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Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of interstitial lung diseases (ILDs), marked by an ongoing, chronic fibrotic process within the lung tissue. IPF leads to an irreversible deterioration of lung function, ultimately resulting in an increased mortality rate.
- idiopathic pulmonary fibrosis
- biomarkers
- Krebs von den Lungen 6
- surfactant proteins
- MUC5B
- Cleaved Cytokeratin 18
1. Introduction
Interstitial lung diseases (ILDs) include a heterogenous group of more than 100 pulmonary disorders distinguished by extensive inflammation and fibrotic changes in the pulmonary tissues. The classification of ILDs involves the analysis of histopathological specimens and radiological criteria correlating with clinical factors. This process typically requires a comprehensive assessment due to their intricate characteristics [1,2,3].
Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of ILDs, marked by an ongoing, chronic fibrotic phenomena causing gradual deterioration of lung function, leading to respiratory failure and death [4,5,6]. IPF frequently affects men; smoking and a history of professional inorganic dust exposure are also risk factors [7].
Early IPF detection is particularly challenging as symptoms are initially mild and non-specific; a definite IPF diagnosis usually relies on high-resolution computed tomography of the chest (HRCT) and lung tissue biopsy with the additional burden of ruling out other morbid conditions that may cause pulmonary fibrosis [7,8,9,10].
With the advent of antifibrotic drugs, early IPF diagnosis became essential in order to preserve the quality of life; therefore, biomarkers usable for risk assessment, early detection, prognosis, or management of the disease became necessary and sought after [11]. Potential biomarkers might reflect various biological phenomena: alveolar epithelial cell damage and dysfunction, extracellular matrix remodeling, fibroblast proliferation, and immune dysfunction/inflammation [12,13].
2. Biomarkers
The advent of biomarkers (also known as biological markers) brought significant changes in clinical practice and the management of some diseases—the impact is evident for, at least, neoplastic disorders—such as digestive solid tumors or prostate cancer [18,19]. An ideal biomarker should be easily obtainable, consistently measurable, and suitable for repeated monitoring while offering several benefits compared to current medical assessments in terms of simplicity, time efficiency, or cost-effectiveness [20].
There is no such ideal biomarker yet identified for idiopathic pulmonary fibrosis, but there are compelling data suggesting that a combination of biomarkers might have a higher rate of success and accuracy in predicting the disease progression [6]. Several biomarkers identified in the peripheral blood exhaled breath condensate or bronchoalveolar lavage offer additional information and should complement imagistics or invasive procedures such as transbronchial or surgical lung biopsy [21].
The disruption of the alveolar basement membrane represents a critical feature of idiopathic pulmonary fibrosis (IPF), triggering abnormal activation of alveolar epithelial cells, increasing the migration of inflammatory and mesenchymal cells into the alveolar space; this process is accompanied by the aberrant cell senescence of the epithelial cells and fibroblasts of the alveoli [22]. Signs of epithelial cell damage hold promise as diagnostic and prognostic biomarkers for IPF, consequently offering valuable support in its clinical care [23]. The main biomarkers associated with alveolar epithelial cell dysfunction are represented by Krebs von den Lungen 6 (KL-6), surfactant proteins, mucin 5B (MUC5B), oncomarkers (CA 15-3, CA 125, CA 19-9, and CEA), Clara cell secretory protein (CC16), telomeres shortening, cleaved cytokeratin 18 (cCK-18), alpha-v beta-6 (αvβ6), and alpha-v beta-1 (αvβ1) integrin [13,22,24].
3. Krebs von den Lungen 6 (KL-6)
Alveolar type 2 epithelial cells (AEC-II) produce a mucinous glycoprotein named Krebs von den Lungen 6, well-known as MUC1, which contains three segments: an extracellular domain (MUC1-N), a singular transmembrane region, and a cytoplasmic tail. The KL-6 epitope domain, elevated in IPF, is included in the extracellular environment. The release of KL-6 following injury is correlated with lung fibrosis and epithelial-mesenchymal transition. While there is evidence associating serum KL-6 levels with IPF progression, a knowledge gap persists regarding the biological activation and cell signaling of the MUC1 cytoplasmic tail in IPF. During the onset of pulmonary fibrosis, the discharge of the extracellular KL6/MUC1 domain results from the proteolytic cleavage near the plasma membrane, mediated by heightened levels of metalloprotease. Fibroblast activity is modulated by the soluble KL6/MUC1, which activates still unidentified targets. Antibodies against KL6 have shown efficacy in alleviating pulmonary fibrosis induced by bleomycin [25].
When AEC-IIs are affected, KL-6 is released into the circulation, enabling it to be detected and measured in the serum [22,23,26]. Even though there are some countries, such as Japan, where KL-6 serves as a diagnostic biomarker for IPF, several studies revealed that this glycoprotein is more effective as a predictive biomarker [22,27].
In IPF, the evolution might be favorable if the initial value of KL-6 in the serum is below 1000 U/mL and there is no continuous rise rather than in situations with ongoing elevated KL-6 or values equal to or higher than 1000 U/mL at the outset [22].
Acute exacerbations of IPF might also be predicted by a high initial serum KL-6 value (more than 1300 U/mL) [28].
KL-6 initial serum level could also potentially serve as a significant means to evaluate the effectiveness of treatment with nintedanib, as its value tends to decrease during therapy [29,30].
4. Surfactant Proteins
Alveolar epithelial type II cells are also responsible for producing lung surfactant, a critical factor not only in preventing lung collapse during the process of breathing, but also in immunity, due to its components (lipids and proteins) and their specific properties [31,32,33,34].
AEC-II cells are accountable for the synthesis, secretion, and recycling of all surfactant components. This process reduces surface tension, enabling normal breathing at transpulmonary pressure. Biochemical studies of the surfactant reveal a composition of approximately 90% lipid and 10% protein. The inquiry arises since, even though AEC-II possesses the capacity for renewal and self-regulation, they still undergo chronic injury in IPF. A pertinent observation from familial IPF suggests that mutations in surfactant proteins, along with telomere mutations, are correlated with chronic AEC-II cell injury and apoptosis [35].
Two of the proteins linked to lung surfactant, SP-A and SP-D, possess significant roles in natural immunity [31,34]. In contrast, the other two proteins (SP-B and SP-C) exhibit strong hydrophobic properties and enhance the reduction in surface tension while interacting with the lipids from the surfactant [31].
Research on variations in SP-A and SP-D concerning polymorphisms and protein levels in bronchoalveolar lavage and blood has revealed a connection with various pulmonary conditions, including IPF [36].
SP-A, encoded by SFTPA1 and SFTPA2 genes, is produced by airway epithelial cells. Mutations in SFTPA are linked to the development of ILDs and susceptibility to pulmonary adenocarcinoma. SP-A, comprising a glycosylate C-terminal lectin domain and an NH2-terminal collagenous domain, forms trimeric complexes and multimers in the airway, essential to ensure the surfactant lipid particle structure. Certain SFTPA mutations lead to misfolded protein responses and epithelial injury. SP-A mutations activate TGF-β signaling, potentially playing a role in the development of alveolar lung disease [36].
Similar to SP-A, SP-D is formed by three polypeptide chains interconnected by disulfide bonds, and all polypeptide chains are replicas of the SFTD gene. The regulatory mechanism governing the transcription of SP-D is not well comprehended, and there is a limited number of studies compared to the extensive research on SP-A regulation.
Polymorphisms in SP-A1, particularly the 6A4 allele markers, have been proposed as potential risk factors for IPF. Conversely, no examined variants of SP-D showed correlations with IPF. The concentration of SP-D was additionally discovered to correlate with the degree of collapse of the pulmonary parenchyma and the annual rate of deterioration in lung function. SP-A and SP-D demonstrate the potential to stimulate the synthesis of collagen and metalloproteinase, suggesting a plausible direct interaction in the pathogenesis of IPF [37].
Alongside Krebs von den Lungen, SP-A and SP-D are valuable biomarkers in prognosis and monitoring therapy progress, as their values decrease after treatment with antifibrotic drugs [38,39].
5. The Mucin MUC5B
Mucus clearance represents the primary mechanical defense process within the lungs [40]. The airway mucus contains several glycosylated proteins of substantial size (up to three million Daltons per monomer), called mucins, which ensure its gel-like structure [40,41,42]. Even though various genes are identified, it is essential to state that only 11 of them are localized in the lungs, and among these, only two exhibit high levels of expression: MUC5B and MUC5AC [42].
In IPF, MUC5B seems to have predictive value and can also be used for prognosis [43]. Previous studies showed that the functionality of the MUC5B promoter in IPF is enhanced by the existence of its rs35705950 variant, found in 38% of the patients suffering from IPF [44,45].
The research observed that in Chinese individuals, this specific polymorphism is not very common, while in European black race patients with IPF, it is entirely absent, which demonstrates that its occurrence is distinct in different target populations [22].
Simultaneously, there are correlations between MUC5B gene polymorphism and the specific imaging of IPF, as this element is related to the existence and predominance of subpleural fibrosis [45]. Several other factors are described to be involved in developing high values of MUC5B in IPF, such as sequence-binding DNA factors, inflammatory agents, or cellular communication pathways [42].
As of yet, the distinct mechanism through which MUC5B is involved in the development of IPF has not been specified. Many opinions tend to converge toward a debate regarding a possible susceptibility to disease based on MUC5B variants in human lungs. The idea, according to the fact that MUC5B overexpression alters not only mucociliary defense but also the complex process of airway epithelial cell repair, becomes increasingly outlined. Because a significant number of transcriptional factors and inflammatory mediators influence and interfere with MUC5B overexpression, many aspects related to their actions remain to be elucidated, when considering MUC5B intervention [42].
In IPF, MUC5B seems to have predictive value and can also be used for prognosis [43]. The early significance of MUC5B presence in the distal airways may indicate the onset of IPF, guiding efforts toward preventive measures for disease progression [46].
Nevertheless, the existence of MUC5B rs35705950 had no concrete effect on the treatment outcome with nintedanib or pirfenidone, but it suggests a higher survival rate in patients affected by IPF [47,48].
6. Oncomarkers (CA 15-3, CA 19-9, CA 125, and CEA)
Up to this point, research has highlighted that IPF and lung cancer are similar in some particular aspects, from genetic and epigenetic markers, to risk factors (age, smoking, and work-related or environmental exposure) and several disturbances that occur in the molecules and cells involved in cellular communication pathways [13,49].
A study developed in China showed that, in patients with IPF, the incidence of developing lung cancer is higher than in the general population (23.1%) [49].
Even though tumor markers are usually assessed in cancers, their values are relevant in some benign pathologies, such as interstitial lung diseases or idiopathic lung fibrosis [49]. In IPF, oncomarkers are correlated with pulmonary function, the progression of the illness, and a heightened risk of mortality [50].
The most specific tumor markers used in the management of patients with IPF are carbohydrate antigen (CA) 15-3, CA 19-9, CA 125, and CEA [13,22].
6.1. CA 15-3
The MUC1 gene produces CA 15-3, a substantial glycosylated molecule that contains a wide extracellular portion, a membrane-penetrating segment, and a cytoplasmic section, which is well-known as one of the most used markers in breast cancer [13,51].
At the same time, CA 15-3 is relevant as a prognostic marker in IPF, quantifying the severity of the disease, and can be used as an alternative to KL-6 due to its accessibility and lower cost [52,53,54].
A study developed by Rusanov et al. concluded that this biomarker might also be helpful in follow-up since the values of CA 15-3 decrease after pulmonary transplant in individuals with IPF [55].
Nevertheless, there are no notable distinctions found in the connection between CA 15-3 levels and pulmonary function tests or in the survival rates between IPF individuals treated with nintedanib and those who underwent therapy with pirfenidone [52].
6.2. CA 19-9
Since Koprowski’s first description of CA 19-9 in 1979 as a carbohydrate produced by the exocrine epithelial cells, this biomarker has been used for the management of gastrointestinal cancers (pancreas, stomach, or colon) [56,57,58]. Nonetheless, increased values of CA 19-9 were detected in several non-malignant conditions, such as IPF or non-tuberculous mycobacterial lung disease [57,58].
On the one hand, higher levels of CA 19-9 were found in end-stage individuals affected by IPF, suggesting that its role could be more specific in predicting the severity rather than the evolution of the disease [59]. On the other hand, the initial serum levels of the biomarker make possible the differentiation between patients affected by progressive disease and those with stable conditions, suggesting that CA 19-9 could also be an effective IPF prognostic biomarker [60].
The reasons behind the increase in CA 19-9 are not fully understood. One theory suggests that injured lungs may release an excess of CA 19-9 as epithelial cells regenerate. Interestingly, significantly affected lungs have been associated with reduced levels of CA-19, potentially linked to the impaired ability to regenerate alveolar epithelium in certain patients. Currently, the molecular involvement of elevated CA 19-9 levels in IPF and other ILDs is speculative [59].
6.3. CA 125
CA 125, encoded by the homonymous MUC16 gene, is a glycoprotein that constitutes a significant part of mucus, offering protection against pathogens to the surfaces of different organs (such as the lungs, pleura, peritoneum, endometrium, and endocervix) and is primarily linked to ovarian cancer [61,62].
It is currently used as a biomarker that indicates epithelial injury in progressive IPF due to its substantial secretion by the metaplastic epithelium [54]. The PROFILE cohort study showed that increasing values of CA 125 reflect not only the disease progression but also the overall survival rate, suggesting the potential development of CA 125 as a theranostic marker for evaluating the efficacy of antifibrotic drugs [63]. This theory was confirmed with another study, which highlighted that CA 125 served as a predictor for transplant-free survival in individuals who received antifibrotic treatment. However, the cutoff values were higher in the subjects who underwent therapy in contrast to those who did not [64].
6.4. CEA
Carcinoembryonic antigen (CEA), first discovered in 1965 by Gold and Freedman, is a glycoprotein originating from the embryonic endodermal epithelium during fetal development that is typically no longer present in the bloodstream after birth. Still, trace amounts may persist in colon tissue [65,66]. Heightened expression of CEA is found in adenocarcinomas, affecting not only the colon but also other organs (including the pancreas, lungs, ovaries, or breasts) [67].
In roughly 50% of IPF individuals, there is an elevated serum CEA level, which is associated with the severity of the disease [68]. Studies demonstrated that patients with IPF have a noteworthy inverse relation between serum CEA levels and lung function [54].
This biomarker could also serve as a potential indicator for predicting the mortality of pulmonary transplant patients with IPF [69]. Even though its role in IPF is not fully known, CEA could become a substantial biomarker for both diagnosis and prognosis in IPF [54].
6. Clara Cell Secretory Protein (CC16)
Max Clara described the human Clara cells (also known as club/goblet/exocrine cells) for the first time in 1937 as epithelial cells that do not have cilia and are involved in secretion while being most commonly found in the terminal and respiratory bronchioles [70,71,72,73]. Usually, they represent 9% of the overall airway epithelial cell population in human lungs and have a defense role by releasing different agents such as Clara cell secretory protein (CCSP) and a regenerative function while serving as progenitor cells in case of lung injury [13,73,74]. Besides this prominent localization, Clara cells can also be found in other organs such as the kidneys, prostate, or uterus [73,74]. Their specialized function in processing toxins is also underlined by the high content of cytochrome P450 mono-oxygenases and flavin-containing monooxygenases, which make these cells sensitive to external pollutants and easily influenced by pathogens and harmful molecules [74].
Activated in response to alveolar injury, club cells contribute to injury repair. Although the specific role of CC16 in this process is not fully elucidated, several studies suggest that migrating club cells replace damaged alveoli during injury resolution. The heightened blood capillary permeability of alveoli may aid the diffusion of CC16, secreted by these migratory club cells, into circulation. Considering that IPF predominantly impacts the alveolar epithelium, an elevated CC16 level in the serum could be a reasonable expectation [75].
An elevation of the club cells’ proteins has been detected in the bronchoalveolar lavage (BAL) and blood serum of individuals suffering from different lung diseases, such as sarcoidosis or pulmonary fibrosis, as well in patients mechanically ventilated with elevated positive end-expiratory pressure (PEEP) settings [73]. While the precise reasons remain unclear, these findings could arise from the prompt stimulation of club cells, promoting CC16 secretion, particularly during the initial phases of lung impairment [75].
While being analyzed through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), CCSP exhibits a detectable molecular size of 10 kiloDaltons, leading to its designation as CC10 [76]. Nonetheless, since the precise molecular mass observed through spectrometry is 15.840, a more accurate abbreviation for CCSP is considered to be CC16 [76].
More recent research has focused on determining if the serum values of CC16 might help to differentiate between IPF and non-IPF ILD, but the results concluded that limited sensitivity hinders its role as a possible biomarker in this matter [77]. Ivette Buendía-Roldán et al. also highlighted that Clara cells influence the process of lung injury recovery by using a factor-related apoptosis-inducing ligand (TRAIL) to trigger the death of the epithelial cells localized at the distal airways and alveoli level [77]. Even though TRAIL-expressing Clara cells were found in individuals with IPF, the involvement of CC16 has not yet been studied, leaving this matter as an opportunity for future research [77].
7. Telomere Shortening
Telomeres represent nucleoprotein complexes composed of repeated TTAGGG hexamer sequences, having as their primary function the protection of the structural integrity and functionality of the terminal regions of linear human chromosomes [78,79]. They contribute to enabling the complete replication of chromosomes, controlling the gene expression and the replicative potential of the cells and their transition into senescence [80].
Telomerase is a ribonucleoprotein complex accountable for the extension of telomeres, which is usually active during pregnancy in immature cells that are not differentiated and in a limited number of lymphocytes [81]. This matter is reflected in considering the shortening of the telomeres as a primary characteristic of the aging process, which happens due to the widely recognized end replication challenge or as a consequence of various biological occurrences [82]. Numerous proteins ensure the proper functioning of the telomerase complex, including telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC) [21].
Telomeres trigger either apoptotic or cellular senescence pathways upon reaching a critical length [83]. Despite the general trend of telomere length diminishing with age, there are significant variations in its average value among different species, and this does not consistently align with life expectancy [82]. A representative indicator of telomere length throughout the body is the length of leukocyte telomeres (LTL), which can be detected in the bloodstream [78].
In IPF, telomere shortening is reported in 25% of the cases, while it is found in 50% of individuals with familial pulmonary fibrosis (FPF); in both situations, its presence suggests a poor prognosis and heightened morbidity post-transplantation [84]. At the same time, in IPF, telomere shortening is linked on its own with a reduced survival rate [22,85]. Shorter telomeres are considered to be a risk factor for the onset of IPF, and their role is also centered on the predictive value and the potential issues associated with transplantation since multiple hematologic complications were detected in patients with both IPF and telomerase mutation [22,81,84]. Aberrant telomere shortening resulting from mutations in TERT and TERC has been identified in 8–15% of individuals with FPF and in up to 3% of sporadic IPF cases [22]. A swifter diminishing in FVC was observed in the IPF cases with shorter telomeres compared to those with longer telomeres, highlighting a notable interplay between the length of telomeres and the reduction in lung function [86]. Future development of new therapies involving telomerase targeting might lead to promising results [86].
8. Cleaved Cytokeratin 18 (cCK-18)
cCK-18 is a structural protein localized in the epithelial tissues, including the alveoli, which is cleaved twice by caspases in the process of epithelial cell apoptosis and is detectable with M30 antibodies [87].
Indicators of endoplasmatic reticulum stress and the unfolded protein response (UPR) show elevated levels of lung AEC-II in IPF individuals. The endoplasmic reticulum contributes to preserving homeostasis in the presence of unfolded or incorrectly folded proteins through the UPR. If the UPR is unsuccessful in ensuring homeostasis, apoptosis follows. Studies have shown that the alveolar epithelium of the lungs in IPF individuals exhibits activation of the UPR.
cCK-18, found in lung AECs in patients suffering from IPF, is produced through the activation of the UPR in vitro and exhibits distinct elevation in the serum of IPF individuals compared to both normal subjects and those with other ILDs. This fact implies that cCK-18 might serve as a biomarker for the UPR and AEC apoptosis, holding potential as a monitoring tool for therapies that regulate them in patients with IPF [87].
9. Alpha-v beta-6 (αvβ6) Integrin
Increased signaling of transforming growth factor-β1 (TGF-β1) facilitates the transition of fibroblasts into myofibroblasts, stimulates collagen gene expression, and leads to the accumulation of fibrous tissue that adversely affects pulmonary function. αv integrins are a group of five heterodimeric transmembrane proteins facilitating the transmission of mechanical force between cells and the extracellular matrix. At the same time, they have been identified as crucial mediators of TGF-β activation in fibrosis [88].
Alpha-v beta-6 (αvβ6) integrin, found only in epithelial cells, acts as an activator of transforming growth factor-β1 (TGF-β1), which represents an underlying mechanism of IPF [89,90]. Since its values are elevated in IPF, αvβ6 integrin seems to be relevant as a prognosis biomarker while assessing the disease progression [91,92]. Increased levels of αvβ6 integrins strongly correlate with an unfavorable prognosis [93].
Although biochemically identified over two decades ago, alpha-v beta-1 (αvβ1) integrin has received limited attention. Its composition, involving α and β subunits found in various heterodimers (5 for αv and 12 for β1), has presented challenges in developing antibodies specific to particular heterodimers or deducing function from gene knockout studies [24].
This entry is adapted from the peer-reviewed paper 10.3390/jpm13111607
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