The mechanisms that mediate the development of fibrosis in patients with Crohn's Disease

Smooth Muscle Cells

Intestinal smooth muscle cells are found in the muscularis propria, which is intimately involved in the development of fibrosis, where there is marked thickening with cellular hyperplasia, hypertrophy and collagen deposition that contribute to fibrosis and stricture formation. Muscularis propria represents the largest mesenchymal cell compartment in the intestine. The muscularis mucosa is also comprised of smooth muscle cells, but this layer becomes effaced in the region of strictured intestine. Smooth muscle cells are characteristically α-smooth muscle actin, desmin and collagen I positive³².

Smooth muscle cells contribute to fibrosis by their ability to produce large amounts of extracellular matrix proteins like collagen, particularly collagen I and III, and fibronectin and vitronectin^{33, 34}.They are highly synthetic and produce large amounts of both cytokines and growth factors including TGF-β1, IL-6, IGF-I, PDGF, and CTGF. In the muscularis propria the master fibrosis cytokine, TGF-β1, can stimulate the expression of all of these factors through autocrine mechanisms.

Myofibroblasts

Myofibroblasts have a phenotype between smooth muscle cells and fibroblasts. Both intestine-resident myofibroblasts including subepithelial myofibroblasts and Interstitial cell of Cajal (ICC) and cells transitioned to myofibroblasts are involved in the process ³⁵. They are α-SMA, vimentin and collagen I positive³². ICCs are also characterized by c-kit positivity. All can be abundant sources of collagen production and other ECM proteins including vitronectin.

Myofibroblasts in patients with Montreal B2 fibrostenotic disease are distinctly phenotypically different than those in patients with non-B2 disease. This is highlighted by distinctly different mobility of B2 myofibroblasts from those in B3 disease. Myofibroblasts from patients with B3 disease have faster rates of mobility in wounding assays than do myofibroblasts isolated from stricturing disease. Mesenchymal stem cells (MSCs) migrate to sites of injury based on homing signals provided by chemotactic factors. Migration is dependent on the interaction between MSCs and ECM ^{36, 37}. Myofibroblast activation and migration are regulated by both autocrine (e.g. TGF-β1, and IGF-I) and paracrine (e.g. TNF-α, IL-6 and IL-13, IFN-γ, and TGF-β1) factors^{37, 38}. Expression of the TNF superfamily member, TL1A (TNSF15) by lymphoid and myeloid cells results in development of mild inflammation that is accompanied by the development of fibrosis in the intestine ³⁹.

The migratory potential of activated myofibroblasts illustrates, on the one hand, a key feature of Crohn’s disease phenotype, as well as our lack of understanding of the process, on the other. The migratory potential of subepithelial myofibroblasts from patients with Crohn’s disease is dependent on the underlying disease phenotype: myofibroblasts from patients with fibrostenotic disease exhibit decreased migration whereas those from patients with penetrating disease exhibit increased migration ³⁶. This correlates with a difference in the levels of the non-receptor protein kinase, focal adhesion kinase (FAK; a regulator of cell migration), the phosphorylation of which increases fibroblast migration ⁴⁰.

Subepithelial myofibroblasts can play a role not only in the development of fibrosis but also in the regulation of the immune response as well. In the normal intestine, myofibroblasts can regulate mucosal tolerance by their ability to support a FoxP3+ Treg response in the intestine ⁴¹. In the intestine of patients with Crohn’s disease, myofibroblasts are activated by numerous paracrine mediators in their environment that promote their production of ECM and proliferation including TGF-β1, PDGF, CTGF, IGFI/II, bFGF and various interleukins: IL-1β, IL:-6 and IL-13. Activated myofibroblasts are highly synthetic and produce not only ECM but are also rich autocrine sources of many of these mediators. This has important implications on the crosstalk between subepithelial myofibroblasts and epithelial cells that regulate each other’s function. Epithelial cells respond to the paracrine stimuli from myofibroblasts to increase their TGF-β1 and TIMP-1 expression ⁴².

Fibroblasts

Fibroblasts are a more heterogeneous group of cells in the intestine than myofibroblasts or smooth muscle cells. They are scattered throughout the wall of the intestine where they are involved in the response to injury and participate in either normal wound healing of the development of fibrosis. They are characteristically vimentin positive, collagen I positive and N-cadherin positive, but α-SMA negative³². In the setting of fibrostenotic Crohn’s disease their expression of N-cadherin is upregulated compared to non-fibrostenotic Crohn’s disease. Increased N-cadherin expression is upregulated in response to TGF-β1 and increases their migratory potential. Mice expressing a dominant-negative N-cadherin develop a spontaneous Crohn’s-like inflammation suggesting this mutation confers defective wound healing in these animal ^{43, 44}. The reduced migration observed in subepithelial myofibroblasts derived from patients with fibrostenotic disease is in contrast with the increased migratory potential of fibroblasts allowing for enhanced invasion of fibroblasts into the injured intestinal wall ^{43, 44}, which suggests that dysfunctional wound healing process may play a role in delayed recovery of physiologic intestine structure and function.

Fibroblasts respond to activation by TGF-β1 and other growth factors such as IGF, CTGF, PDGF and bFGF and cytokines such as IL-1β, IL-6 and TNF-α, with increased cellular proliferation and ECM production. Autocrine production of the ECM protein fibronectin also influences motility of fibroblasts.

Recent evidence suggests that matrix stiffness itself, the product of excess ECM production and fibrosis, is capable of further activation of intestinal fibroblasts ⁴⁵.

Other Mesenchymal cells

The mesenchymal cells compartment can also be expanded by the products of Epithelial-Mesenchymal cell Transition (EMT), Endothelial-Mesenchymal cell Transition (Endo-MT) and invasion of bone-marrow derived circulating fibrocytes^{31, 46}. Each of these processes described results in activated fibroblasts and myofibroblasts within the intestinal wall at sites in injury.

Injury and resultant loss of barrier results in the reprogramming of pluripotent epithelial and endothelial cells. Cells lose epithelial or endothelial markers and initiate synthesis of α-smooth muscle actin. They undergo cytoskeletal rearrangement and assume a spindle shape and migration through basement membrane from the compartment of origin to the interstitial space. Production of ECM proteins, including collagen and fibronectin, by cells following EMT or EndoMT is a significant contributor to the development of fibrosis^{5, 30}.

Circulating fibrocytes derived from bone marrow express markers of hematopoietic cells, but also express collagen I and α-smooth muscle actin characteristic of mesenchymal cells. Once recruited to sites of injury and inflammation they undergo differentiation into fibroblasts and myofibroblasts. They not only respond to local mediators such as TGF-β1, IL-1β, but also are sources of TGF-β1, CTGF which act in paracrine and autocrine fashion on intestinal mesenchymal cells ⁴⁶.

Myofibroblasts in fibrotic tissues can be derived from various sources. These include from resident tissue fibroblasts that evolve from quiescent fibroblasts to cells expressing a myofibroblast phenotype, via transition of epithelial cells into mesenchymal cells (EMT), from tissue migration of bone marrow-derived circulating fibrocytes, and via the recently discovered endothelial to mesenchymal transition (EndoMT) ^{5, 31, 47}. EMT, which is induced by transforming growth factor-β (TGF-β), is not unique to the intestine and occurs in other organs as well ⁴⁸. Fibrocytes are capable of producing fibroblastic proteins and migrating to diseased areas ^{47, 49}. EndoMT is a process in which endothelial cells lose their endothelial cell markers and acquire a mesenchymal phenotype with expression of α-smooth muscle actin (α-SMA), vimentin, and type I collagen ^{5, 50}. In vitro studies have shown that EndoMT can be induced by TGF-β1 via a “non-canonical” (non-Smad) signaling pathway ⁵⁰.

TGF-β

TGF-β is the central regulator in the injured and inflamed intestine. It has both wound healing and Treg properties but its increased expression and activation is a central mediator of fibrosis. Three isoforms of TGFβ exist: TGF-β1, TGF-β, and TGF-β3. Expression of TGF-β1 and TGF-β3 is specifically increased in smooth muscle cells, myofibroblasts, and fibroblasts of strictures as compared with normal intestine in the same patient undergoing resection ⁵². Fibroblasts in the mucosal layer also display increased expression of TGF-β2 ⁵³.

Overexpression of TGF-β1 in transgenic animals is associated with development of fibrosis. As a therapeutic target, however, TGF-β1 and its SMAD signaling pathways are problematic. TGF-βRI/II receptors expressed by mesenchymal cells are linked to canonical Smad2/3 signaling.⁵⁴ Binding of active TGF-β1 to TGF-βRI/II receptors in human intestinal muscle activates canonical Smad signaling, r-Smad2/3 phosphorylation and increased collagen I production which accounts for ~70% of collagen present in the intestine^{33, 55}. With the exception of Smad3 deletion, deletion of TGF-β protein, its receptors or other Smad proteins are generally lethal mutations or a feature of neoplastic cells. In two of the three Smad3 deletion mutants developed, a lethal wasting syndrome develops early in association with immunodeficiency^{56, 57}. In the third Smad3 mutant developed, while the effects of TNBS-induced colitis were reduced, metastatic adenocarcinoma of the colon developed within 4–6 months of age⁵⁸. Overall these observations suggest that pharmacologic targeting of the TGF-β system in Crohn’s disease patients to decrease the development of fibrosis could have untoward effects.

Activation of latent TGF-β1 by smooth muscle and intestinal fibrosis

In our laboratory we have taken an approach to examine paired samples from affected regions of patients not only with Montreal Class B2 fibrostenotic disease but also from Montreal Class B1 inflammatory and Montreal B3 penetrating disease using non-Crohn’s intestine as control to determine the factors involved in the development and progression of fibrosis. Expression and production of TGF-β1, is increased specifically in smooth muscle cells of ileal strictures compared to histologically normal proximal resection margin.^{34, 59} This is in contrast to patients expressing a B1 or B3 phenotype where TGF-β1 expression is unchanged from non-Crohn’s subjects in either affected regions or normal resection margin. TGF-β1, in addition, stimulates expression of other fibrogenic factors including: fibronectin, CTGF and IGF-I.^{33, 60}

Levels of active TGF-β1 associated with muscle cells is increased in strictured regions in comparison to normal intestine in the same patient. Activation of TGF-β1 occurs by the binding of the RGD sequence in Latent TGF-β1 to the RGD binding domain in the extracellular region of αVβ3 integrin. Latent TGF-β3 also possesses and RGD sequence but transcript levels are only slightly increased in regions of stricture; in contrast latent TGF-β2 possesses an SGD sequence that does not bind to and cannot be activated by αVβ3 integrin. The intracellular domain of αVβ3 integrin binds Src kinase via β3(Ser752) ⁶¹. Phosphorylation of β3(Ser752) in response to binding of ligands of the RGD domain (vitronectin, fibronectin or Latent TGF-β1) provides linkage to the actin cytoskeletal⁶². Integrins expressed on smooth muscle cells are key elements of mechano-transduction pathways that communicate with and are regulated by the focal adhesion complex that includes FAK, c-Src, and paxillin, and proteins mediating cytoskeletal remodeling and motility.⁶³ Once bound to αVβ3 integrin, the mechanical activity of the muscle cells, contraction and relaxation, provides for the excess non-proteolytic activation of latent TGF-β1. The increased phasic motor activity in the intestine and colon leads to increased active TGF-β1 liberation ⁶⁴. When binding of latent TGF-β1 to αVβ3 integrin is blocked by use of an RGD inhibitor, Cilengitide, activation of latent TGF-β1 is blocked along with the production of Collagen I, and the development of fibrosis in the TNBS model of Crohn’s disease ⁶⁵.

A unique relationship exists between αVβ3 integrin, not only with respect to activation of TGF-β1 but also the activity of the profibrotic growth factor IGF-I and its binding proteins, IGFBP-3 and IGFBP-5. The expression of all three are increased in muscle cells of strictured intestine ^{34, 59}. Production of fibronectin and vitronectin, activating ligands of αVβ3 integrin (the cognate vitronectin receptor), is increased in stricturing Crohn’s disease. Binding of vitronectin and fibronectin to αVβ3 integrin elicits maximal activation of IGF-I and expression of IGFBP-5. These two factors, in turn, increase expression of collagen IαI. IGFBP-3, on the other hand, binds to TGF-βRI/II receptors and activates canonical Smad signaling via receptor-based Smad2/3 and recruitment of co-Smad4. Translocation of the active r-Smad2/3-co-Smad4 complex to the nucleus regulates expression of numerous genes that contribute to the development of fibrosis including additional fibronectin, IGF-I and CTGF in addition to Collagen I and III. Inhibitory-Smad7 (i-Smad7) is upregulated as the normal counter regulatory signal that inhibits activity of the r-Smad2/3 activity. Increased expression of i-Smad7 has been demonstrated in the epithelium and T-cells of patients with Crohn’s disease where its increase results in inappropriate suppression of the Treg response mediated by TGF-β1⁶⁶. Knockdown of i-Smad7 in animal models of Crohn’s disease and in Phase I clinical trials has demonstrated efficacy to decrease inflammation. In the later study patients expressed exclusively a Montreal B1 inflammatory phenotype⁶⁷. In contrast, our studies have demonstrated a decrease in i-Smad7 in patients with Montreal B2 phenotype Crohn’s disease that contributes to excess TGF-β1 signaling and effects that result in excess collagen I production and fibrosis. These two scenarios with disparate roles for i-Smad7 also illustrate the problematic nature of targeting TGF-β1 and its signaling pathways in Crohn’s disease.

A variety of other cytokines are released by immune cells in Crohn’s disease and in animal models of Crohn’s disease that stimulate fibrosis. In the TNBS-induced model of chronic colitis in BALB/c mice, IL-13 (via IL-13Rα2) increases expression of TGF-β1 and IGF-I and results in fibrosis. Fibrosis was decreased by disruption of IL-13Rα2 signaling^{68, 69}.

IGF-I in development of intestinal fibrosis

The central role of IGF-I in the development of fibrosis in Crohn’s disease was shown by the increased expression of IGF-I, IGFBP-3 and IGFBP-5 in smooth muscle cells of strictured intestine^{34, 59, 70–72}. In the muscularis propria of human intestine, increased autocrine IGF-I production results increased smooth muscle cell proliferation, inhibition of apoptosis as well a increased ECM production including collagen I, fibronectin and vitronectin. The importance of TGF-β1 regulated increased IGF-I expression on the development of fibrosis was confirmed using the Crohn’s disease model, chronic TNBS-colitis, in IGF-I heterozygous mice ⁷³. TGF-β1 also stimulates the expression of IGFBP-3, which further activates TGF-β1 receptors, additionally increases collagen production and also contributes to fibrosis⁵⁹. In the mucosa, IGF-I also stimulates collagen production from subepithelial myofibroblasts ⁷⁴.

Innate immunity and intestinal fibrosis

The innate immune system plays a role both in the pathogenesis of Crohn’s disease and also in initiation of fibrosis. Similarly to immune cells, myofibroblasts express Toll-like receptors (TLRs) that bind bacterial products via pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) ⁴. Activation of TLRs on immune cells activates mitogen-activated protein (MAP) kinases, causes translocation of nuclear factor-κB (NF-κB) to the nucleus, and ultimately results in the secretion of proinflammatory cytokines and chemokines that activate and attract myofibroblasts. Activation of TLRs also stimulates myofibroblast proliferation and collagen production ⁷⁵.

The important role of bacteria in the development of fibrosis in susceptible hosts was examined in the IL-10 mouse model of spontaneous inflammation ⁷⁶. IL10-null mice raised in either conventional housing or under germ-free conditions were randomized to ileocecal resection, intestinal transection without resection, or to no treatment. The post-surgical inflammation and initiation of fibrosis that occurred in genetically susceptible IL10-null mice did not occur in the absence of intestinal bacteria ⁷⁶. The ability of the gut microbiota to alter the expression of profibrotic genes and influence inflammation-associated fibrosis was also demonstrated using the Salmonella typhimurium model of intestinal fibrosis ⁷⁶. Delayed eradication of infection repressed but did not prevent fibrosis. Early eradication of infection and inflammation diminished but did not eradicate the development of subsequent fibrosis.

Development of fibrosis is also a reflection of an abnormal immune response to luminal bacteria. The bacterial product, flagellin, antigen for a commonly observed antibody in patients with Crohn’s disease, is a TLR-5 ligand. Activation of TLR-5 by flagellin in human intestinal fibroblasts in conjunction with the inflammasome leads to increased fibrosis that is mediated directly via hypoxia inducible factor (HIF) and indirectly by eliciting TGF-β1 release from monocytes ⁷⁷.

Adaptive immunity and intestinal fibrosis

Cytokines are integral in the response to inflammation. The immunologic responses in Crohn’s disease include a pro-inflammatory Th1 response involving IL-1β and TNF-α. The Th17 response is characteristic of autoimmunity. TGF-β1 is needed to induce differentiation of this lineage. Th17 and Treg responses are important mediators of the fibrosis observed in Crohn’s disease. While a Th2 response involving IL-4 and IL-13 is most characteristic of ulcerative colitis it can be seen later in the course of Crohn’s disease. Key cytokines in the Th17 pathway include IL-17, IL-23, IL-6 and TNF-α. IL-23, IL-17 and IL-6 induce and activate the transcription factors, STAT3 and RORc.

A general theme emerges from the actions of the cytokines involved in Th1, Th17, Th2 and Treg. They are able to activate mesenchymal cells, stimulate their proliferation and increase collagen production. Il-6, Il-1β, IL-13 and IL-17a are direct mediators of increased latent TGF-β expression in mesenchymal cells. In the proper setting this broad array of cytokines can promote the development of fibrosis in the susceptible patient.

Dysregulation of ECM homeostasis

In the intestine, the components of ECM include structural proteins such as collagen, particularly collagen I, III, and V; specialized proteins, such as vitronectin and fibronectin; and matricellular proteins, such as osteopontin and thrombospondin. These all exert regulatory functions by interacting with cell-surface receptors, including integrins. Within regions of stricturing, expression of collagen, fibronectin and vitronectin, and thrombospondin are all increased [34]. However, regulation of the ECM is a dynamic process involving not only its expression and production, but also its regulated turnover. Turnover of ECM is regulated by the production of 23 human MMPs; these are zinc- and calcium-dependent proteases that are produced by epithelial cells, endothelial cells, MSCs, and macrophages, and activated by proteolytic cleavage. MMPs comprise collagenases, gelatinases, stromolysins, matrilysins, and membrane-type MMPs (MT-MMPs), and thus possess broad substrate specificity^{6, 7, 78, 79}. Increased expression of MMPs, such as the collagenase MMP-8 and the gelatinase MMP-9, is observed subjacent to the intestinal ulcerations of active disease. MMPs, in turn, are regulated by four tissue inhibitors of metalloproteinases (TIMPs) that can be produced by the same cells that produce MMPs.

Cytokines and growth factors, such as TNF-α and TGF-β can regulate MMP expression and promote fibrosis. In intestinal myofibroblasts, for example, TGF-β downregulates MMP-1 and MMP-3 expression and enhances TIMP-1 expression, while TNF-α decreases MMP-2 activity and increases TIMP-1 expression ⁷. IL-21, another T-cell derived cytokine that is increased in IBD, can increase expression of MMPs by intestinal myofibroblasts, contributing to mucosal ulcer formation ⁸⁰. Increased expression of MMP-1, MMP-3, and TIMP-1 has also been demonstrated in the muscularis propria of the intestine in active Crohn’s disease ⁸¹.

The action of MT-MMP-1 exemplifies a unique mechanism of ECM regulation. Through interaction with αVβ8 integrin, MT-MMP-1 regulates the activation of TGF-β1, particularly in the epithelium, thereby increasing the availability of this pro-fibrotic cytokine ⁸².