Fibrosis in IBD: from pathogenesis to therapeutic targets

Abstract

Intestinal fibrosis resulting in stricture formation and obstruction in Crohn’s disease (CD) and increased wall stiffness and symptoms in ulcerative colitis (UC) are among the largest unmet needs in inflammatory bowel diseases (IBD). Fibrosis is caused by a multifactorial and complex process involving immune and non-immune cells, their soluble mediators, and exposure to luminal contents, such as microbiota and environmental factors. To date, no antifibrotic therapy is available. Progress has been made in creating consensus definitions and measurements to quantify stricture morphology for clinical practice and trials, but approaches to determine the degree of fibrosis within a stricture are still lacking. We herein describe the current state of stricture pathogenesis, measuring tools, and clinical trial endpoints development.

Introduction

Inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative colitis (UC), is commonly accompanied by intestinal fibrosis (IF) [1, 2, 3]. In CD, fibrosis presents with strictures and obstructive symptoms [with the majority (>50%) of patients experiencing obstructive symptoms at least once in their lifetime] [4], which often necessitates surgical interventions. In UC, fibrosis may induce urgency and motility abnormalities [5, 6]. Deposition of extracellular matrix (ECM) is a physiological process required for tissue repair, but it becomes excessive and uncontrolled over time in IBD, causing tissue stiffness and functional impairment[7]. Fibroblasts and myofibroblasts are the major source of excessive ECM, which accumulates in a time-dependent manner, but thickening of the muscularis propria is now believed to be the main cause of luminal obstruction and both areas believed to occur in parallel[8, 9] (Figure 1). Currently there is no specific treatment for IF. In this review, we discuss recent advances in basic and clinical science in IF and suggest a roadmap for identifying new therapeutic targets and translating them into improved care.

Figure 1.

Model for progressive accumulation of extracellular matrix and thickening of the muscularis propria, despite a relapsing and remitting chronic inflammatory disease course. Initial inflammation dependent pathways given way to inflammation independent pathways and then both likely occur in parallel. Abbreviations: ECM, extracellular matrix.

Mechanisms of intestinal fibrogenesis

While excessive ECM deposition and scar formation are a hallmark feature of IBD[10, 11], IF is present even in the absence of stricture formation and presents at different degrees of severity [5, 6, 12]. The ECM-producing intestinal mesenchymal cells comprise a heterogenous cell population, which is classically separated into fibroblasts, myofibroblasts, and smooth muscle cells (SMC), but this classification is under revision with the advent of single cell RNA sequencing (scRNAseq) (Figure 2, Table 1). Mesenchymal cells increase in number in IF and their origin and function is detailed below.

Figure 2.

Cellular sources of activated intestinal mesenchymal cells and their capability to transdifferentiate into fibroblasts, myofibroblasts and smooth muscle cells. Abbreviations: EMT, epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition

Table 1: Classical mouse and human cellular markers of cell types contributing to the pool of fibroblasts in intestinal fibrosis.

All cell types function in the context of intestinal repair.

	Markers
Cell type	Present	Absent
Fibroblast	Vimentin, FSP-1, N-cadherin (high), collagen type I, CD90	Markers of endothelial or epithelial cells, α-SMA, desmin
Myofibroblast	α-SMA, vimentin, FSP-1, collagen type I, CD90, desmin (low or absent)	Markers of endothelial or epithelial cells
Smooth muscle cell	Desmin, vimentin (low), α-SMA, collagen type I	Markers of endothelial or epithelial cells, CD90
Pericyte	α-SMA, desmin, HMW-MAA, aminopeptidase N, collagen type I	Markers of endothelial or epithelial cells
Fibrocyte	CD34, CD45, CD11b, CD13, collagen type I, α-SMA	CD90
Endothelial cell	CD31, vWF, VE-cadherin, N-cadherin (low), vimentin (low)	Markers of fibroblasts, myofibroblasts or smooth muscle cells
Epithelial cell	E-cadherin, cytokeratins	Markers of fibroblasts, myofibroblasts or smooth muscle cells

Abbreviations: CD, cluster of differentiation; FSP-1, fibroblast-specific-protein 1; HMW-MAA, high molecular weight myeloma-associated antigen; GFAP, glial fibrillary acidic protein; α-SMA α-smooth muscle actin; VE-cadherin, vascular endothelial cadherin; vWF, von Willebrand Factor. High and low indicate expression level. Updated and adapted from [17].

Mesenchymal cells and their sources

Intestinal mesenchymal cells are non-epithelial, non-endothelial, non-hematopoietic cells originating from mesenchymal stem cells. Their major function is to provide the scaffold in which all other cells reside in, thereby shaping tissue structure [13]. Regardless of their phenotype, all mesenchymal cells are believed to contribute to fibrosis [14].

Fibroblasts, myofibroblasts, and smooth muscle cells.

Cell surface and intracellular markers allow differentiation of intestinal fibroblasts [CD90 (+), vimentin (+), desmin (-)]; myofibroblasts [CD90 (+), vimentin (+), α-smooth muscle actin (SMA) (+), desmin (- or low)]; or SMC [CD90 (-), vimentin (low), α-SMA (+), desmin (+)] [14, 15], but all phenotypes transition into one another in the inflamed intestine[16]. Upon exposure to transforming growth factor (TGF)-β1, α-SMA-poor fibroblasts differentiate into α-SMA-rich, myofibroblast-like cells[17, 18]; SMC can transdifferentiate into myofibroblasts [19]; and myofibroblasts can transdifferentiate into SMC exhibiting enormous plasticity (Figure 2). [9] In IBD, fibroblasts, myofibroblasts, and SMC increase in numbers and migrate to sites of injury where they lay down ECM. Traditionally, myofibroblasts are considered the primary source of ECM [20], but this paradigm may change with recent results from scRNAseq [21].

Pericytes.

Pericytes [α-SMA (+), desmin(+)] surround tiny blood endothelial cells [20, 22], and exhibit an intermediate phenotype between fibroblasts and vascular SMC [14]. Upon activation by inflammation-driven mediators, pericytes differentiate into fibroblasts and contribute to the pool of ECM-producing fibroblasts [23]. The mechanism by which pericytes contribute to IF is unclear, but increased numbers of activated pericytes have been described in UC [24] (Figure 2).

Fibrocytes.

Fibrocytes (bone marrow-derived mesenchymal stem cells) [25] reside in the systemic circulation but traffic to inflamed sites where they differentiate into fibroblasts/myofibroblasts [26, 27]. Data on the functional contribution of fibrocytes to IF are lacking, but their circulating numbers are increased in CD and UC [27, 28, 29] (Figure 2).

Epithelial- and endothelial-to-mesenchymal cell transition (EMT and EndMT).

In EMT and EndMT, mature epithelial or endothelial cells transdifferentiate into mesenchymal cells by expressing the latter’s typical markers, such as neural cadherin (N-cadherin), α-SMA, and vimentin, as well as exhibiting mesenchymal functions like production of fibronectin and collagens and migratory capabilities [30, 31, 32]. Initially established in kidney, liver, heart and lung fibrosis [33], EMT also occurs in IBD[33], irrespective of the degree of inflammation or phenotype (stricturing or penetrating) [34, 35]. Human intestinal microvascular endothelial cells (HIMECs) also undergo EndMT in experimental and human IBD [36] (Figure 2).

Cell heterogeneity in intestinal fibrosis

Single-cell approaches are revolutionizing our understanding of fibrotic diseases [37] by enabling the characterization of multiple cell types’ transcriptional activity [37]. Examples include the identification of a novel mesenchymal cell type [Pdgfrb^high] in lung fibrosis [38], heterogeneity of hepatic stellate cells (HSC), ACKR1+ and PLVAP+ endothelial cells and PDGFRα+ collagen-producing myofibroblasts in liver fibrosis [39, 40, 41, 42], and the contribution of monocytes to the pool of myofibroblasts [PDGFRβ+CD45+] in kidney fibrosis [43].

In IBD, 4 distinct subsets of fibroblasts have been identified in UC, including an activated fibroblast population expressing interleukin (IL)-33, lysyl oxidases (LOX), TNFSF14, and fibroblastic reticular cell-associated genes [44]. In anti-TNF resistant ileal CD, a GIMATS cellular module is present that contains cell positive for THY1 (CD90), podoplanin (PDPN), CHI3L1 and collagen triple-helix repeat-containing 1 (CTHRC1+) fibroblasts [45], the latter also being identified in pulmonary fibrosis [46].

Most recently, the first full-thickness scRNA atlas of stricturing CD was reported. Key observation was robust fibroblast heterogeneity in stricturing CD. Most transcriptional changes occurred in the mucosa and submucosa of stricturing CD compared to controls, with lesser changes in the muscularis propria. Specific fibroblast populations, such as chemokine (C-X-C motif) ligand (CXCL)14+ and matrix metalloprotease (MMP)/WNT5A+ fibroblasts were either increased in number or had upregulated transcriptional activity in CD strictures compared to non-strictured but inflamed and non-inflamed tissues from the same patient. While multiple different cell types acted as signal receivers in a cell-cell interaction analysis, fibroblasts emerged as the major signal-sending cell type in CD strictures and exhibited cell-cell interactions with immune and non-immune cells [21]. This is relevant as it not only opened the door for targeting fibroblast populations overrepresented in CD strictures, but also established fibroblasts as not only a signal receiver, but also as a sender It has to be noted however, that the exact location of the different fibroblast populations still has to be determined. In addition, likely all layers of the intestinal wall, including lamina propria and muscularis mucosa, contribute to fibrogenesis due to the presence of immune and non-immune cells in all intestinal layers. Figure 3 provides a summary of mechanisms of intestinal fibrosis.

Figure 3.

Recently described mechanisms of intestinal fibrogenesis. For clarity of presentation the lamina propria and muscularis mucosa were not delineated separately, which is not meant to diminish their contribution to the process of intestinal fibrosis. Abbreviations: IL, interleukin; TLR, Toll-like receptor; LOX, lysyl oxidases; TNFSF14, tumor necrosis factor superfamily member 14; PDPN, podoplanin; CTHRC1, collagen triple-helix repeat-containing 1; CHI3L1, chitinase-3-like 1.

Fibroblast-activating mediators in intestinal fibrosis

In intestinal inflammation, mesenchymal cells are exposed to a complex microenvironment consisting of luminal contents and multiple inflammatory mediators, such as cytokines and growth factors released by immune cells and non-immune cells. This rich mediator milieu is most likely critical for initiation of IF, but may also perpetuate it [47]. This is supported by the observations that: (a) the location of strictures follows the location of disease, (b) degree of fibrosis correlates with the degree of inflammation and (c) neither in UC nor in CD has fibrosis been reported in areas not affected by inflammation. In the following sections, key mediators of fibrosis are briefly discussed. The most recently described mediators are also delineated in Figure 3 & Box 1.

Box 1. Examples for pro-fibrogenic and anti-fibrogenic mediators in gut fibrosis.

Pro-fibrogenic mediators

TGF-β1 [50, 51, 52, 53]

IL-1 [55, 63]

IL-4 [121, 122]

IL-6 [82, 83, 84]

IL-11 [85, 90]

IL-13 [116, 117, 125]

IL-17 [110, 111, 112]

IL-33 [64, 65, 66, 67] [68]

IL-34 [135]

IL-36 [78, 79]

TL1A [99, 103, 104, 105]

CTGF [171] [3]

IGF-1 [171] [3]

Cadherin 11[21, 148]

Flagellin [68, 167]

Altered ECM composition, e.g. hyaluronan [139, 141, 142]

Increased ECM stiffness [144, 145, 146]

Anti-fibrogenic mediators

IFN-γ [3]

IL-12 [3]

Altered ECM composition, e.g. MFGE8 [140]

Abbreviations: CTGF, connective tissue growth factor; ECM, extracellular matrix; IFN, interferon; IGF, insulin like growth factor; IL, interleukin; MFGE8, milk fat globule-EGF factor 8; TGF, transforming growth factor; TL1A, tumor necrosis factor like cytokine 1A

TGF-β1

TGF-β1 is widely regarded as the dominant pro-fibrotic growth factor, and is upregulated in essentially all fibrotic diseases[48], including CD[49]. In CD, tissue TGF-β1 gene expression levels and phosphorylated SMAD2/3 (positive regulators of TGF-β1) are upregulated in myofibroblasts that overlie strictured (but not non-strictured) areas, while its SMAD7, (negative regulator of TGF-β1) is down-regulated in the same areas [50]. TGF-β1 upregulates α-SMA, collagen I, and fibronectin in human fibroblasts; SMC; and in intestinal organoid-associated mesenchymal cells [51]. Role of TGF-β1 in fibrosis is also underscored by the observation that vaccination against TGF-β1 attenuates fibrosis while inhibiton of SMAD7 increases fibrosis in experimental murine colitis [52, 53].

IL-1 family of cytokines

The cytokines of the IL-1 [IL-1α, IL-1β, IL-33, and IL-1Ra], IL-18 [IL-18 and IL-37], and IL-36 [IL-36Ra, IL-36α, β, γ and IL-38] families also exert pro-fibrotic properties [54]. Epithelial-derived IL-1α is a member of damage-associated molecular patterns (DAMPs) released by multiple cell types [55] that can activate fibroblasts and mediate intestinal inflammation [56]. IL-1α-deficiency or neutralization ameliorate experimental colitis course, while IL-1β neutralization does not [57]. The main source of IL-1β are mononuclear phagocytes, and IL-1β also acts in a pro-inflammatory fashion in the gut [47]. IL-1β signals through IL-1R1 and MyD88 and displays profibrotic activity in experimental lung fibrosis [58, 59, 60], HSC [61], and kidney stromal cells [62]. In the intestine, myofibroblasts exposed to IL-1β upregulate production of collagens I and IV, IL-8, monocyte chemoattractant protein (MCP)-1, and MMP-1 [63], which are the key components of the ECM.

IL-33 is also produced by immune and non-immune cells, including fibroblasts and SMC[64], and mediates pro-inflammatory and type 2 helper T cell (Th2)-associated immune responses through its receptor ST2. Th2 responses have been linked to fibrotic diseases in essentially all organs, and IL-33/ST2-signaling induces EMT and fibroblast proliferation[65]. In the human gut, IL-33 is mainly expressed by fibroblasts, SMC, and epithelial cells, and in UC, expression of IL-33 and ST2 are increased[64, 66, 67]. The IL-33/ST2 axis induces IF in a toll-like receptor (TLR)5- and NOD-like receptor family CARD domain-containing protein (NLRC) 4-dependent manner[68].

All isoforms of IL-36 (α, β, γ) and IL-36 receptor antagonist (IL-36RA) are produced by many different cell types, including fibroblasts and myofibroblasts [69, 70], and act on a wide range of immune and non-immune cells, exerting pro-inflammatory and pro-fibrotic effects [55, 69, 70, 71, 72, 73, 74, 75, 76, 77]. IL-36α and IL-36γ, but not IL-36β, are elevated in UC mucosa [76, 77]. Similarly, IL‐36α and IL-36γ are elevated in CD [78]. Additionally, the IL36 receptor (IL-36R) is increased in stricturing CD[79]. IL-36R activation increased production of collagen VI [79] and IL-36R blockage ameliorated experimental dextran sulfate sodium (DSS) - or trinitrobenzene sulfonic acid (TNBS) [79] (Figure 3).

IL-6 family of cytokines

All members of the IL-6 family of cytokines (IL-6, IL-11, oncostatin M, ciliary neurotrophic factor, cardiotrophin 1, leukemia inhibitory factor, cardiotrophin-like cytokine, and IL-27) activate a common gp130 signaling receptor subunit [80]. The role and sources of IL-6 (mononuclear, epithelial, and mesenchymal cells) are well established in IBD [47, 81].In other fibrotic diseases, IL-6 acts in a pro-inflammatory and pro-fibrotic way, having been implicated in idiopathic pulmonary fibrosis IPF [82] and liver fibrosis [83]. IL-6 is increased in the muscle layer of strictured CD and can be induced by TGF-β1, and IL-6 neutralization normalizes expression of TGF-β1[84].

Mesenchymal cell- and epithelial cell-derived IL-11[85, 86] can act in a pro-fibrogenic fashion through paracrine activation of IL-11RA on mesenchymal cells[87, 88], promoting fibrosis in several organs, including lung, liver, heart, and skin [87, 88, 89]. Recent scRNAseq sequencing studies identified a fibroblast subtype with elevated expression of IL-11, IL-24, and IL-13RA2 in UC, highlighting this signaling pathway in IBD [90]. IL-11 overexpression in either fibroblasts or SMC induces experimental intestinal inflammation and IF [85].

TNF family of cytokines

TNF and TL1A (TNF-like ligand 1A) are known drivers of intestinal inflammation[47, 91]. They also indirectly drive IF by furthering inflammation or by acting directly on mesenchymal cells[47, 91]. TNF promotes fibrosis by increasing collagen I and IV production by intestinal myofibroblasts[63] and upregulating the expression of TGF-β and tissue inhibitor of matrix metalloproteinases 1 (TIMP1) in colonic epithelial cells [92]. Inhibition of TNF has anti-inflammatory properties in CD and UC [93, 94] and can increase myofibroblast migration, decrease collagen production [95], and reduce experimental IF [96]. However, clinically, anti-TNF antibodies do not prevent CD strictures[91]. In pediatric CD patients, early anti-TNF therapy prevents internal penetrating disease (but not strictures) [97].

Most recently, TL1A, which is produced by immune cells, epithelial cells, and fibroblasts and binds to the Death Receptor 3 (DR3) was found to be upregulated in IBD, at both protein and mRNA level[98, 99, 100]. TL1A has gained attention as a therapeutic target due to positive phase 2 results in CD and UC[101]. CD patients with high expression of TL1A in the periphery tend to also exhibit intestinal strictures and worsening ileocecal inflammation with relative sparing of the rectosigmoid [102]. These findings are corroborated by animal studies, as transgenic mice constitutively expressing TL1A in lymphoid or myeloid cells display intestinal and colonic fibrosis [103, 104] and antibody-mediated TL1A blockade can reduce intestinal inflammation and fibrosis[105]. TL1A seems to contribute to IBD pathogenesis via local, but not systemic, induction of IL-17A [106]. Furthermore, there is some evidence that microbes contribute to TL1A-mediated IF and fibroblast activation, which may be dependent on specific microbial populations [99]. Primary intestinal myofibroblasts express DR3 and respond to direct TL1A signaling by increasing collagen production [107]. Moreover, treatment with neutralizing TL1A antibodies results in lowered expression of TGF-β1 and a reduced number of fibroblasts and myofibroblasts, suggesting reversal of established murine colonic fibrosis [107] (Figure 2).

IL-17 and the Th17 response

IL-17 cytokines, primarily produced by Th17 cells, consist of 6 related proteins: IL-17A (also called IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F, all of which signal through 5 receptor subunits (IL-17RA - IL-17RE) [108]. In humans, IL-6, IL-21, IL-23, IL-1β, and TGF-β are critical for Th17 development and, in addition to IL-17A-F, Th17 cells also produce IL-21, IL-22, IL-9, IL-26, TNF-α, and chemokine (C-C motif) ligand 20 (CCL20) [109]. Evidence demonstrates the regulatory role of IL-17 in the development of fibrosis in multiple organs, such as the liver, skin, lung and heart [20, 91], but its contribution to IBD and IF is less clear because of IL-17’s multiple actions in multiple cell types [109].

IL-17A is significantly overexpressed in CD strictures compared with non-strictured areas [110], and IL-17A significantly inhibits myofibroblast migration and promotes the production of MMP-3, MMP-12, TIMP-1, and collagen by myofibroblasts from strictured CD tissues [110]. The high levels of IL-17 in the intestinal mucosa of CD patients may contribute to fibrosis by also inducing EMT in intestinal epithelial cells [111]. In the TNBS-induced IF mouse model, treatment with anti-IL-17 antibody alleviates IF and reduces both mRNA and protein levels of collagen III, TNF-α, TIMP-1, and MMP-2, as well as decreasing the levels of profibrogenic cytokines IL-1β, TGF-β1, and TNF-α [112]. Anti-IL-17A treatment may alleviate IF in mice by reducing EMT [111]. However, these apparently beneficial effects may not be reproducible in humans. In fact, the administration of secukinumab, a human anti-IL-17A monoclonal antibody, failed to show efficacy in CD patients and actually led to worsening of intestinal inflammation in some patients [113]. This may suggest that baseline levels of IL-17 are necessary for intestinal homeostasis, leaving the question of its role in intestinal fibrogenesis unanswered.

IL-4, IL-13 and the Th2 response

After the discovery of the Th1 and Th2 dichotomy of CD4+ T helper cells [114], investigators initially tried to characterize CD as a Th1 condition and UC as a Th2 condition [115]. This characterization later fell into disrepute once Th1 and Th2 cytokines were not found to be restricted to any one type of IBD. As such, Th2 responses could also be detected in late stages of experimental and human CD [116, 117]. Of relevance to fibrosis, numerous in vitro and in vivo studies have shown the pro-fibrotic activity of Th2 cytokines. For example, IL-4 promotes collagen and fibronectin synthesis in normal healing and pathological fibrosis [118] and treatment of human hepatic fibroblasts with IL-4 increases collagen production [119]. However, the role of IL-4 in IBD is still controversial. Anti-IL-4 administration leads to a striking amelioration of experimental colitis [120], prevents the expression of IL-13Rα2 and reduces production of TGF-β1[121, 122]. Early reports claimed that IL-4 mRNA expression in the intestinal mucosa in both CD and UC patients was almost undetectable [123] and that the production of IL-4 was decreased in IBD and could induce impaired immunosuppressive and anti-inflammatory responses [124]. Subsequently, IL-13 and its receptor were found to be overexpressed in areas of fibrosis in CD patients [125]. Anrukinzumab, an anti-IL-13 monoclonal antibody, showed no therapeutic effect in patients with active UC [126], and tralokinumab, another IL-13-neutralizing antibody, was evaluated in adults with moderate-to-severe UC without any significant improvement of clinical response [127]. However, the potential anti-fibrotic effect of both antibodies was not specifically evaluated in these studies, and anti-IL-4 trials in IBD have not been completed.

IL-34

IL-34 is a novel cytokine identified as a tissue-specific ligand of CSF-1 receptor (CSF-1R) [128] and also 2 additional receptors: Receptor-type protein-tyrosine phosphatase (PTP)-ζ and CD138 (syndecan-1) [129]. In humans, IL-34 has a wide distribution in various tissues, including the heart, brain, lung, liver, kidney, spleen, small intestine, colon, and spleen. At cellular level, it can be expressed by various types of cells including synovial fibroblasts, immune, epithelial, endothelial, adipocytes and cancer cells [130]. IL-34 induces lymphocyte differentiation and proliferation and regulates the synthesis of inflammatory components. Aberrant expression of IL‐ 34 has been reported in several autoimmune disorders, such as lupus, arthritis, and systemic sclerosis [128]. IL-34 is overexpressed in chronic hepatitis C liver fibrosis and induces profibrotic macrophages, and serum IL-34 levels can be an indicator of liver fibrosis in patients with chronic hepatitis B, as well as a marker of liver fibrosis in patients with non-alcoholic fatty liver disease [131, 132, 133]. IL-34-deficient mice exhibit less renal fibrosis during the chronic phase after ischemia/reperfusion injury [134]. In the gut, IL-34 is constitutively expressed in human small intestine and colon and shows a higher expression level in stricturing CDs compared to controls [135]. It functions as a direct activator of intestinal fibroblasts, which respond with increased collagen expression (including COL1A1 and COL3A1) in a p38MAPK-dependent manner [135]. IL-34 knockdown results in decreased collagen production in fibroblasts isolated from CD strictures without affecting cell survival [135] (Figure 3).

Inflammation-independent mechanisms of fibrogenesis

Despite impressive progress in controlling inflammation in IBD with novel small molecules and biologics, the incidence of intestinal obstruction and stricture formation remains high [2, 97, 136, 137]. Reasons for this phenomenon are still under investigation, but in vivo and in vitro experimental models of intestinal fibrosis suggest that inflammatory and fibrotic factors increase in parallel during periods of active inflammation [138]. However once inflammation is successfully treated and the level of inflammatory mediators decreases, the level of profibrotic factors remain high[138], indicating that inflammation independent mechanisms for progression of fibrosis may play a role (Figure 4). When and how the disconnect between inflammation-driven and inflammation-independent fibrogenesis occurs in the disease course is unclear, but we speculate that this occurs early in the disease, before both processes start happening in parallel. Potential mechanisms explaining the disconnect between inflammation and fibrosis are ECM mechanoproperties and composition. Examples include hyaluronan degradation which generates damage-associated molecular pattern fragments, that have the ability to sustain inflammation and fibrosis[139]. To the contrary, a recent matrisome analysis of stricturing CD revealed the anti-fibrotic properties of the ECM molecule MFGE8 in vitro and in vivo, despite being upregulated in the stricture ECM[140]. ECM is a strong binding partner for cytokines and fibrotic growth factors, including TGF-β1 [141, 142], ready to release them in active form to promote fibrogenesis. A changed ECM deposition in IBD has a higher adhesive capacity for binding T cells, which is at least partly mediated by collagen VI and may increase immune cell retention in the gut[143]. With increasing stricture severity, the mechanoproperties of the tissue change and cell encounter a stiffer environment. Stiffness alone, in the absence of inflammation, can activate mesenchymal cells towards a profibrogenic phenotype[144, 145, 146]. Abnormal contraction of mesenchymal cells can amplify stiffness[147]. These data gives rise to the concept that inflammation is not the only factor that promotes fibrosis. While inflammation may be important in initiation of fibrogenesis, once established fibrosis may progress on its own in a self-perpetuating manner, which has significant implications on therapeutic approaches (Figure 4). Currently available biologics or small molecule therapies do not target all the inflammatory key players typically involved in fibrogenesis. It hence has to be noted, while the existence of completely inflammation-independent mechanisms of fibrosis has been shown in fibrotic diseases outside the intestine, that this concept is still to be clearly demonstrated in the gut.

Figure 4.

Model for the disconnect of inflammation and fibrosis/extracellular matrix changes. Depicted is a period of active intestinal inflammation before and after treatment.

Homotypic fibroblast interactions

Given the predominant role of fibroblasts in intestinal fibrosis, assessing homotypic fibroblast interactions as a therapeutic target is a reasonable proposition. One recently identified candidate is Cadherin 11 (CDH11), a fibroblast cell-cell adhesion molecule, that was broadly expressed and upregulated in CD strictures [21, 148]. It was discovered in a recent publication of a scRNAseq atlas for stricturing CD as a unifying molecule and the only cell surface receptor shared by the CXCL14+ and MMP/WNT5A+ fibroblasts that were found to be overrepresented in CD strictures [21]. Its pro-fibrotic function was validated by in vitro gain- and loss-of-function experiments, proteomics, as well as knock-out and antibody-mediated CDH11 blockade in experimental colitis. These data point to CDH11 as a key driver of stricturing CD and as a putative therapeutic target. CDH11 has been shown to be upregulated in fibrotic disorders of the lung[149], liver[150], skin[151], intestine[148], and its inhibition attenuates fibrosis in multiple animal models[150]. This raises the prospect of using CDH11 blockade to prevent or treat CD-associated intestinal fibrosis[21] (Figure 3).

Mesenchymal cell senescence

Along the lifespan of a mesenchymal cell, senesecence is considered the stage in which cells reduce their propensity to divide. Senescence may also influence the function of myo-/fibroblasts and hence could be exploited as a therapeutic mechanism. Senotherapeutic and senolytic compounds are emerging as promising new strategies to ameliorate fibrosis [152, 153]. It remains however unclear, if senescence in mesenchymal cells exerts a net pro- or anti-fibrotic effect in tissue repair or fibrosis and this may be highly context dependent on tissue and disease type [153, 154, 155]. Senescence in fibroblasts has shown to increase apoptosis resistance and secretion of inflammatory cytokines, growth factors, immune modulators, and proteases, which then sustained inflammation and fibrosis [152, 153]. One example of targeting senescence in fibrosis is the activation of NK responses through inhibition of NKG2A to eliminate senescent fibroblasts in skin[156]. The concept of mesenchymal cell senescence is just emerging in intestinal fibrosis. RNA sequencing data in human ileal tissues suggested gene expression profiles consistent with resistance to apoptosis and a senescence associated secretory phenotype in complicated ileal CD and that PDGFB was able to induce resistance to apoptosis in fibroblasts in vitro[157]. A different investigation found an increase in senescence signaling in IBD compared to controls[158]. The exact functional characterization and contribution of senescence in intestinal fibrosis still needs to be elucidated.

Host-microbiome interactions and intestinal fibrosis

Compositional and metabolic abnormalities of the gut microbiota (dysbiosis) are key factors involved in IBD pathogenesis [159]. In individuals with a genetic susceptibility to IBD, microbial antigens and products induce functional changes of the intestinal and systemic immune response, which are linked to IBD onset and progression [160]. Initiation of this response depends on recognition of pathogen-associated molecular patterns (PAMPs) by a series of pattern recognition receptors (PRRs) [161]. PRRs are ubiquitous and expressed by intestinal immune (dendritic cells, macrophages, lymphocytes, etc.) and non-immune cells (epithelial cells, fibroblasts, endothelial cells, etc.) [162]. Primary human intestinal myofibroblasts express multiple PRRs, including TLR 1–9, as well as nucleotide-binding oligomerization domain-containing protein (NOD) 1 and NOD2 [163]. The TLR4 ligand lipopolysaccharide (LPS, produced by bacteria) promotes profibrotic activation of intestinal fibroblasts, with enhanced NF-κB promoter activity and increased collagen production [164], and TLR4-deficient mice show ameliorated intestinal fibrosis[165]. Flagellin promotes IF in a TLR5- and NOD-like receptor family CARD domain-containing protein (NLRC) 4-dependent manner [68]. Supporting the notion of microbial-driven fibrogenesis, CD patients carrying NOD2 mutations tend to display a stricturing phenotype [166]. MyD88, which is essential in TLR signaling pathways as an adaptor protein in innate immune responses, was demonstrated to act downstream TLR5 and regulate IF [167]. In a α-SMA-specific MyD88 deletion mouse model, MyD88 deletion prior to, but not after, the induction of experimental colitis resulted in decreased IF [167]. α-SMA(+) HIMFs selectively respond to flagellin with enhanced fibronectin or collagen I production in an MyD88-dependent manner, mediated by eIF2alpha and 4EBP1 [167] (Figure 3).

Bacteria-driven IBD animal models are used to study how microbes induce IF, such as adherent-invasive Escherichia coli (AIEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) infectious mouse models [166, 168]. Research using these models provides evidence of the relationship between gut microbiome and IF and uncovers new molecules and mechanisms involved in intestinal fibrogenesis.

AIEC is a pathotype of E. coli that adheres to the gut epithelium and may cause chronic intestinal inflammation in genetically susceptible hosts, in particular in the ileum [169, 170]. In mice, chronic AIEC infection leads to tissue pathology in predominantly in the large bowel, especially in the cecum, with elevated Th1 and Th17 responses [171] and extensive ECM deposition, higher expression levels of collagen type I and III, and enhanced expression of profibrotic mediators, such as TGF-β1, connective-tissue growth factor (CTGF) and insulin-like growth factor I (IGF-I), all of which are also found to be elevated in CD patients [171]. However, intestinal strictures do not occur in this model, and inflammation and fibrosis are mouse strain-dependent.

In mice, S. Typhimurium induces a chronic colonization of the cecum and colon with transmural inflammation [68], enhanced production of TGF-β and IGF-I, along with extensive collagen I deposition in the cecal mucosa, submucosa, and muscularis mucosa [172], and accumulation of fibroblasts and myofibroblasts in fibrotic areas [173]. A modified Salmonella-induced IF model has been used to investigate the role of flagellin and IL-33 in intestinal fibrosis [68]. In this model, AIEC and an attenuated strain of S. Typhimurium were co-colonized, and IF was induced in a flagellin-dependent manner with the activation of IL-33-ST2 signaling [68].

Human studies demonstrating that the gut microbiota can induce a selective gut fibrotic response (over inflammation only) are not yet available, and no animal model can fully mimic human IBD fibrosis. Nevertheless, further characterization of animal models will help explore fibrotic events in vivo and uncover mechanisms underlying microbe-induced fibrosis in IBD. A summary of commonly used animal models of fibrosis can be found in Table 2. Detailed descriptions of these animal models are available elsewhere [174, 175, 176].

Table 2:

Examples of frequently used mouse models of intestinal fibrosis

	Summary	Relevance to human disease	Advantages	Disadvantages
DSS colitis model [21 , 207]	Multiple cycles of DSS induces chronic colitis and fibrosis	+++	Easy to perform, good reproducibility.	Limited human IBD relevance; limited cell types involved.
TNBS colitis model [208 , 209 , 210]	Intrarectal application of hapten TNBS: results in chronic colitis leading to fibrosis	+++	Widely used for studying intestinal fibrosis	Requires susceptible mice strain to induce colitis; great variability.
AIEC colitis model [68 , 171]	Oral gavaging of AIEC induces gut inflammation in colon	++++	Mimics the inflammation pattern of human CD; Tool for microbiota research in IBD	Require ABSL2 facility; Labor intensive model
Chronic Salmonella induced colitis [172 , 173, 210]	Oral gavaging of S. typhimurium induces gut inflammation and fibrosis (mainly cecum)	++	High degree of fibrosis, very reliable development of fibrosis, transmural inflammation	Requires ABSL2 facility; Labor intensive; Salmonella does not cause IBD
IL-10 knockout colitis model [210, 211 , 212 , 213]	Spontaneous chronic colitis model	+++	Spontaneous colitis model, while chemicals and infectious components could be added to accelerate the onset of disease.	Disease development is slow.
SAMP1/YitFc ileitis [210, 214 , 215 , 216]	Spontaneous inflammation and fibrosis resembles all stages of human CD.	++++	Strain closely mimics all stages of stricture formation in CD patients; symptoms starts as early as 4 weeks.	Low breeding rate; Long experimental duration
TNFΔARE model [176]	Spontaneous ileitis model with overt structural fibrosis development	++++	Ileal fibrosis with luminal narrowing comparable to human CD	Long model duration (24 weeks); high breeding effort needed; reversibility unclear

Abbreviations: AIEC, adherent-invasive E. coli; DSS, dextrane-sodium sulfate; IL, interleukin TNBS, trinitrobenzene sulfonic acid. More detailed information is available [174, 175, 176]. Example references are added to each model.

Creeping fat and smooth muscle hyperplasia in intestinal fibrosis

Creeping fat indicates the wrapping of mesenteric fat tissue around the wall of CD-involved intestinal segments. It was described almost 100 years ago, but its functional implications have only recently received attention [177]. Anatomically, creeping fat is intimately associated with stricturing CD and its extent correlates closely with the degree of transmural inflammation [178] (Figure 3). When the mesentery is extensively removed during ileocolic resections (as opposed to minimal mesenteric resection), recurrence of CD is significantly reduced and fewer reoperations are required [28]. This points to an important pathophysiological role of creeping fat in the events leading to stricture formation in CD patients. The mechanism underlying the formation of creeping fat remains unclear, but one essential factor is adipocyte hyperplasia, which occurs by recruitment and differentiation of adipose tissue-derived stem cells (ASCs) that exhibit high proliferative, invasive, and phagocytic capacities [179]. CD creeping and mesenteric fat have a microbiome signatures not found in non-affected mesenteric or subcutaneous fat, and those signatures have functional relevance [180]. Immune/non-immune cell interactions appear to be critical to the function of creeping fat. Compared with mesenteric tissue from control subjects, the numbers of both T and B memory cells are increased within CD creeping fat [181], and CD mesenteric tissue contains greater amounts of adipocyte-derived chemokines, which may actively recruit T and B lymphocytes [181]. Of critical relevance, CD creeping fat is associated with muscularis propria hyperplasia, which is now believed to be the major factor contributing to luminal narrowing and stricture formation (Figure 3) [177, 182]. Our own preliminary observations suggest a novel role of creeping fat-derived fatty acids on human intestinal muscle cells (HIMCs) hyperplasia [183]; we found that exposure of HIMC to whole creeping fat tissue and fat-conditioned medium dramatically upregulates HIMC proliferation compared with UC and normal mesenteric fat [183]. Creeping fat-derived mediators such as free fatty acids, but not adipokines, induce a differential and selective proliferative response by HIMC[183]. The adipocyte-dependent microenvironment within the creeping fat of patients with CD has been reported to promote an M2 macrophage subtype with secretion of large amounts of pro-fibrotic factors such as TGF-β, leading to IF [184]. This suggests complex cell-cell interactions in creeping fat driving interactions with the intestinal muscularis propria leading to smooth muscle proliferation.

Fibrosis in ulcerative colitis

Until very recently, fibrogenesis and stricture formation have been largely overlooked in UC. Colonic fibrosis in UC has significant clinical consequences, including motility abnormalities, anorectal dysfunction, rectal urgency, and incontinence [5, 6, 185]. As compared with small bowel CD, stricture formation in UC is infrequent (1–10%); however, rates of stricture formation are comparable in colonic CD and UC [185]. UC is traditionally viewed as an inflammatory process confined to the mucosal/submucosal layers. However, one study compared 706 UC tissue cross-sections to CD, diverticular disease, and normal areas from colorectal cancer specimens, and detected submucosal fibrosis in 100% of the UC specimens [5]. Submucosal fibrosis and thickening of the muscularis mucosae were associated with severe and chronic injury, but not active inflammation, suggesting fibrosis and muscularis mucosae thickening are common complications of progressive UC [5]. Another recent investigation showed persistent abnormalities even in endoscopically normal UC mucosa, including ECM remodeling, profibrotic cytokine production, and activated TGF-β signaling pathways. This suggests that while inflammation is necessary to initiate fibrogenesis in UC, suppression of inflammation and subsequent healing do not prevent development of fibrosis [186].

Development of anti-fibrotic therapies in IBD

Despite advances in the field, trials with novel anti-fibrotic drugs in the intestine are lagging for two main reasons: 1) inability to translate recent experimental results into new drugs and 2) lack of consensus on definitions and clinical trial endpoints. This is unfortunate given the very active drug development programs for anti-fibrotics in other organs (recently summarized by us here [187]).

Regarding poor translation into drugs, many disparate cell types and signaling pathways are involved in IF but are yet to be validated in human systems and shown to be functionally relevant. A plethora of cytokines and growth factors is present in inflamed and/or fibrotic IBD tissue, and the variety of biological activity can be either beneficial or detrimental, posing the challenge of differentiating pro- and anti-fibrotic activities in fibrogenesis. One example is the conundrum of developing anti-TGF-β1 strategies: considering its pleotropic functions, direct inhibition of TGF-β1 may constrain immunosuppression, exacerbate inflammation, and increase the risk for malignancy[188]. Thus, different approaches may be needed to target this pathway, such as blocking up- or down-stream regulators or topically delivering TGF-β1 inhibitors to fibrotic sites, as recently started in a clinical development program with a local delivery ALK5 (kinase for transforming growth factor beta receptor 1) inhibitor in stricturing CD (NCT05843578). An example for a bench to bedside approach in intestinal fibrosis is the unfortunately stopped clinical development program of the IL-36R inhibitor spesolimab in patients with fibrostenosing CD (NCT05013385). Strong preclinical data [79] led to exploration of this novel mechanism in patient patients with CD and obstructive symptoms. Targeting inflammation independent mechanisms is being brought forward in fibrostenosing CD with the GI targeted ROCK inhibitor RXC008, an approach that has shown promise in experimental intestinal fibrosis[189]. Overall, it is unlikely that neutralizing the activity of any single cytokine or mechanism would prevent or heal IF. A rational path forward would be harnessing the advent of multiomics technology and applying those technologies to precisely phenotyped patient populations, then spatially locating cell candidates and pathways, comparing this to other fibrotic diseases and ultimately developing a high-resolution gut ‘omics’ map as a resource for the IBD community and industry (Figure 5).

Figure 5.

Deciphering mechanisms of fibrosis using multi-omics approaches. Recent advances in the omics field allow assessment of the transcriptome, genome, epigenome, cell ontogeny and Proteome. Identified cell types and pathways can be spatially located using spatial transcriptomics. Integration of these multi-omics datasets allows high-resolution assessments of cells and their state and already available datasets from other fibrotic disease can inform rational drug development strategies.

In regard to forming a consensus on clinical endpoints, multiple obstacles are present. Various biomarkers have been proposed to stratify complication-free patients at diagnosis; however, none of the proposed biomarkers can discriminate stricturing from penetrating behavior with enough accuracy to allow patients to enroll in fibrosis prevention trials [190, 191, 192]. Additionally, no biomarker can accurately quantify fibrosis in a stricture, which raises questions about the value of endpoints that rely solely on ECM changes. Imaging techniques (ultrasound, computed tomography enterography [CTE], and magnetic resonance enterography [MRE]) can accurately detect intestinal strictures or assess inflammation, but not degree of fibrosis[193], making it impossible to assess responses to anti-fibrotic therapy. One alternative could be to select a population with existing strictures in which previously resected tissues were already evaluated to provide a reliable assessment of the amount of fibrosis, muscle thickening, and inflammation [194]. In these patients, a baseline anti-inflammatory therapy leading to mitigation of obstructive symptoms would permit the randomization into anti-fibrotic therapy versus placebo groups.

Clinically meaningful endpoints could be the absence of obstructive symptoms, observation of stricture morphology changes, or reduced need for subsequent endoscopic and/or surgical interventions. The successful development of biomarkers to predict or quantify fibrosis must be therefore based on solid histopathologic standards. Typically, candidate biomarkers are evaluated in patients with symptomatic strictures, who then undergo imaging followed by bowel resection with grading of inflammation and fibrosis [195, 196, 197, 198, 199, 200, 201, 202]. Validity is then determined by correlating histologic evaluation of fibrosis degree and/or inflammation to imaging results. Multiple histopathology indices have been proposed, but none have been validated following modern methodological standards [203], and their evaluation is highly variable across indices [204]. A reliable and validated histopathologic standard would deliver the discriminatory ability to optimize and compare cross sectional imaging techniques. An international group of IBD pathologists and gastroenterologists under the umbrella of the Stenosis Therapy and Anti-Fibrotic Therapy (STAR) consortium is working on building a validated histopathology index for stricturing IBD [205].

To translate novel anti-fibrotic agents into IBD clinical practice, there is a dire need of reliable definitions; however, systematic reviews reveal tremendous heterogeneity in definitions of stricturing on endoscopy and imaging [193]. To address this problem, the STAR consortium recently created clear definitions for what defines a stricture and what constitutes improvement [205], and multiple projects are now underway to build monitoring tools and endpoints for clinical trials, including a patient-reported outcome tool, a stricture radiology index, and dynamic fibrosis turnover markers. These traditional tools may soon be complemented by new, artificial intelligence-based approaches to discover new therapeutic targets for IF [206].

In conclusion, ultimate progress in the field of IBD-associated fibrosis will be accomplished by combining mechanistic preclinical discovery and clinical validation studies with new validated and regulator-accepted endpoints and state of-the-art drug development programs (Figure 6).

Figure 6.

Necessary steps for developing a pathway to testing anti-fibrotic drugs: Combination of preclinical development, clinical trial endpoint development and the critical step for engaging industry in early drug development, such as first in human studies, to reach the stage of proof of concept clinical trials.

ABBREVIATIONS

AIEC

Adherent-invasive E. Coli

ALK5

transforming growth factor beta receptor 1

ASCs

Adipose tissue-derived stem cells

CCL

Chemokine (C-C motif) ligand

CDH11

Cadherin 11

CTGF

Connective tissue growth factor

CTHRC1

Collagen triple-helix repeat-containing 1

CXCL

Chemokine (C-X-C motif) ligand

CSF-1R

Colony stimulating factor-1 receptor

CTE

Computed tomography enterography

CD

Crohn’s disease

DAMP

Damage-associated molecular pattern

DSS

Dextran sodium sulfate

ECM

Extracellular matrix

EMT

Epithelial to mesenchymal transition

EndMT

Endothelial to mesenchymal transition

HIMCs

Human intestinal smooth muscle cells

HIMECs

Human intestinal microvascular endothelial cells

HSC

Hepatic stellate cells

IBD

Inflammatory bowel disease

IF

Intestinal fibrosis

IGF-I

Insulin-like growth factor I

IL

Interleukin

IPF

Idiopathic pulmonary fibrosis

LOX

Lysyl odxidases

MCP

Monocyte chemoattractant protein

MMP

Matrix metalloprotease

MRE

Magnetic resonance enterography

N-cadherin

Neural cadherin

NLRC

NOD-like receptor family CARD domain-containing protein

NOD

Nucleotide-binding oligomerization domain-containing protein

PAMP

Pathogen-associated molecular patterns

PDPN

Podoplanin

PRR

Pattern recognition receptors

scRNAseq

Single-cell RNA sequencing

SMA

Smooth muscle actin

SMC

Smooth muscle cell

STAR

Stenosis Therapy and Anti-Fibrotic Therapy

S. Typhimurium

Salmonella enterica serovar Typhimurium

TGF

Transforming growth factor

Th

Helper T cell

TIMP-1

Tissue inhibitor of metalloproteases-1

TLR

Toll like receptor

TNBS

Trinitrobenzene sulfonic acid

UC

Ulcerative colitis