This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn’s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.
Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium ΔAroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium ΔAroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.
Ulcerative colitis (UC) and Crohn’s disease (CD) are the two major forms of IBD and are characterized as chronic and relapsing inflammatory disorders of the gastrointestinal tract 1,2. These disorders have a major impact on the quality of life of patients. Symptoms of IBD include abdominal pain, diarrhea, nausea, weight loss, fever, and fatigue3. Recent studies have identified genetic and environmental factors that contribute to disease pathogenesis; it is thought that such risk factors contribute to the disruption of the epithelial barrier resulting in the translocation or oversampling of luminal antigens4. As a consequence, this initiates an aberrant inflammatory response to the commensal flora mediated by intestinal immune cells4. Features of IBD-associated complications may extend to sites beyond the GI tract affecting various organs including joints, skin, and liver1,2. Hallmarks of UC include severe and diffuse inflammation typically localized in the colon1. Disease pathology affects the mucosa and submucosa of the bowel resulting in superficial mucosal ulcerations1. In contrast, CD can affect any part of the GI tract although evidence of disease is commonly found in the colon and distal ileum2. Moreover, the inflammation in CD is transmural, affecting all layers of the bowel wall2.
Several IBD susceptibility genes that have been identified would indicate that dysregulation of the epithelial barrier or immunity are critical contributors to disease progression5. Mutations in nucleotide oligomerization domain 2 (NOD2) expressed by monocytes was found to be associated with increased susceptibility to CD; this highlights a link between altered innate immune detection of bacterial components and the disease6. More recent genome-wide association studies (GWAS) have revealed additional pathways potentially involved in the pathogenesis of IBD including genetic variations in: STAT1, NKX2-3, IL2RA, IL23R dependent pathways linked to adaptive immunity, MUC1, MUC19, and PTGER4 in intestinal barrier maintenance, and ATG16L-mediated autophagy7,8,9. While these population-based genetics studies have enhanced our understanding of IBD, susceptibility alleles alone are likely insufficient in initiating and sustaining chronic disease3. Other non-genetic factors including alterations in gut microbiome composition and a reduction in diversity have been associated with intestinal inflammation. However, it is unclear whether gut dysbiosis precedes or is the consequence of dysregulated immune responses3. Although the etiology of IBD remains unclear, our understanding of the pathogenesis of the disease has been enhanced by experimental mouse models of intestinal inflammation10,11. These models individually do not fully represent the complexity of the human disease, but they are valuable for elucidating pathophysiological pathways that could be relevant to IBD and for the validation of tentative therapeutic strategies10,11. Such mouse models typically rely on the initiation of inflammation by chemical induction or infection, immune cell transfer, or genetic manipulation. Moreover, these strategies often involve perturbations in epithelial integrity or modulation of innate or adaptive immunity.
Salmonella enterica serovars are intestinal pathogens that can infect humans and mice. After ingestion, Salmonella can colonize the gut by direct invasion of epithelia, M cells, or antigen presenting cells12. Mice infected orally with S. Typhimurium results in the colonization primarily of systemic sites such as the spleen and mesenteric lymph nodes with relatively low abundance in the GI tract12. However, pretreatment of mice with streptomycin enhances the efficiency of Salmonella colonization of the gut by diminishing the host protective effects of the normal microbiota13. Pathological features of this model include the disruption or ulceration of the epithelial barrier, granulocyte recruitment, and severe edema13. Alternatively, infection with the vaccine grade S. Typhimurium ΔAroA mutant leads to chronic colonization of the cecum and colon that persists up to day 40 after infection14. The S. Typhimurium ΔAroA strain has a defect in the biosynthesis of aromatic amino acids; this renders the mutant strain avirulent and can be utilized as a highly effective vaccine15. Oral infection in mice leads to a Th1- and Th17-cytokine associated inflammatory response, extensive tissue remodeling, and collagen deposition. Tissue pathology is associated with elevated levels of pro-fibrotic factor such as TGF-β1, CTGF, and IGF14. The transmural fibrotic scarring reported in this model is reminiscent of stricture formations often observed in IBD. The induction of fibrosis by Salmonella requires virulence encoded by Salmonella pathogenicity islands (SPI)-1 and 2 12. Importantly, this S. Tymphimurium ΔAroA infection model is a useful system for the study of fibrotic responses in mutant mice maintained on a C57/Bl6 background. The C57/Bl6 strain is extremely sensitive to S. Typhiumurim SL1344 infection due to a loss-of-function mutation in the gene encoding the natural resistance-associated macrophage protein (NRAMP)-116,17. We have found that IL-17A and RORα-dependent innate lymphoid cells are important contributors to pathogenesis in this model18.
A major complication of CD is the dysregulated and excessive deposition of extracellular matrix (ECM) including collagen2,19. Although the GI tract has a relatively high capacity for regeneration, fibrotic scarring can arise due to unresolved wound healing responses that are associated with chronic and severe inflammation20,21. In CD, this results in deleterious effects on tissue architecture leading to significant organ impairment21,22. The transmural nature of the inflammation observed in CD ultimately precedes the thickening of the bowel wall associated with symptomatic stenosis or stricture formation21. About a third of CD patients require intestinal resection for this complication22. There are no effective anti-fibrotic therapies in IBD given that the use of immunosuppressants such as azathioprine or anti-TNFα biologics have no impact or only modestly reduced the requirement of surgical interventions19,23. While fibrosis is thought be the consequence of chronic inflammation, cells of mesenchymal origin such as fibroblasts and pericytes are thought to be the primary cellular sources of ECM in fibrotic scarring21,24. Chronic S. Typhimurium ΔAroA infection is a robust mouse model of intestinal fibrosis that can offer insights into the pathogenesis of CD-like features.
Our understanding of the pathogenesis of IBD has been greatly enhanced by mouse models of intestinal inflammation. Although such individual models do not recapitulate all features of the complex and multifactorial human disease, they have been useful in identifying key features of disease progression. Fibrotic strictures associated with IBD remains a major unmet clinical need as current treatments are ineffective in reversing disease development. Moreover, intestinal fibrosis is difficult to study in a laboratory setting…
The authors have nothing to disclose.
We thank Ingrid Barta for histology services.
2 ml round bottom safe lock tubes | Eppendorf | 22363344 | |
Stainless steel beads | Qiagen | 69989 | |
PBS | Gibco | 10010031 | |
Large-Orifice Pipet Tips | Fisher | 2707134 | |
2 mL megablock plates | Sarstedt | 82.1972.002 | |
Gavage needles | FST | 18061-22 | |
Streptomycin sulfate | Sigma | S9137 | |
Mixer mill | Retsch | MM |