Chronic Salmonella Infection Induced Intestinal Fibrosis

Summary

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.

Abstract

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.

Introduction

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 oversa....

Protocol

All animal protocols were approved by the Animal Care Committee of the University of British Columbia.

1. Preparation of Salmonella Typhimurium ΔAroA cultures for oral gavage of mice

1. From a frozen glycerol stock of S. Typhimurium ΔAroA, prepare a streak plate using LB agar containing 100 μg/mL streptomycin with a sterile inoculating loop. Incubate overnight at 37 ˚C. Streak plates can be stored up to one week at 4 ˚C.
2. One day prior to infection, prepare antibiotic by dissolving 0.5 g of streptomycin in 2.5 mL of water. After filter sterilizing streptomycin solution, orally gav....

Results

Streptomycin treatment followed by oral infection with S. Typhimurium ΔAroA leads to robust intestinal inflammation and fibrosis especially in the cecum (Figure 1). Typical pathogen burdens of 10⁸ to 10⁹ CFU per 1 g of cecum and 10⁴ CFU per 1 g of spleen can be recovered from infected animals (Figure 2). Assessment of fibrosis in picrosirius red stained cecal sections indicate peak fibrosis 21 days after infection while .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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