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>
	<li>Danese, S., 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=Ulcerative+colitis.">Ulcerative colitis.</a> New England Journal of Medicine. 365 (18), 1713-1725 (2011).</li><li>Baumgart, D. C., Sandborn, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crohn's+disease.">Crohn's disease.</a> The Lancet. 380 (9853), 1590-1605 (2012).</li><li>Knights, D., Lassen, K. G., Xavier, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+inflammatory+bowel+disease+pathogenesis:+linking+host+genetics+and+the+microbiome.">Advances in inflammatory bowel disease pathogenesis: linking host genetics and the microbiome.</a> Gut. 62 (10), 1505-1510 (2013).</li><li>Xavier, R. J., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+pathogenesis+of+inflammatory+bowel+disease.">Unravelling the pathogenesis of inflammatory bowel disease.</a> Nature. 448 (7152), 427-434 (2007).</li><li>Cho, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+immunopathogenesis+of+inflammatory+bowel+disease.">The genetics and immunopathogenesis of inflammatory bowel disease.</a> Nature Reviews Immunology. 8 (6), 458-466 (2008).</li><li>Ogura, 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=A+frameshift+mutation+in+NOD2+associated+with+susceptibility+to+Crohn's+disease.">A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.</a> Nature. 411 (6837), 603-606 (2001).</li><li>Jostins, 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=Host-microbe+interactions+have+shaped+the+genetic+architecture+of+inflammatory+bowel+disease.">Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.</a> Nature. 491 (7422), 119-124 (2012).</li><li>Rivas, M. 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=Deep+resequencing+of+GWAS+loci+identifies+independent+rare+variants+associated+with+inflammatory+bowel+disease.">Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease.</a> Nature Genetics. 43 (11), 1066-1073 (2011).</li><li>Rioux, J. D., 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=Genome-wide+association+study+identifies+new+susceptibility+loci+for+Crohn+disease+and+implicates+autophagy+in+disease+pathogenesis.">Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis.</a> Nature Genetics. 39 (5), 596-604 (2007).</li><li>Uhlig, H. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+enteric+Salmonella+infections.">Pathogenesis of enteric Salmonella infections.</a> Current Opinion in Gastroenterology. 24 (1), 22-26 (2008).</li><li>Barthel, M., 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=Pretreatment+of+mice+with+streptomycin+provides+a+Salmonella+enterica+serovar+Typhimurium+colitis+model+that+allows+analysis+of+both+pathogen+and+host.">Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.</a> Infection and Immunity. 71 (5), 2839-2858 (2003).</li><li>Grassl, G. A., Valdez, Y., Bergstrom, K., Vallance, B. A., 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=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. L., Reid-Yu, S. A., McPhee, J. B., Coombes, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+infection+with+Crohn's+disease-associated+adherent-invasive+Escherichia+coli+leads+to+chronic+inflammation+and+intestinal+fibrosis.">Persistent infection with Crohn's disease-associated adherent-invasive Escherichia coli leads to chronic inflammation and intestinal fibrosis.</a> Nature Communications. 4, 1957 (2013).</li><li>Imai, J., 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=Flagellin-mediated+activation+of+IL-33-ST2+signaling+by+a+pathobiont+promotes+intestinal+fibrosis.">Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis.</a> Mucosal Immunology. , (2019).</li></ol>

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 ...

Tissue fibrosis is the primary way that organs fail over time, in response to chronic inflammation or aging. And in fact, I would argue that if you don't die from an infectious agent or cancer, you'll likely die from organ fibrosis as you age. Unfortunately, there're not a lot of great mouse models to mimic human tissue fibrosis.So what we've developed is a mouse model of Crohn's disease, that's a disease in human that causes fibrosis of the gut and is largely untreatable. In fact, the only way you can treat it is through surgical removal of the fibrotic tissue and re-ligation of the gut. The advantage of our model is that it's fully penetrant in all mouse strains and it very closely mimics human Crohn's disease and the fibrosis persists for over a month, which is really a robust model.The most challenging part of this procedure is the handling of the mice and especially the oral gavage since that requires a lot of practice special certification. When performing this procedure for the first time, one should be aware that working with Salmonella requires proper safety precautions and sterile technique. Disposal of waste and bacteria should all be planned prior to the experiment.The visual demonstration of this procedure is very important because it involves the handling of Salmonella that requires special safety precautions as well as proper sterile technique. To begin this procedure, set out a plate with LB agar containing Streptomycin at a concentration of 100 micrograms per milliliter. Using a sterile inoculating loop, streak the plate with a frozen glycerol stock of S typhimurium delta arrow a.Incubate overnight at 37 degrees Celsius. Streak plates can be stored at four degrees Celsius for up to one week. One day prior to infection, dissolve 0.5 grams of Streptomycin in 2.5 milliliters of water to prepare the antibiotic.Filter sterilize the Streptomycin solution. Then, use a bulb-tipped 22 gauge gavage needle with a one milliliter syringe to orally gavage the mice with 100 microliters of the streptomycin solution. Next, add three milliliters of LB broth containing Streptomycin at a concentration of 50 micrograms per milliliter to a culture tube.Using an inoculation loop, inoculate the LB broth with a single colony. Incubate the culture aerobically overnight at 37 degrees Celsius with shaking at 200 rpm. On the day of infection, perform two consecutive one to 10 dilutions of the overnight Salmonella cultures with sterile PBS to prepare the final infection dose.This results in a 100 microliter inoculum containing approximately three million colony forming units. After this, use a bulb-tipped 22 gauge gavage needle with a milliliter syringe to gavage each mouse with 100 microliters of the prepared Salmonella. First, set out two milliliter safe-lock round bottom microtubes, add one milliliter of sterile PBS and an autoclaved stainless steel bead to each tube.Pre-weigh the tubes prior to tissue collection. After euthanizing mice, resect their cecal and splenic tissues, making sure to collect tissue from individual animals into separate tubes. Weigh each tube to determine the tissue weights.Use a mixer mill apparatus to homogenize the tissues at 30 hertz for 15 minutes. Then, transfer 900 microliters of PBS into each well of a 96-well two milliliter mega block. Pipette 100 microliters of tissue homogenates into the first well, and mix well.Perform serial dilutions by adding 100 microliters to subsequent wells, until a 10 to the negative sixth dilution is obtained. Plate 10 microliters of each dilution in triplicates onto LB agar that contains Streptomycin. Then, count the average colony forming units and determine the colony forming units per gram of tissue as outlined in the text protocol.Open Fiji, which is an open sourced imaging software and drag and drop the tif image file onto the tool bar. On the menu bar, select Image, Type, RGB stack to split the image into red, green and blue channels. Slide the horizontal bar at the bottom of the panel to set the channel to green.Open Image, Adjust, Threshold tool. Adjust minimum and maximum limits to eliminate any background signals. Once the desired threshold is set, close the threshold tool and go to Analyze, Set Measurements, check off Area, Area fraction, Limit to threshold and Display label.Get the tissue selection with either the freehand selections or polygon selections tool and measure the percent area positive for collagen by clicking Analyze, Measure. Then, normalize the absolute area positive for collagen staining to tissue area. Streptomycin treatment followed by oral infection with S typhimurium delta arrow a leads to robust intestinal inflammation and fibrosis, especially in the cecum.Typical pathogen burdens of 100 million to one billion colony forming units can be recovered per gram of cecum from infected animals, while 10 thousand colony forming units can typically be recovered per gram of spleen. Assessment of fibrosis in picrosirius red stained cecal sections indicates peak fibrosis 21 days after infection, while much of the pathology is resolved by day 42 post infection. Thorough preparation of materials is essential as this experiment requires multiple days to set up.Preparation of LB plates and Salmonella and Streptomycin should all be done aseptically. Prior to the experiment, ensure that the experimenter is familiar with the animal protocol as well as safety hazards. Upon assessing the collagen burden, the experimenter may choose to perform other biological assays, such as RNA sequencing to assess gene expression profiles or flow cytometry to assess immune cell subsets or a tissue ELISA to assess cytokine profiles.So our model is particularly good at mimicking a human disease called Crohn's disease where you get fibrosis of the gut, which is largely untreatable and the only way you can treat it is to remove a section of the gut and stitch it back together and hope it doesn't come back. Our model's very robust and we've already shown that targeting innate lymphoid cells type three, a transcription factor called raw alpha, or interleukin 17 ameliorates the disease and prevents the fibrosis. Our model is particularly good at identifying the factors that play in the pathogenesis of Crohn's disease and gut fibrosis and we're already on the road to identifying a number of targets that could be treated therapeutically to alleviate inflammatory bowel disease.Our model has already entered the same ILC3 or alpha NIL17 access may be at play in other fibrotic disease and these include things like psoriasis, multiple sclerosis, idiopathic pulmonary fibrosis. So it's possible that targeting these same factors might alleviate fibrosis in those diseases as well. Although the mutant Salmonella stream isn't virulent, it is important to follow the standard biohazard safety protocols given by the facility.Also, since formaldehyde vapors are known to be carcinogenic, it's important to work behind a fume hood during the fixation step.

chronic-salmonella-infection-induced-intestinal-fibrosis

Research

JoVE Journal

Immunology and Infection

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...