Introduction
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation, epithelial barrier disruption, and immune dysregulation, encompassing Crohn's disease (CD) and ulcerative colitis (UC). Over the past century, IBD has transitioned from a rare condition into a global health issue. While incidence rates have plateaued in Europe and North America, their prevalence continues to rise, with the incidence rapidly increasing in newly industrialized regions such as Asia and South America [1], [2].
This shift is associated with industrialization and westernized lifestyles. In China, rapid urbanization and environmental changes have led to a substantial disease burden. Despite advances in biological therapies, many patients fail to achieve sustained remission [3], highlighting the urgent need to understand the pathogenesis of IBD and discover new therapeutic targets.
The pathogenesis of IBD is shaped by intricate interactions between genetic predispositions, immune dysregulation, and gut microbiota alterations. While the roles of cytokines like tumor necrosis factor-α (TNF-α) and interleukin (IL)–23, as well as immune cell subsets such as T helper (Th) 17 cells, are well-established [4], recent findings suggest that dysregulated protease activity is a crucial yet understudied mechanism. Serine proteases, enzymes characterized by a catalytic triad that includes a serine residue, play vital roles in host defense, cell signaling, and tissue remodeling.
Elevated serine protease levels have been detected in the intestinal tissues and fecal samples of patients with IBD compared with those of healthy controls [5], [6]. These enzymes, derived from both the host and the gut microbiota, exert diverse biological effects. Serine protease inhibitors bind specifically to the catalytic sites of serine proteases, thereby tightly regulating their activity.
In healthy individuals, the serine protease inhibitor network maintains homeostatic balance. Research indicates that host serine protease inhibitors show reduced expression in the intestinal mucosa of patients with IBD, and this reduction correlates with increased disease severity [7]. The gut microbiota is a significant source of intestinal serine proteases and their inhibitors.
Microbiota-derived serine protease inhibitors play a crucial role in the regulatory network of serine proteases and their inhibitors. Recently, serine proteases secreted by gut commensals have attracted considerable attention. Beyond directly compromising the mucosal barrier, these microbial-derived proteases are capable of orchestrating host immune responses and modulating inflammatory signaling pathways, thereby establishing a complex interactive network with host-derived proteases [8], [9].
Nevertheless, this review focuses on the regulatory networks of host-derived serine proteases and inhibitors.
This review synthesizes current knowledge on serine protease-inhibitor interactions in the pathogenesis of IBD and explores emerging therapeutic strategies. First, we describe the mechanism by which dysregulated proteolysis contributes to barrier disruption and immune activation. We also highlight the regulatory roles of host-derived serine protease inhibitors.
Finally, we evaluate emerging therapeutic approaches, including synthetic serine protease inhibitors and microbiome engineering, and discuss the challenges associated with target specificity and delivery.
Serine proteases in IBD
Serine proteases
Serine proteases are enzymatic proteins characterized by the presence of a serine residue within their catalytically active sites. These proteins possess a conserved catalytic triad (His-Asp-Ser) that enables the specific cleavage of peptide bonds through a nucleophilic attack mechanism [10], [11]. Serine proteases are classified into 13 families based on their substrate specificity and evolutionary relationships [12].
They are prevalent in nature and constitute approximately one-third of all known proteases [12]. In the human body, serine proteases are ubiquitously distributed across various systems, including the digestive, coagulation, immune, and epithelial systems. These enzymes play crucial roles in regulating proteolytic activity with precision [10], thereby contributing to essential physiological processes such as digestion, blood clotting, cell death, cell signaling, reproduction, and immune responses (Table 1).Table 1Representative human serine proteases and their physiological roles.CategoryRepresentative EnzymesTissue DistributionNormal Physiological FunctionsReferencesDigestive EnzymesChymotrypsinTrypsinPancreatic elastasePancreas / IntestineFood protein digestion, zymogen activation[22]Coagulation-RelatedFactor VIIaFactor IXaFactor XaFactor XIIaThrombinProtein CBlood / Vascular endotheliumHemostasis and regulation of thrombus formation[147], [148]Immune-RelatedNeutrophil elastaseTryptaseComplement factor DComplement factor BComplement factor CCathepsin GBlood / Inflammatory sitesMediation of immune responses, modulation of inflammatory reactions[14], [48], [149]Fibrinolysis-RelatedUrokinaseTissue-type plasminogen activatorPlasminKidney / Blood / Vascular endotheliumRegulation of thrombus clearance, tissue repair, and pathological processes[150]Reproduction-RelatedProstate-specific antigenAcrosinProstate / Seminal fluidFacilitation of sperm maturation, seminal fluid liquefaction, and fertilization[151], [152]Epithelial BarrierMatriptaseTMPRSS2Epithelial tissuesRegulation of epithelial barrier integrity and trans-epithelial ion transport[153]
Numerous serine proteases are abundantly present in the intestine and are distributed throughout the intestinal lumen and tissues. In addition to their traditional role in food digestion, recent studies have indicated their involvement in various intestinal physiological processes. In addition to their digestive functions, specific proteases play a vital role in maintaining the intestinal barrier.
For example, the transmembrane serine protease matriptase contributes to barrier integrity by modulating the expression of the tight junction protein claudin-2. Matriptase deficiency results in abnormal claudin-2 expression, leading to increased intestinal permeability [13]. Furthermore, certain serine proteases in the intestine play a crucial role in regulating immune responses.
Neutrophil elastase (NE), for instance, facilitates the formation of neutrophil extracellular traps (NETs), reinforcing host defenses against fungal and bacterial pathogens [14].
Serine proteases play a crucial role in maintaining normal intestinal homeostasis and tissue renewal. Under physiological conditions, the activity of serine proteases is tightly regulated, and their imbalance is associated with various gastrointestinal diseases, including IBD [15]. Previous studies have shown that the expression and enzyme activity of host serine proteases in the intestines of patients with CD or UC are significantly higher than those in healthy individuals [16], [17].
This atypical proteolytic activity contributes to IBD development through multiple mechanisms, including the activation of protease-activated receptors (PARs), immune cell modulation, and microbe-host interactions, thereby ultimately disrupting the intestinal barrier and establishing a complex pro-inflammatory network.
Effects of serine proteases on the intestinal barrier
The intestinal barrier is composed of two core components: a physical and a chemical barrier [18]. It serves as the primary defense against both commensal bacterial invasion and intestinal pathogens [19]. The physical barrier comprises intestinal epithelial cells linked by specialized structures, such as tight junctions, desmosomes, and adherens junctions.
The chemical barrier is primarily composed of mucin 2 (MUC2) secreted by goblet cells, which creates a mucus layer that covers the intestinal epithelium via polymerization and glycosylation [18], [19]. The integrity of the intestinal barrier relies on the coordinated action of tight junction proteins (e.g., occludin and claudins), adherens junction proteins (e.g., E-cadherin), and the mucus layer. Disruption of this balance is a key mechanism in the pathogenesis of IBD [20], [21].
Under normal physiological conditions, trypsin and chymotrypsin, which are excreted by the pancreas, are primarily localized in the upper gastrointestinal tract. Their enzymatic functions are typically regulated by specific physiological inhibitors and are influenced by the gut microbiota. Only minimal levels of digestive proteases are present in the lower intestine [22].
However, the inflammatory environment of the gut significantly disrupts this balance, and a notable increase in trypsin activity in the intestines of individuals with IBD has been reported [23]. Additionally, fecal samples containing elevated levels of trypsin and chymotrypsin show enhanced colonic epithelial permeability and reduced transepithelial electrical resistance in vitro, confirming the harmful effects of excessive serine proteases on the integrity of the intestinal barrier [24]. At the level of the chemical barrier, excess trypsin contributes to the mucus degradation in IBD patients [25], [26].
Experimental studies have demonstrated that MUC2 is cleaved by trypsin into two high-molecular-weight glycopeptides, resulting in the structural disruption of the mucus layer [27].
Beyond their detrimental effects on the chemical barrier, serine proteases such as trypsin also compromise the physical barrier. Studies have demonstrated that trypsin cleaves E-cadherin, thereby increasing intestinal permeability [28]. Moreover, chymotrypsin enhances epithelial permeability through redistribution of the tight junction proteins zonula occludens-1 (ZO-1) and occludin [29].
Emerging research has revealed that enterocyte-derived elastase 2A (ELA2A) exhibits markedly elevated activity in both inflamed and non-inflamed intestinal mucosa of patients with IBD. Through concentration-dependent degradation of the tight junction protein occludin and the adherens junction protein E-cadherin, ELA2A directly compromises epithelial barrier integrity [30].
In addition to disrupting the chemical and physical barriers, pro-inflammatory serine proteases can exacerbate tissue damage. NE, the predominant protease released during neutrophil degranulation, contributes to NET formation against pathogen invasion while concurrently acting as a pivotal pro-inflammatory serine protease. Intestinal inflammation in IBD is characterized by marked neutrophil infiltration [31], leading to more than a 10-fold increase in fecal NE activity compared with healthy individuals.
NE levels correlate positively with inflammation severity [32]. NE contributes to tissue disruption by cleaving key extracellular matrix (ECM) components, including collagen and laminin, as well as transmembrane junction proteins [31], [33]. Studies on the mucosa of patients with UC have revealed heightened NE activity alongside reduced levels of intact elastin.
Importantly, the inhibition of NE activity decreased the generation of elastin cleavage fragments, suggesting that dysregulated NE activation is a significant driver of ECM degradation. Consequently, this aberrant proteolytic activity exacerbates the damage to the intestinal mucosal barrier [32].
Notably, excessive endoplasmic reticulum (ER) stress in the intestinal epithelium of patients with IBD is considered a contributing factor for increased trypsin activity, and heightened protease activity exacerbates barrier dysfunction by increased permeability [23]. High temperature requirement A2 (HtrA2) is a mitochondrial serine protease involved in apoptosis and necroptosis [34]. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, it translocated from the mitochondria to the cytoplasm, where it degraded RIPK1 in a serine protease-dependent manner, thereby promoting necroptosis and leading to intestinal epithelial damage [35].
In contrast to their previously mentioned roles in promoting intestinal barrier damage, serine proteases also play a role in maintaining intestinal barrier function. Tissue-type plasminogen activator (tPA) produced by intestinal epithelial cells exerts protective functions by activating the anti-inflammatory cytokine transforming growth factor-β (TGF-β). This pathway suppresses excessive epithelial cell proliferation, mitigates inflammatory responses, and enhances mucosal damage repair [36].
Additionally, thrombin released by the intestinal epithelium plays a protective role on the mucosal surface by degrading microbial biofilm-derived proteins, thereby maintaining mucosal homeostasis, preventing biofilm contact with tissues, and limiting bacterial invasion [37].
Mucosal healing is now recognized as a central therapeutic goal in IBD management [38]. Understanding how elevated serine protease activity damages the intestinal barrier will strengthen our knowledge of IBD pathophysiology and may aid in identifying new therapeutic targets to improve treatment outcomes.
Serine proteases activate PARs
In addition to their direct proteolytic effects on barrier components, serine proteases significantly influence intestinal inflammation by engaging in complex cell signaling processes, primarily through the activation of PARs. PARs are heptahelical transmembrane receptors activated by proteolytic cleavage of their extracellular N-terminal domains. This cleavage exposes a tethered ligand motif that binds intramolecularly to induce intracellular signaling.
The PAR family comprises four members (PAR1-4) that are predominantly activated by serine proteases. Serine proteases unmask cryptic ligand-binding sites by cleaving the extracellular domains of PARs, thereby initiating downstream signaling. This process plays a critical role in various physiological functions of the gut and in the pathogenesis of various intestinal diseases [39].
Current evidence confirms that distinct PAR family members make critical contributions to IBD pathogenesis [40], [41], [42], [43]. Notably, serine proteasome-mediated PAR activation is a pivotal driver of this process (Fig. 1).Fig.
1Dynamic regulatory mechanisms of PARs in IBD. Distinct serine proteases play multifaceted and dynamic roles in IBD by activating specific PARs, thereby triggering downstream signaling pathways. CatG, cathepsin G; IBD, inflammatory bowel disease; NE, neutrophil elastase; PARs, protease-activated receptors; PR3, proteinase 3; TJ, tight junctions.
Serine proteases contribute significantly to the pathogenesis of IBD by activating specific PARs via a dynamically complex regulatory mechanism. Thrombin, a serine protease, exhibits markedly elevated activity in inflamed mucosal areas of patients with IBD, reaching levels up to approximately 100-fold higher than in healthy individuals [5], [44]. As previously mentioned, thrombin contributes to maintaining mucosal homeostasis [37].
However, a recent study reported contradictory results, suggesting that thrombin causes inflammatory damage through PAR activation [44]. This study demonstrated that thrombin induces bacterial translocation and increases intestinal permeability in mice by specifically activating the PAR1 and PAR4 receptors. Notably, when subjected to identical experimental conditions, PAR1-deficient mice were protected from bacterial translocation, epithelial apoptosis, and barrier dysfunction, whereas PAR4-deficient mice remained susceptible.
This indicates that the pathogenic effects of thrombin in IBD are primarily mediated by PAR1 activation, which drives the degradation of tight junction proteins, including ZO-1 and claudin-1 [44]. Neutrophil-derived serine proteases, such as NE and proteinase 3 (PR3), activate the basolaterally expressed protease-activated receptors PAR1 and PAR2 in intestinal epithelial cells. This activation elicits the phosphorylation of the myosin light chain (MLC) through the upregulation of myosin light chain kinase (MLCK), thereby destabilizing tight junctions and compromising epithelial barrier integrity.
Concomitantly, this proteolytic cascade amplified neutrophil transepithelial migration and perpetuated intestinal inflammation [45]. Activation of PAR4 by cathepsin G (CatG) triggers changes in epithelial permeability and inflammation through the activation of MLCK, resulting in the phosphorylation of MLC and instability of tight junctions (TJ) [17], [46]. As a serine protease capable of activating all four PAR subtypes, trypsin has been shown in animal experiments to induce intestinal inflammation through specific engagement of PAR1, PAR2, and PAR4 [43], [47].
Furthermore, mast cell-derived tryptase potentiates PAR2 activation, which subsequently drives fibroblast differentiation via the Akt/mTOR signaling pathway, thereby exacerbating intestinal fibrosis in IBD [48].
Interestingly, while serine proteases exacerbate intestinal inflammation through PAR2 activation in acute inflammatory settings, studies using chronic colitis models have revealed a paradoxically protective effect. Administration of a PAR2 agonist significantly ameliorated inflammation in a 2,4,6-trinitro-benzene sulfonic acid (TNBS)-induced murine model of chronic colitis [49]. A previous study indicated that PAR2 activation enhances epithelial wound healing [50].
Concurrently, the profibrotic effect of tryptase via PAR2 highlights an additional facet of its downstream signaling [48]. This paradoxical nature of PAR2 action epitomizes the complexity of the serine proteases-PARs regulatory network in IBD.
However, the precise role of PAR3 in the pathogenesis of IBD remains unclear. The current understanding of its function in the gastrointestinal tract remains extremely limited [40]. Limited evidence suggests that PAR3 may contribute to barrier integrity preservation [51], with proteases possessing chymotrypsin-like activity identified as putative inhibitors of its activation [47].
Nevertheless, the pathophysiological interplay between serine proteases and PAR3 in IBD is yet to be elucidated. Future studies are warranted to investigate the biological significance and pathological contributions of the serine proteases-PAR3 signaling pathway in IBD.
Collectively, serine proteases profoundly influence the pathogenesis of IBD through the dynamic and complex modulation of signaling networks mediated by distinct PARs. They contribute critically to the pathogenesis, including barrier disruption and increased permeability, while simultaneously exhibiting reparative functions during chronic inflammatory phases. Our current understanding of the serine protease-PAR regulatory network remains limited.
The complex dynamic regulatory mechanisms of the serine proteases-PARs regulatory network require further investigation. Specifically, elucidating the paradoxical role of PAR2 signaling in acute versus chronic inflammation and defining the unknown functions of PAR3 hold substantial promise for advancing our understanding of IBD pathogenesis and developing targeted therapeutic strategies.
Serine proteases regulate cytokines involved in inflammatory signaling networks
The activation of PARs represents a crucial mechanism by which serine proteases modulate inflammatory signaling. However, their influence on the cytokine network extends beyond this receptor-mediated pathway. Research has revealed that NE and CatG potentiate the biological activity of IL-33 approximately 10-fold through proteolytic cleavage, converting it into a highly active mature form [52].
Thus, IL-33 appears to play a dual role in IBD [53]. It can hyperactivate the Th2 inflammatory signaling pathway downstream of the ST2 receptor, triggering disruption of the intestinal barrier and exacerbating the severity of intestinal fibrosis [54]. Conversely, studies utilizing DSS-induced murine colitis models demonstrated that IL-33 facilitated intestinal epithelial restitution by inducing group 2 innate lymphoid cells and the growth factor amphiregulin [55].
Furthermore, within the neutrophil-rich lamina propria microenvironment characteristic of UC patients, elastase and PR3 directly mediate the maturation of pro-IL-1β through proteolytic cleavage, independently of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, thereby promoting intestinal inflammation. Notably, this phenomenon was absent in patients with CD [56]. This differential mechanism may be attributed to the distinct immune polarization profiles of UC and CD.
UC is predominantly associated with Th2/Th17-type responses, whereas CD exhibits a stronger bias toward Th1-type responses [57]. This finding is further supported by a subsequent study, which first identified significantly elevated PR3 activity in the feces of patients with IBD compared with healthy individuals [58]. In addition to their direct role in cytokine maturation, serine proteases can amplify inflammatory signaling by processing chemokines.
For instance, CatG enhances the chemotactic potency of C-X-C motif chemokine ligand 5 (CXCL5) in neutrophils via proteolytic cleavage of this chemokine [59]. Additionally, plasmin promotes colitis progression by activating matrix metalloproteinase (MMP) 9. This activation stimulates the recruitment of myeloid cells to the colonic epithelium and induces the release of chemokine CXCL5, thereby propagating inflammation [60].
Inflammatory cytokines reciprocally regulate serine protease expression, establishing a complex bidirectional regulatory network. The colitis-associated Th2 cytokines, IL-4 and IL-13, activate the transcription factor signal transducer and activator of transcription 6 (STAT6). This activation downregulates the expression of membrane-anchored serine proteases such as matriptase and kallikreins.
Consequently, this diminishes the protease-mediated barrier protective functions. Importantly, the inhibition of STAT6 phosphorylation can restore protease expression and reverse cytokine-induced barrier dysfunction [61], [62]. Granzyme M (GrzM), a serine protease expressed by cytotoxic lymphocytes, particularly natural killer (NK) cells, plays a key role in triggering cell death in eukaryotes.
Its expression is specifically elevated in the inflamed intestinal tissues of patients with UC. Consistent with this finding in humans, GrzM suppresses excessive neutrophil infiltration in a DSS-induced colitis model in wild-type mice, contributing to the maintenance of intestinal epithelial integrity. Notably, compared with their wild-type counterparts, GrzM-deficient mice exhibited significantly exacerbated colitis following DSS administration [63].
The involvement of serine proteases in IBD involves a multifaceted regulatory framework rather than a straightforward pro-inflammatory role. Further exploration is warranted to enhance our understanding of the pathophysiology of IBD as well as the heterogeneity of UC and CD.
Gut microbiota-derived serine proteases in IBD
In healthy individuals, the gut microbiota exists in a state of harmonious symbiosis. The serine proteases they produce are crucial for maintaining intestinal homeostasis and significantly contribute to proteolysis in the intestine, assisting the host in substance metabolism [64]. These proteases are produced predominantly by common gut commensal genera, including Bacteroides, Clostridium, and Streptococcus[64], [65].
In addition, serine proteases generated by commensal microbes may help regulate microbial growth and colonization, thereby indirectly supporting intestinal homeostasis in the host [66].
In patients with IBD, gut dysbiosis alters the bacterial protease profile, consequently elevating intestinal serine protease activity [6]. For example, there is an increase in species capable of producing serine proteases, such as Enterobacteriaceae[67]. Members of this family encode and secrete serine protease autotransporters of Enterobacteriaceae, which act as key virulence factors that contribute to intestinal infection, promote inflammation, and induce epithelial injury [68], [69].
Studies demonstrate that Campylobacter jejuni secretes HtrA, a serine protease that cleaves host intestinal epithelial proteins, including E-cadherin and tight junction components, thereby disrupting the gut barrier [70]. Many serine proteases derived from gut microbes share targets with host proteases, including the intestinal mucus layer, TJ proteins, and PARs, thereby compromising the intestinal barrier and triggering inflammation [69].
Beyond damage patterns similar to those of host proteases, gut microbial serine proteases also possess additional pathogenic mechanisms. A recent study on Campylobacter jejuni reveals that its secreted serine protease HtrA not only directly cleaves E-cadherin to disrupt epithelial adhesions but also opens the paracellular route. This breach enables otherwise noninvasive commensals to translocate across the epithelial barrier, thereby providing a novel potential explanation for aberrant microbial translocation in IBD [71].
Furthermore, extracellular proteases, including serine proteases, secreted by various commensal gut bacteria can degrade host ECM components and aggravate DSS-induced colitis [72]. Additionally, adherent-invasive Escherichia coli (AIEC) secretes the serine protease Vat-AIEC, which promotes bacterial adhesion to and invasion of epithelial cells and thereby contributes to the in vivo pathogenicity of AIEC in the context of CD [73].
Importantly, microbial serine proteases may engage in multilayered interactions with the host protease system rather than merely coexisting passively. At the substrate level, microbial and host serine proteases exhibit overlapping substrate specificity for key components of mucosal structure. For instance, HtrA from Campylobacter jejuni has been shown to cleave tight and adherens junction proteins such as occludin, claudin-8, and E-cadherin.
At the same time, host serine proteases can likewise degrade E-cadherin and other adhesion molecules, indicating that both systems converge on common targets within epithelial junctional complexes [71], [74]. Previous studies indicate that microbial serine protease-mediated thinning of the mucus layer and disruption of epithelial junctions directly weaken the mucosal barrier, thereby rendering the intestinal epithelium more susceptible to inflammatory stimuli [66]. It is reasonable to hypothesize that such structural damage increases the accessibility of the same or adjacent substrates to subsequently infiltrating host serine proteases, further amplifying injury to the intestinal barrier.
Moreover, gut microbial serine proteases may regulate the release and activation of host proteases via PAR signaling. Recent work has shown that the Bacteroides fragilis serine protease 1 secreted by Bacteroides fragilis cleaves and activates PAR2 on the epithelial surface, impairing barrier function and significantly upregulating multiple pro-inflammatory cytokines and chemokines, including TNF-α, IL-1β, and CXCL1, in the mouse colon [75]. The elevated levels of these mediators are closely associated with enhanced neutrophil infiltration, which represents a major source of host serine proteases [76].
Furthermore, several gut bacteria have been found to produce serine protease inhibitors that suppress host serine proteases such as NE and PR3, thereby attenuating inflammation in murine colitis models. These findings suggest that bacterial serine protease inhibitors may help constrain proteolytic imbalance and exert a protective modulatory role in IBD [77].
Current studies on the roles of microbiota-derived serine proteases and their inhibitors in IBD are still limited, and their interactions with host serine protease systems remain poorly characterized. Nonetheless, available evidence indicates that microbial serine proteases contribute substantially to IBD pathogenesis and may form complex regulatory networks with host proteases, ultimately driving dysregulation of the serine protease-inhibitor network balance in the intestine.
Serine protease inhibitors in IBD
Serine protease inhibitors play a crucial role as negative regulators of serine proteases, contributing to the maintenance of a tightly regulated proteolytic equilibrium. While the term “serine protease” typically refers to the evolutionarily conserved serpin superfamily, other families also play a role in inhibiting serine proteases in humans [78]. Notable examples include the serpin, Kazal-type, and the whey acidic protein-type, four-disulfide core (WFDC) inhibitor families [79], which are widely distributed across various biological systems and possess diverse regulatory functions.
This review focused primarily on elucidating the roles of these three families in IBD (Fig. 2).Fig. 2Regulatory roles of the three host-derived serine protease inhibitor families in IBD.
Diagram illustrating the targets and functional roles of representative members from the Serpin, Kazal, and WFDC families in IBD. CatG, cathepsin G; EGFR, epidermal growth factor receptor; IBD, inflammatory bowel disease; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8; LAMB3, laminin β3; NE, neutrophil elastase; NF-κB, nuclear factor kappa-B; PR3, proteinase 3; SLPI, secretory leukocyte protease inhibitors; SPINK4, serine protease inhibitor Kazal-type 4; SPINK7, serine protease inhibitor Kazal-type 7; WFDC, whey acidic protein-type, four-disulfide core.
The serpin family
Serpins, the most extensive and diverse protease inhibitor systems in nature [80], [81], have been identified in over 3,000 variants across various life domains, including animals, plants, fungi, archaea, and bacteria [81]. A typical serpin molecule consists of 350–400 amino acid residues, with a molecular weight ranging from 40 to 100 kDa, and is characterized by a tertiary structure comprising 7–9 α-helices and 3 β-sheets. The core conserved region forms the “reactive center loop”, a key domain for its inhibition function [81], [82].
Phylogenetically, serine protease inhibitors can be divided into 16 clades, named A–P. A total of 37 serpins have been identified in humans, of which 30 are functional protease inhibitors [83]. Serpins participate in the regulation of various processes, including inflammation, coagulation, tissue remodeling, blood pressure, angiogenesis, hormone transport, neuroprotection, and insulin sensitivity [84], thereby contributing significantly to the maintenance of normal physiological functions in humans.
Under physiological conditions, serpins effectively inhibit excessive serine protease activity, thereby preserving protease–antiprotease homeostasis and preventing inadvertent tissue damage. However, patients with IBD exhibit significant dysregulation of the network that governs serine proteases and serpins.
Studies have shown that administering human α-1-antitrypsin (AAT, SerpinA1) can protect mice from both acute colitis and chronic ileitis. This protective effect is mediated by reduced intestinal cytokine (including IL-1β and IL-6) production and diminished inflammatory infiltration [85]. Moreover, AAT is upregulated in the intestinal tissues of IBD patients, which may serve as a compensatory mechanism to counteract the excessive activity of serine proteases associated with IBD [85].
SerpinA3, a chymotrypsin inhibitor, plays a significant role in both physiological and pathophysiological processes in various organs, including the brain, lungs, heart, kidneys, and intestines [86]. Studies in mice demonstrated that SerpinA3n (the murine homolog of SerpinA3) exhibits increased expression during the resolution phase of inflammation. It suppresses inflammatory responses by inhibiting elastase activity, thereby promoting the resolution of intestinal inflammation [87].
Furthermore, a balance between SerpinA3n and NE is observed under mild inflammatory conditions. Conversely, in severe colitis, early NE release disrupts this equilibrium, leading to proteolytic imbalance and exacerbating tissue damage [88]. Subsequent studies have reported that laminin β3 (LAMB3), a key component of the ECM, promotes the expression of SerpinA3 through the integrin α3β1/FAK pathway under inflammatory conditions.
Elevated levels of SerpinA3 can increase the production of pro-inflammatory cytokines, thereby exacerbating intestinal inflammation [89]. This seemingly contradictory effect may be due to differences in the inflammatory stages under investigation, or it could be attributed to species-specific variations. However, further research is required to clarify these aspects.
SerpinA4, also known as kallistatin, is a tissue kallikrein inhibitor that plays a protective role against severe inflammatory responses [90]. In pediatric IBD research, the levels of SerpinA4 in the feces of UC and CD patients were over three times higher than those in healthy individuals [91]. During active IBD, the levels of SerpinA4 in the plasma and intestinal tissues are lower than those in non-inflammatory patients [92], while it is significantly elevated in the intestinal interstitial fluid-extracellular space [93].
This may be due to damage to the intestinal wall, which affects the integrity of tight junctions between the intestinal epithelial cells, leading to their appearance in the feces. In addition to their expected anti-inflammatory role, serpins exhibit pro-inflammatory properties. Elevated expression of plasminogen activator inhibitor-1 (PAI-1 or SerpinE1) has been observed in both IBD patients and DSS-induced colitis [94], [95].
Genetic ablation of PAI-1 prevents the onset and development of DSS-induced colitis in mice. Experimental evidence has shown that PAI-1 contributes to intestinal inflammation by modulating the CXCR2/CXCL1/CXCL5 chemokine axis. Specifically, PAI-1 activates the nuclear factor kappa-B (NF-κB) signaling pathway, leading to the upregulation of neutrophil-mediated chemokines and exacerbation of intestinal inflammation [94].
The precise functional role of the serpin family in IBD pathogenesis remains unclear and warrants further investigation.
Pancreatic secretory trypsin inhibitor (Kazal) family
Kazal-type serine protease inhibitors were first discovered by Kazal et al. in 1948 as pancreatic secretory trypsin inhibitors [96]. Structurally, this family is characterized by the presence of Kazal domains, each of which contains six cysteine residues (Cys) that form three disulfide bonds and a reactive site loop [79], [97]. Kazal-type serine protease inhibitors are widely expressed in diverse species [79].
Under physiological conditions, the serine protease inhibitor Kazal-type 4 (SPINK4) is expressed in intestinal goblet cells (GCs) within the digestive tract [98]. SPINK4 expression was significantly elevated in the intestinal tissues of patients with IBD and exhibited dynamic alterations in a DSS-induced murine colitis model [99]. SPINK4 expression levels are linked to disease severity, with low expression during the active phase and increased expression during periods of remission.
Mechanistically, SPINK4 binds to epidermal growth factor receptor (EGFR) via its Kazal-like motif, activating the EGFR pathway. This activation subsequently upregulates Wnt/β-catenin signaling while suppressing the Hippo pathway, ultimately promoting GC regeneration and restoration of the mucus layer. The significant reduction in SPINK4 expression during the active phase suggests its potential occurrence within specific clinical stages of acute colitis or preclinical phases of IBD, which may be attributed to the acute depletion of GCs during colitis flares [99].
A recent study further elucidated the mechanism regulating SPINK4 during mucosal injury, wherein PGE2 derived from mesenchymal stromal cells upregulated the prostaglandin E receptor 4 signaling pathway. This signaling cascade enhances the activity of the histone deacetylases HDAC4, HDAC5, and HDAC7 within epithelial cells, ultimately elevating SPINK4 mRNA levels and facilitating its extracellular release [100].
Additionally, an analysis utilizing the GEO database revealed that although SPINK7 mRNA levels in patients with IBD were lower than those in healthy individuals, the difference was not statistically significant [101]. Evidence also suggests that in cutaneous wounds, SPINK7 promotes the resolution of inflammation during wound healing by inhibiting multiple protease activities and modulating macrophage polarization and cytokine secretion [102]. These findings offer insights into intestinal mucosal healing, suggesting that SPINK7 promotes IBD mucosal repair by regulating protease activity and the immune microenvironment.
The WFDC family
WFDC family proteins are defined by the presence of one or more WFDC domains, each composed of 40–50 amino acids, and containing eight conserved cysteine residues that form four disulfide bonds [103]. This protein family is widely expressed in humans and exhibits diverse functions such as antiprotease activity, antimicrobial properties, and immune regulation [104]. Representative members of the human WFDC family include elafins and secretory leukocyte protease inhibitors (SLPI).
Elafin, an endogenous serine protease inhibitor predominantly secreted by epithelial cells, plays a significant role in IBD by inhibiting NE and PR3 activity. Elafin administration reduces fecal PR3 and NE activity in patients with IBD [58], [105]. Overexpression of elafin in transgenic mice significantly inhibits NE/PR3 and elastase-like proteolytic activity induced by colitis.
Furthermore, the genetic deletion of NE/PR3 or transgenic overexpression of elafin protected against the development of colitis. These findings indicated that the protective effect of elafin against IBD progression was primarily attributable to its inhibition of elastase and PR3 activity [106]. Additionally, elafin independently mitigates inflammatory responses by modulating NF-κB activation and regulating cytokine and chemokine production [106].
In addition to modulating serine protease activity, elafin may participate in IBD pathogenesis through interactions with other proteases. Studies in animal models have demonstrated that elafin exerts antifibrotic effects in the intestine by inhibiting cathepsin S activity, thereby suppressing PAR2 signaling [107]. Another study reported that WFDC2, a serine protease inhibitor secreted by colonic GCs, maintains epithelial barrier integrity by inhibiting the activity of MMP12 and MMP13.
Downregulation of WFDC2 in UC contributes to a protease–antiprotease imbalance, facilitating the disruption of tight junctions and the invasion of commensal bacteria [108].
However, reports of elafin expression in patients with IBD are inconsistent. Several studies have demonstrated that elafin expression is significantly downregulated in both the serum and inflamed colonic mucosa of patients with active IBD, which correlates negatively with disease activity indices (e.g., modified Mayo Score and Crohn's Disease Activity Index, CDAI) [7]. Conversely, other studies have reported elevated elafin transcript and protein levels in the intestinal tissues of patients with IBD compared with healthy controls [32], [109].
Nevertheless, this increase appears to be insufficient to compensate for the increased serine protease activity. Heterogeneity in elafin regulation also appears to exist between IBD subtypes. Compared with non-inflamed UC tissues, inflamed UC tissues exhibited significantly elevated elafin mRNA levels and immunostaining intensity.
In contrast, this increase in elafin expression was less pronounced in inflamed CD tissues than in their non-inflamed counterparts [110]. A recent study revealed reduced elafin expression in the inflamed colonic mucosa of both patients with UC and CD, which correlated negatively with the degree of tissue damage [111]. Furthermore, a study on pediatric patients with IBD observed increased serum elafin levels during the active disease phases in both UC and CD.
However, elevated levels were detected only during the remission phase of UC [112].
SLPI, another key member of the WFDC family, is secreted by epithelial cells in healthy intestines [113]. SLPI exhibits inhibitory activity toward NE and CatG and can also suppress NF-κB signaling to modulate monocyte/macrophage function [114]. Thus, SLPI plays a significant role in the regulation of inflammatory responses and maintenance of intestinal immune homeostasis [113].
In UC, SLPI expression is elevated in inflamed mucosal tissue. However, no such elevation was observed in the non-inflamed UC mucosa or colonic tissues of CD patients [110]. Animal models have further elucidated the protective roles of SLPI.
During DSS-induced acute colitis in wild-type mice, SLPI expression increased in the colonic epithelium [115], [116]. Conversely, in thymic stromal lymphopoietin-deficient mice, impaired recovery from colitis occurs due to diminished colonic SLPI expression and the consequent inability to effectively inhibit NE, resulting in excessive NE activity [115]. Notably, in contrast to prior studies reporting no elevation of SLPI expression in the colonic tissues of CD patients, another investigation demonstrated that SLPI expression was elevated in the inflamed mucosa during active disease in both colonic-type CD and ileal-type CD patients compared with healthy controls.
Furthermore, this study revealed site-specific differences in SLPI expression in CD, with significantly lower levels detected in ileal CD compared with colonic CD [117].
Collectively, research on WFDC family members other than elafin and SLPI in the context of IBD is lacking. Although potential associations exist for certain members, their precise mechanistic roles and functional contributions to IBD pathogenesis require further investigation for a comprehensive understanding and characterization.
The diagnostic and therapeutic implications of serine protease/inhibitor regulatory networks in IBD
The investigation of the serine protease/inhibitor regulatory network has become pivotal in IBD diagnosis, disease monitoring, and therapy (Fig. 3). In the diagnostic domain, fecal NE has shown superior diagnostic accuracy compared with C-reactive protein (CRP).
However, it does not outperform CRP in correlation with endoscopic inflammation [118]. Two-dimensional difference gel electrophoresis (2D-DIGE) to quantify fecal chymotrypsin revealed a diagnostic efficacy comparable to that of fecal calprotectin (FC) [119]. Transitioning to circulatory biomarkers, studies have suggested elastase as a potential plasma biomarker for diagnosing pediatric IBD [120].
Although the expression profile of elafin in patients with IBD warrants further investigation, current evidence indicates that serum elafin has a moderate discriminatory capacity to distinguish UC from healthy controls in pediatric cohorts, surpassing its ability to differentiate CD from non-IBD individuals [112]. For IBD monitoring, serum dipeptidyl peptidase-4 (DPP-4), a membrane-anchored serine protease expressed in diverse cell types, serves as a novel biomarker for assessing the inflammatory activity in IBD. Evidence suggests its ability to differentiate between clinically active diseases and remission states [121].
Plasma NE levels in patients with IBD can serve as biomarkers of disease activity, effectively identifying patients in remission [122]. Although serum NE has the potential to assist in diagnosing IBD and assessing its activity, evidence suggests that NE levels in blood are not suitable as predictive biomarkers for disease progression and treatment outcomes [123]. Urinary biomarker levels have also progressed significantly.
The urinary NE/elafin ratio demonstrated a robust discriminatory capacity for distinguishing patients with CD from healthy controls, achieving an area under the curve (AUC) of 0.896 [124]. In UC cohorts, this biomarker ratio also exhibited significant diagnostic utility (AUC = 0.815) with superior specificity (90.9 %), suggesting its enhanced potential for excluding UC diagnoses [124]. Within the realm of immunodiagnostics, PR3-antineutrophil cytoplasmic antibody (ANCA), an autoantibody targeting PR3, reportedly has potential diagnostic utility for distinguishing UC from CD during early disease stages [125].
Within the immunodiagnostic sphere, a study conducted in Chinese IBD cohorts further demonstrated the discriminatory capacity of PR3-ANCA for differentiating UC from CD, revealing its correlation with UC disease activity [126].Fig. 3Diagnostic and therapeutic strategies targeting the serine protease/inhibitor system in IBD. Diagram illustrating the translational medicine applications of the serine protease/inhibitor network.
CD, Crohn's disease; GZMB PET, granzyme B positron emission tomography; IBD, inflammatory bowel disease; PBS, phosphate buffered saline; UC, ulcerative colitis.
Complementing these biomarker-based approaches, significant progress has also been made in the field of molecular imaging for direct visual detection. Positron emission tomography (PET) imaging using granzyme B (GZMB) has shown promise for assessing inflammatory activity in IBD by targeting a serine protease released by immune cells. In DSS-induced murine colitis models, GZMB PET detects active intestinal inflammation with high temporal resolution [127], representing a novel strategy for noninvasive IBD monitoring.
In addition, a novel dual-lock chemiluminescent probe, BPan-CL, was designed based on chymotrypsin and vanin-1, demonstrating high sensitivity for imaging in a DSS-induced mouse colitis model. BPan-CL not only permits accurate in vivo differentiation between colitis mice and healthy controls, but also enables early prediction of colitis through noninvasive fecal assays [128].
In addition to these diagnostic and monitoring advancements, novel therapeutic strategies targeting serine protease/inhibitor regulatory networks warrant further investigation. Experimental studies using murine models have shown that serine protease inhibitors ameliorate intestinal inflammation [32], [87], [99], [115]. This evidence underpins a promising treatment paradigm: targeted delivery of these inhibitors to antagonize pathologically elevated serine protease activity, thereby rebalancing the protease–antiprotease axis in patients.
Dabigatran, a direct thrombin inhibitor, ameliorates experimental colitis in murine models [44]. Nafamostat mesilate, a synthetic serine protease inhibitor, suppresses trypsin activity [129]. Studies in murine models of DSS- and TNBS-induced IBD have consistently demonstrated their ability to attenuate intestinal inflammation by inhibiting MC-derived tryptase activity [130], [131].
A novel serine protease inhibitor, UAMC-00050, was developed and validated in a rat IBD model. It effectively suppressed intestinal serine protease activity and ameliorated visceral hypersensitivity, yet failed to significantly attenuate intestinal inflammation [132]. Notably, in another study employing murine models of chronic colitis T-cell transfer, UAMC-00050 demonstrated efficacy in reducing intestinal inflammation and barrier impairment [133].
These divergent outcomes may reflect variations in administration protocols or distinct phases of inflammatory progression. In addition to novel drug discovery, these investigations have provided innovative therapeutic strategies for IBD. Human NE causes dose-dependent degradation of biological agents, including infliximab, adalimumab, etanercept, and vedolizumab.
Recombinant elafin inhibits human NE-mediated proteolysis [32], supporting the therapeutic viability of combining serine protease inhibitors with biologics for synergistic management of IBD. In addition to strategies employing combined biological agent therapies, a recent study has reported that antibiotic treatment in patients with IBD markedly elevates fecal trypsin and chymotrypsin activity [24]. Since excessive serine protease activity impairs intestinal barrier function, this observation implies that greater caution is necessary when administering antibiotics to patients with IBD to prevent disruption of the serine protease-protease inhibitor balance.
A previous study engineered two food-grade lactic acid bacterial strains, Lactococcus lactis and Lactobacillus casei, to express the human elafin gene. Oral administration of these elafin-expressing bacteria reduced proteolytic activity and effectively ameliorated symptoms in murine colitis models [16]. Furthermore, the therapeutic efficacy of delivering this serine protease inhibitor surpasses that of delivering IL-10 or TGF-β1 [134].
This delivery approach enables the stable local release of elafin at sites of intestinal injury, while also preventing the suppression of digestive enzymes essential for normal gastrointestinal function that is inherent to direct oral elafin administration.
Although certain therapeutic effects have been observed in mouse models, only a limited number of serine protease inhibitors have advanced into human studies, most of which remain in early or exploratory stages. In line with the previously discussed preclinical findings, therapeutic benefits have been observed in patients with UC who received serine protease inhibitors, including urinary trypsin inhibitor and camostat mesylate [135], [136]. A phase I study (NCT03522935) of elafin further represents these early efforts, having primarily assessed its safety and pharmacokinetics [137].
Notably, a phase II clinical trial of the trypsin-like protease inhibitor APC-2059 demonstrated acceptable safety and clinical improvement in patients with UC [138]. In contrast, the development of UAMC-00050 has been hampered by its broad-spectrum inhibitory profile, which resulted in off-target effects and inconsistent efficacy under inflammatory conditions, thereby limiting its therapeutic potential [139]. The contrasting outcomes observed with these two candidate inhibitors illustrate translational challenges of targeting serine proteases in IBD.
The high selectivity of APC-2059 for trypsin-like proteases provides the rationale for its superior clinical feasibility.
Targeting the serine protease network represents a promising therapeutic strategy. Nevertheless, its clinical translation faces significant challenges, particularly those related to target specificity, drug delivery, and molecular stability. Achieving high target selectivity remains a major hurdle.
Although different serine proteases have distinct physiological roles and unique substrates, their catalytic sites are highly conserved. Consequently, developing inhibitors that target these catalytic domains remains difficult, as the structural similarity of the catalytic sites across serine proteases limits the ability to achieve adequate specificity [12]. Furthermore, as noted previously, protease activity varies substantially between UC and CD, across acute and chronic inflammation stages, and even in different gut regions.
This heterogeneity further compounds the difficulty of selecting a viable target and increases the likelihood of off-target effects. Consequently, nonselective inhibition may interfere with digestive enzymes like trypsin or chymotrypsin, leading to malabsorption, or suppress immune proteases essential for host defense, thereby potentially increasing susceptibility to infection [24], [140]. Therefore, overcoming this challenge requires a shift from broad-spectrum inhibition toward precision therapeutics.
Future research should leverage structure-based drug design and virtual screening to develop inhibitors that target alternative domains.
Another major challenge is drug delivery and stability. Oral administration must overcome degradation in the gastrointestinal tract, whereas systemic administration requires precise targeting of inflamed intestinal tissue to enhance efficacy and minimize systemic toxicity [141]. The inflammatory microenvironment in IBD poses additional hurdles for drug delivery.
The altered intestinal pH microenvironment in IBD may affect the release kinetics of pH-dependent formulations [142]. Furthermore, elevated protease levels in the gut may prematurely degrade peptide- or protein-based delivery carriers [143]. Several emerging delivery strategies offer promising avenues to enhance targeting specificity.
These strategies include engineered probiotics that locally release protease inhibitors and nanotechnology-based approaches [16], [144]. These nanocarriers are designed to activate specifically at sites of intestinal inflammation in response to pathophysiological stimuli such as oxidative stress [145], [146]. Nonetheless, most of these approaches remain in early preclinical development, and extensive in vivo evaluation is required to establish their safety and efficacy.
Current research on serine protease inhibitors in IBD remains limited, with the majority of studies conducted in animal models and scant clinical evidence from human studies. The human intestinal microenvironment is significantly more complex than that of animal models, involving broader protease networks, intricate microbiome interactions, and multifaceted host immune responses. Therefore, the pharmacokinetics, tissue distribution, stability, and therapeutic efficacy of serine protease inhibitors in humans require further investigation.
Conclusion
Serine proteases participate in essential physiological processes, including digestion, hemostasis, and immune responses. Serine protease inhibitors maintain homeostasis by regulating protease activity. The dysregulated serine protease-inhibitor network is critically implicated in the pathogenesis of IBD, influencing mucosal homeostasis, immune responses, and barrier function.
Despite compelling preclinical validation of its diagnostic and therapeutic potential, clinical translation remains constrained by the limited predictive power of animal models and the complexity of the human intestinal microenvironment. To bridge this gap, future efforts must prioritize: 1) developing highly selective inhibitors to overcome target specificity challenges, 2) validating pathophysiological mechanisms and therapeutic candidates in human-relevant systems like organoids, 3) exploring innovative combination therapies, such as pairing protease inhibitors with biologics to improve efficacy, and 4) advancing promising biomarker candidates into rigorous clinical validation stages to improve diagnosis and monitoring. Focusing on these strategic directions is crucial to translate the mechanistic understanding of this network into tangible clinical benefits for IBD patients.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.