Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, refers to chronic immune-mediated conditions associated with damage to the intestinal barrier and impaired immune response regulation [1]. In recent years, the mucosal barrier molecular components, such as еру fatty acid binding protein (I-FABP), mucin 2, and trefoil factors (TFF2, TFF3). These components play a key role in preserving the intestinal epithelium integrity and the inflammatory response modulation [2, 3]. Gut microbiota plays an important role in maintaining the mucosal barrier [4].

Current research shows that I-FABP is a sensitive enterocyte damage marker, and serum I-FABP levels are correlated to the inflammatory activity associated with IBD [5]. Mucin 2, the main intestinal mucus component, forms a protective layer inhibiting the adhesion of pathogens and penetration of those into the epithelium. In IBD, the mucin 2 synthesis and structure are disturbed, which results in the increased intestinal barrier permeability [6]. Trefoil factors TFF2 and TFF3 are involved in the intestinal mucosa repair; TFF3 is considered as a promising marker of the inflammatory activity associated with ulcerative colitis [7, 8]. TFF peptides also form part of chronic inflammation associated with various disorders, such as ulcerative colitis, diverticulitis, cholecystitis, IBD, gastroesophageal reflux disease, pancreatitis, and chronic obstructive pulmonary disease [9].

Current research emphasizes the importance of comprehensive assessment of the mucosal barrier state and molecular components in IBD. It has been shown that the intestinal epithelium barrier function impairment, including at the level of mitochondrial mechanisms, contributes greatly to the IBD pathogenesis and can be assessed based on serum marker levels. In this regard, the search for available laboratory indicators reflecting the mucosal barrier state is still a relevant task [10, 11].

The study aimed to perform the comprehensive comparative assessment of the mucosal barrier serum components in healthy donors and patients with IBD.

METHODS

A total of 20 patients with IBD aged 20–70 years undergoing inpatient treatment due to exacerbation of ulcerative colitis and 20 healthy donors aged 18–65 years with no signs of IBD were included in the study. Inclusion criteria for patients with IBD: established diagnosis of IBD confirmed by clinical, endoscopic, and morphological data; exacerbation of the disease. Exclusion criteria: pregnancy or breastfeeding, decompensation of the concomitant liver or kidney disease, ileostomy or colostomy, previous major small bowel or colon resection surgery, subtotal or total colectomy, congenital or acquired immunodeficiency, malignant neoplasms, signs of active inflammation in a patient, fact of taking antimicrobial drugs for three months before enrollment, patient’s refusal to submit the informed consent to take part in the study. Individuals having no IBD were included in the group of healthy donors. Exclusion criteria: 1) concomitant disorders and conditions affecting gut microbiota: IBD, malabsorption syndrome, cancer, gastrointestinal surgery (except appendectomy), functional intestinal disorders, celiac disease, allergy, type 1 and 2 diabetes mellitus, metabolic syndrome, non-alcoholic fatty liver disease, mental disorders, fact of taking immunosuppressive, cytostatic, and antibacterial agents, glucocorticosteroids, and probiotics/prebiotics, regular use of NSAIDs throughout three months before the study, obesity or underweight; 2) alcohol and substance abuse; chronic disorders affecting internal organs; 3) infectious and parasitic diseases; 4) diarrhea (with a stool frequency of more than three times a day) for at least three consecutive days within the last month; 5) pregnancy or lactation. The study sample was selected randomly; the study was single-center and crosssectional. There was no preliminary sample size calculation.

Biomaterial collection and testing method

Venous blood samples were collected in vacuum test tubes with the coagulation activator. Blood serum was obtained by centrifugation at 2000 g for 10 min at +4 °C, then aliquoted and stored at – 80 °C before conducting the analysis.

I-FABP, TFF2, TFF3, and mucin 2 (MUC2) concentrations were determined by the enzyme-linked immunosorbent assay (ELISA) using the commercially available reagent kits (CloudClone Corp., USA) in strict accordance with the manufacturer's instructions. Measurement ranges: I-FABP — 15.6–1000 pg/mL, TFF2 — 6.25–400 pg/mL, TFF3 — 12.5–800 pg/mL, MUC2 — 0.156–10 ng/mL.

The experimental results were analyzed using the statistical software tool on the MedCalc® Statistical Software version 20 platform (MedCalc Software Ltd., Belgium). The data were presented as the median and interquartile range (Me [Q25–Q75]). The differences were considered significant at p ≤ 0.05, the nonparametric Mann–Whitney U-test was used for comparison.

RESULTS

Comparative analysis of serum concentrations of mucosal barrier components in patients with IBD and healthy donors revealed some alterations (table).

I-FABP and mucin 2 levels in the group of patients with IBD matched the values of the control group. At the same time, alteration of the levels of trefoil factors was reported.

The TFF2 concentration in patients with IBD was significantly higher compared to the values of the control group (5 times).

Median TFF2 levels were 83.30 pg/mL in patients with IBD and 16.60 pg/mL in healthy donors. It should be noted that the lower quartile of TFF2 values w as at the zero mark in the group of healthy donors, while in patients with IBD the lowest factor level was 46.29 pg/mL.

Significant differences were also reported for TFF3, the level of which was 4.2 times higher compared to values in the  control group (specifically, 253.12 pg/mL vs. 59.90 pg/mL), and the lower quartile for TFF3 in healthy donors was also was at the zero mark.

DISCUSSION

The data obtained suggest changes in the content of the key mucosal barrier components in patients with IBD, which is consistent with the current ideas about the pathogenesis of this group of disorders.

The most pronounced shifts are reported for trefoil factors, suggesting the important role of those in maintaining the mucosal barrier integrity and the mucous membrane repair [1].

A significant (4.2-fold) TFF3 level increase in patients with IBD deserves special attention. It is well known that such a marked TFF3 concentration increase represents a compensatory response aimed at restoring the damaged epithelium. It is noteworthy that even the lowest TFF3 values  in patients with IBD were significantly higher than those in healthy individuals, which may indicate the constant activation of repair mechanisms in chronic inflammation [2].

The 4.2-fold TFF3 level increase in patients with IBD reported in our study is of great pathogenetic and clinical significance. Moreover, current papers confirm the fact that the TFF3 level increase decree is correlated to the epithelial damage severity and the damaged mucosa area [6]. Such significant TFF3 concentration increase is likely to reflect activation of the compensatory mechanisms aimed at restoring the intestinal barrier integrity. This is confirmed by experimental data, according to which TFF3 stimulates the epithelial cell migration and accelerates the mucosal lesion regeneration [12].

Along with the generally accepted role of TFF3 in the stabilization of the mucin layer secreted by goblet cells of the intestine, its ability to stimulate mucosal repair via activation of the ERK pathway is well known [7]. The TFF3 expression is activated in response to damage to the mucous membrane, as well as under the exposure to pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα) and various interleukins.

The detected considerable TFF3 concentration increase may be determined by two major mechanisms. First, this is a compensatory response aimed at restoring the damaged epithelium and activating anti-inflammatory mechanisms. Second, this is an increase in its entry into the systemic circulation resulting from the breach in the mucosal barrier integrity, caused both by the transcapillary permeability increase and by dysfunction of the gap junctions between enterocytes.

The current data expand the knowledge about TTF functions. TFF peptides play not only mitogenic, but also structural function as part of the insoluble gastric mucus bicarbonate barrier (TFF2/ MUC6 complex) and in the hypothesized mucosal clearance system for various microorganisms. These can trap viruses on the mucosal surface using immunoglobulins and participate in the innate immune defense [13]. The TFF3 expression is regulated by commensal bacteria through toll-like receptors. It has been shown that TFF1 can play a protective role during inflammation, including in the brain, and TFF3 expressed in active microglial cells can act as a neuronal chemotactic factor [13]. The literature reports that the trefoil factor concentration increase can be observed in individuals with inflammation of other localization [14, 15]. Such conditions also include periodontitis, in which both local and systemic TFF-1 and TFF-3 level increase is reported, which suggests that these peptides are involved in the response to the periodontal tissue disruption [15].

Our data are consistent with the papers considering TFF3 as a mucosal regeneration factor in ulcerative colitis [16] and TFF2 as a factor stabilizing the mucin barrier that is involved in protection against damage [17, 18]. In experimental models, TFF2/SP-deficient mice showed the increased sensitivity to the NSAID-induced damage, which confirms the TFF2 protective role [17]. 

The TFF2 immunomodulatory properties, which have been actively studied in recent years, are worth noting. It has been determined that TFF2 can suppress production of the key pro-inflammatory cytokines, including IL1β and TNFα, through inhibition of the NF-κB signaling pathway [9]. It is also noted that macrophages promote epithelial proliferation via a TFF2-dependent mechanism [19]. These data suggest that elevated TFF2 levels associated with IBD may represent not only a consequence of epithelial damage but also an active compensatory mechanism aimed at limiting the inflammatory response [15]. The TFF2 interaction with gut microbiota is an important aspect. According to the latest studies, this protein can modulate the gut microbiome composition and functional activity [20].

The results for mucin 2 are also interesting. Despite the fact that no significant intergroup differences were revealed, the reported upward trend of its concentration in patients with IBD is consistent with the current ideas about the compensatory mucin hyperproduction in the context of chronic inflammation [21]. The fact that mucin 2 represents a highmolecular-weight glycoprotein, which significantly limits its passive diffusion across the intestinal barrier, is an important pathophysiological aspect. In this regard, serum mucin 2 levels canbe a less sensitive marker of the barrier function impairment compared to low-molecular-weight proteins, such as I-FABP. No significant increase in blood mucin 2 concentrations, despite active inflammation, can be explained by the fact that the increased mucin 2 secretion associated with IBD is aimed primarily at forming a protective mucin layer in the intestinal lumen. Furthermore, the reported upward trend can result from the compensatory enhancement of its synthesis, while considerable amounts enter the systemic circulation only in cases of the extreme damage to the mucosal barrier.

Study limitations

The study results should be considered taking into account some natural limitations. First, the sample size (20 people per group) was relatively small, which could reflect a certain limitation in the analysis statistical power. Second, the single-center and cross-sectional design of the study makes it impossible to assess the marker dynamic changes over time and the marker association with long-term outcomes. Third, the studied group included patients with exacerbation of ulcerative colitis, so the findings apply primarily to this category of patients.

CONCLUSIONS

Elevated serum TFF2 and TFF3 concentrations were revealed in patients with exacerbation of IBD compared to healthy donors. The median TFF2 level was 83.30 pg/mL vs. 16.60 pg/mL (p = 0.0104), TFF3 — 253.12 pg/mL vs. 59.90 pg/mL (p = 0.0109). Serum I-FABP and mucin 2 concentrations of patients with IBD showed no significant differences from the values of healthy donors. This can be due to the fact that the I-FABP levels reflecting the acute damage to enterocytes not always increase in chronic inflammation, and mucin 2 is a large glycoprotein that can hardly enter the systemic circulation, so its serum levels poorly reflect the intestinal inflammation activity. The TFF3 minimum levels in patients with IBD were higher, than that in healthy individuals, which can reflect the constant activation of repair processes in chronic inflammation. Determination of serum TFF2 and TFF3 levels can be used as one more laboratory criterion for the IBD-associated inflammation activity.