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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
Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
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Xiaomin Wu , Xiaoxuan Lin , Jinyu Tan , Zishan Liu , Jinshen He , Fan Hu , Yu Wang , Minhu Chen , Fen Liu , Ren Mao
Correspondence to: Ren Mao
ORCID https://orcid.org/0000-0002-5523-8185
E-mail maor5@mail.sysu.edu.cn.
Fen Liu
ORCID https://orcid.org/0000-0001-9214-0016
E-mail liuf237@mail.sysu.edu.cn
Xiaomin Wu and Xiaoxuan Lin contributed equally to this work as first authors.
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 2023;17(3):360-374. https://doi.org/10.5009/gnl220045
Published online March 10, 2023, Published date May 15, 2023
Copyright © Gut and Liver.
Intestinal fibrosis associated stricture is a common complication of inflammatory bowel disease usually requiring endoscopic or surgical intervention. Effective anti-fibrotic agents aiming to control or reverse intestinal fibrosis are still unavailable. Thus, clarifying the mechanism underpinning intestinal fibrosis is imperative. Fibrosis is characterized by an excessive accumulation of extracellular matrix (ECM) proteins at the injured sites. Multiple cellular types are implicated in fibrosis development. Among these cells, mesenchymal cells are major compartments that are activated and then enhance the production of ECM. Additionally, immune cells contribute to the persistent activation of mesenchymal cells and perpetuation of inflammation. Molecules are messengers of crosstalk between these cellular compartments. Although inflammation is necessary for fibrosis development, purely controlling intestinal inflammation cannot halt the development of fibrosis, suggesting that chronic inflammation is not the unique contributor to fibrogenesis. Several inflammation-independent mechanisms including gut microbiota, creeping fat, ECM interaction, and metabolic reprogramming are involved in the pathogenesis of fibrosis. In the past decades, substantial progress has been made in elucidating the cellular and molecular mechanisms of intestinal fibrosis. Here, we summarized new discoveries and advances of cellular components and major molecular mediators that are associated with intestinal fibrosis, aiming to provide a basis for exploring effective anti-fibrotic therapies in this field.
Keywords: Inflammatory bowel diseases, Intestinal fibrosis, Immune system, Creeping fat, Gastrointestinal microbiota
Fibrosis is a dysregulated outcome of wound healing, especially during chronic inflammatory disorders.1 When inflammation is persistent, the severity of the damage may exceed the ability of the affected tissue to completely heal, which then initiates fibrotic response that eventually results in fibrosis.2,3 The gastrointestinal tract is a tubular structure and therefore fibrosis is presented with the narrowing of lumen and intestinal stricture.4
Intestinal stricture is a common complication of inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC).5,6 CD is a transmural disease that can affect the entire gastrointestinal tract, while UC is a superficial inflammatory disease, restricted to the colonic mucosa and submucosa layer.7 At initial diagnosis, at least 10% of CD patients are presented with a fibrostenosis phenotype.8 However, up to 50% of CD patients ultimately progress to stricturing or penetrating complications and 70% of patients require surgery within their life time.5,9 Even though stricture formation is rather infrequent in UC, recent evidence suggested fibrosis occurs in both acute and chronic UC.10 In the past decades, despite the availability and efficacy of biological therapies in IBD, the incidence of intestinal stricture does not achieve a significant reduction.11 This implies that pure anti-inflammatory treatments do not necessarily alleviate the associated fibrosis.
The review would provide a cellular and molecular biology of intestinal fibrosis (Fig. 1). Considering the close association between intestinal fibrosis and stricturing complications, understanding the pathogenesis of intestinal fibrosis is crucial to identify new anti-fibrotic targets for patients with intestinal strictures.
Intestinal fibrosis is driven by multiple cellular compartments including mesenchymal cells and immune cells.12 The histological feature of intestinal stricture is thickening of the muscularis mucosa and muscularis propria owing to the activation and proliferation of mesenchymal cells. Activated mesenchymal cells not only produce matrix components, but also secrete chemokines to recruit cells from the immune system (e.g., macrophages and T cells), thus perpetuating chronic inflammation. Reciprocal interaction between mesenchymal and immune cell populations in the intestine create a unique pro-fibrotic microenvironment, eventually resulting in fibrosis formation.13
Intestinal fibrosis results from sustained activation and proliferation of myofibroblasts.14 The activated myofibroblasts, as the final effector cells, can produce extracellular matrix (ECM) proteins and secrete cytokines such as interleukin (IL)-6 and IL-11, which facilitates formation of a fibrogenic milieu.15-17 The majority of myofibroblasts derive from resident fibroblasts and smooth muscle cells (SMCs). However, they can also originate from other cell types like epithelial and endothelial cells, pericytes, bone marrow stem cells and bone marrow-derived circulating fibrocytes.18,19 The various types of cells weave together in the inflamed intestine and contribute to the development of intestinal fibrosis.20
Fibroblasts are characterized by an elongated or spindle-shaped morphology, which are the most abundant cell type in connective tissue. Their main function is to maintain tissue integrity.21 Fibroblasts can be activated and multiply in response to pro-inflammatory mediators, such as insulin-like growth factor (IGF)-I, fibroblast growth factor, and IL-1β.22 The growth of fibroblasts can also be induced by immune cells or inflammatory cells through a cell-to-cell contact mechanism.23,24 In addition, fibroblasts can migrate to the site of inflammation foci along the concentration gradient of pro-inflammatory cytokines via activation of NF-κB and JAK-STAT signaling pathways.16,25,26 A previous study reported that fibroblasts isolated from inflamed or fibrotic CD tissue, or inflamed UC mucosa exhibited an increased proliferation, when compared with normal tissue.27 Recently, Wohlfahrt
Myofibroblasts are characterized by the expression of α-smooth muscle actin (α-SMA), with enhanced production of collagen and increased capacity of contraction.30 Although the exact molecular mechanism of myofibroblasts in fibrosis remains incompletely understood, mediators acting on myofibroblasts are clearly demonstrated, including pro-inflammatory cytokines, paracrine and autocrine factors (e.g., IGF-1), and pathogen or damage-associated molecular patterns.31 The activated myofibroblasts initiate fibrotic process in the following ways. Firstly, myofibroblasts secrete ECM components, as well as various cytokines and chemokines, directly or indirectly contributing to the thickening of mesenchymal cell layer.32,33 Secondly, myofibroblasts participate in tissue remodeling through mechanical contractions.34,35 Thirdly, the mechanic contraction of myofibroblasts can activate latent transforming growth factor-β1 (TGF-β1) released from ECM.36 TGF-β1 and its related pathways are major drivers in the process of fibrosis.37 Recently, de Bruyn
SMCs are one of the three interrelated cell phenotypes (the other two being fibroblasts and myofibroblasts).33 SMCs and fibroblasts are derived from the same primitive mesenchymal cells.39 SMCs are regarded as the progenitors of myofibroblasts.39 A dynamic equilibrium exists between SMCs and myofibroblasts phenotypes.39 SMCs can change their phenotypes in response to environmental stimulation.40 A previous study found that SMCs isolated from CD ileum presented alterations in morphology and contractile activity.41 It was demonstrated that SMCs isolated from CD ileum had an overexpression of platelet-derived growth factor (PDGF)-β, which drove the myogenic phenotype switch to synthetic one. The effect of PDGF-β was paralleled to a reduced encoding of contractile genes that were responsible for quiescent smooth muscle.41 SMCs are also able to release significant amounts of IL-6, contributing to inflammatory process.42 Besides, these cells actively contribute to the development of intestinal fibrosis by inducing the production of collagens and MMPs.41 These evidences suggested that the phenomenon of smooth muscle hyperplasia/hypertrophy in CD may be a driving force, rather than simply a passive increase of stricture formation.
Epithelial or endothelial-mesenchymal transition (EMT or EndMT) represents a dynamic entity where epithelial or endothelial cells transform to mesenchymal cells in response to inflammatory cytokines, oxidative stress and hypoxia.43-45 EMT is implicated in CD-associated fistulas and intestinal fibrosis.46,47 During formation of CD-associated fistulas, intestinal epithelial cells start with the dissociation from the base membrane and then migrate to the lining of the fistula tracts, where they convert to mesenchymal cells.47 In CD-associated intestinal fibrosis, EMT serves as a reservoir that can generate new fibroblasts and consequently result in fibrosis formation.46,48 Results from Iwano
Telocytes (TCs) are a novel type of interstitial cells characterized by CD34/PDGFRα, and have been demonstrated to be involved in several disorders including CD.54-56 The function of TCs is widely linked with other cells including mast cells, macrophages, myofibroblasts, and fibroblasts.57 Milia
Fibrocytes are bone marrow-derived mesenchymal progenitors, with the features of both hematopoietic (CD34) and fibroblast markers (collagen-I). Fibrocytes play a critical role in fibrotic diseases.59 Previous studies revealed that fibrocytes could be triggered by several inflammatory cytokines.59 Specifically, within four-day following injury, activated fibrocytes typically migrate into injured sites and then participate in fibrotic reactions through a direct way by production of ECM proteins and fibrogenic cytokines, or an indirect way by differentiation into myofibroblasts.60-62 Sazuka
A variety of key innate (macrophages) and adaptive (T cell subsets) immune cell types have been well-established in orchestrating the fibrotic microenvironment in intestine. The immune cell skewing in fibrosis niche probably perpetuates inflammation and exacerbates the process of wound healing.65 Here, we will discuss several immune cell subsets, pointing toward novel immune-based therapeutic strategies in fibrosis.
T helper 2 (Th2) cells are hallmarked by the secretion of cytokines IL-4, IL-5, and IL-13, which are responsible for type 2 immune responses.66 The type 2-associated cytokines are actively engaged in wound healing and fibrosis.65 At inflammatory sites, activated innate immune cells, such as group 2 innate lymphoid cells (ILC2) and basophils, are usually the early sources of local cytokines IL-4, IL-5, and IL-13, which trigger the activation and accumulation of Th2 cells.67,68 Th2 cells-derived IL-4 and IL-13 further promote the accumulation and proliferation of ILC2, thus creating a vicious cycle.69 Activated Th2 cells orchestrate the process of tissue fibrosis directly and indirectly by acting on immune or non-immune cells including local M2 macrophages, fibroblasts, endothelial cells and epithelial cells.70,71 Besides, as a well-known opponent of Th1 cells, Th2 cells can reverse the expression levels of Th1-associated anti-fibrotic cytokines such as interferon γ (IFN-γ).72 However, randomized controlled trials showed that blockade of IL-13 to target Th2 responses while administration of IFN-γ to stimulate Th1 responses failed to attenuate pulmonary fibrosis.73-75 Conversely, in a phase II trial, neutralization of IL-4/IL-13 effectively improved early skin fibrosis.76 The inconsistent results suggested targeting Th2 response as a therapeutic strategy for fibrosis requires further investigation.
Macrophages are highly heterogeneous and plastic cell populations, and are key regulators of tissue fibrosis in several organs.66,77 Generally, macrophages are classified into two subtypes: M1 macrophages with pro-inflammatory roles, and M2 macrophages with pro-fibrotic properties. The latter is activated by IL-4 and IL-13, and characterized by effects of inflammation resolution and tissue restoration.66 In intestine, STAT6-dependent M2 macrophages promote mucosal repair through activating Wnt signaling pathway.78 Moreover, macrophages from CD patients showed a significant enrichment in the expressions of M2-related as well as fibrotic-related genes, implying that M2 macrophages potentially exacerbated fibrosis formation.79 Results from STAT6 deficient colitis mice showed that the frequency of CD16+ macrophages was enhanced in the damaged mucosa of CD patients with stenotic or penetrating complications, and were also associated with the expression of fibrotic-related markers.80 Blockade of the interactions between inflamed macrophages and stromal cells has been proven to potentially ameliorate aberrant wound repair in zebrafish IBD model.81 Recently, the advanced scRNA-seq has revealed a novel macrophage subgroup, named with CX3CR1+SiglecF+ transitional macrophages, which are abundant in fibrotic niche and exhibit a pro-fibrotic effect in bleomycin-induced lung fibrosis.82 ScRNA-seq will be a promising technique to reveal the cellular heterogeneity of macrophages in fibrosis.
T helper 17 (Th17) cells are characterized by RAR-related orphan receptor γt (ROR-γt) expression and signatured by producing IL-17, IL-21, and IL-22 cytokines,83,84 which have fibrogenic properties. IL-17A, the predominant Th17-assciated cytokines, exerts its fibrotic effects via acting on myofibroblasts and regulating EMT.85-87 In gut, elevated levels of tissue Th17 cells and IL-17 production are observed in patients with intestinal stenosis.87 Recently, Paul
ILCs are a functionally diverse but developmentally related family of innate lymphocytes, with phenotypes and functions having striking similarities to T helper (Th) cells.95 According to cytokine signatures and transcription factors expression, ILCs are divided into three groups.96 Group 1 ILCs (ILC1) subsets share common properties with Th1 cells and express the transcription factor T-bet.97 Group 2 ILCs (ILC2) express the transcription factors RORα and GATA-3, which resembles Th2 cells functionally.98 Group 3 ILCs (ILC3), expressing transcription factor RORγt, are analogous to Th17 cells.99,100 ILC2 can respond rapidly to tissue damage, followed by an increase of Th2-like cytokines.101 An increased frequency of ILC2 has been detected in intestinal tissues from CD patients.102 Lo
Molecules are messengers of crosstalk between immune and non-immune cells and actively contribute to persistent inflammation.13 Although inflammation is a prerequisite of fibrosis, purely controlling intestinal inflammation cannot hold back the progression of fibrosis.8 This implies that inflammation is not the exclusive driver of fibrosis. In the following section, a detailed discussion concerning inflammation-dependent, but with a focus on inflammation-independent factors of fibrosis, will be reported.
Cytokines and chemokines secreted by immune and non-immune cells are orchestrators of sustained inflammatory microenvironment, and are also observed to possess pro-fibrotic effects, which lay foundations for uncovering novel therapeutic targets in fibrotic disorders.142 Several novel cytokines involved in fibrogenesis will be detailly described in this part, and the elaborate profile of cytokines and chemokines are shown in Table 1.
Table 1 Cytokine and Chemokine Profiles Involved in Intestinal Fibrosis
Cytokine | Pro- or anti-fibrosis | Effects on intestinal fibrosis | References |
---|---|---|---|
IL-1 | Pro | Induce fibroblasts activation; enhance pro-fibrotic cytokines production; inhibit ECM degradation; enhance collagens expression | 104-106 |
IL-4 | Pro | Promote myofibroblasts activation; promote type 2 immunity-induced fibrosis | 107,108 |
IL-6 | Pro | Promote SMCs activation; promote fibroblasts activation and proliferation; enhance ECM production | 109-111 |
IL-10 | Uncertain | Inhibit collagens deposition; no effects on fibroblasts and myofibroblasts | 112,113 |
IL-11 | Pro | Enhance collagens expression; may promote SMCs hyperplasia | 114 |
IL-12 | Pro | Promote inflammation | 115,116 |
IL-13 | Pro | Promote TGF-β1 production; initiate fibrosis | 117,118 |
IL-17 | Pro | Stimulate myofibroblasts activation; enhance collagens expression; decrease ECM degradation; induce EMT | 85,89,119 |
IL-21 | Uncertain | Facilitate Th2 and Th17 development; enhance MMPs secretion | 120,121 |
IL-22 | Pro | Inhibit inflammation; enhance MMPs secretion; promote myofibroblasts differentiation | 122-124 |
IL-23 | Pro | Promote inflammation; promote fibrotic responses | 115,116,124 |
IL-25 | Non | IL-13 production depends on IL-25; induce type 2 immunity; promote inflammation | 125,126 |
IL-33 | Pro | Promote pro-fibrotic type 2 immunity | 127,128 |
IL-34 | Pro | Enhance collagens expression; promote fibroblasts activation | 129 |
IL-36 | Pro | Enhance collagens expression; promote fibroblasts activation | 130 |
TL1A | Pro | Enhance collagens expression and pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 131,132 |
IFN-γ | Anti | Inhibit TGF-β signaling; inhibit fibroblasts migration and myofibroblasts differentiation | 133-135 |
TGF-β | Pro | Enhance pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 43,136-138 |
CXCR4 | Pro | Mediate pro-fibrotic effects of PDGF-C | 139 |
CCL11 | Pro | Induce eosinophils recruitment and in turn promote fibroblasts activation | 140 |
CXCL8 | Pro | Promote pro-fibrotic growth factors production; enhance MMPs secretion | 141 |
IL, interleukin; ECM, extracellular matrix; SMCs, smooth muscle cells; TGF, transforming growth factor; EMT, epithelial-mesenchymal transition; Th, T helper; MMPs, matrix metalloproteinases; TL1A, tumor necrosis factor-like ligand 1A; IFN, interferon; CXCR4, C-X-C motif chemokine receptor 4; PDGF-C, platelet-derived growth factor-C; CCL11, C-C motif chemokine ligand 11; CXCL8, C-X-C motif chemokine ligand 8.
IL-11, a member of IL-6 family, is recognized as a pro-fibrotic cytokine secreted by stromal cells, as well as epithelial cells during tissue injuries.143 IL-11 is upregulated in various fibro-inflammation disorders.143 Ng
IL-33, a member of IL-1 superfamily, is passively released upon cellular damage and necrosis and is thus considered as an alert of inflammation.147 IL-33 is also involved in the process of fibrogenesis.148 Binding of IL-33 to its receptor ST2 triggers activation of Th2 cells to produce amphiregulin, which then drove osteopontin production by eosinophils, thus forming IL-33-amphiregulin-osteopontin axis. The axis conferred to fibrotic responses in eosinophilic airway inflammation.149 With regard to intestine, the expression of IL-33 and ST2 were upregulated in mucosa from UC patients and dextran sulfate sodium (DSS) colitis model.136,137 In particular, elevated epithelial expression of IL-33 was strongly associated with fibrosis progression in pediatric Crohn’s ileitis.138 A recent study by Imai
IL-34, a member of 4-helical cytokine family, is produced by a wide range of cells including fibroblasts, immune cells, epithelial cells, endothelial cells and adipocytes.150 The association of aberrant high expression of IL-34 and fibrosis has been identified in several organs, including liver, kidney, and gut.129,151,152 Production of IL-34 was enhanced in inflamed mucosa in IBD patients and in DSS-induced colitis, which was regulated by tumor necrosis factor-α (TNF-α) via NF-κB signaling.153,154 Notably, the expression of IL-34 was elevated in fibrostrictures sites of CD.129 It was observed that activated fibroblasts by TNF-α exhibited increased expression of IL-34.129,155 Besides, fibroblasts stimulated with IL-34 could enhance expression of COL1A1 and COL3A1, while this effect disappeared in fibroblast-specific IL-34 knockout mice.129 These evidences raise a possibility that fibroblast is a cellular target of IL-34.
IL-36, also belonging to IL-1 superfamily, consists of five isoforms: IL-36α, IL-36β, IL-36γ, IL-36Ra, and IL-38.156,157 Among them, IL-36α, IL-36β, and IL-36γ play pro-inflammatory effects through activating IL-36 receptor (IL-36R) signaling, while IL-36Ra and IL-38 have opposing effects as they are inhibitors of IL-36R signaling.156,158 It is demonstrated that IL-36α and IL-36γ are elevated in both CD and UC mucosa under inflammation stimuli.158,159 Of note, IL-36α had an increased expression in tissues of CD fibrostenosis.130 Stimulation of IL-36α and IL-36γ can induce fibroblasts activation and epithelial cells proliferation,159,160 which is associated with an enhancement of collagen-VI secretion and ultimately fibrosis development.130 Importantly, both IL-36R blockade and IL-36 genes knockout are sufficient to attenuate intestinal fibrosis, which highlights the therapeutic values of IL-36 in fibrosis.130
Tumor necrosis factor-like ligand 1A (TL1A) is a member of TNF superfamily and interacts with death receptor-3 (DR3) to form TL1A/DR3 co-stimulatory system.161 Aberrant TL1A/DR3 signaling is involved in chronic inflammation and fibrogenesis.162-165 CD patients with higher expression of serum TL1A were prone to develop stricture.166 Another study found that CD patients presenting with specific TL1A genotype rs6478108 were susceptible to forming stricturing phenotype.167
Recently, several inflammation-independent mechanisms including ECM interaction, creeping fat (CrF), gut microbiota, as well as metabolic reprogramming, have attracted much attention because of their unique roles in intestinal fibrosis, which will be discussed in the following part.14,172,173
ECM is a highly specialized and dynamic three-dimensional scaffold in tissue, which is an active player rather than a purely passive player in fibrosis.14 ECM comprises a variety of fibrous components such as collagens, hyaluronan (HA) and fibronectin.174 HA exists as a high-molecular-weight polymer in normal conditions. During excessive inflammation, the polymer is cleaved to fragments of lower molecular weight, which promotes fibroblasts proliferation and myofibroblasts differentiation, thus contributing to fibrosis process. Besides, HA in low molecular weight fragments aids in recruiting immune cells to inflammatory sites, which in turn release a variety of inflammatory mediators and growth factors to initiate fibrosis.175-177 Fibronectin can enhance the susceptibility of SMCs to proliferation through combining with αVβ3 integrin.178 Additionally, the phenotype and function of myofibroblasts are altered along with the increased ECM stiffness. Myofibroblasts isolated from stenotic intestine display enhanced contractility of ECM and decreased activity of MMP-3, resulting in a vicious circle that further leads to tissue rigidity.38 Increased ECM stiffness is also able to drive fibroblasts to produce more ECM proteins through the Hippo and yes-associated protein pathway.14
CrF indicates that mesenteric fat wrapping around more than 50% of the intestinal circumference, which is the unique hallmark of CD.179 Although CrF was first described nearly 100 years ago, whether CrF is pathogenic or protective is still a controversy.179 Previous studies found that CrF was associated with the severity of intestinal inflammation and strictures.180 Inclusion mesentery in ileocolic resection achieved a reduction of stricture recurrence and reoperation.181,182 However, Ha
The relationship between CrF and intestinal fibrosis is still underexplored. Our previous study has uncovered a positive feedback loop between CrF and intestinal muscularis propria.184 Firstly, CrF-derived long-chain free fatty acids significantly enhanced proliferation and activation of intestinal muscle cells, with increased production of ECM proteins and strictures formation subsequently.184,185 Vise versa, hypertrophic muscularis propria triggered migration of preadipocytes out of MAT by fibronectin production, which facilitated CrF development.186 Another study observed that adipocytes within CrF were capable to convert to fibroblasts, whereas selective ablation of CrF adipocytes attenuated collagen deposition and bowel wall thickening.187 Noteworthily, new animal models via repeated colonic biopsy or antimesenteric enterotomy have been recently established,188,189 which will make a big difference for uncovering the complex relationship between CrF and intestinal fibrosis in the future.
Accumulating evidence indicates that gut microbiota plays crucial roles in fibrosis.8 The direct evidence was that experimental mice when reared under specific-pathogen-free conditions displayed a minimal inflammation, whereas when injected bacteria or bacterial wall components they exhibited inflammation and fibrosis.190 The terminal ileum is the most common site of intestinal stricture, where AIEC mainly colonize, giving us a hint that AIEC may be correlated with fibrosis development.191 Indeed, research using mice with AIEC inoculation found that flagellin of AIEC via IL-33-ST2 signaling facilitated intestinal fibrosis.127 Intestinal fibroblasts isolated from CD patients are observed to have an increased expression of several Toll-like receptors (TLRs) including 2, 3, 4, 6, 7.192 It is known that TLRs can be activated by perceiving microbial components, which are called pathogen-associated molecular patterns.172 The activated TLRs then promote the differentiation of fibroblasts into myofibroblasts.192 For example, TLR-3 activation in fibroblasts can augment α-SMA expression and TGF-β1 production via NF-κB signaling.193 Additionally, lipopolysaccharide activating TLR-4 can also stimulate α-SMA expression and collagen synthesis in fibroblasts.194,195 In conclusion, when exposed to pathogen-associated molecular patterns, intestinal myofibroblasts expressed upregulated levels of α-SMA and increased production of ECM proteins, thus confirming the link between gut microbiota and intestinal fibrosis.172,174,192
Metabolic reprogramming has been widely described in fibrotic diseases. Generally, increased glycolysis and decreased fatty acid metabolism in fibroblasts are the main manifestations of metabolic reprogramming,173 Succinate is a key regulator of glycolysis.173 A recent study has reported that the expression levels of succinate and its specific receptor SUCNR1 in both serum and intestinal tissue are significantly increased in CD patients when compared with non-CD patients. Additionally, fibroblasts isolated from damaged intestine of CD patients also displayed an enhanced expression of SUCNR1. Furtherm ore, fibroblasts treated with succinate dose-dependently increased mRNA expressions of pro-fibrotic factor (e.g., TGF-β), as well as fibrotic markers (e.g., COL1A1, α-SMA), implying that succinate may be a potential target for intestinal fibrosis.196
With regard to lipid metabolism, peroxisome proliferator-activated receptor-γ (PPAR-γ) is responsible for the uptake and oxidation of fatty acids and is recognized as an anti-fibrotic factor.173,197 Results from a mice model with intestinal fibrosis showed that the expression of PPAR-γ was significantly decreased in fibrotic colon. Additionally, the administration of PPAR-γ agonist (GED-0507-34 Levo) was able to reduce the production of collagens and the expression of pro-fibrotic molecules, as well as prevent TGF-β-induced EMT, thus attenuating fibrosis.197
Despite substantial progress have been achieved over the past decades in the understanding of cellular and molecular pathogenesis of fibrosis, ideal anti-fibrotic agents that specifically target intestinal fibrosis without significant side-effects have not been identified yet. Unravelling the inflammatory-independent mechanisms concerning pathogenesis of intestinal fibrosis, such as intestinal muscularis propria thickening, microbiota colonization and mesenteric fat hypertrophy, may open a new avenue to this perplexing issue.172,198 In addition, emerging methodology such as scRNA-seq has brought about new discoveries. For example, a deeper understanding of cell populations like fibroblasts or macrophages may reveal novel therapeutic points towards fibrosis.29,82 The past few years have also witnessed a rapid evolution of multi-omics analyses, which are able to integrate data across different levels of cellular organization, including genomes, epigenomes, transcriptomes, as well as proteomes. These multifaceted approaches provide an unprecedented opportunity to decode the complex mechanisms underlying intestinal fibrosis. Promising anti-fibrotic agents targeting intestine should be available in the near future.
This work was supported by the National Natural Science Foundation of China (NSFC grant numbers 81970483, and 82170537 to R.M.).
No potential conflict of interest relevant to this article was reported.
Gut and Liver 2023; 17(3): 360-374
Published online May 15, 2023 https://doi.org/10.5009/gnl220045
Copyright © Gut and Liver.
Xiaomin Wu , Xiaoxuan Lin , Jinyu Tan , Zishan Liu , Jinshen He , Fan Hu , Yu Wang , Minhu Chen , Fen Liu , Ren Mao
Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China
Correspondence to:Ren Mao
ORCID https://orcid.org/0000-0002-5523-8185
E-mail maor5@mail.sysu.edu.cn.
Fen Liu
ORCID https://orcid.org/0000-0001-9214-0016
E-mail liuf237@mail.sysu.edu.cn
Xiaomin Wu and Xiaoxuan Lin contributed equally to this work as first authors.
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.
Intestinal fibrosis associated stricture is a common complication of inflammatory bowel disease usually requiring endoscopic or surgical intervention. Effective anti-fibrotic agents aiming to control or reverse intestinal fibrosis are still unavailable. Thus, clarifying the mechanism underpinning intestinal fibrosis is imperative. Fibrosis is characterized by an excessive accumulation of extracellular matrix (ECM) proteins at the injured sites. Multiple cellular types are implicated in fibrosis development. Among these cells, mesenchymal cells are major compartments that are activated and then enhance the production of ECM. Additionally, immune cells contribute to the persistent activation of mesenchymal cells and perpetuation of inflammation. Molecules are messengers of crosstalk between these cellular compartments. Although inflammation is necessary for fibrosis development, purely controlling intestinal inflammation cannot halt the development of fibrosis, suggesting that chronic inflammation is not the unique contributor to fibrogenesis. Several inflammation-independent mechanisms including gut microbiota, creeping fat, ECM interaction, and metabolic reprogramming are involved in the pathogenesis of fibrosis. In the past decades, substantial progress has been made in elucidating the cellular and molecular mechanisms of intestinal fibrosis. Here, we summarized new discoveries and advances of cellular components and major molecular mediators that are associated with intestinal fibrosis, aiming to provide a basis for exploring effective anti-fibrotic therapies in this field.
Keywords: Inflammatory bowel diseases, Intestinal fibrosis, Immune system, Creeping fat, Gastrointestinal microbiota
Fibrosis is a dysregulated outcome of wound healing, especially during chronic inflammatory disorders.1 When inflammation is persistent, the severity of the damage may exceed the ability of the affected tissue to completely heal, which then initiates fibrotic response that eventually results in fibrosis.2,3 The gastrointestinal tract is a tubular structure and therefore fibrosis is presented with the narrowing of lumen and intestinal stricture.4
Intestinal stricture is a common complication of inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC).5,6 CD is a transmural disease that can affect the entire gastrointestinal tract, while UC is a superficial inflammatory disease, restricted to the colonic mucosa and submucosa layer.7 At initial diagnosis, at least 10% of CD patients are presented with a fibrostenosis phenotype.8 However, up to 50% of CD patients ultimately progress to stricturing or penetrating complications and 70% of patients require surgery within their life time.5,9 Even though stricture formation is rather infrequent in UC, recent evidence suggested fibrosis occurs in both acute and chronic UC.10 In the past decades, despite the availability and efficacy of biological therapies in IBD, the incidence of intestinal stricture does not achieve a significant reduction.11 This implies that pure anti-inflammatory treatments do not necessarily alleviate the associated fibrosis.
The review would provide a cellular and molecular biology of intestinal fibrosis (Fig. 1). Considering the close association between intestinal fibrosis and stricturing complications, understanding the pathogenesis of intestinal fibrosis is crucial to identify new anti-fibrotic targets for patients with intestinal strictures.
Intestinal fibrosis is driven by multiple cellular compartments including mesenchymal cells and immune cells.12 The histological feature of intestinal stricture is thickening of the muscularis mucosa and muscularis propria owing to the activation and proliferation of mesenchymal cells. Activated mesenchymal cells not only produce matrix components, but also secrete chemokines to recruit cells from the immune system (e.g., macrophages and T cells), thus perpetuating chronic inflammation. Reciprocal interaction between mesenchymal and immune cell populations in the intestine create a unique pro-fibrotic microenvironment, eventually resulting in fibrosis formation.13
Intestinal fibrosis results from sustained activation and proliferation of myofibroblasts.14 The activated myofibroblasts, as the final effector cells, can produce extracellular matrix (ECM) proteins and secrete cytokines such as interleukin (IL)-6 and IL-11, which facilitates formation of a fibrogenic milieu.15-17 The majority of myofibroblasts derive from resident fibroblasts and smooth muscle cells (SMCs). However, they can also originate from other cell types like epithelial and endothelial cells, pericytes, bone marrow stem cells and bone marrow-derived circulating fibrocytes.18,19 The various types of cells weave together in the inflamed intestine and contribute to the development of intestinal fibrosis.20
Fibroblasts are characterized by an elongated or spindle-shaped morphology, which are the most abundant cell type in connective tissue. Their main function is to maintain tissue integrity.21 Fibroblasts can be activated and multiply in response to pro-inflammatory mediators, such as insulin-like growth factor (IGF)-I, fibroblast growth factor, and IL-1β.22 The growth of fibroblasts can also be induced by immune cells or inflammatory cells through a cell-to-cell contact mechanism.23,24 In addition, fibroblasts can migrate to the site of inflammation foci along the concentration gradient of pro-inflammatory cytokines via activation of NF-κB and JAK-STAT signaling pathways.16,25,26 A previous study reported that fibroblasts isolated from inflamed or fibrotic CD tissue, or inflamed UC mucosa exhibited an increased proliferation, when compared with normal tissue.27 Recently, Wohlfahrt
Myofibroblasts are characterized by the expression of α-smooth muscle actin (α-SMA), with enhanced production of collagen and increased capacity of contraction.30 Although the exact molecular mechanism of myofibroblasts in fibrosis remains incompletely understood, mediators acting on myofibroblasts are clearly demonstrated, including pro-inflammatory cytokines, paracrine and autocrine factors (e.g., IGF-1), and pathogen or damage-associated molecular patterns.31 The activated myofibroblasts initiate fibrotic process in the following ways. Firstly, myofibroblasts secrete ECM components, as well as various cytokines and chemokines, directly or indirectly contributing to the thickening of mesenchymal cell layer.32,33 Secondly, myofibroblasts participate in tissue remodeling through mechanical contractions.34,35 Thirdly, the mechanic contraction of myofibroblasts can activate latent transforming growth factor-β1 (TGF-β1) released from ECM.36 TGF-β1 and its related pathways are major drivers in the process of fibrosis.37 Recently, de Bruyn
SMCs are one of the three interrelated cell phenotypes (the other two being fibroblasts and myofibroblasts).33 SMCs and fibroblasts are derived from the same primitive mesenchymal cells.39 SMCs are regarded as the progenitors of myofibroblasts.39 A dynamic equilibrium exists between SMCs and myofibroblasts phenotypes.39 SMCs can change their phenotypes in response to environmental stimulation.40 A previous study found that SMCs isolated from CD ileum presented alterations in morphology and contractile activity.41 It was demonstrated that SMCs isolated from CD ileum had an overexpression of platelet-derived growth factor (PDGF)-β, which drove the myogenic phenotype switch to synthetic one. The effect of PDGF-β was paralleled to a reduced encoding of contractile genes that were responsible for quiescent smooth muscle.41 SMCs are also able to release significant amounts of IL-6, contributing to inflammatory process.42 Besides, these cells actively contribute to the development of intestinal fibrosis by inducing the production of collagens and MMPs.41 These evidences suggested that the phenomenon of smooth muscle hyperplasia/hypertrophy in CD may be a driving force, rather than simply a passive increase of stricture formation.
Epithelial or endothelial-mesenchymal transition (EMT or EndMT) represents a dynamic entity where epithelial or endothelial cells transform to mesenchymal cells in response to inflammatory cytokines, oxidative stress and hypoxia.43-45 EMT is implicated in CD-associated fistulas and intestinal fibrosis.46,47 During formation of CD-associated fistulas, intestinal epithelial cells start with the dissociation from the base membrane and then migrate to the lining of the fistula tracts, where they convert to mesenchymal cells.47 In CD-associated intestinal fibrosis, EMT serves as a reservoir that can generate new fibroblasts and consequently result in fibrosis formation.46,48 Results from Iwano
Telocytes (TCs) are a novel type of interstitial cells characterized by CD34/PDGFRα, and have been demonstrated to be involved in several disorders including CD.54-56 The function of TCs is widely linked with other cells including mast cells, macrophages, myofibroblasts, and fibroblasts.57 Milia
Fibrocytes are bone marrow-derived mesenchymal progenitors, with the features of both hematopoietic (CD34) and fibroblast markers (collagen-I). Fibrocytes play a critical role in fibrotic diseases.59 Previous studies revealed that fibrocytes could be triggered by several inflammatory cytokines.59 Specifically, within four-day following injury, activated fibrocytes typically migrate into injured sites and then participate in fibrotic reactions through a direct way by production of ECM proteins and fibrogenic cytokines, or an indirect way by differentiation into myofibroblasts.60-62 Sazuka
A variety of key innate (macrophages) and adaptive (T cell subsets) immune cell types have been well-established in orchestrating the fibrotic microenvironment in intestine. The immune cell skewing in fibrosis niche probably perpetuates inflammation and exacerbates the process of wound healing.65 Here, we will discuss several immune cell subsets, pointing toward novel immune-based therapeutic strategies in fibrosis.
T helper 2 (Th2) cells are hallmarked by the secretion of cytokines IL-4, IL-5, and IL-13, which are responsible for type 2 immune responses.66 The type 2-associated cytokines are actively engaged in wound healing and fibrosis.65 At inflammatory sites, activated innate immune cells, such as group 2 innate lymphoid cells (ILC2) and basophils, are usually the early sources of local cytokines IL-4, IL-5, and IL-13, which trigger the activation and accumulation of Th2 cells.67,68 Th2 cells-derived IL-4 and IL-13 further promote the accumulation and proliferation of ILC2, thus creating a vicious cycle.69 Activated Th2 cells orchestrate the process of tissue fibrosis directly and indirectly by acting on immune or non-immune cells including local M2 macrophages, fibroblasts, endothelial cells and epithelial cells.70,71 Besides, as a well-known opponent of Th1 cells, Th2 cells can reverse the expression levels of Th1-associated anti-fibrotic cytokines such as interferon γ (IFN-γ).72 However, randomized controlled trials showed that blockade of IL-13 to target Th2 responses while administration of IFN-γ to stimulate Th1 responses failed to attenuate pulmonary fibrosis.73-75 Conversely, in a phase II trial, neutralization of IL-4/IL-13 effectively improved early skin fibrosis.76 The inconsistent results suggested targeting Th2 response as a therapeutic strategy for fibrosis requires further investigation.
Macrophages are highly heterogeneous and plastic cell populations, and are key regulators of tissue fibrosis in several organs.66,77 Generally, macrophages are classified into two subtypes: M1 macrophages with pro-inflammatory roles, and M2 macrophages with pro-fibrotic properties. The latter is activated by IL-4 and IL-13, and characterized by effects of inflammation resolution and tissue restoration.66 In intestine, STAT6-dependent M2 macrophages promote mucosal repair through activating Wnt signaling pathway.78 Moreover, macrophages from CD patients showed a significant enrichment in the expressions of M2-related as well as fibrotic-related genes, implying that M2 macrophages potentially exacerbated fibrosis formation.79 Results from STAT6 deficient colitis mice showed that the frequency of CD16+ macrophages was enhanced in the damaged mucosa of CD patients with stenotic or penetrating complications, and were also associated with the expression of fibrotic-related markers.80 Blockade of the interactions between inflamed macrophages and stromal cells has been proven to potentially ameliorate aberrant wound repair in zebrafish IBD model.81 Recently, the advanced scRNA-seq has revealed a novel macrophage subgroup, named with CX3CR1+SiglecF+ transitional macrophages, which are abundant in fibrotic niche and exhibit a pro-fibrotic effect in bleomycin-induced lung fibrosis.82 ScRNA-seq will be a promising technique to reveal the cellular heterogeneity of macrophages in fibrosis.
T helper 17 (Th17) cells are characterized by RAR-related orphan receptor γt (ROR-γt) expression and signatured by producing IL-17, IL-21, and IL-22 cytokines,83,84 which have fibrogenic properties. IL-17A, the predominant Th17-assciated cytokines, exerts its fibrotic effects via acting on myofibroblasts and regulating EMT.85-87 In gut, elevated levels of tissue Th17 cells and IL-17 production are observed in patients with intestinal stenosis.87 Recently, Paul
ILCs are a functionally diverse but developmentally related family of innate lymphocytes, with phenotypes and functions having striking similarities to T helper (Th) cells.95 According to cytokine signatures and transcription factors expression, ILCs are divided into three groups.96 Group 1 ILCs (ILC1) subsets share common properties with Th1 cells and express the transcription factor T-bet.97 Group 2 ILCs (ILC2) express the transcription factors RORα and GATA-3, which resembles Th2 cells functionally.98 Group 3 ILCs (ILC3), expressing transcription factor RORγt, are analogous to Th17 cells.99,100 ILC2 can respond rapidly to tissue damage, followed by an increase of Th2-like cytokines.101 An increased frequency of ILC2 has been detected in intestinal tissues from CD patients.102 Lo
Molecules are messengers of crosstalk between immune and non-immune cells and actively contribute to persistent inflammation.13 Although inflammation is a prerequisite of fibrosis, purely controlling intestinal inflammation cannot hold back the progression of fibrosis.8 This implies that inflammation is not the exclusive driver of fibrosis. In the following section, a detailed discussion concerning inflammation-dependent, but with a focus on inflammation-independent factors of fibrosis, will be reported.
Cytokines and chemokines secreted by immune and non-immune cells are orchestrators of sustained inflammatory microenvironment, and are also observed to possess pro-fibrotic effects, which lay foundations for uncovering novel therapeutic targets in fibrotic disorders.142 Several novel cytokines involved in fibrogenesis will be detailly described in this part, and the elaborate profile of cytokines and chemokines are shown in Table 1.
Table 1 . Cytokine and Chemokine Profiles Involved in Intestinal Fibrosis.
Cytokine | Pro- or anti-fibrosis | Effects on intestinal fibrosis | References |
---|---|---|---|
IL-1 | Pro | Induce fibroblasts activation; enhance pro-fibrotic cytokines production; inhibit ECM degradation; enhance collagens expression | 104-106 |
IL-4 | Pro | Promote myofibroblasts activation; promote type 2 immunity-induced fibrosis | 107,108 |
IL-6 | Pro | Promote SMCs activation; promote fibroblasts activation and proliferation; enhance ECM production | 109-111 |
IL-10 | Uncertain | Inhibit collagens deposition; no effects on fibroblasts and myofibroblasts | 112,113 |
IL-11 | Pro | Enhance collagens expression; may promote SMCs hyperplasia | 114 |
IL-12 | Pro | Promote inflammation | 115,116 |
IL-13 | Pro | Promote TGF-β1 production; initiate fibrosis | 117,118 |
IL-17 | Pro | Stimulate myofibroblasts activation; enhance collagens expression; decrease ECM degradation; induce EMT | 85,89,119 |
IL-21 | Uncertain | Facilitate Th2 and Th17 development; enhance MMPs secretion | 120,121 |
IL-22 | Pro | Inhibit inflammation; enhance MMPs secretion; promote myofibroblasts differentiation | 122-124 |
IL-23 | Pro | Promote inflammation; promote fibrotic responses | 115,116,124 |
IL-25 | Non | IL-13 production depends on IL-25; induce type 2 immunity; promote inflammation | 125,126 |
IL-33 | Pro | Promote pro-fibrotic type 2 immunity | 127,128 |
IL-34 | Pro | Enhance collagens expression; promote fibroblasts activation | 129 |
IL-36 | Pro | Enhance collagens expression; promote fibroblasts activation | 130 |
TL1A | Pro | Enhance collagens expression and pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 131,132 |
IFN-γ | Anti | Inhibit TGF-β signaling; inhibit fibroblasts migration and myofibroblasts differentiation | 133-135 |
TGF-β | Pro | Enhance pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 43,136-138 |
CXCR4 | Pro | Mediate pro-fibrotic effects of PDGF-C | 139 |
CCL11 | Pro | Induce eosinophils recruitment and in turn promote fibroblasts activation | 140 |
CXCL8 | Pro | Promote pro-fibrotic growth factors production; enhance MMPs secretion | 141 |
IL, interleukin; ECM, extracellular matrix; SMCs, smooth muscle cells; TGF, transforming growth factor; EMT, epithelial-mesenchymal transition; Th, T helper; MMPs, matrix metalloproteinases; TL1A, tumor necrosis factor-like ligand 1A; IFN, interferon; CXCR4, C-X-C motif chemokine receptor 4; PDGF-C, platelet-derived growth factor-C; CCL11, C-C motif chemokine ligand 11; CXCL8, C-X-C motif chemokine ligand 8..
IL-11, a member of IL-6 family, is recognized as a pro-fibrotic cytokine secreted by stromal cells, as well as epithelial cells during tissue injuries.143 IL-11 is upregulated in various fibro-inflammation disorders.143 Ng
IL-33, a member of IL-1 superfamily, is passively released upon cellular damage and necrosis and is thus considered as an alert of inflammation.147 IL-33 is also involved in the process of fibrogenesis.148 Binding of IL-33 to its receptor ST2 triggers activation of Th2 cells to produce amphiregulin, which then drove osteopontin production by eosinophils, thus forming IL-33-amphiregulin-osteopontin axis. The axis conferred to fibrotic responses in eosinophilic airway inflammation.149 With regard to intestine, the expression of IL-33 and ST2 were upregulated in mucosa from UC patients and dextran sulfate sodium (DSS) colitis model.136,137 In particular, elevated epithelial expression of IL-33 was strongly associated with fibrosis progression in pediatric Crohn’s ileitis.138 A recent study by Imai
IL-34, a member of 4-helical cytokine family, is produced by a wide range of cells including fibroblasts, immune cells, epithelial cells, endothelial cells and adipocytes.150 The association of aberrant high expression of IL-34 and fibrosis has been identified in several organs, including liver, kidney, and gut.129,151,152 Production of IL-34 was enhanced in inflamed mucosa in IBD patients and in DSS-induced colitis, which was regulated by tumor necrosis factor-α (TNF-α) via NF-κB signaling.153,154 Notably, the expression of IL-34 was elevated in fibrostrictures sites of CD.129 It was observed that activated fibroblasts by TNF-α exhibited increased expression of IL-34.129,155 Besides, fibroblasts stimulated with IL-34 could enhance expression of COL1A1 and COL3A1, while this effect disappeared in fibroblast-specific IL-34 knockout mice.129 These evidences raise a possibility that fibroblast is a cellular target of IL-34.
IL-36, also belonging to IL-1 superfamily, consists of five isoforms: IL-36α, IL-36β, IL-36γ, IL-36Ra, and IL-38.156,157 Among them, IL-36α, IL-36β, and IL-36γ play pro-inflammatory effects through activating IL-36 receptor (IL-36R) signaling, while IL-36Ra and IL-38 have opposing effects as they are inhibitors of IL-36R signaling.156,158 It is demonstrated that IL-36α and IL-36γ are elevated in both CD and UC mucosa under inflammation stimuli.158,159 Of note, IL-36α had an increased expression in tissues of CD fibrostenosis.130 Stimulation of IL-36α and IL-36γ can induce fibroblasts activation and epithelial cells proliferation,159,160 which is associated with an enhancement of collagen-VI secretion and ultimately fibrosis development.130 Importantly, both IL-36R blockade and IL-36 genes knockout are sufficient to attenuate intestinal fibrosis, which highlights the therapeutic values of IL-36 in fibrosis.130
Tumor necrosis factor-like ligand 1A (TL1A) is a member of TNF superfamily and interacts with death receptor-3 (DR3) to form TL1A/DR3 co-stimulatory system.161 Aberrant TL1A/DR3 signaling is involved in chronic inflammation and fibrogenesis.162-165 CD patients with higher expression of serum TL1A were prone to develop stricture.166 Another study found that CD patients presenting with specific TL1A genotype rs6478108 were susceptible to forming stricturing phenotype.167
Recently, several inflammation-independent mechanisms including ECM interaction, creeping fat (CrF), gut microbiota, as well as metabolic reprogramming, have attracted much attention because of their unique roles in intestinal fibrosis, which will be discussed in the following part.14,172,173
ECM is a highly specialized and dynamic three-dimensional scaffold in tissue, which is an active player rather than a purely passive player in fibrosis.14 ECM comprises a variety of fibrous components such as collagens, hyaluronan (HA) and fibronectin.174 HA exists as a high-molecular-weight polymer in normal conditions. During excessive inflammation, the polymer is cleaved to fragments of lower molecular weight, which promotes fibroblasts proliferation and myofibroblasts differentiation, thus contributing to fibrosis process. Besides, HA in low molecular weight fragments aids in recruiting immune cells to inflammatory sites, which in turn release a variety of inflammatory mediators and growth factors to initiate fibrosis.175-177 Fibronectin can enhance the susceptibility of SMCs to proliferation through combining with αVβ3 integrin.178 Additionally, the phenotype and function of myofibroblasts are altered along with the increased ECM stiffness. Myofibroblasts isolated from stenotic intestine display enhanced contractility of ECM and decreased activity of MMP-3, resulting in a vicious circle that further leads to tissue rigidity.38 Increased ECM stiffness is also able to drive fibroblasts to produce more ECM proteins through the Hippo and yes-associated protein pathway.14
CrF indicates that mesenteric fat wrapping around more than 50% of the intestinal circumference, which is the unique hallmark of CD.179 Although CrF was first described nearly 100 years ago, whether CrF is pathogenic or protective is still a controversy.179 Previous studies found that CrF was associated with the severity of intestinal inflammation and strictures.180 Inclusion mesentery in ileocolic resection achieved a reduction of stricture recurrence and reoperation.181,182 However, Ha
The relationship between CrF and intestinal fibrosis is still underexplored. Our previous study has uncovered a positive feedback loop between CrF and intestinal muscularis propria.184 Firstly, CrF-derived long-chain free fatty acids significantly enhanced proliferation and activation of intestinal muscle cells, with increased production of ECM proteins and strictures formation subsequently.184,185 Vise versa, hypertrophic muscularis propria triggered migration of preadipocytes out of MAT by fibronectin production, which facilitated CrF development.186 Another study observed that adipocytes within CrF were capable to convert to fibroblasts, whereas selective ablation of CrF adipocytes attenuated collagen deposition and bowel wall thickening.187 Noteworthily, new animal models via repeated colonic biopsy or antimesenteric enterotomy have been recently established,188,189 which will make a big difference for uncovering the complex relationship between CrF and intestinal fibrosis in the future.
Accumulating evidence indicates that gut microbiota plays crucial roles in fibrosis.8 The direct evidence was that experimental mice when reared under specific-pathogen-free conditions displayed a minimal inflammation, whereas when injected bacteria or bacterial wall components they exhibited inflammation and fibrosis.190 The terminal ileum is the most common site of intestinal stricture, where AIEC mainly colonize, giving us a hint that AIEC may be correlated with fibrosis development.191 Indeed, research using mice with AIEC inoculation found that flagellin of AIEC via IL-33-ST2 signaling facilitated intestinal fibrosis.127 Intestinal fibroblasts isolated from CD patients are observed to have an increased expression of several Toll-like receptors (TLRs) including 2, 3, 4, 6, 7.192 It is known that TLRs can be activated by perceiving microbial components, which are called pathogen-associated molecular patterns.172 The activated TLRs then promote the differentiation of fibroblasts into myofibroblasts.192 For example, TLR-3 activation in fibroblasts can augment α-SMA expression and TGF-β1 production via NF-κB signaling.193 Additionally, lipopolysaccharide activating TLR-4 can also stimulate α-SMA expression and collagen synthesis in fibroblasts.194,195 In conclusion, when exposed to pathogen-associated molecular patterns, intestinal myofibroblasts expressed upregulated levels of α-SMA and increased production of ECM proteins, thus confirming the link between gut microbiota and intestinal fibrosis.172,174,192
Metabolic reprogramming has been widely described in fibrotic diseases. Generally, increased glycolysis and decreased fatty acid metabolism in fibroblasts are the main manifestations of metabolic reprogramming,173 Succinate is a key regulator of glycolysis.173 A recent study has reported that the expression levels of succinate and its specific receptor SUCNR1 in both serum and intestinal tissue are significantly increased in CD patients when compared with non-CD patients. Additionally, fibroblasts isolated from damaged intestine of CD patients also displayed an enhanced expression of SUCNR1. Furtherm ore, fibroblasts treated with succinate dose-dependently increased mRNA expressions of pro-fibrotic factor (e.g., TGF-β), as well as fibrotic markers (e.g., COL1A1, α-SMA), implying that succinate may be a potential target for intestinal fibrosis.196
With regard to lipid metabolism, peroxisome proliferator-activated receptor-γ (PPAR-γ) is responsible for the uptake and oxidation of fatty acids and is recognized as an anti-fibrotic factor.173,197 Results from a mice model with intestinal fibrosis showed that the expression of PPAR-γ was significantly decreased in fibrotic colon. Additionally, the administration of PPAR-γ agonist (GED-0507-34 Levo) was able to reduce the production of collagens and the expression of pro-fibrotic molecules, as well as prevent TGF-β-induced EMT, thus attenuating fibrosis.197
Despite substantial progress have been achieved over the past decades in the understanding of cellular and molecular pathogenesis of fibrosis, ideal anti-fibrotic agents that specifically target intestinal fibrosis without significant side-effects have not been identified yet. Unravelling the inflammatory-independent mechanisms concerning pathogenesis of intestinal fibrosis, such as intestinal muscularis propria thickening, microbiota colonization and mesenteric fat hypertrophy, may open a new avenue to this perplexing issue.172,198 In addition, emerging methodology such as scRNA-seq has brought about new discoveries. For example, a deeper understanding of cell populations like fibroblasts or macrophages may reveal novel therapeutic points towards fibrosis.29,82 The past few years have also witnessed a rapid evolution of multi-omics analyses, which are able to integrate data across different levels of cellular organization, including genomes, epigenomes, transcriptomes, as well as proteomes. These multifaceted approaches provide an unprecedented opportunity to decode the complex mechanisms underlying intestinal fibrosis. Promising anti-fibrotic agents targeting intestine should be available in the near future.
This work was supported by the National Natural Science Foundation of China (NSFC grant numbers 81970483, and 82170537 to R.M.).
No potential conflict of interest relevant to this article was reported.
Table 1 Cytokine and Chemokine Profiles Involved in Intestinal Fibrosis
Cytokine | Pro- or anti-fibrosis | Effects on intestinal fibrosis | References |
---|---|---|---|
IL-1 | Pro | Induce fibroblasts activation; enhance pro-fibrotic cytokines production; inhibit ECM degradation; enhance collagens expression | 104-106 |
IL-4 | Pro | Promote myofibroblasts activation; promote type 2 immunity-induced fibrosis | 107,108 |
IL-6 | Pro | Promote SMCs activation; promote fibroblasts activation and proliferation; enhance ECM production | 109-111 |
IL-10 | Uncertain | Inhibit collagens deposition; no effects on fibroblasts and myofibroblasts | 112,113 |
IL-11 | Pro | Enhance collagens expression; may promote SMCs hyperplasia | 114 |
IL-12 | Pro | Promote inflammation | 115,116 |
IL-13 | Pro | Promote TGF-β1 production; initiate fibrosis | 117,118 |
IL-17 | Pro | Stimulate myofibroblasts activation; enhance collagens expression; decrease ECM degradation; induce EMT | 85,89,119 |
IL-21 | Uncertain | Facilitate Th2 and Th17 development; enhance MMPs secretion | 120,121 |
IL-22 | Pro | Inhibit inflammation; enhance MMPs secretion; promote myofibroblasts differentiation | 122-124 |
IL-23 | Pro | Promote inflammation; promote fibrotic responses | 115,116,124 |
IL-25 | Non | IL-13 production depends on IL-25; induce type 2 immunity; promote inflammation | 125,126 |
IL-33 | Pro | Promote pro-fibrotic type 2 immunity | 127,128 |
IL-34 | Pro | Enhance collagens expression; promote fibroblasts activation | 129 |
IL-36 | Pro | Enhance collagens expression; promote fibroblasts activation | 130 |
TL1A | Pro | Enhance collagens expression and pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 131,132 |
IFN-γ | Anti | Inhibit TGF-β signaling; inhibit fibroblasts migration and myofibroblasts differentiation | 133-135 |
TGF-β | Pro | Enhance pro-fibrotic molecules production; promote fibroblasts activation; induce EMT | 43,136-138 |
CXCR4 | Pro | Mediate pro-fibrotic effects of PDGF-C | 139 |
CCL11 | Pro | Induce eosinophils recruitment and in turn promote fibroblasts activation | 140 |
CXCL8 | Pro | Promote pro-fibrotic growth factors production; enhance MMPs secretion | 141 |
IL, interleukin; ECM, extracellular matrix; SMCs, smooth muscle cells; TGF, transforming growth factor; EMT, epithelial-mesenchymal transition; Th, T helper; MMPs, matrix metalloproteinases; TL1A, tumor necrosis factor-like ligand 1A; IFN, interferon; CXCR4, C-X-C motif chemokine receptor 4; PDGF-C, platelet-derived growth factor-C; CCL11, C-C motif chemokine ligand 11; CXCL8, C-X-C motif chemokine ligand 8.