Name: chronic salmonella infection induced intestinal fibrosis
Uploaded: 2019-09-22T14:00:00
Description: Watch this Scientific Journal Video about Chronic Salmonella Infection Induced Intestinal Fibrosis at JoVE.com

We thank Ingrid Barta for histology services.

All animal protocols were approved by the Animal Care Committee of the University of British Columbia.
1. Preparation of Salmonella Typhimurium &#916;AroA cultures for oral gavage of mice
<ol>
	<li>From a frozen glycerol stock of S. Typhimurium &#916;AroA, prepare a streak plate using LB agar containing 100 &#956;g/mL streptomycin with a sterile inoculating loop. Incubate overnight at 37 &#730;C. Streak plates can be stored up to one week at 4 &#730;C.</li>
	<li>One day pri...

<ol>
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H., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+intestinal+inflammation+as+tools+to+understand+the+pathogenesis+of+inflammatory+bowel+disease.">Mouse models of intestinal inflammation as tools to understand the pathogenesis of inflammatory bowel disease.</a> European Journal of Immunology. 39 (8), 2021-2026 (2009).</li><li>Nell, S., Suerbaum, S., Josenhans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+the+microbiota+on+the+pathogenesis+of+IBD:+lessons+from+mouse+infection+models.">The impact of the microbiota on the pathogenesis of IBD: lessons from mouse infection models.</a> Nature Reviews Microbiology. 8 (8), 564-577 (2010).</li><li>Grassl, G. A., Finlay, B. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Enteric+Salmonella+Infection+in+Mice+Leads+to+Severe+and+Persistent+Intestinal+Fibrosis.">Chronic Enteric Salmonella Infection in Mice Leads to Severe and Persistent Intestinal Fibrosis.</a> Gastroenterology. 134 (3), 768-780 (2008).</li><li>Hoiseth, S. K., Stocker, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aromatic-dependent+Salmonella+typhimurium+are+non-virulent+and+effective+as+live+vaccines.">Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines.</a> Nature. 291 (5812), 238-239 (1981).</li><li>Valdez, Y., Ferreira, R. B., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+Salmonella+virulence+and+host+resistance.">Molecular mechanisms of Salmonella virulence and host resistance.</a> Current Topics in Microbiology and Immunology. 337, 93-127 (2009).</li><li>Valdez, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nramp1+drives+an+accelerated+inflammatory+response+during+Salmonella-induced+colitis+in+mice.">Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice.</a> Cellular Microbiology. 11 (2), 351-362 (2009).</li><li>Lo, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orphan+nuclear+receptor+RORalpha+and+group+3+innate+lymphoid+cells+drive+fibrosis+in+a+mouse+model+of+Crohn's+disease.">The orphan nuclear receptor RORalpha and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease.</a> Science Immunology. 1 (3), eaaf8864 (2016).</li><li>Burke, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrogenesis+in+Crohn's+disease.">Fibrogenesis in Crohn's disease.</a> American Journal of Gastroenterology. 102 (2), 439-448 (2007).</li><li>Hogan, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+and+regeneration+of+the+respiratory+system:+complexity,+plasticity,+and+mechanisms+of+lung+stem+cell+function.">Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.</a> Cell Stem Cell. 15 (2), 123-138 (2014).</li><li>Fiocchi, C., Lund, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Themes+in+fibrosis+and+gastrointestinal+inflammation.">Themes in fibrosis and gastrointestinal inflammation.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 300 (5), G677-G683 (2011).</li><li>Rieder, F., Fiocchi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+fibrosis+in+IBD&#8212;a+dynamic,+multifactorial+process.">Intestinal fibrosis in IBD&#8212;a dynamic, multifactorial process.</a> Nature Reviews Gastroenterology &amp; Hepatology. 6 (4), 228-235 (2009).</li><li>Bouguen, G., Peyrin-Biroulet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+for+adult+Crohn's+disease:+what+is+the+actual+risk?.">Surgery for adult Crohn's disease: what is the actual risk?.</a> Gut. 60 (9), 1178-1181 (2011).</li><li>Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+fibrosis.">Cellular and molecular mechanisms of fibrosis.</a> The Journal of Pathology. 214 (2), 199-210 (2008).</li><li>Junqueira, L. C., Bignolas, G., Brentani, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picrosirius+staining+plus+polarization+microscopy,+a+specific+method+for+collagen+detection+in+tissue+sections.">Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.</a> Histochem J. 11 (4), 447-455 (1979).</li><li>Lo, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+Preserves+Gut+Epithelial+Integrity+and+Promotes+Disease+Remission+during+Chronic+Salmonella+Infection.">IL-22 Preserves Gut Epithelial Integrity and Promotes Disease Remission during Chronic Salmonella Infection.</a> Journal of Immunology. 202 (3), 956-965 (2019).</li><li>Fichtner-Feigl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+IL-13+triggers+TGF-beta1-dependent+tissue+fibrosis+in+chronic+2,4,6-trinitrobenzene+sulfonic+acid+colitis.">Induction of IL-13 triggers TGF-beta1-dependent tissue fibrosis in chronic 2,4,6-trinitrobenzene sulfonic acid colitis.</a> Journal of Immunology. 178 (9), 5859-5870 (2007).</li><li>Fichtner-Feigl, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-13+signaling+via+IL-13R+alpha2+induces+major+downstream+fibrogenic+factors+mediating+fibrosis+in+chronic+TNBS+colitis.">IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis.</a> Gastroenterology. 135 (6), e2001-e2007 (2013).</li><li>Johnson, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+fibrosis+is+reduced+by+early+elimination+of+inflammation+in+a+mouse+model+of+IBD:+impact+of+a+&amp;#34;Top-Down&amp;#34;+approach+to+intestinal+fibrosis+in+mice.">Intestinal fibrosis is reduced by early elimination of inflammation in a mouse model of IBD: impact of a &amp;#34;Top-Down&amp;#34; approach to intestinal fibrosis in mice.</a> Inflammatory Bowel Diseases. 18 (3), 460-471 (2012).</li><li>Darfeuille-Michaud, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+adherent-invasive+Escherichia+coli+associated+with+ileal+mucosa+in+Crohn's+disease.">High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease.</a> Gastroenterology. 127 (2), 412-421 (2004).</li><li>Small, C. 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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...

Ulcerative colitis (UC) and Crohn&#8217;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 &#916;AroA mutant leads to chronic colonization of the cecum and colon that persists up to day 40 after infection14. The S. Typhimurium &#916;AroA 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-&#946;1, 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 &#916;AroA 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 ROR&#945;-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-TNF&#945; 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 &#916;AroA infection is a robust mouse model of intestinal fibrosis that can offer insights into the pathogenesis of CD-like features.

<table>
	<tbody>
		<tr>
			<td>2 ml round bottom safe lock tubes</td>
			<td>Eppendorf</td>
			<td>22363344</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel beads</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Large-Orifice Pipet Tips</td>
			<td>Fisher</td>
			<td>2707134</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL megablock plates</td>
			<td>Sarstedt</td>
			<td>82.1972.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Gavage needles</td>
			<td>FST</td>
			<td>18061-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Sigma</td>
			<td>S9137</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer mill</td>
			<td>Retsch</td>
			<td>MM</td>
			<td></td>
		</tr>
	</tbody>
</table>

chronic salmonella infection induced intestinal fibrosis

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 &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA 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.

Streptomycin treatment followed by oral infection with S. Typhimurium &#916;AroA 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 w...

Watch this Scientific Journal Video about Chronic Salmonella Infection Induced Intestinal Fibrosis at JoVE.com

Chronic Salmonella Infection Induced Intestinal Fibrosis

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;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 ...

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