Therapeutic potential of elafin in airway inflammatory disease

Abstract

Elafin, which is derived from trappin-2 or pre-elafin by proteolysis, is an endogenous serine protease inhibitor with a low molecular weight. Its inhibitory activity is dependent on anchoring to the extracellular matrix by forming covalent bonds with its distinctive N-terminal domain via tissue transglutaminases. In addition to inhibiting proteases, it also exhibits anti-inflammatory, antibiotic, antifungal, antiviral and immunomodulatory functions. Elafin plays an important role in inflammatory disease and is a promising candidate for the anti-inflammatory treatment of respiratory diseases. This review will discuss the therapeutic potential of elafin in airway inflammation and provide evidence and suggestions for the future treatment of airway inflammatory diseases. In addition, the therapeutic potential of elafin in lung cancer is also discussed.

Introduction

Elafin, also known as PI3, has a predicted molecular mass of 11.3 kD and is a skin-derived antileucoproteinase (SKALP) or elastase-specific inhibitor (ESI), an endogenous low molecular weight inhibitor of neutrophil serine proteases. Elafin was originally isolated from the skin of psoriasis patients and is considered an “alert” antiproteinase. Elafin has been shown to affect a number of factors that are important to the inflammatory process, such as cell recruitment, the nuclear factor κB (NF-κB) pathway, and cytokine release. ¹ Elafin is expressed in bronchial secretions and was termed ESI. Subsequent studies revealed its numerous biological functions, including the modulation of inflammatory cytokine release, as well as innate and adaptive immunity. Elafin may provide a therapeutic option for chronic respiratory diseases given its critical role in cellular homeostasis and humoral immunity.

Expression and structure

Elafin is encoded by the PI3 gene, which is located on human chromosome 22 q12-13. The PI3 gene encodes a 117 amino acid elafin precursor protein, and the first 22 amino acids (signal peptide) of the N-terminal are degraded to form a precursor protein (pre-elafin/trappin-2). ² The precursor protein consists of two domains, the N-terminal and C-terminal domains. The C-terminus is composed of 57 amino acids and has a lactalbumin (WAP) domain, which is composed of four disulfide bonds. ³ It has good thermal stability, can exist stably in strong acid and alkali environments, and has complete anti-peptidase activity. Including the WAP domain, the C-terminus is mainly responsible for the inhibition of elastase. The N-terminus is composed of 38 amino acids and contains five repeat sequences that are rich in glutamine and lysine, which provide a substrate for transglutaminase, and glutamine and lysine serve as acyl groups. Donor and acceptor elafin in the extracellular matrix (ECM) can be cross-linked by tissue-specific transglutaminase and may exist in the ECM as a tissue-binding inhibitor of human neutrophil elastase (HNE). This binding of the transglutaminase substrate domain to WAP/4-disulfide is similar to that of other proteins called “trappins”.

Elafin is expressed constitutively by the epithelia of barrier tissues, including the oral cavity, trachea, lung, gut, esophagus, vagina, and epidermis, as well as some immune cells, such as monocytes, alveolar macrophages, neutral granulocytes and T cells.^4,5 Trappin paralogs vary in number and content among mammalian species. There are now 20 known trappin genes. Only eutherians and the majority of mammals have typical trappin genes in their genome sequences, and different species have different numbers and combinations of paralogs. For instance, humans have only one trappin-2 gene,^6–8 whereas pigs, cows, bovines, and hippopotamus have multiple trappin genes, and at the other extreme, the trappin gene is completely absent from mice and rats.^7,9

In comparison, eight cysteine residues in the 57 residues of the WAP domain are positioned in certain ways to produce a core of four intracellular disulfides, which is why it is sometimes called a four-disulfide core domain. ¹⁰ By binding to proteases, the trappin-2 WAP domain exerts antiprotease activity. The PI3 gene is approximately 20 kb in length and has three exons and two introns with a conventional TATA box and CACA box at the 5′-terminus. The first few amino acids of the mature protein, the signal peptide, and the 5′-untranslated region are all encoded by exon 1. The 3′-untranslated region is encoded by the third exon, whereas the second exon contains the bulk of the mature protein sequence. The NF-κB binding site in the 5′-untranslated region regulates the expression of elafin. The primary translation product is a 117 amino acid (12.3kD) immature protein that upon breakdown of a 22 amino acid hydrophobic signal sequence, yields the active trappin-2 elafin, which is then liberated from the C-terminus of trappin-2 via proteolysis and retains the TGS domain that can cross-link with various ECM components. Mast cell tryptase was identified as a possible physiological elafin-releasing enzyme because, in contrast to most other examined proteases, it can promptly and precisely produce elafin from its precursor trappin-2 in vitro.^11,12

Function

Antiprotease activity

Elafin and its precursor have a narrow antiprotease spectrum of neutrophil and pancreatic elastase, as well as neutrophil proteinase 3. ¹³ Serine proteases, including tryptase, cathepsin G, plasminogen, chymotrypsin, and granzyme, are not inhibited by this compound. As a result, it is possible that the main purpose of elafin is to protect tissues against excessive proteolysis caused by the activation of neutrophils and the synthesis of proteases.^14,15

Trappin-2 is preferentially cleaved in the cementoin domain at the N-terminus and at the junction among the N-terminal domain and the C-terminal elafin-containing region in vitro by a variety of serine or cysteine proteases, although not by acids or matrix metalloproteinases (MMPs). ¹⁶ Elafin’s four-disulfide core makes it significantly more resistant to proteolysis by enzymes such as neutrophil-produced MMP-8. ¹⁷ Elafin can play a critical role in developing novel therapeutic strategies for neutrophil-induced chronic airway disease.^10,18 However, Brown et al. ¹⁹ reported that the house dust mite Dermatophagoides pteronyssinus, a frequent allergen source associated with atopic asthma, was capable of inactivating elafin by proteolysis. Based on this study, we can present a more nuanced understanding of the pathophysiology of asthma.

As previously described, cross-linking of the transglutaminases (TGS) domain of the elafin N-terminus to various ECM components, including laminin, fibronectin, β-crystallin, collagen Ⅳ, fibrinogen ²⁰ and elastin, ²¹ increases the concentration of the elafin molecule at the site of action, thereby protecting it against elastase-mediated degradation. Although cross-linking can position the protease inhibitor precisely where it is needed to work, it also comes with a cost: the modification of the components of elastic fiber, glutamines and lysines. Such modifications can eventually threaten the biochemical and biological characteristics of these elastic fibers, including their capacity to resist breakdown and normal turnover and tendency for the accumulation and activation of fibroblasts to increase tropoelastin synthesis.^22,23

Antimicrobial activity

Elafin has antimicrobial activity against Gram-negative and Gram-positive pathogens. ²⁴ Recently, Liu et al. ²⁵ found that mice exposed to dextran sodium sulfate were protected from developing acute colitis when Elafin-expressing Escherichia coli nissle1917 (EcN) was present, suggesting its use in the treatment of inflammatory bowel disease. Meyer-Hoffert et al. ²⁶ found that Pseudomonas aeruginosa supernatants could induce elafin mRNA and protein expression in human keratinocytes. This shows that elafin is an innate immune response factor that is induced by the secretions of P. aeruginosa. Simpson et al. ²⁷ demonstrated that intratracheal transfer of adenovirus encoding a human gene with dual antielastase and antimicrobial properties could protect against acute inflammatory injury in murine lungs caused by Pseudomonas aeruginosa, a bacterium that usually resists common antibiotics, and the concentration of elafin in bronchoalveolar lavage fluid (BALF) was inversely correlated with the number of P. aeruginosa. Taken together, the protective effect of elafin was dependent on its antimicrobial property but not on its resistance to neutrophil elastase, which is consistent with the expanding research showing that lung anti-neutrophil elastases have uses beyond inhibiting neutrophil elastase. According to some researchers, the expression and secretion of elafin were increased in respiratory tract epithelial cells following exposure to Pseudomonas aeruginosa. In addition to its antibacterial activity, elafin is also a potent bactericidal agent and can promote epithelial cell proliferation and tissue repair in alveolar units.^28,29 The antifungal activity of elafin against Aspergillus fumigatus and Candida albicans has also been reported.^29,30The underlying mechanism of the antibacterial function of elafin is currently unknown. Pathways unrelated to its anti-proteinase activity have been demonstrated despite its ability to inhibit pathogen translocation via exogenous proteases. The cementoin N- and C-terminal regions have both been shown to exert significant antibacterial effects at low concentrations. ³¹ The cationic nature of elafin is thought to be a critical factor in its antimicrobial activity.^32,33 In addition to facilitating more effective, CD14-dependent macrophage clearance and aiding in the synthesis of chemokines that encourage neutrophil recruitment, trappin-2 appears to opsonize P. aeruginosa.

Therefore, trappin-2 may be crucial for innate detection and removal of pathogens in the very early phases of pulmonary infection. ³² Elafin has since been shown to protect T cells from HIV-1 infection in vitro. Although the specific mechanism of elafin’s antiviral activity is unknown, it is most likely that exogenously added N-terminus-unmodified E was able to enter the nucleus and to reduce viral attachment/entry and transcytosis. ³⁴ Additionally, another study identified possible connections, methods, and object at elafin’s antiviral effects on genital epithelial cells, which may be essential in reducing vulnerability to HIV-1 mucosal infection and may be exploited in studies of bactericides. ³⁴ Drannik et al. ³⁵ first demonstrated the anti-HSV-2 activity of elafin/trappin-2 in the female vaginal mucosa. Taken together, these results indicated the antibacterial and antiviral properties of elafin.

Anti-inflammatory activity

The structure of elafin determines its function. Its C-terminus contains a structure that inhibits elastase. Neutrophils are activated during the inflammatory response to promote the release of elastase, which is critical for initiating and maintaining inflammation. The inhibition of elastase by elafin can maintain a healthy inflammatory response and avoid an excessive inflammatory response that results in tissue and organ damage. Numerous studies have suggested this anti-inflammatory activity of elafin in conditions such as atherosclerosis, myocardial infarction, and lung emphysema; moreover, this activity appears to be unaffected by its antiprotease activity.^8,36,37 According to Marcus et al., ³⁸ elafin inhibits the activation of the transcription factors AP-1 and NF-κB in lipopolysaccharides (LPS) -induced U937 monocytic cells via an inhibitory effect on the ubiquitin‒proteasome pathway. Previous studies have suggested that the expression level of elafin in patients with vigorous celiac disease (CD) was decreased, as indicated by the reduction in 33-mer peptide deamidation by elafin, and combined with its anti-inflammatory and protective effects on barriers in a gluten sensitivity mouse model, this finding suggests that this molecule may play a therapeutic and pathological role in gluten-related illnesses. ³⁹

Additionally, previous studies have confirmed that elafin can reduce inflammatory cells in BALF by inhibiting the activity of NF-κB, thereby alleviating mechanical ventilation-induced acute lung injury. ⁴⁰ In recent years, it has also been found that elafin can effectively reduce damage to chronic hyperoxia-induced lung injury in neonatal mice with bronchopulmonary dysplasia, inhibit the release of inflammatory mediators and improve the basic lung function of mice, thereby promoting lung development.^41–43 The possible mechanism involves protecting the lung structure from elastin degradation by regulating the expression of multiple inflammatory factors by inhibiting the overactivation of the NF-κB signaling pathway, reducing the excessive synthesis of tropolastin and fibulin-5, and improving the state of alveolar development. Based on neonatal mice and cell culture experiments, researchers conducted a study on premature infants born at less than 32 weeks of gestational age in the neonatal ICU of a children’s hospital, and the results further confirmed that recombinant elafin promoted lung growth by inhibiting the activity of mouse lung elastase, which is required for the expression of p-EGFR and Kruppel-like factor 4 (klf4). ⁴⁴ These studies suggest that elafin exerts a significant anti-inflammatory effect and plays a vital role in inhibiting excessive inflammatory damage.

Role in the innate immune response

Numerous studies have suggested that elafin plays a role in modulating the adaptive immune response and stimulating the innate immune response. It is a crucial molecule in the innate immune system that may be necessary for the convergence of this mechanism with the protective immune system. As previously demonstrated, trappin-2 improves the activation of CD14-dependent macrophages and promotes neutrophil recruitment by inducing chemokines. The strong murine cytomegalovirus (MCMV) promoter was used by Sallenave et al ⁴⁵ to create a unique transgenic mouse strain that expressed human elafin, and then the reaction to bacterial LPS was examined when it was limited to the lungs or systemically administered. Following intratracheal LPS administration, more elafin animals had fewer inflammatory cells in their BALF and lower serum/BALF ratios of cytokines than wild-type animals, suggesting that the lungs may be primed for innate responses. Mice expressing elafin had lower serum TNF-α levels than wild-type mice after systemic LPS administration. Additionally, elafin has been shown to suppress extracellular protease generation during neutrophil hyperactivation in an inflammatory setting in chronic pulmonary illness. ⁴⁶ These results suggest that elafin may have dual effects, upregulating the innate immunity of the lungs while suppressing the circulatory capacity for unintended systemic inflammatory reactions.

Expression in Lung Cancer

The persistent chronic inflammatory response is one of the major causes of the progression of chronic diseases to cancer and plays a major role in tumor initiation, development, malignant transformation, invasion, and metastasis.^47,48 Neutrophils are an important component in the inflammatory environment and can promote tumor progression in tissues by degrading the ECM, increasing tumor cell proliferation and metastasis, and promoting angiogenesis.^49–51 Elafin plays an important role in the anti-inflammatory response, and this anti-inflammatory effect has a significant impact on the inhibition of tumor incidence and growth. ⁵² Elafin can inhibit matrix degradation by neutrophil-derived elastase, the invasion and metastasis of malignant cells, and tumor proliferation.^53,54 However, a few studies have reported that elafin plays a role in promoting tumor development in lung cancer, and elafin can drive poor prognosis in patients. ⁵⁵ Therefore, elafin could be a double-edged sword in the treatment of lung cancer because it has been found to suppress the development of lung cancer and can promote cancer cell migration and invasion. Elafin’s role in cancer progression is controversial because it has a complicated regulatory mechanism associated with cell physiology.

Some studies have shown that elafin is expressed in lung cancer and is related to poor prognosis. SenthilKumar and colleagues examined the expression of elafin in lung cancer. ⁵⁶ They found high serum elafin levels in non-small cell lung cancer, suggesting that elafin could be part of the inflammatory immune environment of lung cancer and regulate intracellular redox homeostasis. A study showed that elafin could be a secreted protein that plays a role in the development of tumors. ⁵⁵ Combined with the putative roles of elafin in inflammation and immunity, ⁵⁷ elafin may be secreted by lung cancer cells and extracellularly modulate the tumor microenvironment through its peptidase-regulating activity. In addition, compared with the group with lower elafin expression, the group with higher elafin expression had shorter overall survival from lung cancer, suggesting that elafin could be a prognostic factor for lung cancer. ⁵⁷ Current research has confirmed that elafin is involved in the occurrence and development of lung cancer, but the molecular mechanism by which elafin regulates the occurrence, development, invasion and drug resistance of lung cancer is still unclear. Elafin may be a beneficial molecular target for treating lung cancer; elafin can be used to predict the prognosis of lung cancer, and further research is necessary to develop a novel cancer therapy.

Effects on airway inflammatory diseases

In the respiratory system, elafin is predominantly synthesized and released by type I alveolar epithelial cells in the airway, and the secretion of elafin by Clara cells and alveolar macrophages as a defense can occur in response to inflammatory stimuli such as LPS, IL-1β, TNF-α, and NE, which play crucial roles in the initiation and propagation of the inflammatory response. ⁵⁸ Additionally, this active molecule can protect lung tissue from inflammatory damage by inhibiting the NF-κB signaling pathway, and elafin can reduce lung cell apoptosis and protect lung function. ⁵² A study showed that elafin levels in patients with chronic obstructive pulmonary diseases (COPD) were lower than those in healthy people, and the acute aggravation of COPD by elafin was shorter than the clinical relief period. ⁵⁹ Additionally, elafin was confirmed to be an independent factor that affects acute aggravation and can be used as a predictive factor for acute aggravation. Lower elafin levels were associated with higher risk of acute aggravation. Furthermore, lower elafin levels were associated with a worse condition after acute aggravation.

Elafin and its biologically bioactive precursor trappin-2 are believed to be involved in preventing the effect of excessive inflammation on tissue. An imbalance between protease and antiprotease activity is recognized as a key mechanism that is fundamental to a variety of lung inflammatory diseases, such as COPD, cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), and pulmonary fibrosis with asthma.^58,60–62 The accumulation and activation of neutrophils, macrophages, and other inflammatory cells result in the secretion of several proteases, such as neutrophil elastase, cathepsin G and proteinase 3, which degrade a variety of ECM components in lung tissues. ⁶³ Apart from the direct injury to lung tissues, unbridled elastase production showed additional biological effects. It acts as a secretagogue for mucin production ⁶⁴ and an activating agent of MMPs. ⁶⁵ Active neutrophils and NE, in particular, perform critical functions. NE is the most powerful mucus secretion factor and the most important effector in the pneumonic injury cascade. NE is extremely destructive to lung tissue, and it easily causes lung fibrosis and remodeling during the recovery of lung tissue. The NE inhibitor elafin can inhibit NE’s destruction of lung tissue and prevent fibrosis and remodeling. ⁶⁶ Thus, this anti-proteinase property is essential for therapeutic strategies for chronic airway inflammatory diseases. In addition, the anti-inflammatory properties of elafin/trappin-2 have been evaluated in a variety of animal models, including hamster and mouse acute lung injury (ALI), LPS-induced inflammation, and porcine pancreatic elastase (PPE)-induced emphysema. ⁶⁷ The results demonstrated a significant decrease in PPE-induced emphysematous-like lesions, and there was an increase in the number of neutrophils in bronchoalveolar lavage fluid and lung bleeding, indicating the alleviation of inflammation in these models.

Elafin plays multiple roles in lung diseases. For example, in allergic asthma, elafin mainly reduces or completely abrogates protease activity through proteolysis. In some bacterial pneumonia diseases, elafin has a protective effect on airway epithelial cells to alleviate the damaged biofilm caused by Pseudomonas aeruginosa.^68,69 Elastin may be a key endogenous defense molecule against local airway infections, and the antibacterial properties of elafin may not be related to its antiprotease ability but to its cationic nature.^60,65 In COPD, ARDS and pulmonary fibrosis, elafin, which is an inhibitor of NE, alleviates the inflammatory response, acts as an important protective barrier against proteases, and plays an important role in combating NE-mediated lung diseases. It has been reported that an imbalance between HNE and elafin in blood is associated with ARDS progression. Therefore, the NE/elastin ratio may be a useful clinical biomarker to track ARDS progression.^70,71

Therapeutic effects of elafin

The pharmacological suppression of neutrophil-derived elastase has shown considerable therapeutic benefits in a variety of preclinical constructs. Numerous clinical tests using neutrophil elastase inhibitors in pulmonary disease are currently ongoing, forming the basis for clinical therapeutic strategies. For example, sivelestat sodium hydrate is a polymorphonuclear neutrophil elastase inhibitor with a low molecular weight that is approved for use in Japan for the management of acute lung injury related to sepsis and systemic inflammatory reactions. A meta-analysis of randomized clinical studies in Japan showed a nonsignificant decrease in the death rate. ⁷² This finding was later confirmed by a meta-analysis of randomized controlled studies, which demonstrated that sivelestat therapy may increase PaO₂/FiO₂ levels but had little or no influence on mortality during 28-30 days, the days of ventilation, or ICU stays. ⁷³ In the future, research will need to focus innovative drug delivery methods and particular indicators of neutrophil-derived elastase activity to inhibit regions of inflammatory tissue damage.

Conclusion and outlook

This review describes the biological activities of elafin and summarises the positive role played by elafin in airway inflammatory diseases. Elafin has unique structural domains and regulatory properties, mainly inhibiting neutrophil-derived elastase to a certain extent to attenuate HNE-induced injury, and its expression is affected by the inflammatory state of the host and further influences the inflammatory response. In the development of tumors, elafin can inhibit neutrophil-derived elastase from degrading the matrix, inhibit the invasion and metastasis of tumor cells, and inhibit the spread of tumors. On the other hand, it may protect cyclin E from being hydrolyzed by cellular elastase, thereby regulating cell cycle progression.

As a small molecule NE inhibitor, elafin’s structure and biological properties are especially suitable for purification into an aerosol, which can be directly inhaled and act on the respiratory tract and alveoli to exert therapeutic effects, which not only enhances local active drug molecules but also minimizes systemic side effects. However, there is still much knowledge of elafin that is lacking. The receptor of elafin has not yet been described. It is not clear whether elafin has a single target or inhibits other proteases. Several application challenges are present. First, the aerosol production process is complicated, and further research is needed to find a suitable elafin carrier. Second, it is necessary to consider the reasonable cost, establish the optimal dosing scheme, and develop an effective inhalation device. Regardless, the role of elafin in fighting microorganisms and inflammation has been confirmed by a large number of studies. In addition, the regulatory role of elafin in lung cancer has also been confirmed in recent years, which is helpful for the development of new targeted drugs that can further treat lung cancer. The current research is biased toward in vitro studies, and there have been fewer in vivo studies, so the challenges of how to screen suitable carriers, how to improve drug delivery methods, and how to better serve patients who are suitable for elafin treatment remains to be determined.

However, there are still some limitations and shortcomings in this paper. Elafin’s role in different diseases is different. This study mainly only discusses the role of elafin in airway diseases, and does not compare it with the mechanism of other disease episodes. The next step in the future could be to continue to expand and deepen the role played by elafin in different diseases, with a view to providing an effective target for the treatment of the disease.