol. 2020; 63: 490-501https://doi.org/10.1165/rcmb.2019-0374OCCrossref PubMed Scopus (13) Google Scholar Besides looking into the above-mentioned issues, future preclinical studies should also be directed at assessing possible therapeutic interventions targeting club cells or with CCSP-supplementation in lung transplantation, for instance using the murine lung transplant model of CLAD as a proof of concept. As recombinant (rh) CCSP is easy to synthesize and has a plasma half-life of > 10 h, human clinical administration should also be feasible, as was reported in a pilot study in neonatal respiratory disease via intratracheal administration.26Levine CR Gewolb IH Allen K et al.The safety, pharmacokinetics, and anti-inflammatory effects of intratracheal recombinant human Clara cell protein in premature infants with respiratory distress syndrome.Pediatr Res. 2005; 58: 15-21https://doi.org/10.1203/01.PDR.0000156371.89952.35Crossref PubMed Scopus (92) Google Scholar In lung transplant recipients, an elegant mode of administration for targeted treatment of the allograft could be via nebulized rhCCSP. In conclusion, CCSP is another - yet interesting - protein joining the crowded club of possible novel biomarkers to assess "graft quality", however many questions remain before it can be readily incorporated in clinical monitoring or used for treatment after lung transplantation. Current findings nevertheless do shed more light on the underlying pathobiology of CLAD, which remains the major hurdle for long-term posttransplant survival, and may open the door for early therapeutic interventions aimed at CLAD prevention, which remains a significant unmet medical need. None of the authors of this manuscript have any conflicts of interest to disclose in relation to this manuscript. The authors confirm that the work described has not been published previously, that it is not under consideration for publication elsewhere, that its publication is approved by all authors, and that, if accepted, it will not be published elsewhere in the same form in English or in any other language, without the written consent of the copyright holder. All authors contributed in an important manner to the writing of the paper according to the guidelines of the International Committee of Medical Journal Editors (ICMJE). All authors have read and approved the manuscript, all authors take responsibility for the manuscript, and the submitting author has permission from all authors to submit the manuscript on their behalf. RV is a Senior Clinical Research Fellow of the Research Foundation – Flanders (FWO) and is supported by a Research Grant from the FWO (G060322N). SB is supported by the Paul Corris International Clinical Research Training Scholarship. SL is supported by the Wallenberg Foundation.