Introduction

Inflammatory bowel diseases (IBD) are chronic systemic inflammatory conditions that primarily affect the gastrointestinal tract (GI) and include Crohn’s disease (CD) and ulcerative colitis (UC). Although the exact etiopathogenesis of IBD remains incompletely understood, evidence points to a disrupted interplay between the mucosal immune barrier and the gut microbiome in genetically susceptible individuals. This interaction results in an inappropriate inflammatory response mediated by both innate and adaptive immunity [1]. Increased intestinal epithelial permeability appears to contribute to chronic intestinal inflammation as well. Disruption of intercellular lateral junctions permits a greater proportion of luminal antigens, including bacterial antigens, traverse the epithelium paracellularly, evading degradation into smaller, non-immune peptides, and potentially triggering heightened mucosal immunoreactivity [2,3].

In a subset of patients, IBD is associated with primary sclerosing cholangitis (PSC), a chronic inflammatory disorder of unknown etiology characterized by progressive fibrosing stenosis and obliteration of the extrahepatic and larger intrahepatic bile ducts [4]. The association between PSC and IBD is substantial, with 50% to 80% of PSC patients developing IBD over time [5]. PSC-IBD presents more commonly as pancolitis, as observed in both endoscopic and microscopic evaluations, and carries a fourfold higher risk of colorectal cancer compared to patients with UC alone [6]. The etiopathogenesis of PSC-IBD and its distinctions from UC remain largely unexplored.

Advances in diagnostic techniques and the implementation of modern therapeutic approaches have significantly improved the overall clinical management of patients. However, accurate diagnosis remains a considerable challenge, and many patients continue to face an unfavorable clinical course marked by frequent disease recurrences or severe complications [7]. The variable clinical presentation, inconsistent therapeutic response, and diverse genetic background suggest significant interindividual variability in the pathogenic mechanisms driving disease progression. Consequently, there remains a critical need for novel biomarkers to enhance diagnostic precision, facilitate personalized treatment strategies, accurately assess inflammation severity, and improve the prediction of disease progression and severe complications. Proteomics offers a promising approach for identifying potential new biomarkers through the analysis of a wide array of proteins in tissue samples. However, proteomics studies addressing IBD are limited [8], often constrained by small patient cohorts, and studies specifically focused on PSC-IBD are scarce [9].

The primary goal of this project is to identify new biomarkers to improve diagnostic precision for PSC-IBD and UC, as well as to optimize the assessment of disease activity. The secondary objectives are to enhance our understanding of the etiopathogenesis and immunopathogenic background of PSC-IBD and UC, and to correlate the severity of the histological inflammatory activity and the abundance of specific proteins with the degree of intestinal wall permeability.

Materials and methods

This is a prospective, observational, single-center biomarker study. The study follows the standard protocols of STROBE guidelines [10] and the SPIRIT reporting guidelines [11].

Study setting and timeline

This project is structured as a sequence of consecutive steps (Fig 1). Initially, candidate biomarkers associated with the diagnosis and/or disease activity will be identified through untargeted proteomic characterization of the bowel mucosa in patients with PSC-IBD and UC. Subsequently, these candidate biomarkers will be validated in external cohorts of PSC-IBD and UC patients, with visualization in the bowel mucosa via targeted proteomics and immunohistochemistry, and in peripheral blood through enzyme-linked immunosorbent assay (ELISA). Targeted proteomic analysis will also be employed to precisely quantify the levels of specific proteins within the tissue.

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Fig 1. A flow-chart showing a design of the study.

ELISA = enzyme-linked immunosorbent assay; IBD = inflammatory bowel disease; PSC = primary sclerosing cholangitis.

https://doi.org/10.1371/journal.pone.0341885.g001

The project timeline is divided into several consecutive phases. The recruitment phase, including endoscopic sampling of patients and healthy controls started on 01/07/2024 and will be completed by December 2025. In parallel, histopathological evaluation of intestinal samples is being performed during this recruitment phase. Upon completion of sampling, an untargeted proteomic analysis of intestinal mucosal samples will follow, with an anticipated duration of approximately six months, i.e., by March 2026. All data will be obtained and subsequent bioinformatic analysis of proteomic data focused on identifying candidate biomarkers will be performed by June 2026. The publication of the results is expected by December 2026.

In subsequent studies, we plan to verify the predictive significance of the newly identified biomarkers in relation to the long-term clinical outcomes. These future studies will therefore include a long-term follow-up of patients lasting at least two years from the time of biopsy, during which it will be monitored whether the patients develop predefined complications and, if so, after what period of time. The predefined complications will include the need for therapy intensification, hospitalization, surgical intervention, development of Acute Severe Colitis and the occurrence of dysplasia or invasive malignancy.

Discussion

IBD were traditionally regarded as conditions limited to the GI tract, with CD and UC recognized as the two primary phenotypes. However, they are now perceived as systemic disorders with a strong predilection for the GI tract. Their phenotypes are considered part of a continuous spectrum of conditions, ranging from CD involving the small bowel and upper GI tract, through colonic CD and IBD-unclassified, to distal UC [13,25]. As previously mentioned, the precise etiopathogenesis remains incompletely understood. Nonetheless, an imbalance in homeostasis between the intestinal epithelial barrier, immune barrier, and commensal intestinal microbiota in a genetically susceptible host is considered a key mechanism in the disease’s onset and progression [1]. Inflammation is primarily driven by T cells, with T helper 1 and 2 subpopulations playing dominant roles. Recent studies have also demonstrated significant involvement of Th17 cells, natural killer cells, and antigen-presenting dendritic cells [26]. Additionally, the role of impaired mucosal integrity is increasingly recognized as a contributing factor. The intestinal lumen is lined by a single layer of columnar epithelial cells bound by tight junctions, adherent junctions, and desmosomes [27]. Under normal conditions, up to 90% of all antigens are transported transcellularly from the intestinal lumen into the bloodstream, with associated degradation into small, non-immunogenic peptides. Only 10% of antigens are transported paracellularly, preserving their antigenic properties and playing an essential role in inducing immune tolerance [2]. Increased intestinal permeability allows a larger proportion of immunologically active antigens to translocate into the bloodstream, potentially contributing to the risk of various immunopathological diseases, including IBD [3].

Over the past decades, advancements in the understanding of pathogenesis, morphology, and clinical presentation have resulted in increasingly refined subclassifications of IBD. Diagnosis now requires a multidisciplinary approach, integrating the clinical course of the disease with laboratory, radiological, endoscopic, and histopathological findings [14,18]. Despite these advancements, up to 6% of adult and 13% of pediatric IBD cases remain unclassified [7]. Accurate diagnosis of CD or UC, along with precise phenotypic subclassification, is essential for guiding optimal medical and surgical management. This underscores the ongoing effort to identify novel biomarkers that not only enhance diagnostic precision and enable a more accurate assessment of disease activity, but also help to stratify patients according to disease behavior, predict therapeutic response, and guide personalized treatment decisions. Biomarkers reflecting immune activation, epithelial barrier dysfunction, or fibrosis could assist in identifying patients at higher risk of aggressive or treatment-refractory disease. These advancements are expected to facilitate the earlier initiation of appropriate therapy, improve therapeutic monitoring, and ultimately reduce the long-term incidence of severe complications. Numerous promising serum, fecal, and tissue biomarkers have been identified over time, though only a small fraction have been integrated into routine clinical practice. An ideal biomarker would be easily detectable by non-invasive methods, highly sensitive, specific, and cost-effective. Currently, no biomarker fulfills all these criteria. Among the most widely used are several conventional serological markers such as ASCA and pANCA, yet their clinical utility is restricted by suboptimal sensitivity and specificity. Notably, ASCA positivity has also been observed in non-IBD conditions, including untreated celiac disease [28], where it likely reflects a nonspecific immune response to microbial antigens associated with increased intestinal permeability. This overlap emphasizes that ASCA or pANCA positivity alone should not be interpreted as definitive evidence of IBD, particularly in patients with atypical or overlapping clinical features. In this study we seek to overcome these limitations by identifying biomarkers with greater disease specificity and a closer mechanistic link to intestinal inflammation and mucosal barrier dysfunction.

As noted above, IBD are often tied to PSC, which is a debilitating disease with many patients progressing to liver cirrhosis and requiring liver transplantation. The connection between PSC and IBD is well established, with most PSC patients developing IBD during their lifetime [5]. PSC-associated IBD has traditionally been classified as UC and, less commonly, as CD. However, it is now increasingly recognized as a distinct form of IBD, and many specialists avoid further subclassification when the disease occurs in the context of PSC. Unlike conventional UC, PSC-IBD more commonly presents as pancolitis on both endoscopic and microscopic examination. Morphological changes typically involve the right colon with a characteristic reverse gradient of inflammation, common occurrence of backwash ileitis, and frequent sparing of the rectum [29]. Importantly, the condition carries a substantially higher risk of colorectal cancer compared to conventional IBD, despite a milder clinical course [6]. It is therefore clinically imperative to distinguish PSC-associated IBD from conventional IBD, a distinction further emphasized by the potential for biliary disease to manifest long after the onset of intestinal inflammation [30]. Unfortunately, no reliable biomarker has been identified to distinguish PSC-associated IBD from conventional IBD, apart from the presence of advanced biliary disease.

When searching for new reliable biomarkers of a disease or specific clinical condition, it is essential to determine whether the biomarker should be identified at the level of DNA, transcriptomics, or protein expression. Genetic predisposition clearly plays an essential role in the etiology of IBD, and advances in molecular genetics, particularly genome-wide association studies, have identified multiple candidate genes whose pathogenic mutations or single nucleotide polymorphisms correlate with an increased risk of CD or UC or with a higher likelihood of aggressive disease phenotypes. However, genetic alterations do not always correlate with downstream protein expression. Additionally, environmental factors play a significant role in IBD etiology, impacting transcriptomic and protein expression without affecting the DNA level. Biomarker discovery at the protein level is therefore more clinically relevant, as proteins directly represent functional and dynamic pathological processes, such as inflammation and fibrosis, offering valuable insights into disease activity and progression. In contrast, DNA-based biomarkers, while valuable for understanding genetic predispositions, are static and may not accurately reflect the current disease state. Proteomics offers a dynamic approach, linking genomic and transcriptomic changes to the phenotypic manifestations of IBD, providing insights into the mechanisms driving disease initiation and progression. Consequently, proteomics represents an ideal approach for identifying extensive sets of candidate biomarkers by enabling broad-spectrum protein analysis from tissue samples or body fluids [31]. Proteomic studies in IBD remain limited, often involving small patient cohorts, with only a few identified biomarkers advancing to clinical application. A 2019 review by Assadsangabi et al. [8] highlighted only 19 studies, of which 13 analyzed tissue samples (endoscopic biopsies or resection specimens), while the remainder focused on body fluids. Even more limited information is available on proteomics in PSC-IBD. To our knowledge, the only relevant study by Vessby et al. [9] analyzed nine PSC-IBD patients and seven UC patients in clinical and histologic remission, and seven healthy controls, identifying AGPAT1 protein as a potential PSC-IBD marker.

From a practical point of view, the potential clinical implementation of the identified biomarkers will also depend on their cost-effectiveness and accessibility. Although proteomic discovery phases rely on advanced and resource-intensive technologies, subsequent validation using immunohistochemistry or ELISA offers a cost-efficient and scalable means for clinical adoption. By focusing on biomarkers measurable through routine laboratory techniques, this study aims to facilitate the development of affordable diagnostic tools that can be integrated into everyday clinical practice without substantial increases in healthcare costs.

In conclusion, our proposed study aims to identify novel biomarkers to enhance the diagnostic accuracy for PSC-IBD and UC and improve assessments of the disease activity. This could enable better risk stratification and personalized treatment plans. Our study realistically anticipates including 150 subjects, making it one of the largest IBD proteomic studies to date. We believe the study is well-founded, timely, and serves a meaningful purpose.

Supporting information