Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease

Serge Dionne; Sophie Restellini; Jamie Koenekoop; Pedro Salvador Escribano; Ciriaco A. Piccirillo; Patrick Charlebois; A. Sender Liberman; Barry Stein; Carl Frederic Duchatellier; Ernest Gerald Seidman

doi:10.12688/f1000research.13906.1

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Research Article

Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease

[version 1; peer review: 1 approved, 1 approved with reservations]

Serge Dionne¹, Sophie Restellini¹, Jamie Koenekoop¹, [...] Pedro Salvador Escribano^1,2, Ciriaco A. Piccirillo³, Patrick Charlebois⁴, A. Sender Liberman⁴, Barry Stein⁴, Carl Frederic Duchatellier¹, Ernest Gerald Seidman ¹

Serge Dionne¹, Sophie Restellini¹, [...] Jamie Koenekoop¹, Pedro Salvador Escribano^1,2, Ciriaco A. Piccirillo³, Patrick Charlebois⁴, A. Sender Liberman⁴, Barry Stein⁴, Carl Frederic Duchatellier¹, Ernest Gerald Seidman ¹

PUBLISHED 11 Mar 2019

Author details Author details

¹ Division of Gastroenterology, Research Institute, McGill University Health Center, Faculty of Medicine, McGill University, Montréal, QC, H3G 1A4, Canada
² Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, 46010, Spain
³ Department of Microbiology and Immunology, Research Institute, McGill University Health Center, Faculty of Medicine, McGill University, Montréal, QC, H4A 3J1, Canada
⁴ Colorectal Surgery, Research Institute, McGill University Health Center, Faculty of Medicine, McGill University, Montréal, QC, H4A 3J1 , Canada

Serge Dionne
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Sophie Restellini
Roles: Investigation, Project Administration, Resources, Visualization, Writing – Review & Editing

Jamie Koenekoop
Roles: Investigation, Project Administration, Resources, Visualization, Writing – Review & Editing

Pedro Salvador Escribano
Roles: Funding Acquisition, Investigation, Resources, Writing – Review & Editing

Ciriaco A. Piccirillo
Roles: Conceptualization, Investigation, Methodology, Supervision, Writing – Review & Editing

Patrick Charlebois
Roles: Resources, Writing – Review & Editing

A. Sender Liberman
Roles: Investigation, Writing – Review & Editing

Barry Stein
Roles: Investigation, Writing – Review & Editing

Carl Frederic Duchatellier
Roles: Investigation, Project Administration, Resources, Writing – Review & Editing

Ernest Gerald Seidman
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Intestinal fibrosis represents a serious complication of inflammatory bowel diseases (IBD), often necessitating surgical resections. Myofibroblasts are primarily responsible for interstitial matrix accumulation in fibrotic diseases. However intestinal myofibroblasts (IMF) remain inadequately characterized. The aim was to examine fibroblast markers and fibrosis-associated gene expression in IMF isolated from resected intestine from IBD and control patients. As well as determining the effect of the fibrogenic cytokine TGFβ.
Methods: Intestinal resections were obtained (n =35) from consenting patients undergoing elective surgery (2014-16). Primary cultures of IMF were isolated using DTT and EDTA and cultured. Viability and phenotypic characterization of IMF was carried out by flow cytometry and fluorescence microscopy. IMF (passages 3-8) were treated for 24 hours. Cytokines were quantified in IMF by real time PCR and in supernatants using the human pro-inflammatory cytokine panel
Results: All markers and most fibrosis mediators studied were preferentially expressed by IMF compared to mucosal tissue. Metalloproteinases (MMP) 2 and 3, as well as their inhibitor TIMP1, are highly expressed by IMF. They also highly expressed inflammatory mediators, including IL-6, IL-8, CCL2 and PTGS2. Whereas mucosal expression of pro-inflammatory cytokines such as TNFα and IL-17 is increased in IBD, that of fibrosis mediators was not different. Fibrosis-related gene expression in IMF from IBD patients and controls was similar, but IMF from IBD expressed higher levels of several inflammatory genes. IMF from CD and UC had mostly similar expression profiles. TGFβ induced expression of fibrogenic genes αSMA, COL1A1, CTGF, FN1 and LOX. TGFβ-stimulated IMF released increased levels of IL-6, whereas IL-6, IL-8, as well as small amounts of IFN-γ and IL12p70 were produced following stimulation with IL-1β+IL-23.
Conclusions: This study extends knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required.

Keywords

intestinal myofibroblasts, inflammatory bowel disease, fibrosis, crohn’s disease, ulcerative colitis, inflammation, cytokines gene expression

Corresponding author: Ernest Gerald Seidman

Competing interests: No competing interests were disclosed.

Grant information: Support for this research was in the form of funds provided to EG Seidman as a Tier 1 Canada Research Chair in immune mediated gastrointestinal disorders. PS Escobar was provided salary support through the Spanish government with a predoctoral training grant Resolución de la Presidencia de la Agencia Estatal de Investigación, por la que se conceden ayudas a la movilidad predoctoral para la realización de estancias breves en Centros de I+D, convocatoria 2016. S Restillini was provided salary support by the Government of Switzerland through her home institute Geneva University Hospital.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2019 Dionne S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Dionne S, Restellini S, Koenekoop J et al. Characterization of myofibroblasts isolated from the intestine of patients with inflammatory bowel disease [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2019, 8:275 (https://doi.org/10.12688/f1000research.13906.1) First published: 11 Mar 2019, 8:275 (https://doi.org/10.12688/f1000research.13906.1) Latest published: 11 Mar 2019, 8:275 (https://doi.org/10.12688/f1000research.13906.1)

Introduction

The two major forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by chronic and relapsing intestinal inflammation that develop as the result of an abnormal regulation of immune responses, likely directed at least in part, against the host commensal gut microbiota^1–3. The prevalence of IBD is estimated to be over 3.6 million persons in North America and Europe, and the incidence is increasing in Asia and Africa^4,5. Genetic studies have identified over 200 susceptibility loci for IBD, mostly shared between CD and UC^6–8. These loci are enriched for pathways that interact with environmental factors to modulate intestinal homeostasis, including IL23R, ATG16L1, IRGM and NOD2⁹.

The natural history and clinical course of IBD is extremely heterogeneous. Up to 16% of UC patients require a colectomy within 10 years, whereas about 50% of patients with CD develop complications such as strictures, fistulas, and abscesses that frequently require surgery within the same period^10,11. Intestinal fibrosis is a common and potentially serious complication of CD, but not UC, that results from the reaction of intestinal tissue to the damage inflicted by chronic inflammation¹². NOD2 has been long considered the most important genetic predictor of the evolution toward a fibrostenosing phenotype^13,14. Fibrosis in CD is also associated with polymorphisms of the Jak2, ATG16L1, CX3CR1 and MMP3 genes¹⁴.

An improved understanding of the cellular and molecular mechanisms that underlie the pathogenesis of intestinal fibrosis is needed. The mechanisms are complex, dynamic, and likely involve multiple cell types and soluble factors¹². Intestinal myofibroblasts (IMF) play important roles in inflammation and tissue remodeling¹⁵. The development of fibrosis results from an imbalance in extracellular matrix (ECM) deposition and degradation. Alpha-smooth muscle actin (α-SMA) positive myofibroblasts were identified as the primary cell type responsible for interstitial matrix accumulation in fibrotic diseases^16,17. A hallmark of mesenchymal cell activation is the acquisition of a myofibroblast phenotype, whereby fibroblasts transform into myofibroblasts acquiring smooth muscle features, most notably the expression of α-SMA and synthesis of mesenchymal cell related matrix proteins. TGF-β1, a prototype of the TGF-β superfamily, is widely considered to be the major profibrogenic cytokine that is responsible for the myofibroblast differentiation and subsequent matrix synthesis^16,18. Although the TGF-β/Smad pathway is considered a driving force of fibrosis, myofibroblasts are activated by numerous paracrine mediators in their environment that promote their production of ECM and proliferation including, TGF-β1, PDGF, CTGF, IGFI/II, bFGF and various interleukins: IL-1β, IL-6 and IL-13^17,19.

Although inflammation is necessary for fibrosis, recent evidence indicates that once initiated, fibrosis in CD can progress independently of inflammation¹⁷. Consequently, current anti-inflammatory treatments may not prevent fibrosis once excessive ECM deposition has commenced^17,20. Matrix stiffness is capable of further activation of intestinal fibroblasts and can contribute to progression of fibrosis independently of inflammation^21,22.

There is currently little information about the identity, abundance and characteristics of intestinal mesenchymal cells such as fibroblasts and IMF under normal and pathological conditions. In this study, we examined the expression of fibroblast and IMF molecular markers in the intestine from patients with CD, UC and from non-IBD control patients.

Methods

Human intestinal tissue acquisition and consent

The McGill University Health Center’s research ethics board approved the study design and consent forms. Intestinal resections were obtained (2014–2016) from patients undergoing elective intestinal surgery who voluntarily gave written informed consent to participate. Written informed consent for publication of the participants/patients’ details and/or their images was obtained from the participants/patients/parents/guardian/relative of the participant/patient Resected tissue was obtained from 15 CD and 6 UC patients, as well as from uninvolved surgical specimens (>5 cm from the tumor margin) of 14 control patients undergoing colectomy for carcinoma or polyps. The characteristics of the patient groups are shown in Table 1.

Table 1. Description of the IBD patients and controls studied.

	CD	UC	Controls
Number	15	6	14
Females, n (%) Age at resection, median (range)	8 (53.3) 36.9 (18-68)	3 (50) 41.9 (19-56)	6 (42.9) 60.4 (44-78)
Age at diagnosis • A1 (<16) • A2 (17-40) • A3 (>40)	6 7 2	2 4 0	- - -
Treatment • None, n (%) • 5-ASA alone • Thiopurine or methotrexate • TNF inhibitor • Oral corticosteroids	6 (40) 0 (0) 4 (26.7) 5 (33) 3 (20)	3 (50) 0 (0) 0 (0) 3 (50) 1 (16.7)	14 (100) - - - -
Disease location: CD • L1-terminal ileum • L2-colon • L3-ileocolonic • L4-upper GI tract	7 4 4 0	- - - -	- - - -
Disease location: UC • E1-proctitis • E2-left side colitis • E3-pancolitis • E4-proximal colitis	- - - -	0 0 6 0	- - - -
Disease behavior: CD • B1- Inflammatory • B2-Stricturing • B3-Penetrating Perianal disease, n (%)	4 7 4 2(13.3)	- - - -	- - - -

CD: Crohn’s disease, IBD: inflammatory bowel disease, UC: ulcerative colitis

Isolation and culture of human intestinal subepithelial fibroblasts and myofibroblasts

Primary cultures of IMF were isolated and cultured according to the method reported by Mahida et al.²³. Briefly, tissue specimens were trimmed of fat and thoroughly washed. The mucosa was cut into 0.5 cm pieces and incubated in 1.5 mM DTT (Sigma-Aldrich Canada Ltd, Oakville, ON, Canada) in HBSS for 15 min to remove mucus. Tissue was washed and then incubated in HBSS containing 2 mM EDTA for 2 × 30 min in a shaker at 37°C.

The resulting mucosal samples denuded of epithelial cells were cultured at 37°C, in RPMI-1640 supplemented with 10% fetal calf serum (FCS) to allow myofibroblasts to migrate out, in order to establish primary cultures. Established colonies of IMF were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 1% non-essential amino acids (Gibco, Carlsbad, CA, USA), and 200 mM glutamine (Sigma). Experiments were performed on passages 3–8.

Viability and Phenotypic Characterization of Primary Myofibroblast Cultures

Cells obtained as above were prepared for flow cytometry as follows. After trypsinization, cells were counted and distributed in 1x10⁶ per sample tubes. Cells were washed twice in PBS and blocked with Human BD Fc Block (BD Biosciences, Mississuagua, ON) according to the manufacturer’s instructions. Samples were then incubated with eBioscience^TM Fixable Viability Dye eFluor 450 (Life Technology Inc., Burlington, ON) for 30 min in ice, washed again in PBS and incubated in PBS containing the surface marker antibodies for 20 min in ice (Table 2). Samples were then permeabilized with BD Cytofix/Cytoperm Buffer (BD Biosciences) for 20 min on ice and then washed with BD Perm/Wash (BD Biosciences) and incubated with BD Perm/Wash Buffer containing intracellular marker antibodies for 20 min on ice (Table 2). Samples were washed with BD Perm/Wash and analyzed by flow cytometry using the BD LSRFortessa™ X-20 (BD Biosciences). Machine compensation was performed using unstained cells, viability dye stained cells and UltraComp ebeads (eBioscience) conjugated with each antibody. Dead cells and debris were gated out and purity of the cells was calculated on live cells. Fluorescence microscopy was performed with an Olympus IX71 microscope (Olympus Canada, Toronto, ON) equipped with an Olympus LUCPLFLN20X/0.45 lens.

Table 2. Specific antibodies used for immunofluorescence and flow cytometry.

Marker	Intracellular/Extracellular	Sample ul (1:100)	Manufacturer/Catalogue #
Alpha Smooth Muscle Actin	Intracellular	5	Abcam/ab197240
FSP-1 (S100A4)	Intracellular	5	Biolegend/370005
CD90	Extracellular	2.5	BD Biosciences/563804
EpCAM	Extracellular	5	BD Biosciences/347199
CD45	Extracellular	2.5	BD Biosciences/560566
FAP PE	Extracellular/Intracellular	10	R & D Systems/ FAB3715P-025
CD271 PE-Cy7 (LNGFR)	Extracellular/Intracellular	5	BD Biosciences/ 562122

In some experiments, cells were incubated with SMAD (5uM, x 2hr) and/or TGFα (3 ng/ml, overnight).

Multiplex cytokine array

IMF were plated on 6 well-plates and treated for 24 hours. The medium was collected and stored at -80°. Supernatants of the cultures were tested for IFNγ, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70 and IL-13, using the human pro-inflammatory cytokine panel (Meso Scale Diagnostics, Rockville, MD).

Real time polymerase chain reaction (qPCR)

mRNA from IMF was isolated with RNeasy Mini Plus kit (Qiagen, Toronto, ON, Canada) according to the manufacturer’s instructions. cDNA was generated from 1 µg of total RNA using Transcriptor First Strand cDNA Synthesis kit (Sigma-Aldrich Canada Ltd). Real-time RT-PCR was performed by a “StepOnePLus” RT-PCR (Life Technologies, Burlington, ON) using PerfeCTa SYBR green Fast Mix (Quanta Biosciences, Beverly, MA). Primers were as described in Table 3.

Table 3. Sequence of primers used.

GENE NAME		PRIMER SEQUENCES
*FAP*	forward	5’-TAT TCA GAG TAA CAC AGG ATT CA-3’
	reverse	5’-ACT TCT TGC TTG GAG GAT AG-3’
*FSP-1/S100A4*	forward	5’-CTT CTT CTT TCT TGG TTT GGT-3’
	reverse	5’-AGC AGT CAG GAT CAA CAC-3’
*CD90*	forward	5'-AGA GAC TTG GAT GAG GAG-3’
	reverse	5'-CTG AGA ATG CTG GAG ATG-3’
*ACTA2/αSMA*	forward	5’-CTG TTC CAG CCA TCC TTC AT-3’
	reverse	5’-CCG TGA TCT CCT TCT GCA TT-3’
*COL1A1*	forward	5’-GAG AGC ATG ACC GAT GGA TT-3’
	revers	5’-CCT TCT TGA GGT TGC CAG TC-3’
*CTGF*	forward	5'-CTC CTG CAG GCT AGA GAAGC-3’
	reverse	5'-GAT GCA CTT TTT GCC CTT CTT-3’
*CCN1*	forward	5’-AAT GGA CCT CGC ATC CTA TA-3’
	reverse	5’-TTC TTT CAC AAG GCG GCA-3’
*TIMP1*	forward	5’TTG ACT TCT GGT GTC CCC AC-3’
	reverse	5′-CTG TTG TTG CTG TGG CTG AT-3’
*MMP9*	forward	5'-TTG GTC CAC CTG GTT CAA CT-3’
	reverse	5’-ACG ACG TCT TCC AGT ACC GA-3’
*MMP2*	forward	5′-GCC CCA GAC AGG TGA TCT TG-3′
	reverse	5′-GCT TGC GAG GGA AGA AGT TGT-3′
*MMP3*	forward	5′-CAA TTT CAT GAG CAG CAA CG-3′
	reverse	5′-AGG GAT TAA TGG AGA TGC CC-3
*IL-6*	forward	5′-CAA AGA TGG CTG AAA AAG ATG GA-3’
	reverse	5′-CTG TTC TGG AGG TAC TCT AGG T-3’
*IL-8*	forward	5-CCT TCC TGA TTT CTG CAG CTC-3’
	reverse	5′-GTC CAC TCT CAA TCA CTC TCA G-3’
*IL-17*	forward	5′-ACT ACA ACC GAT CCA CCT CAC-3’
	reverse	5′-ACT TTG CCT CCC AGA TCA CAG-3′
*TNFA*	forward	5′-AGG CGG TGC TTG TTC CTC AG-3′,
	reverse	5′-GGC TAC AGG CTT GTC ACT CG-3′
*IL23A*	forward	5′-GGG ACA CAT GGA TCT AAG AG-3′
	reverse	5′-GCA AGC AGA ACT GAC TGT TG-3′
*PTGS2*	forward	5′-ATA TGT TCT CCT GCC TAC TGG AA-3′
	reverse	5′-GCC CTT CAC GTT ATT GCA GAT G-3′
*FN1*	forward	5′-CCC CTG GGG TCA CCT ATT AC-3′
	reverse	5′-CGG TCA GTC GGT ATC CTG TT-3′
*NOX4*	forward	5′-GGT AAG CCA AGA GTG TTC GG-3′
	reverse	5-GCT GTA TAA CCA AGG GCC AG-3′
*LOX*	forward	5′-TGG CAG TCT ATG TCT GCA CC-3′
	reverse	5′-CTA TGG CTA CCA CAG GCG AT-3′

Primers were ordered from Integrated DNA Technologies (Coralville, IA). Gene expression was standardized using GAPDH expression. Results were quantified using the ΔCt or 2ΔCt method.

Statistical analysis

Data were analyzed using GraphPad and presented as median. Statistical analysis of the results between groups was determined by the Mann-Whitney test. Significance was set as p<0.05.

Results and discussion

Characterization of primary IMF cultures

The viability of IMF cells as determined by flow cytometry was 97.8% (n= 3/group; passages 3-8). No difference was observed between UC, CD and control groups. Immunofluroescence microscopy revealed that the IMF were aSMA+, CD90+, FSP1 +, and negative for hematopoietic and epithelial markers CD45 and EpCAM. Representative images are illustrated in Figure 1.

Figure 1. Immunostaining of IMF primary cultures.

A) Representative photomicrograph of colonic myofibroblasts isolated from a resection of a control patient. Cells have a fibroblast shape and express expressed a marker of myofibroblast, a-SMA (in green). B) IMF cultured from a colonic resection in a patient with ulcerative colitis. Cells also express CD 90 (in red).

Expression of fibroblast markers by primary IMF cultures

We examined the gene expression of fibroblast markers in IMF cultures and compared their levels with those in the mucosa. The expression of fibroblast activation protein (FAP) was more than 200 times higher in IMF cultures than in mucosal tissue. Levels of Fibroblast specific protein 1 (FSP1, also known as S100A4) and those of the stromal marker CD90/Thy-1 were 5–8 times greater in IMF than in the mucosa. The myofibroblast marker αSMA in primary IMF cultures was 2-fold the mucosal level (Figure 2A). We then examined the expression of different mediators implicated in the fibrotic process. As illustrated in Figure 2A, IMF expressed high levels of type 1 collagen (COL1A1) and fibronectin 1 (FN1). Connective tissue growth factor (CTGF/CCN2), a matricellular protein recognized to promote matrix protein deposition and fibrogenesis, was similarly expressed by IMF and mucosa. CCN1, another matricellular protein, was preferentially expressed by IMF.

Figure 2. Comparative expression of fibrosis-related mediators and fibroblast markers by intestinal myofibroblasts (IMF) compared to intestinal mucosa levels in human bowel resections.

Gene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005, *** p, 0.00

Fibrosis is a pathological condition characterized by the deposition of excessive or abnormal ECM components, including collagen type I. Metalloproteinases (MMP) are responsible for the degradation of ECM components and are thus play a central role in ECM remodeling. Their activity is controlled by tissue inhibitors of metalloproteinase (TIMP). MMP-2 and MMP-3 are highly expressed in IMF (over 100 times the mucosal levels). However, MMP-9 transcript levels were higher in the mucosa (p=0.05). IMF were found to be an important source of TIMP1, with expression largely exceeded that of the mucosa (Figure 2B). Accumulating evidence points toward the NAD(P)H oxidase of the Nox family and particularly Nox4 as the predominant enzyme source for ROS generation in fibrotic disease²⁴. As shown in Figure 2B, IMF expressed significant levels of NOX4 transcripts.

The inflammatory potential of IMF was then investigated. IMF expressed high levels of pro-inflammatory cytokines IL-6 and IL-8, more than 40 and 30 times the mucosal levels, respectively (Figure 3). The chemokine CCL2 was also more expressed by IMF cultures than mucosal tissue. Transcripts of cytokines such as TNFα, IFNγ, IL-17A and IL-23A were undetectable in most IMF samples analyzed. PTGS2, also known as cyclooxygenase-2 or COX-2, is highly expressed by IMF, approximately 200 times the mucosa levels.

Figure 3. Inflammation-related gene expression in intestinal myofibroblasts (IMF) and intestinal mucosa resections.

Gene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005, *** p < 0.001

Mucosal gene expression of fibrosis mediators

We then examined mRNA level of fibrosis related genes in the mucosa of IBD patients and of controls. No significant differences were found in fibroblast markers and fibrosis gene expression in mucosa from IBD patients compared to controls. MMP3, MMP9 and TIMP1 expression tended to be higher in IBD tissue, but the differences were not statistically different due to the large variation in IBD mucosa (data not shown). There was no clear expression profile related to the pathology except for TIMP1 which was more expressed in UC than in CD (Figure 4). Pro-inflammatory cytokine expression was greater in mucosa isolated from IBD patients. Expression of IL-17 and TNFα, but not IL-23A, were significantly greater in the intestine from IBD patients (Figure 4). Values for IL-6 and IL-8 were too dispersed to achieve significance.

Figure 4. Mucosal gene expression from control and inflammatory bowel disease (IBD) patients.

Gene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05, ** p< 0.005

Fibrosis-related gene expression in IMF from control and IBD patients.

Expression of fibroblast markers and fibrosis-related genes was not different between IMF generated from IBD and control patients (Figure 5). Increased CTGF expression by IBD IMF was marginally (significant p=0.053). FSP-1 expression was not different between IBD and control groups, but IMF from UC patients expressed lower transcript levels compared to those from controls (p=0.0357).

Figure 5. Fibrosis-related gene expression in intestinal myofibroblasts (IMF) cultures obtained from resected bowel in control and inflammatory bowel disease (IBD) patients.

Gene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians.

Inflammatory gene expression in IMF from control and IBD patients

IMF from IBD resections expressed higher levels of IL-6 than those from controls (p=0.0357, Figure 6). IL-8 was nearly 100 times more expressed by IMF from IBD but due to the small number of samples the difference did not reach statistical significance. CCL2 expression was also 30 times more in IMFs from IBD patients. There was a trend towards higher PTGS2 expression in IMF from CD patients compared to those from controls and UC patients.

Figure 6. Inflammatory gene expression in intestinal myofibroblasts (IMF) obtained from control and inflammatory bowel disease (IBD) patients.

Gene expression was determined by real-time qPCR, and data normalized compared to GAPDH expression. Data are presented as medians. * p< 0.05

Effect of TGF-β on fibrosis-related gene expression in IMF from control & IBD patients

TGFβ stimulation of IMF resulted in an increased expression of profibrotic genes COL1A1 and CTGF. CCN1 as well as FN1 expression were also upregulated following stimulation with TGFβ (Figure 7). Lysyl oxidase (LOX), a collagen modifying enzyme required for the cross-linking of collagen, was also induced. There was marked upregulation of αSMA of IMF. On the other hand, TGFβ down-regulated expression of fibroblast markers FSP1 and CD90. TGFβ slightly induced MMP9 and TIMP1 expression, although not significantly. No significant effect of TGFβ was observed on NOX4, IL-6, IL-8 and PTGS2 expression, although IL-6 expression was increased in 4 of 6 IMF cultures.

Figure 7. Effect of TGFβ on fibrosis-related gene expression intestinal myofibroblasts (IMF) obtained from control and inflammatory bowel disease (IBD) patients.

* p< 0.05, ** p< 0.005, *** p < 0.001.

Modulation of inflammation-related gene expression in IMF by fibrogenic and inflammatory signals

To assess the inflammatory properties of IMF, we determined their cytokine release following fibrogenic and inflammatory stimulation. IMF spontaneously released IL-6 and IL-8 (Figure 8a). TGFβ induced IL-6 release (Figure 8b). Stimulation with IL-1β+IL-23 increased IL-6 and IL-8 production. In addition, small amounts of IFNγ and IL-12p70 were released (Figure 8c).

Figure 8. Effect of TGFβ and IL-1β+IL-23 on cytokine production by intestinal myofibroblasts (IMF) obtained from control and inflammatory bowel disease (IBD) patients.

Data are mean ± SEM from 3–5 different IMF cultures from IBD and control, respectively (A and B) and 2 IMF cultures from both IBD and control IMF cultures (C).

Fibrosis is a chronic, progressive process characterised by an excessive deposition of collagen and other ECM components. Intestinal fibrosis is a common complication of CD, an incurable disorder, forcing patients to undergo bowel resections over their lifetime. More than 40% of CD patients with ileal involvement will require one or more resections of strictures²⁵. Although less common in UC, longstanding disease is believed to cause fibrosis resulting in altered bowel function^26,27. In this study, isolation techniques used yielded multiple cell populations present in the intestinal lamina propria including: immune cells, epithelial cells, and MF. This permitted the establishment of primary IMF cultures with a high rate of viability. After the first passage, only MF remained, as identified by their unique phenotype (Figure 1).

A variety of signals promote fibroblast differentiation into IMF and augment ECM expression. TGFβ1 represents the prototype of the profibrogenic mediators, with its unique ability to drive myofibroblast activation through both canonical and non-canonical signaling pathways leading to expression and deposition of ECM¹⁸.

There is little information about the identity, localization, and abundance of the different intestinal mesenchymal cell types. Various studies show that fibroblasts isolated from different tissues are morphologically and functionally heterogeneous subpopulations²⁸. Several fibroblast markers have been described, but none of them is unique to fibroblasts, and not all fibroblasts express the proposed markers. This lack of specific markers has impeded the characterization of IMF and their putative precursors. Moreover, characterization of these markers under normal and pathological conditions is still lacking. This study aimed to determine the expression of several markers as well as fibrosis related mediators in IMF derived from the CD, UC and control patients.

Among the markers available to identify fibroblasts, Thy-1 (CD90), FAP and FSP1 (S100A4) are the most extensively studied¹⁵. Our results show that all three markers are highly expressed by IMF compared to the intestinal mucosa. IMF also express higher levels of the myofibroblast marker αSMA.

Heterogeneous expression of surface receptor Thy-1 in fibroblasts from several tissues is well established. Normal lung fibroblasts express Thy-1, whereas myofibroblasts in the fibroblastic foci in idiopathic pulmonary fibrosis lack Thy-1 expression²⁹. It has been shown that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype³⁰. In contrast to lung fibroblasts, TGFβ up-regulated αSMA expression only in Thy-1+ myometrial and orbital fibroblasts³¹. These results show that the presence rather than the absence of CD90 apparently favors the appearance of a myofibroblast phenotype in response to TGFβ. In this study, we did not observe any difference in CD90 expression between IMF from IBD patients and controls.

Our results reveal that IMF are also enriched in several mediators. COL1A1 and FN1 were highly expressed in IMF. However, expression of the fibrogenic gene CTGF, also known as CCN2, a key mediator of ECM production in pathological fibrotic conditions, was similar to that in the mucosa. This is likely because the epithelium is an important source of CTGF³². CCN1/CYR61 expression was augmented in IMF compared to the intestinal mucosa. To our knowledge, this is the first time that CCN1 expression is reported in IMF. CCN1 levels in parenchymal liver cells were relatively low compared to that in hepatic stellate cells and portal myofibroblasts³³. The same study found that overexpressed CCN1 significantly inhibited production of collagen type I, attenuated TGFβ signaling and induced production of reactive oxygen species (ROS), leading to dose-dependent cellular senescence and apoptosis. On the contrary, CCN1 has been shown to augment TGF-β signaling and contribute to fibrogenic responses to lung injury³⁴. Its role in intestinal fibrosis is still unknown.

It was recently demonstrated that CCN1 promotes mucosal healing in murine colitis. Mechanistically, CCN1 induced IL-6 in macrophages and fibroblasts and promoted intestinal epithelial healing³⁵. IL-6 is produced by several cell types in the lamina propria. Our data indicates that IMF cultures spontaneously released IL-6 and IL-8. In addition, we found that both fibrogenic and inflammatory stimuli can up-regulate IL-6 production. IL-6 has been shown to induce production of collagen 1 and fibronectin in fibroblasts from normal lungs and in idiopathic pulmonary fibrosis. In vivo neutralization of IL-6 trans-signaling resulted in a reduction in pulmonary inflammation and fibrosis, associated with improvement in respiratory function³⁶. Neutralization of autocrine IL-6 reversed STAT3 phosphorylation and normalized expression of TGFβ1 in structured intestinal muscle³⁷.

Dataset 1.The following raw data sets are provided as comma separated values (.csv) files.

Conclusions

We demonstrated that TGFβ induced several pro-fibrotic genes in IMF. Although IMF are reported to be activated and to express αSMA^18,22,28, stimulation with TGFβ resulted in a 20-fold increase of αSMA expression. This is accompanied by upregulation of COL1A1, FN1 and CTGF. Interestingly, TGFβ increased expression of LOX, an enzyme required to modify collagen, a pre-requisite for the cross-linking of collagen. Inhibition of LOX has recently been reported to alleviate lung fibrosis by modulating the inflammatory response preceding myofibroblast accumulation³⁸. NOX4 expression was also induced by TGFβ. NOX4 modulates TGFβ/SMAD-signaling via intracellular ROS production. Increased expression of NOX4 has been reported in idiopathic pulmonary fibrosis, suggesting its role in pathogenesis³⁹. FSP1 and CD90 expression were decreased by TGFβ. αSMA expression by IMF and down-regulated by IFNγ⁴⁰. IL-1 and TNFα induced loss of fibroblast Thy-1 surface expression in vitro²⁹. IFNγ, in combination with TNFα, has been associated with the loss of pericryptal intestinal myofibroblasts⁴¹. Whether decreased FSP1 and CD90 expression by TGFβ has a functional consequence on IMF is currently unknown. A recent study provided evidence of HIF-1 dependent induction of Notch ligands associated with M1 macrophages⁴². In contrast to M2 macrophages, M1 cells activate Notch signaling pathway in epithelial cells. It was suggested that the prevalence of M2 over M1 macrophages in the mucosa of CD patients may mediate the diminished enterocyte differentiation and impaired mucosal regeneration observed in these patients⁴². The current study extends our knowledge about the pathogenesis of fibrosis in IBD. Further research in the identification of mechanisms involved in IMF activation and fibrogenesis are required. A better understanding of the reciprocal regulation of macrophage phenotype and mucosal repair following intestinal damage will help to establish new approaches to CD therapy.

Data availability

Underlying data

F1000Research: Dataset 1. The following raw data sets are provided as comma separated values (.csv) files:, https://doi.org/10.5256/f1000research.13906.d231722⁴³.

Figure 1 dataset on immunostaining of IMF primary cultures.
Figure 2 dataset on the expression of fibrosis-related mediators and fibroblast markers by intestinal myofibroblasts (IMF) compared to intestinal mucosa levels in human bowel resections.
Figure 3 dataset on the inflammation-related gene expression in intestinal myofibroblasts (IMF) and intestinal mucosa resections.
Figure 4 dataset on the mucosal gene expression from control and inflammatory bowel disease patients.
Figure 5 dataset on the fibrosis-related gene expression in intestinal myofibroblasts cultures obtained from resected bowel in control and inflammatory bowel disease patients.
Figure 6 dataset on the inflammatory gene expression in intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.
Figure 7 dataset on the effect of TGFβ on fibrosis-related gene expression intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.
Figure 8 dataset on the effect of TGFβ and IL-1β+IL-23 on cytokine production by intestinal myofibroblasts obtained from control and inflammatory bowel disease patients.

Grant information

Support for this research was in the form of funds provided to EG Seidman as a Tier 1 Canada Research Chair in immune mediated gastrointestinal disorders. PS Escobar was provided salary support through the Spanish government with a predoctoral training grant Resolución de la Presidencia de la Agencia Estatal de Investigación, por la que se conceden ayudas a la movilidad predoctoral para la realización de estancias breves en Centros de I+D, convocatoria 2016. S Restillini was provided salary support by the Government of Switzerland through her home institute Geneva University Hospital.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Faculty Opinions recommended

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VERSION 1 PUBLISHED 11 Mar 2019