Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases

Abstract Background and Aims Crohn’s disease [CD] and ulcerative colitis [UC] require lifelong treatment and patient monitoring. Current biomarkers have several limitations; therefore, there is an unmet need to identify novel biomarkers in inflammatory bowel disease [IBD]. Previously, the role of plasminogen activator inhibitor 1 [PAI-1] was established in the pathogenesis of IBD and suggested as a potential biomarker. Therefore, we aimed to comprehensively analyse the selectivity of PAI-1 in IBD, its correlation with disease activity, and its potential to predict therapeutic response. Methods Blood, colon biopsy, organoid cultures [OC], and faecal samples were used from active and inactive IBD patients and control subjects. Serpin E1 gene expressions and PAI-1 protein levels and localisation in serum, biopsy, and faecal samples were evaluated by qRT-PCR, ELISA, and immunostaining, respectively. Results The study population comprised 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients. We demonstrated that the serum, mucosal, and faecal PAI-1 concentrations are elevated in IBD patients, showing clinical and endoscopic activity. In responders [decrease of eMayo ≥3 in UC; or SES-CD  50% in CD], the initial PAI-1 level decreased significantly upon successful therapy. OCs derived from active IBD patients produced higher concentrations of PAI-1 than the controls, suggesting that epithelial cells could be a source of PAI-1. Moreover, faecal PAI-1 selectively increases in active IBD but not in other organic gastrointestinal diseases. Conclusions The serum, mucosal, and faecal PAI-1 concentration correlates with disease activity and therapeutic response in IBD, suggesting that PAI-1 could be used as a novel, non-invasive, disease-specific, faecal biomarker in patient follow-up.


Introduction
Crohn's disease [CD] and ulcerative colitis [UC], two forms of inflammatory bowel disease [IBD], are chronic, tissue-destructive conditions developing as a consequence of uncontrolled activation of effector immune cells in the intestinal mucosa.The continuing increase in IBD incidence worldwide is associated with a rapidly increasing global CD and UC burden. 1 The impact of symptoms and complications on the patients can be profound, leading to impaired quality of life and social and occupational dysfunction. 2,3The past decades have brought substantial advances in the pharmacological management of IBD by introducing immunosuppressive agents, biologics, and lately small molecules.However, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal.Moreover, an effective treatment may achieve clinical remission, but the subclinical inflammation can persist within the mucosa of the gut, contributing to a risk of 'symptomatic' relapse.The tight disease monitoring strategy based on objective inflammation markers and timely therapy escalation leads to improved clinical and endoscopic outcomes, compared with symptomdriven management alone. 4In the long term, this approach would decrease the disease burden on patients and the costs of the health care system. 5Therefore, an effective monitoring strategy enabling physicians to make difficult decisions to prevent severe disease complications is still an unmet clinical need.
C-reactive protein [CRP] and faecal calprotectin [FC] 6 are the most widely studied inflammatory biomarkers for disease monitoring.Although CRP is commonly used, it has insufficient sensitivity and specificity for intestinal inflammation. 7Recently, faecal biomarkers have emerged as non-invasive, rapid, simple, and low-cost diagnostic tools to detect intestinal inflammation. 8ost studies have been performed with FC, which is today the gold standard among faecal markers 9 ; however, significant limitations hinder its use in everyday practice. 10Therefore, alternative biomarkers must be developed to overcome these shortcomings, precisely monitor the disease course, and predict therapeutic response.In a recent paper, Kaiko et al. identified a novel protein-plasminogen activator inhibitor 1 [PAI-1]showing an important role in controlling key inflammatory modulators during mucosal damage in colitis. 11They also highlighted that the mucosal SerpinE1 gene [the gene encoding PAI-1] expression is elevated in active, more severe IBD patients who do not respond to anti-tumour necrosis factor [TNF]  therapy.In another study, Wang et al. showed that PAI-1 induces neutrophil-mediated chemokine expression by activating the NF-κB pathway in dextran sodium suphate [DSS]-induced colitis model in mice. 12These studies emphasised the potential role of PAI-1 in IBD pathogenesis; however, the protein levels of PAI-1 in different biological samples and their correlation with the disease activity or therapeutic response remained unknown.
Therefore, in this study, we aimed to comprehensively analyse the expression profile of PAI-1 in IBD patients' serum, mucosa, and faeces to investigate its selectivity, correlation with the disease activity, and potential to predict therapeutic response in IBD.

Patient population and disease activity measurements
Consecutive patients diagnosed with CD and UC who underwent colonoscopy between 2020 and 2021 at the Department of Medicine, University of Szeged, were enrolled in the study.All IBD patients were eligible for the study except those under 18 years of age, pregnant women, and those who did not consent to take part in the study.The inactive IBD group consisted of patients showing no sign of activity at the index colonoscopy.For patients in the active IBD group, new therapeutic agent[s] such as conventional anti-inflammatory drugs (mesalazine, corticosteroid, budesonide, azathioprine, ciclosporin], anticytokine (anti TNF-α [infliximab, adalimumab], anti-IL12/23 [ustekinumab]), α4β7 anti-integrin agent [vedolizumab], or the small molecule JAK inhibitor [tofacitinib] were introduced.Alternatively the current therapy was modified according to the step-up or accelerated step-up strategy, and/ or combination therapy was used to increase the therapeutic effectiveness.Throughout the 12-month follow-up period, patients were strictly controlled at regular visits performed every 2 months.Demographic data, data on disease activity, and therapeutic response were documented at every visit.In CD patients, clinical disease activity was determined using the Crohn's Disease Activity Index [CDAI] and in patients with UC, clinical disease activity was measured using the partial Mayo score [pMayo]. 13,14Clinically active disease was defined as CDAI ≥150 in CD and pMayo ≥2 in UC.Colonoscopy was performed at inclusion and Week 52, except for the case of uncertain disease exacerbation requiring unscheduled endoscopy to clarify symptoms.Endoscopically active disease was defined as SES-CD ≥3 in CD and eMayo ≥2 in UC in any examined bowel segment. 15Patients were grouped into the following categories based on the clinical and endoscopic activity of their disease, and for simplicity, these groups were used later: inactive, mildly, moderately, and severely active.Regarding the clinical condition of the patients, inactive disease was defined as CDAI˂150 in CD and as pMayo˂2 in UC.Mildly active disease was defined as CDAI between 150 and 219 and pMayo between 2 and 4. CDAI between 220 and 450 and pMayo 5-6 corresponded to moderately active disease, and CDAI ˃450 and pMayo 7--9 to severe disease. 14,15egarding endoscopic disease activity, SES-CD between 0 and 2 suggested inactive, 3-6 mildly active, 7-15 moderately active, and ≥16 severely active CD. 16In UC, eMayo 0-1 expressed inactive, 2 moderately active, and 3 severely active disease. 15In the case of active disease, a decrease of the score by 3 or more was taken as a therapeutic response in UC, and a decrease in SES-CD by >50% was considered an endoscopic response in CD. 16 The control group consisted of non-IBD subjects who underwent colonoscopy.

Sample collection
Biopsies were obtained from the inflamed [from the edge of the ulcer or, in the absence of an ulcer, from the most inflamed region: n = 6] and from the non-inflamed part of the colon [n = 6] of IBD patients and the healthy sigmoid colon of controls.The biopsies were discharged from the forceps into the transport media.The tissue samples were placed immediately in ice-cold NaHCO 3 containing Hank's Balanced Salt Solution [HBSS, Sigma, H9269] and transferred to the laboratory.Serum and faecal specimens were collected within 1-3 days before endoscopy.Serum samples were obtained to determine the routine inflammatory parameters [CRP, total blood count] at every appointment.CRP was considered to be elevated above 5 mg/L.At the same time, additional blood and stool samples were collected for the cytokine profile determination.Blood specimens were centrifuged, and the serum was snap-frozen and stored at -80°C until use.Faecal samples were also frozen until further investigations.

Human colonic organoid culture
Human colonic organoid cultures [OC] were generated using 3-4 colon biopsy samples. 17First, colonic crypts were isolated as previously described. 18Briefly, biopsy samples were washed three times with HBSS supplemented with an antibiotic and antimycotic mix [Supplementary Table 1].Then, biopsies were minced into ~1 mm 3 pieces, transferred to the sterile, 30-ml centrifuge tube [Greiner, 201170], and washed with HBSS 8-10 times.The tissue pieces were placed into 3 ml 10 mM dithiothreitol [DTT, Roche, 11583786001] in completed HBSS to reduce the disulphide bonds, and were incubated in a shaking incubator at 165 rpm at 37°C for 15 min.Next, DTT was removed, and the tissue was washed with HBSS 3 times.The samples were put into 3 ml completed HBSS containing 0.8 mg/ml Collagenase A [Roche, 37170821] and incubated for 50 min at 165 rpm at 37°C in a shaking incubator.At the end of the incubation, the samples were resuspended, and the isolated crypts in the supernatant were checked with a Primovert light microscope [Zeiss].If necessary, the 15 min digestion step was repeated to maximise the number of isolated crypts.Then, the supernatant was collected into a sterile 1.5 ml tube and centrifuged for 5 min at 2000 rpm at 4°C.The isolated crypts were resuspended in Matrigel [Corning, 356232] diluted with HBSS in a 1:4 ratio, and 10 μl domes were placed into a 24 well plate [Greiner, 662160], two domes per well.After the polymerisation of Matrigel, 1 ml feeding medium [Supplementary Table 2], completed with 10 µM Rho kinase inhibitor [Tocris, 1254], was added per well.The medium was changed every other day.OCs were passaged after 7 days of culture using TrypLE Express Enzyme, completed with 10 µM Rho kinase inhibitor.

Protein isolation from biological samples
For total protein isolation, colonic biopsy samples were minced into small pieces with a razor blade in a Petri dish and were transferred into 1 ml RIPA lysis buffer [Millipore, 20- After that, the samples were centrifuged at 3500 rpm for 10 min at 4°C.The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use.For total protein isolation from stool samples, 100 mg stool was vortexed in 300 µl RIPA lysis buffer with protease inhibitor for 5 min.After that, the samples were centrifuged at 4000 g for 10 min at 4°C.The supernatant was transferred to a new, sterile tube, and the centrifuge step was repeated at 12000 g for 10 min at 4°C.The supernatant was transferred into a new 1.5 ml tube, snap-frozen, and stored at -80°C until use.

Determination of the cytokine expression in serum, colonic biopsy, and faecal samples
To define the cytokine expression pattern of the serum and the colonic mucosa, the Proteome Profiler Human Cytokine Array Kit [R&D Systems, ARY005B] was used according to the manufacturer's protocol.Briefly, 200 µl sera were used without dilution in all serum cytokine determination measurements; for the mucosal cytokine expression investigation, 500 µg total protein was applied at all membranes after total protein isolation.The total protein concentrations of biopsy samples were determined by Bradford assay.Blots were imaged with a ChemiDoc MP [BioRad].

Determination of PAI-1 in serum, colonic biopsy, organoids, and faecal samples
To determine the plasminogen activator inhibitor 1 [PAI-1] concentration in the serum, mucosa, and faecal samples, a commercially available enzyme-linked immunosorbent assay [ELISA] kit [Abcam, ab269373] was used according to the manufacturer's protocol.Multiskan FC [Thermo Scientific] plate reader was used for the ELISA and Protein Bradford measurements.

Serum
Blood specimens were collected and stored as described above.For serum ELISA measurements, samples were diluted to 20-100 x with the Sample Diluent NS buffer of the ELISA kit.

Tissue and organoids
For the tissue and organoid samples, the total protein concentrations were defined by the Protein Bradford method and protein levels were normalised to the total protein concentration.

Faeces
For the faecal samples, the total protein concentrations were defined by the Protein Bradford method, and protein levels were normalised to the total protein concentration.For the measurement of PAI-1 in tissue and faecal samples, the ab269373 ELISA kit was validated.Recombinant human PAI-1 protein from the ab269373 ELISA kit was used for the recovery measurements, and the following quantities of PAI-1 were added to the samples: 0, 15.6, 31.25,62.5, 125, 250, 500, and 1000 pg.Three biological and three technical measurements were done at all sample types according to the manufacturer's protocol.The results of the validation are summarised in Supplementary Figure 1.

Gene expression analyses of colonic biopsy samples and organoid cultures
Gene expression analyses were performed as previously described. 17Briefly, total RNA was isolated from homogenised biopsies with NucleoSpin RNA Plus Kit [Macherey-Nagel, 740984], whereas for homogenised organoids NucleoZOL [Macherey-Nagel, 740404.200]was used according to the instructions of the manufacturer.iScript cDNA Synthesis Kit [Bio-Rad, 1708891] was used for the reverse transcription of mRNA samples.The cDNA concentration was 50 ng/μl at the tissue and 25 ng/μl at the OC samples.The primers were designed in the NCBI Primer-BLAST [Supplementary Table 3].Except for BACT, all primers were designed to the exon-exon junction, and all polymerare chain reaction [PCR] products were less than 200 bp.Amplicons were detected by a Lightcycler 96 [Roche, 05815916001] using SYBR Green [Bio-Rad, 1725274].GAPDH and BACT were used as housekeeping genes for normalisation, and the Livak Method was applied to the evaluation.The gene expression of non-IBD control patients was applied to define the relative gene expression fold changes of IBD patients.

Immunofluorescence staining
Immunostainings were performed as previously described. 19riefly, colon biopsies were fixed in 4% PFA-PBS for 2 h and then incubated overnight in 30% sucrose for cryoprotection.Fixed biopsy samples and organoids were embedded into Cryomatrix [Thermo Scientific, 6769006], frozen at -20°C, and 7-μm thick sections were cut.Organoids were fixed after sectioning in 4% PFA-DPBS for 20 min at room temperature.Biopsies were permeabilised with 0.02% Triton-X100-TBS for 10 min, followed by 10 min of washing in TBS three times.For organoids, the permeabilisation was performed using citrate-Tween 20 solution (0.001 M sodium citrate buffer [Sigma, C8532], pH 6.0, and 0.05% Tween 20 [Sigma, P1379-100]) and boiled for 30 min.Unspecific antibody binding was blocked for 2 h at 37°C with 10% BSA-TBS containing 1% goat serum.Mouse monoclonal anti PAI-1 primary antibody [Invitrogen, MA-33H1F7] was diluted 1:200 in 10% BSA-TBS, and sections were incubated overnight at 4°C.The primary antibody was washed three times as described, and then an Alexa Fluor Plus 488 conjugated goat anti-mouse secondary antibody [Invitrogen, A48286] was applied in 1:2000 dilution for 2 h at room temperature.Samples were washed three times, and then nuclei were labelled with DAPI [Sigma, MBD0015-5ML] in 1:500 dilution for 30 min.For isotype control, mouse polyclonal IgG [ab37355] under the same conditions as the primary antibody was used.Sections were placed into Fluoromount [Sigma, F4680-25ML], and images were obtained by Zeiss LSM 880 confocal microscope with a 40 x oil immersion objective.

PAI-1 stability in faecal samples
The stability of the PAI-1 was defined in stool samples; 100 mg faeces was diluted into 1.5 ml sterile tubes and incubated at room temperature and 4°C for up to 7 days.Total protein isolation was performed every day.The isolation protocol and the PAI-1 level determination were the same as described above.

Data and statistical analysis
All software used for data analysis is summarised in Supplementary Table 4. Statistical analysis was performed with GraphPad Prism software [GraphPad, 9.5.0].For all data with non-normal distribution, non-parametric tests were employed.Between two groups, unpaired Mann-Whitney or paired Wilcoxon tests were performed.The Kruskal-Wallis test with Dunn's multiple comparisons test was used for three or more groups.The significance was accepted at p <0.05.Data are imaged by box plot and presented with the median, interquartile range [IQR], and minimum and maximum points.For the correlation analysis, non-parametric Spearman's correlation was used.MedCalc statistical software [MedCalc Software, 20.211] was used for the receiver operating characteristic [ROC] analysis.The area under the curve [AUC] value, sensitivity, and specificity were defined, and the cut-off value was determined according to the maximisation criterion of the Youden index.

Ethical considerations
Ethical approval for the study was obtained from the Regional and Institutional Human Medical Biological Research Ethics Committee, University of Szeged [247/2018-SZTE; 4412].The research was carried out according to the Code of Ethics of the World Medical Association [Declaration of Helsinki], and written informed consent was obtained from the enrolled patients.

Patient population
The study population consisted of 132 IBD patients [56 CD and 76 UC] and 40 non-IBD patients [10 without abnormalities, seven with diverticulosis, 14 with colon polyps, and nine with colorectal cancer].The demographic and clinical characteristics of the IBD patients are presented in Table 1 and Table 2.The mean age was 39 years for both CD [22-72] and UC [18-72].Patients in clinical remission accounted for 37.5% of CD and 28.9% of UC, and endoscopic remission was confirmed in 30.4% of CD and 25% of UC patients.New therapy was introduced in 21.4% of CD and 21.1% of UC patients.Switch and dose escalation of biological therapy was carried out in 16.1% and 14.3% of patients with CD and 17.1% and 9.2% of patients with UC.

Serpin E1 gene expression in the colonic biopsies was significantly elevated in IBD patients and decreased in responders
The primary sources of the secreted PAI-1 in the blood circulation are the endothelial cells, but other cell types, including epithelial and immune cells or adipocytes, also express this protein, which may be more significant in the local inflammation in IBD.Therefore we determined the relative gene expression fold changes [Fc] of Serpin E1 [the gene encoding PAI-1] in colonic biopsy samples from non-IBD control subjects, therapy-naive, active IBD patients, and responders and non-responders.Compared with the control samples, mucosal Serpin E1 expression was significantly higher in therapy-naive, active IBD patients [2.585 vs 18.17, p = 0.006; Figure 2A] but showed no significant difference between CD and UC patients [Figure 2B].We also determined the relative expression of four well-known inflammatory genes, TNF-α, IL-1β, IL-6, and TGF-β, from the same samples.In these experiments, IL-1β and TGF-β showed elevated expression in the untreated IBD biopsies, whereas TNF-α and IL-6 were not significantly elevated [Supplementary Figure 4A, B].In addition, Serpin E1 gene expression was significantly lower in endoscopically inactive patients compared with active subjects [0.96 vs 14.32, p <0.0001, Figure 2C] and in responders compared with the untreated patients [0.9326 vs 18.17, p <0.0001], or with non-responders [0.9326 vs 8.487, p = 0.002 Figure 2D].Among the four other genes, only TGF-β and IL-1β were upregulated in the endoscopically active IBD patients and decreased in responder subjects [Supplementary Figure 4C and D].6.651%, p = 0.0126], whereas the non-inflamed part showed no difference when compared with control samples [5.716% vs 6.641%, p >0.9999; Figure 3B].Notably, the majority of the PAI-1 positive cells were observed primarily in the epithelial cell layer.Next, we isolated the total protein from the biopsy samples and determined the PAI-1 concentration in the colonic tissue, which was significantly higher in the IBD samples [0.00 vs 55.96 pg/mg, p = 0.001; Figure 3C].However, no significant difference was detected between CD and UC patients [29.24 vs 40.55 pg/mg, p = 0.7903; Figure 3D].Moreover, PAI-  3I].Finally, when patients were categorised into responder and non-responder groups based on their response to the selected anti-inflammatory therapy, we found that the initial PAI-1 level was significantly higher in the responder patients compared with the non-responders [62.77 vs 34.15 pg/mg, p = 0.03; Figure 3J].Notably, mucosal PAI-1 concentration decreased significantly in responders, and this is not the case in non-responders [responders: 62.77 vs 0.23 pg/mg, p = 0.0001; non-responders: 34.15 vs 24.58 pg/mg, p = 0.58; Figure 3K].These results suggest that the tissue concentration of PAI-1 correlates with the disease activity and decreases in response to therapy.

PAI-1 expression is higher in IBD patientderived colonic organoids compared with controls
To further investigate whether epithelial cells could be the primary sources of PAI-1 in the colonic tissue, we established colonic organoid cultures [OC] from biopsy samples obtained from control subjects and IBD patients with active disease.Adult stem cell-based organoids consist of epithelial cells that display apical to basal polarity and maintain features of the original tissue in 3D cell cultures. 20In our experiments, OCs were used for experiments between the first and second passage to avoid changes in the inflammatory phenotype.The analysis of Serpin E1 revealed that the relative gene expression Fc was higher in the inflamed IBD organoids; however, the difference was not significant [2.034 vs 1.003 ng/g, p = 0.3095; Figure 4A].On the other hand, immunostaining revealed that the expression of PAI-1 was higher in IBD organoids [Figure 4B].Moreover, the PAI-1 concentration in the organoids [35.29 vs 0.00 ng/g, p = 0.0286; Figure 4C] and in the medium [31.28 vs 28.50 ng/g, p = 0.0423; Figure 4D] were significantly higher in the IBD organoids compared with the control.

PAI-1 as a potential faecal biomarker in IBD
The widely accepted tight disease monitoring strategy in IBD requires rapid, cheap, and easily accessible biomarkers, allowing patient self-testing.Faecal disease markers are suitable for these goals.Therefore, in the next step we analysed the faecal concentration of PAI-1 in IBD patients.First, we assessed the stability of PAI-1 in the faeces at room temperature and 4°C up to 7 days, as this is a crucial parameter for a faecal biomarker.Our results suggest that PAI-1 remains stable for 7 days at 4°C in faecal samples collected from IBD patients [Figure 5].In addition, we showed that PAI-1 concentration decreased in the first 24 h at room temperature but remained stable for 7 days.Notably, in these experiments, we did not use any stabiliser or enzyme inhibitor agent, which presumably could further improve the stability of PAI-1.In the next step, we compared the concentration of PAI-1 in faecal samples collected from controls and IBD patients.Our results demonstrated that the level of PAI-1 was significantly higher in the faecal samples of IBD patients in general [0.00 vs 0.84 ng/g, p <0.0001; Figure 6A].Similar to the serum and tissue PAI-1 levels, we could not find any significant difference between UC and CD patients [0.77 vs 1.15 ng/g, p = 0.73; Figure 6B].Importantly, the faecal PAI-1 concentration correlated with the endoscopic and clinical disease activity, and it was significantly higher in patients with endoscopically or clinically active disease compared with inactive patients and controls [endoscopically active IBD vs inactive IBD: 1.535 vs 0.215 ng/g, p <0,0001; endoscopically active IBD vs control: 1.535 vs 0.02 ng/g, p <0.0001; clinically active IBD vs inactive IBD: 1.45 vs 0.01 ng/g, p <0,0001; clinically active IBD vs control: 1.45 vs 0.02 ng/g, p <0.0001; Figure 6C, D].The increased faecal PAI-1 level correlated with the endoscopic activity [r = 0.5501, p <0.0001; Figure 6E], whereas ROC curves demonstrated that faecal PAI-1 showed a relatively high power to distinguish between active and inactive IBD patients [AUC = 0.82; specificity: 80%; sensitivity: 74%; cut-off: 0.6 ng/g; Figure 6F] and between controls and active IBD patients [AUC = 0.83; specificity: 62%; sensitivity: 88%; cut-off: 0.2 ng/g; Figure 6G].In the same patient cohort, CRP was significantly increased in the clinically or endoscopically active patients as well.However, no correlation was detected between CRP and faecal PAI-1 concentration [Supplementary Figure 5A-C].Finally, the faecal PAI-1 concentration was significantly lower in responders vs non-responders after the treatment [0.33 vs 1.1 pg/g, p = 0.047; Figure 6H].Importantly, CRP did not show a significant difference between the responder and non-responder patients [Supplementary Figure 5D].These results suggest that faecal PAI-1 could be used as a novel biomarker in the clinical follow-up of IBD patients.

PAI-1 as a potential faecal marker for differential diagnosis of colorectal diseases
The controversial selectivity is one of the most significant limitations of the current gold standard FC, which could also be elevated in other organic gastrointestinal diseases. 21To assess the selectivity of faecal PAI-1, we collected samples from negative controls, IBD patients, and patients who had organic lesions detectable at colonoscopy except for IBD, and allocated them to six groups: negative, active IBD, inactive IBD, adenoma, colorectal cancer, and diverticulosis [without inflammation].We found that faecal PAI-1 was elevated only in active IBD.In contrast, in other gastrointestinal [GI] diseases such as adenoma, colorectal cancer, or diverticulosis, it did not show a significant elevation compared with the control patients [Figure 7].To gain information about the dependency on disease localisation, we compared the faecal concentration of PAI-1 in patients with ileal, colonic, or ileocolonic localisations.In this cohort, the faecal PAI-1 concentration was significantly higher in patients with colonic and ileocolonic localisation.Although patients with ileal localisation displayed elevated faecal PAI-1 concentration, the difference was not significant compared with the controls.Notably, there were no significant differences among the faecal PAI-1 concentrations in the ileal, colonic, or combined localisations [Supplementary Figure 6].Due to the low numbers, these observations require further validation.

Discussion
PAI-1 emerged recently as a critical link between the epithelium and inflammation, which showed elevated mucosal gene expression in patients with IBD who did not respond to anti-TNF biologic therapy. 11The present study demonstrated that PAI-1, encoded by the gene Serpin E1, is a member of the serine protease inhibitor [serpin] family, and its primary function is to inhibit the tissue-and urokinase-type plasminogen activator, which has a major role in the fibrinolysis and the homeostasis of the extracellular matrix. 22The expression of PAI-1 is regulated by multiple growth factors, hormones, and inflammatory cytokines [eg, TNF-α, IL-1β, IL-6, and TGFβ], [23][24][25][26][27] and it can be secreted by different cell types [eg, endothelial, epithelial, and immune cells] and act as a pleiotropic cytokine. 28Clinical studies have established that patients with IBD have an approximately 3-fold increased risk of thrombotic events, and those with active disease have abnormal blood coagulation parameters. 29A recent study identified an immune-coagulation gene axis in IBD in several independent cohorts where elevated PAI-1 was central in controlling key inflammatory modulators and mucosal damage in colitis. 11he study described that PAI-1 exacerbated mucosal damage in experimental colitis by blocking tissue plasminogen activator-mediated cleavage and activation of the anti-inflammatory TGF-β.In contrast, the inhibition of PAI-1 reduced mucosal damage and inflammation in mice.The authors also demonstrated that the colonic mucosal Serpin E1 gene expression is elevated in active, more severe IBD and patients who do not respond to anti-TNF therapy.Our results also confirmed the elevated mucosal Serpin E1 gene expression in active IBD patients.Additionally, we detected a significantly increased concentration of PAI-1 in the serum and mucosal samples of active IBD patients.Importantly, the serum and mucosal PAI-1 concentration and the Serpin E1 gene expression in the colonic tissue decreased significantly in patients who responded to anti-inflammatory therapy.Our study and the previous study by Kaiko et al. 11 showed that PAI-1positive cells were detected in the epithelium of the colonic crypts and colonic organoids, which were significantly enriched in the inflamed colon.The costs of overall care for IBD have also increased in the past 5 years, and the annual mean health care costs are over 3-fold higher than for patients without IBD. 30The key cost drivers for IBD patients are treatment with specific therapeutics, including biologics, emergency department use, and health care services associated with relapsing disease, anaemia, or mental health comorbidity.Biologic, therapy-based, individualised treatment that focuses more on preventing disease progression is expensive at the outset but may ultimately lead to decreased rates of surgeries and hospitalisations, potentially yielding lower long-term costs for treatment. 31o achieve these goals, reliable biomarkers that are easy and relatively cheap to measure are crucially needed.Serological laboratory parameters, total leukocyte count, CRP, and erythrocyte sedimentation rate offer indirect, objective, but non-specific markers for IBD. 32,33In this study, both serum PAI-1 and CRP were elevated in active IBD patients.Additionally, serum PAI-1 concentration decreased significantly in therapy responder patients, whereas CRP remained elevated.Notably, a large number of studies have demonstrated relatively poor sensitivity and specificity of serum biomarkers in IBD diagnosis and patient monitoring. 32,34,35For example, studies showed that CRP is in the normal range in ~50% of UC patients. 35,36This was also confirmed in our cohort, as the patients with UC displayed significantly lower CRP levels than those with CD.Additionally, CRP can be elevated due to many factors unrelated to IBD, including infection and rheumatoid and autoimmune diseases. 35ecent studies indicated that faecal biomarkers strongly correlate with mucosal inflammation in IBD. 36,37Among many potential faecal biomarkers, most studies have been performed with FC, which is today the gold standard among faecal markers. 10FC is a small, calcium-binding protein found in abundance in neutrophil granulocytes. 9The presence of calprotectin in faeces is a consequence of neutrophil migration into the gastrointestinal tissue due to an inflammatory process; therefore, FC concentrations demonstrate a good correlation with intestinal inflammation.Several recent studies reported that the AUC of FC is between 0.8 and 0.9 in different patient populations, [38][39][40] which increased above 0.9 when FC was combined with other markers, such as oncostatin M. 40 In our experiments, faecal PAI-1 concentration was significantly higher in active IBD patients compared with the controls [AUC = 0.83] and in active versus inactive IBD and control samples [AUC = 0.82].These results suggest that faecal PAI-1 is comparable to FC.
However, there are considerable limitations of FC in clinical settings.First, several studies demonstrated the lack of specificity of FC for IBD, and it is increased in colorectal cancer, gastroenteritis, irritable bowel syndrome, diverticulitis, food intolerance, and non-steroidal enteropathy as well. 21In our patient cohort, faecal PAI-1 demonstrated remarkable selectivity as it was elevated only in active IBD patients.In contrast in other GI diseases, such as adenoma, colorectal cancer, or diverticulosis, it was not significantly higher than in the control patients.In addition, FC concentration is affected by disease-independent factors such as age and comorbidities and shows a considerable day-to-day variability. 41There are no optimal cut-off values to define active and inactive disease or to predict clinical and endoscopic remission or treatment response. 9,42FC level is localisation dependent as it frequently shows normal values in CD located in the small bowel and cannot differentiate between CD and UC. 10,43Our preliminary results showed that faecal PAI-1 was higher in patients with colonic and ileocolonic disease localisation.Additionally it was higher in patients with ileal localisation, but the difference was not statistically significant.Therefore, this parameter requires further investigation.A recent study highlighted that FC is unstable at room temperature, which may lead to falsenegative results and under-treatment in children with IBD. 44n contrast, faecal PAI-1 showed remarkable stability for up to 1 week.
There are some limitations to this study.First, the sample size did not allow subgroup analysis by disease type, location, and treatment type.To confirm the value of faecal PAI-1 in predicting relapses, longitudinal sample collection will be needed, which is currently ongoing.To further validate the results, multicentric clinical trials with large patient populations will be required.On the other hand, there are several strengths of the current study.First, this is the first comprehensive evaluation of PAI-1 in different human biological samples [serum, colon mucosa, and faeces] for gene and protein expression levels.In contrast, the previous report focused solely on colonic mucosal gene expression.Another strength is the identification of a potential selective faecal biomarker, which could be used in patient follow-up in IBD.
In conclusion, correct monitoring of IBD, which is necessary to guide treatment decisions in the treat-to-target era properly, can be difficult in daily clinical practice, and there is still an unmet need for biomarkers that could assess the duration of disease remission and the risk of relapse. 45Our results suggest that PAI-1 could be a potential novel biomarker in IBD based on the obtained data.In addition, determination of PAI-1 in the serum and faeces can overcome several limitations of the currently used biomarkers such as CRP and FC.

Table 1 .
Demographic and clinical characteristics of the study population.

Table 2 .
Summary table of the medications applied in the study population.