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    Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE

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    Veterans Affairs Medical Center, Univ. California San Francisco
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Molecular Activity of Inflammation and Epithelial-Mesenchymal Transition in the Microenvironment of Ulcerative Colitis

Yu Kyung Jun1 , Nayoung Kim1,2 , Hyuk Yoon1 , Ji Hyun Park2 , Hyung Kyung Kim3,4 , Yonghoon Choi1 , Ji Ae Lee3 , Cheol Min Shin1 , Young Soo Park1 , Dong Ho Lee1,2

1Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea; 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea; 3Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea; 4Department of Pathology and Translational Genomics, Samsung Medical Center, Seoul, Korea

Correspondence to: Nayoung Kim
ORCID https://orcid.org/0000-0002-9397-0406
E-mail nakim49@snu.ac.kr

Received: July 22, 2023; Revised: December 7, 2023; Accepted: December 20, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gut Liver.

Published online February 22, 2024

Copyright © Gut and Liver.

Background/Aims: The genetic expression in the active inflammatory regions is increased in ulcerative colitis (UC) with endoscopic activity. The aim of this study was to investigate the molecular activity of inflammation and tissue remodeling markers in endoscopically inflamed and uninflamed regions of UC.
Methods: Patients with UC (n=47) and controls (n=20) were prospectively enrolled at the Seoul National University Bundang Hospital. Inflamed tissue was obtained at the most active lesion, and uninflamed tissue was collected from approximately 15 cm above the upper end of the active lesion via colonoscopic biopsies. The messenger RNA expression levels of transforming growth factor β (TGF-β), interleukin (IL)-1β, IL-6, IL-17A, E-cadherin, olfactomedin-4 (OLFM4), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), vimentin, fibroblast-specific protein-1 (FSP1), and α-smooth muscle actin (SMA) were evaluated. Mucosal healing (MH) was defined according to a Mayo endoscopic score of 0, 1 or non-MH (Mayo endoscopic score of 2 or 3).
Results: The messenger RNA expressions of TGF-β, IL-1β, OLFM4, FSP1, vimentin, and α-SMA were significantly higher, and that of E-cadherin was significantly lower in inflamed and uninflamed regions of patients with UC than those in controls. In the inflamed regions, patients in the non-MH group had significantly increased genetic expression of TGF-β, FSP1, vimentin, and α-SMA compared to patients in the MH group. Similarly, the non-MH group had significantly higher genetic expression of TGF-β, IL-1β, IL-6, vimentin, and α-SMA than the MH group in the uninflamed regions.
Conclusions: Endoscopic activity in UC suggests inflammation and tissue remodeling of uninflamed regions similar to inflamed regions (ClinicalTrials.gov, NCT05653011).

Keywords: Colitis, ulcerative, Epithelial-mesenchymal transition, Fibrosis, Cytokines

Ulcerative colitis (UC), a chronic inflammatory disorder of the gastrointestinal tract, is characterized by continuous and superficial inflammation. The extent of UC is limited to the colon and rectum, and extends from the rectum to the proximal colon.1 Endoscopy is widely used to evaluate the extent and activity of disease in patients with UC.2 The Mayo endoscopic score (MES) is the major scoring system used to evaluate the disease activity of UC. In addition, mucosal healing (MH), which means an MES of 0 or 1, is known to be related to a reduction in relapse, hospitalization, colectomy, and colitis-associated dysplasia.3,4

The transcript levels of proinflammatory cytokines and chemokines, such as transforming growth factor β (TGF-β), interleukin (IL)-6, IL-8, IL-10, CXC motif chemokine ligand 2, and CXC motif chemokine ligand 10, in the biopsied samples from patients with UC, are positively correlated with endoscopic activity.5,6 Chronic and relapsing intestinal inflammation leads to tissue remodeling, but tissue remodeling in UC remains largely unexplored.7 Chronic inflammation is induced and aggravated by immune cells, including macrophages, neutrophils, and lymphocytes, which release cytokines, chemokines, and inflammatory mediators. Repetitive chronic inflammation results in tissue remodeling, a dynamic and coordinated process involving epithelial-to-mesenchymal transition (EMT). EMT is a process in which epithelial cells transform into migratory mesenchymal cells with a more invasive phenotype.8,9 There are three types of EMT: type 1 EMT occurs during embryonic development and promotes organogenesis, type 2 EMT is related to wound healing and tissue repair, and type 3 EMT is associated with tumor progression and metastasis.10 Among these three types, our study focused on type 2 EMT in the inflammatory microenvironment of UC.

Inflammatory microenvironment refers to a complex molecular environment that exists within and around a site of active inflammation and is dynamically regulated by immune cells, stromal cells, signaling molecules, and extracellular matrix components. Based on this background, we hypothesized that, in UC, endoscopically uninflamed regions may not be free from chronic inflammation or tissue repair (EMT), and genetic expression profiles of the uninflamed regions may be associated with endoscopic activity. Thus, this study aimed to investigate the relationship between endoscopic activity and the transcript levels of inflammation and tissue remodeling markers in uninflamed regions as well as in inflamed regions in patients with UC.

1. Patient population

All patients diagnosed with UC were prospectively enrolled at the Digestive Disease Center of Seoul National University Bundang Hospital between May 2013 and August 2022. This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB number: B-1305-201-001). Written informed consent was obtained from all the participants. All subjects who provided informed consent were asked to complete a questionnaire requesting data regarding age, sex, smoking and drinking habits, family history of inflammatory bowel disease (IBD), and colon cancer.

The biopsy samples were obtained from the most endoscopically active region (inflamed region) and approximately 15 cm (10 to 20 cm) above the upper end of the active lesion via colonoscopy, which was considered endoscopically normal (uninflamed region). Tissue sampling of the uninflamed regions could not be obtained in patients with pancolitis involving the cecum to the rectum. Only biopsied samples at the uninflamed region were obtained in patients with endoscopic remission. The inflamed and uninflamed regions were identified by one endoscopist (N.K.), an expert gastroenterologist over 30 years. Patients were categorized according to endoscopic activity: patients with MES 0 or 1 were included in the MH group, and the others in the non-MH group.

Subjects who had undergone colonoscopy to check the colon polyps were enrolled as controls when they did not show any evidence of colitis or colon cancer. Biopsy samples from controls were obtained 20 cm above the anal verge by one endoscopist (N.K.). Hematoxylin and eosin staining was performed to confirm that the biopsied samples did not show any evidence of colitis or dysplasia. The study protocol was registered at ClinicalTrials.gov (NCT05653011).

2. Data collection

Data regarding sex, age at UC diagnosis, age, and disease duration were also obtained. The disease extent was classified according to the Montreal classification as proctitis, left-sided colitis, and extensive colitis.11 Clinical and endoscopic activities were evaluated according to the Mayo score.12 Treatment at the endoscopic examination was categorized as follows: topical and oral 5-aminosalicylic acid, immunomodulators, and biologics or small molecules.

3. mRNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted from biopsy samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. The extracted RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from 1 μg of RNA using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). The expression of mRNA encoding markers of inflammation and tissue repair was determined using quantitative real-time polymerase chain reaction. The expression of the markers, including TGF-β, IL-1β, IL-6, IL-17A, E-cadherin, olfactomedin-4 (OLFM4), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), vimentin, fibroblast-specific protein-1 (FSP1), and α-smooth muscle actin (SMA), was evaluated using the primers listed in Supplementary Table 1. Thermal cycling reactions were performed using a StepOnePlus Real-Time PCR (Applied Biosystems) with SYBR Premix Ex Taq (Takara Bio, Shiga, Japan). The amplification protocol consisted of an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing and extension at 60°C to 65°C for 33 seconds. Relative quantification of gene expression using quantitative real-time polymerase chain reaction data was calculated relative to β-actin using the 2−ΔΔCt method.

4. Histologic evaluation and immunohistochemical staining

Biopsied samples taken during endoscopy were fixed with 10% neutral buffered formalin and embedded in paraffin blocks. The formalin-fixed paraffin-embedded tissues were cut into 4 μm-thick sections for the hematoxylin and eosin stain and immunohistochemistry. The histologic activity of inflamed regions was assessed by Nancy histologic index,13,14 and biopsied samples were reviewed by gastrointestinal pathologists (H.K.K. and J.A.L.).

For immunohistochemistry, tissue sections were incubated with primary antibodies (E-cadherin [1:100 dilution], vimentin [1:3,000 dilution], α-SMA [1:6,000 dilution], FSP1 [1:3,000 dilution]; Santa Cruz Biotechnology, Santa Cruz, CA, USA). An OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used to detect primary antibodies. The tissue sections were counterstained with hematoxylin and post-counterstained with bluing reagent.

Immunohistochemical scores of E-cadherin, vimentin, FSP1, and α-SMA were measured using intensity and percentage of area. The intensity of staining was scored as 0, no staining; 1+, weakly positive staining; 2+, moderately positive staining; +3, strongly positive staining.15,16 The intensities were multiplied by the percentages of area, and the possible scores ranged from 0 to 300. Two pathologists (H.K.K. and J.A.L.) independently scored Nancy histologic index and immunoreactivity.

5. Statistical analysis

Data were statistically analyzed using SPSS software (version 25.0; SPSS Inc., Armonk, NY, USA) and GraphPad Prism software (version 9.0; GraphPad Software Inc., San Diego, CA, USA). The Mann-Whitney U test was used to compare continuous variables because the distribution of variables was nonparametric. The chi-square test and Fisher exact test were used for categorical variables (mean±standard deviation). The correlation between MES and the expression levels of molecular markers was estimated using the Spearman correlation coefficient. Statistical significance was set at p-value <0.05.

1. Baseline characteristics of patients

Forty-seven UC patients were prospectively included, with a median disease duration of 34 months and a median age at diagnosis of 44 years (Table 1). Most patients (n=41) had already been diagnosed with UC and had index endoscopic examination because their symptoms (stool frequency, rectal bleeding, or abdominal pain) did not respond to treatment. The others (n=6) had the endoscopic examination for diagnosis. Patients were divided into two groups according to MH. Nine patients were included in the MH group (MES 0 or 1), and the other 38 patients were included in the non-MH group (MES 2 or 3).

Table 1. Baseline Characteristics, Disease Extent and Activity of UC According to MH

CharacteristicsAll patientsPatients with MH (MES 0 or 1)Patients without MH (MES 2 or 3)p-value
No. of patients47 (100)9 (19.1)38 (80.9)
Male sex25 (53.2)4 (44.4)21 (55.3)0.715
Age at UC diagnosis, yr41.43±15.0446.13±13.9240.45±15.260.496
Age, yr52.57±15.0056.00±12.7751.76±15.520.655
Disease duration, mo68.13±72.1956.95±91.9270.48±68.610.177
Disease extent
Proctitis19 (40.4)6 (66.7)13 (34.2)0.190
Left-sided colitis15 (31.9)2 (22.2)13 (34.2)
Extensive colitis13 (27.7)1 (11.1)12 (31.6)
Stool frequency
012 (25.5)5 (55.6)7 (18.4)0.051
111 (23.4)1 (11.1)10 (26.3)
212 (25.5)3 (33.3)9 (23.7)
312 (25.5)012 (31.6)
Rectal bleeding
08 (17.0)3 (33.3)4 (10.5)0.163
120 (42.6)4 (44.4)16 (42.1)
219 (40.4)2 (22.2)18 (47.4)
Physician's assessment
127 (57.4)7 (77.8)19 (50.0)0.310
212 (25.5)1 (11.1)12 (31.6)
38 (17.0)1 (11.1)7 (18.4)
MES
01 (2.1)1 (11.1)0<0.001
18 (17.0)8 (88.9)0
227 (57.4)027 (71.1)
311 (23.4)011 (28.9)
Mayo score8.09±2.694.78±1.998.87±2.20<0.001
NHI*
28 (18.2)1 (11.1)7 (20.0)0.357
325 (56.8)7 (77.8)18 (51.4)
411 (25.0)1 (11.1)10 (28.6)
Medication at the endoscopic examination
Topical 5-ASA17 (36.2)4 (44.4)13 (34.2)0.704
Oral 5-ASA41 (87.2)6 (66.7)35 (92.1)0.075
Immunomodulator30 (63.8)4 (44.4)26 (68.4)0.252
Biologics or small molecule3 (6.4)03 (7.9)1.000

Data are presented as number (%) or mean±SD.

UC, ulcerative colitis; MH, mucosal healing; MES, Mayo endoscopic subscore; NHI, Nancy histologic index; 5-ASA, 5-aminosalicylic acid.

*NHI from inflamed regions are calculated.



The two groups had similar baseline demographics, disease characteristics, and medication at the colonoscopy, except for Mayo scores and MES (Table 1). The mean Mayo score of the non-MH group was significantly higher than that of the MH group (8.87±2.20 vs 4.78±1.99, p<0.001). Histologic evaluation using Nancy histologic index confirmed that all enrolled patients had histologic activity, although there was no significant difference in histologic scores between MH and non-MH groups. Most patients were treated with oral 5-aminosalicylic acid (MH group, n=6, 66.7%; non-MH group, n=35, 92.1%). All patients treated with biologics or small molecules were included in non-MH group (7.9%).

2. Comparative analyses between inflamed and uninflamed colon tissues of patients with UC and normal colon tissues from healthy controls

In inflamed regions, the genetic expression of TGF-β and IL-1β was upregulated compared to that in the controls (TGF-β, p=0.004; IL-1β, p=0.002) (Fig. 1A). Also, the genetic expression of TGF-β and IL-1β was significantly higher in uninflamed regions than that in controls (TGF-β, p=0.002; IL-1β, p=0.014). However, there was no significant difference in the genetic expression of IL-6 and IL-17A among the inflamed and uninflamed regions and controls.

Figure 1.Comparison of messenger RNA (mRNA) expression levels of inflammation and tissue repair markers between inflamed and uninflamed regions of patients with ulcerative colitis (UC) and normal tissues from controls. (A) The genetic expression levels of TGF-β and IL-1β were significantly elevated in patients with UC, regardless of biopsied regions compared to that in the controls. (B) The genetic expression of E-cadherin was significantly suppressed, while that of OLFM4, FSP1, vimentin, and α-SMA was significantly elevated in the inflamed and uninflamed regions compared to that in the controls. The mRNA expression of LGR5 in the uninflamed regions was significantly higher than that in the controls. TGF, transforming growth factor; IL, interleukin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin.

The genetic expression of E-cadherin was significantly lower, and that of OLFM4, FSP1, vimentin, and α-SMA was significantly higher in the inflamed regions than that in the controls (E-cadherin, p=0.002; OLFM4, p=0.001; FSP1, p=0.001; vimentin, p=0.036; α-SMA, p<0.001) (Fig. 1B). In the uninflamed regions, the genetic expression of E-cadherin was significantly lower and that of OLFM4, LGR5, FSP1, vimentin, and α-SMA was significantly higher than that in the controls (E-cadherin, p=0.012; OLFM4, p<0.001; LGR5, p=0.036; FSP1, p=0.001; vimentin, p= 0.008; α-SMA, p=0.001). Notably, there was no difference in the genetic expression of all inflammation and tissue repair markers between the inflamed and uninflamed regions.

Additionally, immunohistochemical staining was performed to identify the tissue expression level of E-cadherin, vimentin, FSP1, and α-SMA (Supplementary Fig. 1A and B). Inflamed regions from patients with UC had a decreased mean immunohistochemical score of E-cadherin and increased mean immunohistochemical scores of vimentin, FSP1, and α-SMA compared to normal tissues from controls. However, there were no statistically significant differences.

3. Inflammatory markers expressed in both inflamed and uninflamed regions

We compared the genetic expression levels of inflammatory markers (TGF-β, IL-1β, IL-6, and IL-17A) in the inflamed and uninflamed regions of the MH and non-MH group and controls. In the inflamed regions, colon mucosal TGF-β levels in the non-MH group were significantly higher than those in the MH group and controls (non-MH group vs control, p<0.001; non-MH group vs MH group, p=0.001) (Fig. 2A). Transcription levels of IL-1β in the MH and non-MH groups were significantly higher than those in the controls (MH group vs control, p=0.004; non-MH group vs control, p=0.009). There was no significant difference in the genetic expression of IL-6 and IL-17A in the inflamed regions among the three groups.

Figure 2.Comparison of messenger RNA (mRNA) expression levels of inflammatory markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to the endoscopic activity. (A) In inflamed regions, the genetic expression of TGF-β in the non-mucosal healing (MH) group was significantly higher than that in the MH group and controls. The genetic expression of IL-1β in the non-MH and MH groups was significantly upregulated compared to the controls. (B) In uninflamed regions, the genetic expression of TGF-β and IL-1β in the non-MH group was significantly higher than that in the MH group and controls. The genetic expression of IL-6 in the non-MH group was significantly higher than that in the MH group. TGF, transforming growth factor; IL, interleukin; MES, Mayo endoscopic score.

In uninflamed regions, the genetic expression of TGF-β and IL-1β was significantly higher in the non-MH group than in the MH group and controls (TGF-β, non-MH group vs MH group, p=0.029, non-MH group vs control, p<0.001; IL-1β, non-MH group vs MH group, p=0.023, non-MH group vs control, p=0.002) (Fig. 2B). The genetic expression of IL-6 was significantly higher in the non-MH group than in the MH group (non-MH group vs MH group, p=0.024). There was no significant difference in the genetic expression of IL-17A in the uninflamed regions among the three groups.

4. Tissue repair markers correlated with the prognosis of patients with UC

Next, the expression levels of tissue repair markers were evaluated. The genetic expression of E-cadherin was significantly decreased and that of OLFM4, FSP1, vimentin, and α-SMA was significantly increased in inflamed regions with non-MH group compared to that in the controls (E-cadherin, p=0.002; OLFM4, p<0.001; FSP1, p<0.001; vimentin, p=0.003; α-SMA, p<0.001) (Fig. 3A). In inflamed regions, the genetic expression of FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the MH group (FSP1, p=0.018; vimentin, p=0.049; α-SMA, p=0.009). However, the immunoreactivity of E-cadherin, vimentin, FSP, and α-SMA was not different among the MH and non-MH groups and controls (Supplementary Fig. 1C).

Figure 3.Comparison of messenger RNA (mRNA) expression levels of tissue remodeling (epithelial-to-mesenchymal transition) markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to their prognosis. (A) In inflamed regions, the genetic expression of E-cadherin was significantly suppressed, and that of OLFM4, FSP1, vimentin, and α-SMA in the non-mucosal healing (MH) group compared to the controls. The genetic expression of FSP1, vimentin, and α-SMA in the non-MH group was significantly higher than that in the MH group. (B) Uninflamed regions in the non-MH group showed high activity of tissue remodeling compared to the control: the genetic expression of E-cadherin was significantly lower, and that of OLFM4, LGR5, FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the controls. Moreover, the genetic expression levels of vimentin and α-SMA were significantly elevated in uninflamed regions of the non-MH group compared to those of the MH group. OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; MES, Mayo endoscopic score.

In uninflamed regions, the genetic expression of E-cadherin was significantly lower and that of OLFM4, FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the controls (E-cadherin, p=0.030; OLFM4, p<0.001; LGR5, p=0.012; FSP1, p<0.001; vimentin, p<0.001; and α-SMA, p<0.001) (Fig. 3B). The genetic expression of vimentin and α-SMA was significantly increased in the non-MH group compared to that in the MH group (vimentin, p=0.004; α-SMA, p=0.049). However, there were no differences in the expression of tissue repair markers between the MH group and controls in both inflamed and uninflamed regions.

Also, we evaluated the correlation between the expression levels of molecular markers and MES. The expression levels of vimentin, LGR5, α-SMA, and FSP1 in the uninflamed regions were highly correlated with MES (vimentin, ρ=0.503, p=0.014; LGR5, ρ=0.418, p=0.047; α-SMA, ρ=0.415, p=0.049; FSP1, ρ=0.444, p=0.034). However, the expression levels of the other markers in the uninflamed regions and those of all markers in the inflamed regions were not correlated with MES.

Our study revealed that the genetic expression of inflammation and tissue repair markers was correlated with endoscopic activity in patients with UC but no difference in histologic activity according to MH. In the inflamed regions, the non-MH group had significantly higher expression levels of TGF-β, FSP1, vimentin, and α-SMA than the MH group. Moreover, endoscopically normal-looking regions showed different genetic expression levels according to endoscopic activity. In the uninflamed regions, the genetic expression of TGF-β, IL-1β, vimentin, and α-SMA in the non-MH group was significantly higher than that in the MH group. These results suggest that, in patients with UC, the activity of colitis and tissue remodeling can be sensitively reflected by the genetic expression levels of biopsied samples. In addition, the trend was similar between endoscopically inflamed regions and nearby normal-looking uninflamed regions.

Proliferated and activated immune cells invade the microenvironment of UC, cause local increase of inflammatory markers, such as TGF-β, IL-1β, IL-6, and IL-17A, and contribute to the development of dysregulated immune responses.17,18 The non-MH group had significantly higher expression levels of TGF-β than the MH group and controls, regardless of biopsied regions. For example, the expression levels of IL-1β in the uninflamed regions from the non-MH group were significantly higher than those in the controls and uninflamed regions from the MH group.

IL-6 is involved in the activation of immune cells, mucosal damage, and tissue remodeling in the intestines, and IL-6 expression is elevated in the colonic mucosa of patients with IBD compared to that in controls.19 In our study, unlike the inflamed region, the uninflamed regions from the non-MH group had significantly higher IL-6 expression than the uninflamed regions from the MH group. The number of participants might be insufficient to demonstrate the relationship between the expression levels of IL-6 in the inflamed regions and the endoscopic activity in UC.

The excessive and uncontrolled activation of T helper 17 cells increases IL-17A and contributes to the development of UC. T helper 17 cells secrete IL-17A and promote fibroblast proliferation, transformation of fibroblasts to myofibroblasts, EMT, and MH.9 Previous studies demonstrated that inflamed regions from patients with UC had a high genetic expression of IL-17A,20 and the serum and tissue IL-17A levels were upregulated in patients with UC compared to the controls.21,22 Therefore, it was expected that IL-17A antagonists could be developed as new therapeutic agents for UC. However, secukinumab and ixekizumab, IL-17A antagonists widely prescribed for psoriasis, psoriatic arthritis, and ankylosing spondylitis, can cause IBD.23 Also, our study cannot show the positive relationship between the genetic expression of IL-17A and endoscopic activity in patients with UC. Although the exact mechanism of IL-17A antagonists-related IBD is not known, IL-17A might not just be a proinflammatory cytokine but play a balancing role in the intestinal microenvironment. Moreover, the small number of participants might be associated with irrelevance between the expression levels of IL-17A and the endoscopic activity in UC.

Although EMT-related studies have been conducted more on patients with CD rather than on those with UC, upregulation of EMT markers was identified in colonic mucosal tissues from patients with UC and was related to cancer progression in UC.24,25 Unlike previous studies, we focused on type 2 EMT, which promotes tissue remodeling and fibrosis in UC. During type 2 EMT, epithelial cells lose their cell-cell adhesion and gain migratory properties, which induce an increase in adult stem cell (OLFM4 and LGR5) and mesenchymal cell markers (FSP1, vimentin, and α-SMA) and a decrease in the epithelial cell marker (E-cadherin).26-29 There are two types of stem cells in the intestine: OLFM4+ and LGR5+ intestinal stem cells. OLFM4+ stem cells are located in the upper region of intestinal crypts, while LGR5+ stem cells are located at the base of intestinal crypts. OLFM4 is highly expressed in active UC, and LGR5 is activated in human colonic epithelial organoids.30,31 LGR5+ stem cells show high cellular turnover in colonic MH process.32 Therefore, our result that mRNA expression of LGR5 was prominent in the uninflamed regions from the non-MH group suggests that vigorous tissue repairing occurred to avoid the extension of inflamed regions. FSP1, vimentin, and α-SMA are the main markers of fibrosis.33,34 The inflamed and uninflamed regions from the non-MH group show higher expression of FSP1, vimentin, and α-SMA and lower expression of E-cadherin than those from controls. It is very understandable that EMT tended to occur more actively in the non-MH group than in the controls. Notably, the non-MH group has higher genetic expression of vimentin and α-SMA than the MH group, regardless of biopsied regions. Therefore, the genetic expression of EMT markers is related to the endoscopic activity in both inflamed and uninflamed regions.

Sex-related differences in the incidence/prevalence, extra-intestinal manifestations, and treatment responses have been reported in UC and immune responses.35,36 However, our results did not show any differences in the activity of inflammation and tissue repair in uninflamed regions between males and females (data not shown). This may be attributed to the small sample size.

The colon mucosa microenvironment is important for the development, progression, and remission of UC. The uninflamed region near the upper margin of the inflamed region shares its microenvironment with the inflamed region. Like our study, the study by Dobre et al.20 also compared transcript profiles between the inflamed and uninflamed regions of UC, and the two regions share the molecular activity patterns of cytokines and transcription factors related to immune responses. Molecular and histological activities can persist in endoscopically healed mucosa of patients with UC,37,38 and be related to a risk of clinical relapse.39,40 Histological activity, recently focused as a new therapeutic target for the deep remission in UC,41 in the uninflamed regions, is associated with patients’ prognosis.42 However, not all histological scoring systems are validated, and there are possible discrepancies within or/and between interpreters.43,44 In our study, the discrepancy in the genetic expression of inflammation and tissue remodeling (EMT) was shown between non-MH and MH groups whose histological activities did not differ. Therefore, the mRNA expression profile of the inflammatory and EMT markers can be more sensitive for predicting the disease activity of colon mucosa microenvironments than histological evaluation. Fig. 4 summarizes the key cell populations and markers involved in inflammation and tissue remodeling in UC.9,18,19,26,33,34 The molecular activity profiles in the microenvironment of UC help us gain a better understanding of the pathophysiology of UC and develop new therapeutic agents that effectively modulate the microenvironment.

Figure 4.Schematic figure regarding the microenvironment of inflammation, epithelial-to-mesenchymal transition (EMT), and tissue remodeling in ulcerative colitis. Dendritic cells identify microbial products and produce cytokines, such as TGF-β, IL-1β, and IL-6, which stimulate the proliferation and differentiation of naïve T cells. Naïve T cells are differentiated into Th1, Th2, and Th17 cells. Th17 cells, which play a role in the regulation of inflammation, produce IL-17, a proinflammatory cytokine that contributes to activating macrophages and EMT. In response to microbial products and other cytokines, macrophages produce proinflammatory cytokines, including IL-1β and IL-6. The proinflammatory cytokines can induce EMT in intestinal epithelial cells. In turn, there is a loss of epithelial characteristics and an acquisition of mesenchymal features. During EMT, the expression of epithelial marker (E-cadherin) decreases, and that of mesenchymal markers (FSP1, vimentin, α-SMA, OLFM4, and LGR5) increases. EMT can promote the formation of fibroblasts and myofibroblasts, which are important in tissue remodeling. Fibroblasts can proliferate and migrate to the site of injury, where they secrete extracellular matrix proteins. Myofibroblasts can arise from fibroblasts, and both fibroblasts and myofibroblasts express FSP1 and vimentin. Myofibroblasts, expressing α-SMA, induce wound contraction and control tissue architecture. FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; DC, dendritic cell; MQ, macrophage; IL, interleukin; TGF, transforming growth factor; Th, T helper.

The strength of this study is that it first elucidated the genetic expression levels of inflammation and tissue remodeling in the uninflamed regions in patients with UC and proposed a concept of molecular activity in the colon mucosa microenvironment. However, this study has several limitations. First, the definition of microenvironment is rather ambiguous. We regarded the uninflamed region when the endoscopic finding did not show any inflammation sign, 15 cm separated from the upper margin of the inflamed region. Second, the lack of participants may affect the results. Third, the uninflamed regions might share a microenvironment with the inflamed regions but histological activity in the uninflamed regions was not proven in the present study. However, it is already known that the microenvironment in patients with UC can promote inflammation and play a defensive role.45

In conclusion, our findings suggest that the molecular activity of inflammation and tissue remodeling in the colon mucosa microenvironment, which includes the uninflamed as well as inflamed regions, was associated with the endoscopic activity in UC.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A2C2085149).

We appreciate the help of Ryoung Hee Nam (Seoul National University Bundang Hospital), Soo In Choi (Seoul National University Bundang Hospital), Jae Young Jang (Seoul National University Bundang Hospital), and Eun Hye Kim (Seoul National University Bundang Hospital) for the collection of samples, and Su Kyung Ha (Seoul National University Bundang Hospital) for helping patients complete the questionnaire.

Study concept and design: N.K. Data acquisition: N.K. Data analysis and interpretation: Y.K.J., N.K., J.H.P., H.K.K., J.A.L. Drafting of the manuscript: Y.K.J. Critical revision of the manuscript for important intellectual content: N.K., H.Y. Statistical analysis: Y.K.J. Obtained funding: N.K. Administrative, technical, or material support; study supervision: N.K., Y.C., C.M.S., Y.S.P., D.H.L. Approval of final manuscript: all authors

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Article

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Gut and Liver

Published online February 22, 2024

Copyright © Gut and Liver.

Molecular Activity of Inflammation and Epithelial-Mesenchymal Transition in the Microenvironment of Ulcerative Colitis

Yu Kyung Jun1 , Nayoung Kim1,2 , Hyuk Yoon1 , Ji Hyun Park2 , Hyung Kyung Kim3,4 , Yonghoon Choi1 , Ji Ae Lee3 , Cheol Min Shin1 , Young Soo Park1 , Dong Ho Lee1,2

1Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea; 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea; 3Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea; 4Department of Pathology and Translational Genomics, Samsung Medical Center, Seoul, Korea

Correspondence to:Nayoung Kim
ORCID https://orcid.org/0000-0002-9397-0406
E-mail nakim49@snu.ac.kr

Received: July 22, 2023; Revised: December 7, 2023; Accepted: December 20, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims: The genetic expression in the active inflammatory regions is increased in ulcerative colitis (UC) with endoscopic activity. The aim of this study was to investigate the molecular activity of inflammation and tissue remodeling markers in endoscopically inflamed and uninflamed regions of UC.
Methods: Patients with UC (n=47) and controls (n=20) were prospectively enrolled at the Seoul National University Bundang Hospital. Inflamed tissue was obtained at the most active lesion, and uninflamed tissue was collected from approximately 15 cm above the upper end of the active lesion via colonoscopic biopsies. The messenger RNA expression levels of transforming growth factor β (TGF-β), interleukin (IL)-1β, IL-6, IL-17A, E-cadherin, olfactomedin-4 (OLFM4), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), vimentin, fibroblast-specific protein-1 (FSP1), and α-smooth muscle actin (SMA) were evaluated. Mucosal healing (MH) was defined according to a Mayo endoscopic score of 0, 1 or non-MH (Mayo endoscopic score of 2 or 3).
Results: The messenger RNA expressions of TGF-β, IL-1β, OLFM4, FSP1, vimentin, and α-SMA were significantly higher, and that of E-cadherin was significantly lower in inflamed and uninflamed regions of patients with UC than those in controls. In the inflamed regions, patients in the non-MH group had significantly increased genetic expression of TGF-β, FSP1, vimentin, and α-SMA compared to patients in the MH group. Similarly, the non-MH group had significantly higher genetic expression of TGF-β, IL-1β, IL-6, vimentin, and α-SMA than the MH group in the uninflamed regions.
Conclusions: Endoscopic activity in UC suggests inflammation and tissue remodeling of uninflamed regions similar to inflamed regions (ClinicalTrials.gov, NCT05653011).

Keywords: Colitis, ulcerative, Epithelial-mesenchymal transition, Fibrosis, Cytokines

INTRODUCTION

Ulcerative colitis (UC), a chronic inflammatory disorder of the gastrointestinal tract, is characterized by continuous and superficial inflammation. The extent of UC is limited to the colon and rectum, and extends from the rectum to the proximal colon.1 Endoscopy is widely used to evaluate the extent and activity of disease in patients with UC.2 The Mayo endoscopic score (MES) is the major scoring system used to evaluate the disease activity of UC. In addition, mucosal healing (MH), which means an MES of 0 or 1, is known to be related to a reduction in relapse, hospitalization, colectomy, and colitis-associated dysplasia.3,4

The transcript levels of proinflammatory cytokines and chemokines, such as transforming growth factor β (TGF-β), interleukin (IL)-6, IL-8, IL-10, CXC motif chemokine ligand 2, and CXC motif chemokine ligand 10, in the biopsied samples from patients with UC, are positively correlated with endoscopic activity.5,6 Chronic and relapsing intestinal inflammation leads to tissue remodeling, but tissue remodeling in UC remains largely unexplored.7 Chronic inflammation is induced and aggravated by immune cells, including macrophages, neutrophils, and lymphocytes, which release cytokines, chemokines, and inflammatory mediators. Repetitive chronic inflammation results in tissue remodeling, a dynamic and coordinated process involving epithelial-to-mesenchymal transition (EMT). EMT is a process in which epithelial cells transform into migratory mesenchymal cells with a more invasive phenotype.8,9 There are three types of EMT: type 1 EMT occurs during embryonic development and promotes organogenesis, type 2 EMT is related to wound healing and tissue repair, and type 3 EMT is associated with tumor progression and metastasis.10 Among these three types, our study focused on type 2 EMT in the inflammatory microenvironment of UC.

Inflammatory microenvironment refers to a complex molecular environment that exists within and around a site of active inflammation and is dynamically regulated by immune cells, stromal cells, signaling molecules, and extracellular matrix components. Based on this background, we hypothesized that, in UC, endoscopically uninflamed regions may not be free from chronic inflammation or tissue repair (EMT), and genetic expression profiles of the uninflamed regions may be associated with endoscopic activity. Thus, this study aimed to investigate the relationship between endoscopic activity and the transcript levels of inflammation and tissue remodeling markers in uninflamed regions as well as in inflamed regions in patients with UC.

MATERIALS AND METHODS

1. Patient population

All patients diagnosed with UC were prospectively enrolled at the Digestive Disease Center of Seoul National University Bundang Hospital between May 2013 and August 2022. This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (IRB number: B-1305-201-001). Written informed consent was obtained from all the participants. All subjects who provided informed consent were asked to complete a questionnaire requesting data regarding age, sex, smoking and drinking habits, family history of inflammatory bowel disease (IBD), and colon cancer.

The biopsy samples were obtained from the most endoscopically active region (inflamed region) and approximately 15 cm (10 to 20 cm) above the upper end of the active lesion via colonoscopy, which was considered endoscopically normal (uninflamed region). Tissue sampling of the uninflamed regions could not be obtained in patients with pancolitis involving the cecum to the rectum. Only biopsied samples at the uninflamed region were obtained in patients with endoscopic remission. The inflamed and uninflamed regions were identified by one endoscopist (N.K.), an expert gastroenterologist over 30 years. Patients were categorized according to endoscopic activity: patients with MES 0 or 1 were included in the MH group, and the others in the non-MH group.

Subjects who had undergone colonoscopy to check the colon polyps were enrolled as controls when they did not show any evidence of colitis or colon cancer. Biopsy samples from controls were obtained 20 cm above the anal verge by one endoscopist (N.K.). Hematoxylin and eosin staining was performed to confirm that the biopsied samples did not show any evidence of colitis or dysplasia. The study protocol was registered at ClinicalTrials.gov (NCT05653011).

2. Data collection

Data regarding sex, age at UC diagnosis, age, and disease duration were also obtained. The disease extent was classified according to the Montreal classification as proctitis, left-sided colitis, and extensive colitis.11 Clinical and endoscopic activities were evaluated according to the Mayo score.12 Treatment at the endoscopic examination was categorized as follows: topical and oral 5-aminosalicylic acid, immunomodulators, and biologics or small molecules.

3. mRNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted from biopsy samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer. The extracted RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from 1 μg of RNA using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). The expression of mRNA encoding markers of inflammation and tissue repair was determined using quantitative real-time polymerase chain reaction. The expression of the markers, including TGF-β, IL-1β, IL-6, IL-17A, E-cadherin, olfactomedin-4 (OLFM4), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), vimentin, fibroblast-specific protein-1 (FSP1), and α-smooth muscle actin (SMA), was evaluated using the primers listed in Supplementary Table 1. Thermal cycling reactions were performed using a StepOnePlus Real-Time PCR (Applied Biosystems) with SYBR Premix Ex Taq (Takara Bio, Shiga, Japan). The amplification protocol consisted of an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing and extension at 60°C to 65°C for 33 seconds. Relative quantification of gene expression using quantitative real-time polymerase chain reaction data was calculated relative to β-actin using the 2−ΔΔCt method.

4. Histologic evaluation and immunohistochemical staining

Biopsied samples taken during endoscopy were fixed with 10% neutral buffered formalin and embedded in paraffin blocks. The formalin-fixed paraffin-embedded tissues were cut into 4 μm-thick sections for the hematoxylin and eosin stain and immunohistochemistry. The histologic activity of inflamed regions was assessed by Nancy histologic index,13,14 and biopsied samples were reviewed by gastrointestinal pathologists (H.K.K. and J.A.L.).

For immunohistochemistry, tissue sections were incubated with primary antibodies (E-cadherin [1:100 dilution], vimentin [1:3,000 dilution], α-SMA [1:6,000 dilution], FSP1 [1:3,000 dilution]; Santa Cruz Biotechnology, Santa Cruz, CA, USA). An OptiView DAB IHC Detection Kit (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used to detect primary antibodies. The tissue sections were counterstained with hematoxylin and post-counterstained with bluing reagent.

Immunohistochemical scores of E-cadherin, vimentin, FSP1, and α-SMA were measured using intensity and percentage of area. The intensity of staining was scored as 0, no staining; 1+, weakly positive staining; 2+, moderately positive staining; +3, strongly positive staining.15,16 The intensities were multiplied by the percentages of area, and the possible scores ranged from 0 to 300. Two pathologists (H.K.K. and J.A.L.) independently scored Nancy histologic index and immunoreactivity.

5. Statistical analysis

Data were statistically analyzed using SPSS software (version 25.0; SPSS Inc., Armonk, NY, USA) and GraphPad Prism software (version 9.0; GraphPad Software Inc., San Diego, CA, USA). The Mann-Whitney U test was used to compare continuous variables because the distribution of variables was nonparametric. The chi-square test and Fisher exact test were used for categorical variables (mean±standard deviation). The correlation between MES and the expression levels of molecular markers was estimated using the Spearman correlation coefficient. Statistical significance was set at p-value <0.05.

RESULTS

1. Baseline characteristics of patients

Forty-seven UC patients were prospectively included, with a median disease duration of 34 months and a median age at diagnosis of 44 years (Table 1). Most patients (n=41) had already been diagnosed with UC and had index endoscopic examination because their symptoms (stool frequency, rectal bleeding, or abdominal pain) did not respond to treatment. The others (n=6) had the endoscopic examination for diagnosis. Patients were divided into two groups according to MH. Nine patients were included in the MH group (MES 0 or 1), and the other 38 patients were included in the non-MH group (MES 2 or 3).

Table 1 . Baseline Characteristics, Disease Extent and Activity of UC According to MH.

CharacteristicsAll patientsPatients with MH (MES 0 or 1)Patients without MH (MES 2 or 3)p-value
No. of patients47 (100)9 (19.1)38 (80.9)
Male sex25 (53.2)4 (44.4)21 (55.3)0.715
Age at UC diagnosis, yr41.43±15.0446.13±13.9240.45±15.260.496
Age, yr52.57±15.0056.00±12.7751.76±15.520.655
Disease duration, mo68.13±72.1956.95±91.9270.48±68.610.177
Disease extent
Proctitis19 (40.4)6 (66.7)13 (34.2)0.190
Left-sided colitis15 (31.9)2 (22.2)13 (34.2)
Extensive colitis13 (27.7)1 (11.1)12 (31.6)
Stool frequency
012 (25.5)5 (55.6)7 (18.4)0.051
111 (23.4)1 (11.1)10 (26.3)
212 (25.5)3 (33.3)9 (23.7)
312 (25.5)012 (31.6)
Rectal bleeding
08 (17.0)3 (33.3)4 (10.5)0.163
120 (42.6)4 (44.4)16 (42.1)
219 (40.4)2 (22.2)18 (47.4)
Physician's assessment
127 (57.4)7 (77.8)19 (50.0)0.310
212 (25.5)1 (11.1)12 (31.6)
38 (17.0)1 (11.1)7 (18.4)
MES
01 (2.1)1 (11.1)0<0.001
18 (17.0)8 (88.9)0
227 (57.4)027 (71.1)
311 (23.4)011 (28.9)
Mayo score8.09±2.694.78±1.998.87±2.20<0.001
NHI*
28 (18.2)1 (11.1)7 (20.0)0.357
325 (56.8)7 (77.8)18 (51.4)
411 (25.0)1 (11.1)10 (28.6)
Medication at the endoscopic examination
Topical 5-ASA17 (36.2)4 (44.4)13 (34.2)0.704
Oral 5-ASA41 (87.2)6 (66.7)35 (92.1)0.075
Immunomodulator30 (63.8)4 (44.4)26 (68.4)0.252
Biologics or small molecule3 (6.4)03 (7.9)1.000

Data are presented as number (%) or mean±SD..

UC, ulcerative colitis; MH, mucosal healing; MES, Mayo endoscopic subscore; NHI, Nancy histologic index; 5-ASA, 5-aminosalicylic acid..

*NHI from inflamed regions are calculated..



The two groups had similar baseline demographics, disease characteristics, and medication at the colonoscopy, except for Mayo scores and MES (Table 1). The mean Mayo score of the non-MH group was significantly higher than that of the MH group (8.87±2.20 vs 4.78±1.99, p<0.001). Histologic evaluation using Nancy histologic index confirmed that all enrolled patients had histologic activity, although there was no significant difference in histologic scores between MH and non-MH groups. Most patients were treated with oral 5-aminosalicylic acid (MH group, n=6, 66.7%; non-MH group, n=35, 92.1%). All patients treated with biologics or small molecules were included in non-MH group (7.9%).

2. Comparative analyses between inflamed and uninflamed colon tissues of patients with UC and normal colon tissues from healthy controls

In inflamed regions, the genetic expression of TGF-β and IL-1β was upregulated compared to that in the controls (TGF-β, p=0.004; IL-1β, p=0.002) (Fig. 1A). Also, the genetic expression of TGF-β and IL-1β was significantly higher in uninflamed regions than that in controls (TGF-β, p=0.002; IL-1β, p=0.014). However, there was no significant difference in the genetic expression of IL-6 and IL-17A among the inflamed and uninflamed regions and controls.

Figure 1. Comparison of messenger RNA (mRNA) expression levels of inflammation and tissue repair markers between inflamed and uninflamed regions of patients with ulcerative colitis (UC) and normal tissues from controls. (A) The genetic expression levels of TGF-β and IL-1β were significantly elevated in patients with UC, regardless of biopsied regions compared to that in the controls. (B) The genetic expression of E-cadherin was significantly suppressed, while that of OLFM4, FSP1, vimentin, and α-SMA was significantly elevated in the inflamed and uninflamed regions compared to that in the controls. The mRNA expression of LGR5 in the uninflamed regions was significantly higher than that in the controls. TGF, transforming growth factor; IL, interleukin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin.

The genetic expression of E-cadherin was significantly lower, and that of OLFM4, FSP1, vimentin, and α-SMA was significantly higher in the inflamed regions than that in the controls (E-cadherin, p=0.002; OLFM4, p=0.001; FSP1, p=0.001; vimentin, p=0.036; α-SMA, p<0.001) (Fig. 1B). In the uninflamed regions, the genetic expression of E-cadherin was significantly lower and that of OLFM4, LGR5, FSP1, vimentin, and α-SMA was significantly higher than that in the controls (E-cadherin, p=0.012; OLFM4, p<0.001; LGR5, p=0.036; FSP1, p=0.001; vimentin, p= 0.008; α-SMA, p=0.001). Notably, there was no difference in the genetic expression of all inflammation and tissue repair markers between the inflamed and uninflamed regions.

Additionally, immunohistochemical staining was performed to identify the tissue expression level of E-cadherin, vimentin, FSP1, and α-SMA (Supplementary Fig. 1A and B). Inflamed regions from patients with UC had a decreased mean immunohistochemical score of E-cadherin and increased mean immunohistochemical scores of vimentin, FSP1, and α-SMA compared to normal tissues from controls. However, there were no statistically significant differences.

3. Inflammatory markers expressed in both inflamed and uninflamed regions

We compared the genetic expression levels of inflammatory markers (TGF-β, IL-1β, IL-6, and IL-17A) in the inflamed and uninflamed regions of the MH and non-MH group and controls. In the inflamed regions, colon mucosal TGF-β levels in the non-MH group were significantly higher than those in the MH group and controls (non-MH group vs control, p<0.001; non-MH group vs MH group, p=0.001) (Fig. 2A). Transcription levels of IL-1β in the MH and non-MH groups were significantly higher than those in the controls (MH group vs control, p=0.004; non-MH group vs control, p=0.009). There was no significant difference in the genetic expression of IL-6 and IL-17A in the inflamed regions among the three groups.

Figure 2. Comparison of messenger RNA (mRNA) expression levels of inflammatory markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to the endoscopic activity. (A) In inflamed regions, the genetic expression of TGF-β in the non-mucosal healing (MH) group was significantly higher than that in the MH group and controls. The genetic expression of IL-1β in the non-MH and MH groups was significantly upregulated compared to the controls. (B) In uninflamed regions, the genetic expression of TGF-β and IL-1β in the non-MH group was significantly higher than that in the MH group and controls. The genetic expression of IL-6 in the non-MH group was significantly higher than that in the MH group. TGF, transforming growth factor; IL, interleukin; MES, Mayo endoscopic score.

In uninflamed regions, the genetic expression of TGF-β and IL-1β was significantly higher in the non-MH group than in the MH group and controls (TGF-β, non-MH group vs MH group, p=0.029, non-MH group vs control, p<0.001; IL-1β, non-MH group vs MH group, p=0.023, non-MH group vs control, p=0.002) (Fig. 2B). The genetic expression of IL-6 was significantly higher in the non-MH group than in the MH group (non-MH group vs MH group, p=0.024). There was no significant difference in the genetic expression of IL-17A in the uninflamed regions among the three groups.

4. Tissue repair markers correlated with the prognosis of patients with UC

Next, the expression levels of tissue repair markers were evaluated. The genetic expression of E-cadherin was significantly decreased and that of OLFM4, FSP1, vimentin, and α-SMA was significantly increased in inflamed regions with non-MH group compared to that in the controls (E-cadherin, p=0.002; OLFM4, p<0.001; FSP1, p<0.001; vimentin, p=0.003; α-SMA, p<0.001) (Fig. 3A). In inflamed regions, the genetic expression of FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the MH group (FSP1, p=0.018; vimentin, p=0.049; α-SMA, p=0.009). However, the immunoreactivity of E-cadherin, vimentin, FSP, and α-SMA was not different among the MH and non-MH groups and controls (Supplementary Fig. 1C).

Figure 3. Comparison of messenger RNA (mRNA) expression levels of tissue remodeling (epithelial-to-mesenchymal transition) markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to their prognosis. (A) In inflamed regions, the genetic expression of E-cadherin was significantly suppressed, and that of OLFM4, FSP1, vimentin, and α-SMA in the non-mucosal healing (MH) group compared to the controls. The genetic expression of FSP1, vimentin, and α-SMA in the non-MH group was significantly higher than that in the MH group. (B) Uninflamed regions in the non-MH group showed high activity of tissue remodeling compared to the control: the genetic expression of E-cadherin was significantly lower, and that of OLFM4, LGR5, FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the controls. Moreover, the genetic expression levels of vimentin and α-SMA were significantly elevated in uninflamed regions of the non-MH group compared to those of the MH group. OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; MES, Mayo endoscopic score.

In uninflamed regions, the genetic expression of E-cadherin was significantly lower and that of OLFM4, FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the controls (E-cadherin, p=0.030; OLFM4, p<0.001; LGR5, p=0.012; FSP1, p<0.001; vimentin, p<0.001; and α-SMA, p<0.001) (Fig. 3B). The genetic expression of vimentin and α-SMA was significantly increased in the non-MH group compared to that in the MH group (vimentin, p=0.004; α-SMA, p=0.049). However, there were no differences in the expression of tissue repair markers between the MH group and controls in both inflamed and uninflamed regions.

Also, we evaluated the correlation between the expression levels of molecular markers and MES. The expression levels of vimentin, LGR5, α-SMA, and FSP1 in the uninflamed regions were highly correlated with MES (vimentin, ρ=0.503, p=0.014; LGR5, ρ=0.418, p=0.047; α-SMA, ρ=0.415, p=0.049; FSP1, ρ=0.444, p=0.034). However, the expression levels of the other markers in the uninflamed regions and those of all markers in the inflamed regions were not correlated with MES.

DISCUSSION

Our study revealed that the genetic expression of inflammation and tissue repair markers was correlated with endoscopic activity in patients with UC but no difference in histologic activity according to MH. In the inflamed regions, the non-MH group had significantly higher expression levels of TGF-β, FSP1, vimentin, and α-SMA than the MH group. Moreover, endoscopically normal-looking regions showed different genetic expression levels according to endoscopic activity. In the uninflamed regions, the genetic expression of TGF-β, IL-1β, vimentin, and α-SMA in the non-MH group was significantly higher than that in the MH group. These results suggest that, in patients with UC, the activity of colitis and tissue remodeling can be sensitively reflected by the genetic expression levels of biopsied samples. In addition, the trend was similar between endoscopically inflamed regions and nearby normal-looking uninflamed regions.

Proliferated and activated immune cells invade the microenvironment of UC, cause local increase of inflammatory markers, such as TGF-β, IL-1β, IL-6, and IL-17A, and contribute to the development of dysregulated immune responses.17,18 The non-MH group had significantly higher expression levels of TGF-β than the MH group and controls, regardless of biopsied regions. For example, the expression levels of IL-1β in the uninflamed regions from the non-MH group were significantly higher than those in the controls and uninflamed regions from the MH group.

IL-6 is involved in the activation of immune cells, mucosal damage, and tissue remodeling in the intestines, and IL-6 expression is elevated in the colonic mucosa of patients with IBD compared to that in controls.19 In our study, unlike the inflamed region, the uninflamed regions from the non-MH group had significantly higher IL-6 expression than the uninflamed regions from the MH group. The number of participants might be insufficient to demonstrate the relationship between the expression levels of IL-6 in the inflamed regions and the endoscopic activity in UC.

The excessive and uncontrolled activation of T helper 17 cells increases IL-17A and contributes to the development of UC. T helper 17 cells secrete IL-17A and promote fibroblast proliferation, transformation of fibroblasts to myofibroblasts, EMT, and MH.9 Previous studies demonstrated that inflamed regions from patients with UC had a high genetic expression of IL-17A,20 and the serum and tissue IL-17A levels were upregulated in patients with UC compared to the controls.21,22 Therefore, it was expected that IL-17A antagonists could be developed as new therapeutic agents for UC. However, secukinumab and ixekizumab, IL-17A antagonists widely prescribed for psoriasis, psoriatic arthritis, and ankylosing spondylitis, can cause IBD.23 Also, our study cannot show the positive relationship between the genetic expression of IL-17A and endoscopic activity in patients with UC. Although the exact mechanism of IL-17A antagonists-related IBD is not known, IL-17A might not just be a proinflammatory cytokine but play a balancing role in the intestinal microenvironment. Moreover, the small number of participants might be associated with irrelevance between the expression levels of IL-17A and the endoscopic activity in UC.

Although EMT-related studies have been conducted more on patients with CD rather than on those with UC, upregulation of EMT markers was identified in colonic mucosal tissues from patients with UC and was related to cancer progression in UC.24,25 Unlike previous studies, we focused on type 2 EMT, which promotes tissue remodeling and fibrosis in UC. During type 2 EMT, epithelial cells lose their cell-cell adhesion and gain migratory properties, which induce an increase in adult stem cell (OLFM4 and LGR5) and mesenchymal cell markers (FSP1, vimentin, and α-SMA) and a decrease in the epithelial cell marker (E-cadherin).26-29 There are two types of stem cells in the intestine: OLFM4+ and LGR5+ intestinal stem cells. OLFM4+ stem cells are located in the upper region of intestinal crypts, while LGR5+ stem cells are located at the base of intestinal crypts. OLFM4 is highly expressed in active UC, and LGR5 is activated in human colonic epithelial organoids.30,31 LGR5+ stem cells show high cellular turnover in colonic MH process.32 Therefore, our result that mRNA expression of LGR5 was prominent in the uninflamed regions from the non-MH group suggests that vigorous tissue repairing occurred to avoid the extension of inflamed regions. FSP1, vimentin, and α-SMA are the main markers of fibrosis.33,34 The inflamed and uninflamed regions from the non-MH group show higher expression of FSP1, vimentin, and α-SMA and lower expression of E-cadherin than those from controls. It is very understandable that EMT tended to occur more actively in the non-MH group than in the controls. Notably, the non-MH group has higher genetic expression of vimentin and α-SMA than the MH group, regardless of biopsied regions. Therefore, the genetic expression of EMT markers is related to the endoscopic activity in both inflamed and uninflamed regions.

Sex-related differences in the incidence/prevalence, extra-intestinal manifestations, and treatment responses have been reported in UC and immune responses.35,36 However, our results did not show any differences in the activity of inflammation and tissue repair in uninflamed regions between males and females (data not shown). This may be attributed to the small sample size.

The colon mucosa microenvironment is important for the development, progression, and remission of UC. The uninflamed region near the upper margin of the inflamed region shares its microenvironment with the inflamed region. Like our study, the study by Dobre et al.20 also compared transcript profiles between the inflamed and uninflamed regions of UC, and the two regions share the molecular activity patterns of cytokines and transcription factors related to immune responses. Molecular and histological activities can persist in endoscopically healed mucosa of patients with UC,37,38 and be related to a risk of clinical relapse.39,40 Histological activity, recently focused as a new therapeutic target for the deep remission in UC,41 in the uninflamed regions, is associated with patients’ prognosis.42 However, not all histological scoring systems are validated, and there are possible discrepancies within or/and between interpreters.43,44 In our study, the discrepancy in the genetic expression of inflammation and tissue remodeling (EMT) was shown between non-MH and MH groups whose histological activities did not differ. Therefore, the mRNA expression profile of the inflammatory and EMT markers can be more sensitive for predicting the disease activity of colon mucosa microenvironments than histological evaluation. Fig. 4 summarizes the key cell populations and markers involved in inflammation and tissue remodeling in UC.9,18,19,26,33,34 The molecular activity profiles in the microenvironment of UC help us gain a better understanding of the pathophysiology of UC and develop new therapeutic agents that effectively modulate the microenvironment.

Figure 4. Schematic figure regarding the microenvironment of inflammation, epithelial-to-mesenchymal transition (EMT), and tissue remodeling in ulcerative colitis. Dendritic cells identify microbial products and produce cytokines, such as TGF-β, IL-1β, and IL-6, which stimulate the proliferation and differentiation of naïve T cells. Naïve T cells are differentiated into Th1, Th2, and Th17 cells. Th17 cells, which play a role in the regulation of inflammation, produce IL-17, a proinflammatory cytokine that contributes to activating macrophages and EMT. In response to microbial products and other cytokines, macrophages produce proinflammatory cytokines, including IL-1β and IL-6. The proinflammatory cytokines can induce EMT in intestinal epithelial cells. In turn, there is a loss of epithelial characteristics and an acquisition of mesenchymal features. During EMT, the expression of epithelial marker (E-cadherin) decreases, and that of mesenchymal markers (FSP1, vimentin, α-SMA, OLFM4, and LGR5) increases. EMT can promote the formation of fibroblasts and myofibroblasts, which are important in tissue remodeling. Fibroblasts can proliferate and migrate to the site of injury, where they secrete extracellular matrix proteins. Myofibroblasts can arise from fibroblasts, and both fibroblasts and myofibroblasts express FSP1 and vimentin. Myofibroblasts, expressing α-SMA, induce wound contraction and control tissue architecture. FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; DC, dendritic cell; MQ, macrophage; IL, interleukin; TGF, transforming growth factor; Th, T helper.

The strength of this study is that it first elucidated the genetic expression levels of inflammation and tissue remodeling in the uninflamed regions in patients with UC and proposed a concept of molecular activity in the colon mucosa microenvironment. However, this study has several limitations. First, the definition of microenvironment is rather ambiguous. We regarded the uninflamed region when the endoscopic finding did not show any inflammation sign, 15 cm separated from the upper margin of the inflamed region. Second, the lack of participants may affect the results. Third, the uninflamed regions might share a microenvironment with the inflamed regions but histological activity in the uninflamed regions was not proven in the present study. However, it is already known that the microenvironment in patients with UC can promote inflammation and play a defensive role.45

In conclusion, our findings suggest that the molecular activity of inflammation and tissue remodeling in the colon mucosa microenvironment, which includes the uninflamed as well as inflamed regions, was associated with the endoscopic activity in UC.

ACKNOWLEDGEMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A2C2085149).

We appreciate the help of Ryoung Hee Nam (Seoul National University Bundang Hospital), Soo In Choi (Seoul National University Bundang Hospital), Jae Young Jang (Seoul National University Bundang Hospital), and Eun Hye Kim (Seoul National University Bundang Hospital) for the collection of samples, and Su Kyung Ha (Seoul National University Bundang Hospital) for helping patients complete the questionnaire.

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Study concept and design: N.K. Data acquisition: N.K. Data analysis and interpretation: Y.K.J., N.K., J.H.P., H.K.K., J.A.L. Drafting of the manuscript: Y.K.J. Critical revision of the manuscript for important intellectual content: N.K., H.Y. Statistical analysis: Y.K.J. Obtained funding: N.K. Administrative, technical, or material support; study supervision: N.K., Y.C., C.M.S., Y.S.P., D.H.L. Approval of final manuscript: all authors

SUPPLEMENTARY MATERIALS

Supplementary materials can be accessed at https://doi.org/10.5009/gnl230283.

Fig 1.

Figure 1.Comparison of messenger RNA (mRNA) expression levels of inflammation and tissue repair markers between inflamed and uninflamed regions of patients with ulcerative colitis (UC) and normal tissues from controls. (A) The genetic expression levels of TGF-β and IL-1β were significantly elevated in patients with UC, regardless of biopsied regions compared to that in the controls. (B) The genetic expression of E-cadherin was significantly suppressed, while that of OLFM4, FSP1, vimentin, and α-SMA was significantly elevated in the inflamed and uninflamed regions compared to that in the controls. The mRNA expression of LGR5 in the uninflamed regions was significantly higher than that in the controls. TGF, transforming growth factor; IL, interleukin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin.
Gut and Liver 2024; :

Fig 2.

Figure 2.Comparison of messenger RNA (mRNA) expression levels of inflammatory markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to the endoscopic activity. (A) In inflamed regions, the genetic expression of TGF-β in the non-mucosal healing (MH) group was significantly higher than that in the MH group and controls. The genetic expression of IL-1β in the non-MH and MH groups was significantly upregulated compared to the controls. (B) In uninflamed regions, the genetic expression of TGF-β and IL-1β in the non-MH group was significantly higher than that in the MH group and controls. The genetic expression of IL-6 in the non-MH group was significantly higher than that in the MH group. TGF, transforming growth factor; IL, interleukin; MES, Mayo endoscopic score.
Gut and Liver 2024; :

Fig 3.

Figure 3.Comparison of messenger RNA (mRNA) expression levels of tissue remodeling (epithelial-to-mesenchymal transition) markers in inflamed and uninflamed regions of patients with ulcerative colitis (UC) according to their prognosis. (A) In inflamed regions, the genetic expression of E-cadherin was significantly suppressed, and that of OLFM4, FSP1, vimentin, and α-SMA in the non-mucosal healing (MH) group compared to the controls. The genetic expression of FSP1, vimentin, and α-SMA in the non-MH group was significantly higher than that in the MH group. (B) Uninflamed regions in the non-MH group showed high activity of tissue remodeling compared to the control: the genetic expression of E-cadherin was significantly lower, and that of OLFM4, LGR5, FSP1, vimentin, and α-SMA was significantly higher in the non-MH group than in the controls. Moreover, the genetic expression levels of vimentin and α-SMA were significantly elevated in uninflamed regions of the non-MH group compared to those of the MH group. OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; MES, Mayo endoscopic score.
Gut and Liver 2024; :

Fig 4.

Figure 4.Schematic figure regarding the microenvironment of inflammation, epithelial-to-mesenchymal transition (EMT), and tissue remodeling in ulcerative colitis. Dendritic cells identify microbial products and produce cytokines, such as TGF-β, IL-1β, and IL-6, which stimulate the proliferation and differentiation of naïve T cells. Naïve T cells are differentiated into Th1, Th2, and Th17 cells. Th17 cells, which play a role in the regulation of inflammation, produce IL-17, a proinflammatory cytokine that contributes to activating macrophages and EMT. In response to microbial products and other cytokines, macrophages produce proinflammatory cytokines, including IL-1β and IL-6. The proinflammatory cytokines can induce EMT in intestinal epithelial cells. In turn, there is a loss of epithelial characteristics and an acquisition of mesenchymal features. During EMT, the expression of epithelial marker (E-cadherin) decreases, and that of mesenchymal markers (FSP1, vimentin, α-SMA, OLFM4, and LGR5) increases. EMT can promote the formation of fibroblasts and myofibroblasts, which are important in tissue remodeling. Fibroblasts can proliferate and migrate to the site of injury, where they secrete extracellular matrix proteins. Myofibroblasts can arise from fibroblasts, and both fibroblasts and myofibroblasts express FSP1 and vimentin. Myofibroblasts, expressing α-SMA, induce wound contraction and control tissue architecture. FSP1, fibroblast-specific protein-1; SMA, smooth muscle actin; OLFM4, olfactomedin-4; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; FSP1, fibroblast-specific protein-1; DC, dendritic cell; MQ, macrophage; IL, interleukin; TGF, transforming growth factor; Th, T helper.
Gut and Liver 2024; :

Table 1 Baseline Characteristics, Disease Extent and Activity of UC According to MH

CharacteristicsAll patientsPatients with MH (MES 0 or 1)Patients without MH (MES 2 or 3)p-value
No. of patients47 (100)9 (19.1)38 (80.9)
Male sex25 (53.2)4 (44.4)21 (55.3)0.715
Age at UC diagnosis, yr41.43±15.0446.13±13.9240.45±15.260.496
Age, yr52.57±15.0056.00±12.7751.76±15.520.655
Disease duration, mo68.13±72.1956.95±91.9270.48±68.610.177
Disease extent
Proctitis19 (40.4)6 (66.7)13 (34.2)0.190
Left-sided colitis15 (31.9)2 (22.2)13 (34.2)
Extensive colitis13 (27.7)1 (11.1)12 (31.6)
Stool frequency
012 (25.5)5 (55.6)7 (18.4)0.051
111 (23.4)1 (11.1)10 (26.3)
212 (25.5)3 (33.3)9 (23.7)
312 (25.5)012 (31.6)
Rectal bleeding
08 (17.0)3 (33.3)4 (10.5)0.163
120 (42.6)4 (44.4)16 (42.1)
219 (40.4)2 (22.2)18 (47.4)
Physician's assessment
127 (57.4)7 (77.8)19 (50.0)0.310
212 (25.5)1 (11.1)12 (31.6)
38 (17.0)1 (11.1)7 (18.4)
MES
01 (2.1)1 (11.1)0<0.001
18 (17.0)8 (88.9)0
227 (57.4)027 (71.1)
311 (23.4)011 (28.9)
Mayo score8.09±2.694.78±1.998.87±2.20<0.001
NHI*
28 (18.2)1 (11.1)7 (20.0)0.357
325 (56.8)7 (77.8)18 (51.4)
411 (25.0)1 (11.1)10 (28.6)
Medication at the endoscopic examination
Topical 5-ASA17 (36.2)4 (44.4)13 (34.2)0.704
Oral 5-ASA41 (87.2)6 (66.7)35 (92.1)0.075
Immunomodulator30 (63.8)4 (44.4)26 (68.4)0.252
Biologics or small molecule3 (6.4)03 (7.9)1.000

Data are presented as number (%) or mean±SD.

UC, ulcerative colitis; MH, mucosal healing; MES, Mayo endoscopic subscore; NHI, Nancy histologic index; 5-ASA, 5-aminosalicylic acid.

*NHI from inflamed regions are calculated.


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Gut and Liver

Vol.18 No.2
March, 2024

pISSN 1976-2283
eISSN 2005-1212

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