Using Salmonella enterica Serotype Typhimurium to Model Intestinal Fibrosis

Abstract

See “Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis” by Grassl GA, Valdez Y, Bergstrom KSB, et al on page 768. See “Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis” by Grassl GA, Valdez Y, Bergstrom KSB, et al on page 768. Crohn’s disease (CD) and ulcerative colitis (UC) are life-long, relapsing illnesses that primarily affect the gastrointestinal tract of the young adult. Such conditions are characterized by chronic inflammation, mucosal damage, and epithelial cell destruction resulting from a complex interplay among genetic, immunologic, and microbial factors. Unlike UC, in which inflammation is limited to the mucosa, the inflammation that occurs during CD is transmural and granulomatous, and affects not only the intestinal mucosa but also the muscularis and serosa. Although inflammatory changes in intestinal physiology cause much of the symptomatology associated with CD, significant morbidity results from the irreversible tissue injury and fibrosis that frequently occur as a common complication.1Becker J.M. Surgical therapy for ulcerative colitis and Crohn’s disease.Gastroenterol Clin North Am. 1999; 28: 371-390Google Scholar, 2Graham M.F. Pathogenesis of intestinal strictures in Crohn’s disease: an update.Inflamm Bowel Dis. 1995; 1: 220-227Google Scholar In fact, more than one third of patients with CD develop a distinct fibrostenosing phenotype that results in recurrent intestinal stricture formation.3Burke J.P. Muslow J.J. O’Keane C. et al.Fibrogenesis in Crohn’s disease.Am J Gastroenterol. 2007; 102: 439-448Google Scholar Fibrosis in CD is believed to result from an overzealous healing response to injury.4Pucilowska J. Williams K.L. Lund P.K. Fibrogenesis IV Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659Google Scholar For reasons that remain unclear, the reparative process associated with CD progresses uncontrollably, leading to the proliferation of mesenchymal cells and the unrestrained deposition of extracellular matrix (ECM).2Graham M.F. Pathogenesis of intestinal strictures in Crohn’s disease: an update.Inflamm Bowel Dis. 1995; 1: 220-227Google Scholar, 5Geboes K.P. Cabooter L. Geboes K. Contribution of morphology for the comprehension of mechanisms of fibrosis in inflammatory enterocolitis.Acta Gastroenterol Belg. 2000; 63: 371-376Google Scholar Abnormal contraction of this ECM leads to scar formation and tissue distortion. Ultimately, this fibrotic process thickens the wall of the gut, reducing flexibility and narrowing the bowel lumen, frequently resulting in obstructive strictures.2Graham M.F. Pathogenesis of intestinal strictures in Crohn’s disease: an update.Inflamm Bowel Dis. 1995; 1: 220-227Google Scholar, 5Geboes K.P. Cabooter L. Geboes K. Contribution of morphology for the comprehension of mechanisms of fibrosis in inflammatory enterocolitis.Acta Gastroenterol Belg. 2000; 63: 371-376Google Scholar, 6Assche G.V. Can we influence fibrosis in Crohn’s disease?.Acta Gastroenterol Belg. 2001; 64: 193-196Google Scholar In intestinal fibrosis, mechanisms to degrade ECM are seemingly not operative at appropriate levels and fibrogenic cells are not only maintained but expanded in number. This is most likely caused by an imbalance between the synthesis of matrix and its degradation by matrix metalloproteinases (MMP).4Pucilowska J. Williams K.L. Lund P.K. Fibrogenesis IV Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659Google Scholar, 7Schuooan D. Hahn E.G. MMPs in the gut: inflammation hits the matrix.Gut. 2000; 47: 12-14Google Scholar Furthermore, CD is characterized by hyperplasia of the muscle layers and in severely fibrotic areas there is a change in smooth muscle cells toward a fibrogenic phenotype.8Pucilowska J.B. McNaughton K.K. Mohapatra N.K. et al.IGF-1 and procollagen alpha 1 are coexpressed in a subset of mesenchymal cells in active Crohn’s disease.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1307-G1322Google Scholar Although excessive ECM depositions, as well as the proliferation of mesenchymal cells, are key contributory factors to stricture development, a hallmark feature of intestinal fibrosis is that strictures characteristically display thickening of all layers of the intestinal wall (Figure 1). Histologically, areas of fibrosis are principally composed of islands of smooth muscle cells in the submucosa that are associated with dense collagen deposition, and disruption and expansion of the muscularis mucosa. Collagen appears to be the major ECM component associated with intestinal fibrosis and a number of subtypes, each with different functions have been identified.2Graham M.F. Pathogenesis of intestinal strictures in Crohn’s disease: an update.Inflamm Bowel Dis. 1995; 1: 220-227Google Scholar, 3Burke J.P. Muslow J.J. O’Keane C. et al.Fibrogenesis in Crohn’s disease.Am J Gastroenterol. 2007; 102: 439-448Google Scholar, 4Pucilowska J. Williams K.L. Lund P.K. Fibrogenesis IV Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659Google Scholar, 9Matthes H. Herbst H. Schuppan D. et al.Cellular localization of procollagen gene transcripts in inflammatory bowel diseases.Gastroenterology. 1992; 102: 431-442Crossref Scopus (82) Google Scholar In normal intestine, the major collagen subtypes are type 1 (70%), type III (20%), and type V (12%), whereas in intestinal fibrosis there is an increase in total collagen (types I, III, IV, and V) in addition to perhaps the specific and relative increase of types III and V. Furthermore, collagen type I is the predominant collagen subtype found at sites of stricture. In addition, thickening of the muscularis layers (muscularis overgrowth) seems to be associated with an increase in the number of vimentin-positive (V+) cells in areas of fibrosis.3Burke J.P. Muslow J.J. O’Keane C. et al.Fibrogenesis in Crohn’s disease.Am J Gastroenterol. 2007; 102: 439-448Google Scholar, 4Pucilowska J. Williams K.L. Lund P.K. Fibrogenesis IV Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659Google Scholar In severely fibrotic tissue, entire layers of histologically normal muscularis are populated largely by V+/α-smooth muscle actin (A) – and V+/A+ fibroblasts rather than normal V−/A+ smooth muscle cells. This suggests that muscularis overgrowth in CD involves a change in enteric smooth muscle cells toward a fibroblast or myofibroblast phenotype. Intestinal fibrosis presents a significant complication of CD since more than half of all procedures in patients with CD are performed to relieve intestinal obstruction. Surgery is rarely curative; 70% of patients have endoscopic evidence of stricture recurrence at 1 year, and at 4 years, 40% have symptomatic recurrence that requires further intervention.3Burke J.P. Muslow J.J. O’Keane C. et al.Fibrogenesis in Crohn’s disease.Am J Gastroenterol. 2007; 102: 439-448Google Scholar, 10Rutgeerts P. Geboes K. Vantrappen G. et al.Natural history of recurrent Crohn’s disease at the ileocolonic anastomosis after curative surgery.Gut. 1984; 25: 665-672Google Scholar, 11Rutgeerts P. Geboes K. Vantrappen G. et al.Predictability of the postoperative cause of Crohn’s disease.Gastroenterology. 1990; 99: 956-963Crossref Scopus (1351) Google Scholar, 12Whelan G. Farmer R.G. Fazio V.W. et al.Recurrence after surgery in Crohn’s disease Relationship to localization of disease (clinical pattern) and surgical indication.Gastroenterology. 1985; 88: 1826-1833Google Scholar, 13Dietz D.W. Laurerti S. Strong S.A. et al.Safety and long term efficacy of strictureplasty in 314 patients with obstructing small bowel Crohn’s disease.J Am Cell Surg. 2001; 192: 330-337Google Scholar Despite advances in CD management over the past 25 years, the incidence of strictures and the requirement for intestinal resection have not diminished.3Burke J.P. Muslow J.J. O’Keane C. et al.Fibrogenesis in Crohn’s disease.Am J Gastroenterol. 2007; 102: 439-448Google Scholar This largely reflects our limited understanding of the molecular mechanisms that underlie intestinal fibrogenesis. The current mechanistic framework proposes that stricture formation is promoted, in part, by fibroblast overexpression of adhesion molecules, such as intracellular adhesion molecule-1 and profibrotic growth factors. For example, the transforming growth factor (TGF)-β family plays a universal role in the development of fibrosis with the TGF-β1 subtype, specifically being implicated in pathogenic fibrosis.2Graham M.F. Pathogenesis of intestinal strictures in Crohn’s disease: an update.Inflamm Bowel Dis. 1995; 1: 220-227Google Scholar, 6Assche G.V. Can we influence fibrosis in Crohn’s disease?.Acta Gastroenterol Belg. 2001; 64: 193-196Google Scholar, 14McKaig B.C. Hughes K. Tighe P.J. et al.Differential expression of TGF-beta isoforms by normal and inflammatory bowel disease intestinal myofibroblasts.Am J Physiol Cell Physiol. 2002; 282: C172-C182Google Scholar Overexpression of TGF-β and its receptors in both the intestinal wall and in fibroblast cultures taken from sites of intestinal stricture in patients with CD suggests a regulatory role for this cytokine in intestinal fibrogenesis.15diMola F.F. Friess H. Scheuren A. et al.Transforming growth factor-betas and their signaling receptors are coexpressed in Crohn’s disease.Ann Surg. 1999; 229: 67-75Google Scholar, 16diMola F.F. DiSebastiano P. Gardini A. et al.Differential expression of connective tissue growth factor in inflammatory bowel disease.Digestion. 2004; 69: 245-253Google Scholar These studies suggest that active TGF-β1 causes fibrosis through the induction of connective tissue growth factor (CTGF). In turn, collagen production is positively regulated by TGF-β1, insulin growth factor-1, and CTGF, whereas matrix degradation is restrained by the ability of TGF-β1 to inhibit the expression of MMP-1 and -3 and to stimulate tissue inhibitors of metalloproteinases. To date, the knowledge of the mechanisms that lead to intestinal fibrosis and stricture formation are rather incomplete. This is because mechanistic studies of fibrosis are difficult to perform in humans. Furthermore, despite the large number of murine models of inflammatory bowel disease currently available, such models have so far proven of little use in studying the pathogenesis of stricture formation because they are accompanied by limited intestinal fibrosis. Rats have been shown to develop fibrosis after injection of peptidoglycan-polysaccharide into their gut walls, and after repeated bouts of trinitrobenzene sulfonic acid (TNBS) colitis.17van Tol E.A. Holt L. Li Fl. et al.Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts.Am J Physiol Gastrointest Liver Physiol. 1999; 227: G245-G255Google Scholar, 18Moris G.P. Beck P.L. Herridge M.J. et al.Hapten-induced model of chronic inflammation and ulceration in the rat colon.Gastroenterology. 2003; 125: 1750-1761Google Scholar However, although mice are more resistant to developing fibrotic disease than any other species,4Pucilowska J. Williams K.L. Lund P.K. Fibrogenesis IV Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659Google Scholar the number of research tools available makes the mouse the most desirable animal species to model human disease and test therapies. Progress has been made within the past 5 years as models of intestinal fibrosis in mice have increased in the literature. For example, intestinal fibrosis in mice has been shown to occur after repeated induction of TNBS colitis19Lawrance I.C. Wu F. Leite A.Z. et al.A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Google Scholar and after adenoviral gene transfer and overexpression of TGF-β120Vallance B.A. Gunawan M.I. Hewlett B. et al.TGF-beta1 gene transfer to the mouse colon leads to intestinal fibrosis.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G116-G128Google Scholar and monocyte chemoattractant protein-1 (MCP-1) in the colon.21Motomura Y. Khan W.I. El-Sharkawy R.T. et al.Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein-1.Gut. 2006; 55: 662-670Google Scholar Despite these advances, such models are inadequate because they are labor intensive, lead to frequent mortality, and the fibrosis can vary in the intensity and position within the gastrointestinal tract. Although the overexpression of fibrotic cytokines has proven useful in determining their potential role in fibrosis, these models are further limited in their ability to address how fibrotic processes develop following inflammatory events. In this issue of Gastroenterology, Grassl et al22Grassl G.A. Valdez Y. Bergstrom K.S.B. et al.Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis.Gastroenterology. 2008; 134: 768-780Abstract Full Text Full Text PDF Scopus (111) Google Scholar describe a mouse model of severe and persistent intestinal fibrosis that mimics the pathology seen in Crohn’s patients and trumps all previous models (Figure 1). This model is unique in that the intestinal fibrosis is initiated by a bacteria-induced colitis. In this model, Grassl et al pretreated mice with 20 mg/ml streptomycin 24 hours before oral infection with Salmonella enterica serovar Typhimurium (S typhimurium), an intracellular bacterial pathogen that is responsible for causing food poisoning and gastroenteritis in humans. Mice are more resistant to the development of gastroenteritis phenotype and, after oral inoculation with S typhimurium, mice typically experience symptoms of typhoid fever. However, when mice are pretreated with streptomycin before infection with S typhimurium, the resulting enteric phase of the infection is far more successful, leading to heavy colonization of the cecum and colon with significant colitis. The findings reported by Grassl et al detail two important advances. First, the study documents that infection with S typhimurium leads to chronic infection and colitis in association with extensive transmural ECM deposition within the cecum and colon. For instance, at early time points after infection with S typhimurium (day 7), the fibrosis spanned the entire cecal wall, whereas from day 21 postinfection and onward to day 40, the fibrosis was mainly localized to the muscle layers and the submucosa. Remarkably, although such observations have been made in CD, S typhimurium infections in humans are not associated with intestinal fibrosis. In addition, fibrosis and extensive transmural inflammation was noted along the entire length of the colon, with the most severe and extensive fibrosis found in the cecum. With such consistent localization of fibrotic areas, it is hoped that this model may serve to aid future investigation of mechanisms underlying the fibrotic response. During active clinical disease, the affected gut tissue often exhibits extensive damage to the epithelium and underlying tissues along with the expression of a characteristic cytokine profile dominated by interleukin (IL)-1, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. In keeping with this profile, the second significant finding of the Grassl et al22Grassl G.A. Valdez Y. Bergstrom K.S.B. et al.Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis.Gastroenterology. 2008; 134: 768-780Abstract Full Text Full Text PDF Scopus (111) Google Scholar model is that TNF-α, IFN-γ, MCP-1, and IL-17 were detected in the S typhimurium-infected cecae in concert with transmural infiltration of inflammatory and immune cells. The increased expression of Th-1 cytokines in this model was also accompanied by an increased expression of profibrotic growth factors, such as TGF-β1, CTGF, and IGF-1. TGF-β1 and IGF-1 are both potent inducers of collagen type I, which were strongly up-regulated in the submucosa and external muscle layers of the infected cecae. Examination of the cell types in areas marked by fibrosis further revealed that the majority of the cells were A−/desmin (D)−, identifying them as fibroblasts. Regions rich in fibroblasts also correlated to areas of strong collagen deposition, suggesting that, in this model, fibroblasts are the chief producers of collagen. Again, this observation is consistent with the fact that fibroblasts seem to be the predominant mesenchymal cell type within fibrotic tissues associated with CD. Strong collagen staining also appeared adjacent to V−A+D+ cells (smooth muscle) and V+A+ cells (putative myofibroblast cells). Thus, this bacterial-induced model of intestinal fibrosis established by Grassl et al22Grassl G.A. Valdez Y. Bergstrom K.S.B. et al.Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis.Gastroenterology. 2008; 134: 768-780Abstract Full Text Full Text PDF Scopus (111) Google Scholar introduces a novel mouse model of chronic colitis and intestinal fibrosis that mimics many of the features of CD-induced fibrosis. In addition, for those who study intestinal fibrosis, one should be encouraged by this model for its apparent ease of induction, reproducibility, and a much more manageable time frame to monitor fibrotic illness. However, like any new model system, many questions remain to be answered: Can this model be manipulated by intervention? What knowledge can be gained from the study of bacterial virulence factors and strategies, which are apparently necessary to initiate both chronic inflammation and the fibrosis? Will this model offer a nexus toward mechanistic study? Nevertheless, this is an important contribution and represents an exciting significant advance for the study of intestinal fibrosis. Chronic Enteric Salmonella Infection in Mice Leads to Severe and Persistent Intestinal FibrosisGastroenterologyVol. 134Issue 3PreviewBackground & Aims: Intestinal fibrosis and stricture formation are serious complications of Crohn’s disease, often requiring surgical intervention. Unfortunately, the mechanisms underlying intestinal fibrosis development are poorly understood, in part because of the lack of relevant animal models. Here, we present a novel murine model of severe and persistent intestinal fibrosis caused by chronic bacterial-induced colitis. Methods: Mice were treated with streptomycin 24 hours prior to oral infection with Salmonella enterica serovar Typhimurium. Full-Text PDF