Name: therapeutic evaluation of fecal microbiota transplantation in an interleukin 10-deficient mouse model
Uploaded: 2022-04-06T13:10:00
Description: Watch this Scientific Journal Video about Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model at JoVE.com

This work was supported, in part, by grants from the National Institutes of Health (R01 HL152683 and R21 AI126097 to Q. Li) and by American Heart Association Grant-in-Aid 17GRNT33460395 (to Q. Li) (heart.org).

All procedures performed on animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (Protocol # 1512071A).
1. Collection of fresh fecal pellets
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	<li>Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes.
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		<li>Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 &#176;C in dry heat for 30 min. Use sterile conical tubes as well. Weigh the c...

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	<li>D'Argenio, V., Salvatore, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+gut+microbiome+in+the+healthy+adult+status.">The role of the gut microbiome in the healthy adult status.</a> Clinica Chimica Acta. 451, 97-102 (2015).</li><li>Baquero, F., Nombela, 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+microbiome+as+a+human+organ.">The microbiome as a human organ.</a> Clinical Microbiology and Infection. 18, 2-4 (2012).</li><li>Hawrelak, J. A., Myers, S. 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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=Suppression+of+tumorigenesis+in+the+Apc(min)+mouse:+down-regulation+of+beta-catenin+signaling+by+a+combination+of+tea+plus+sulindac.">Suppression of tumorigenesis in the Apc(min) mouse: down-regulation of beta-catenin signaling by a combination of tea plus sulindac.</a> Carcinogenesis. 24 (2), 263-267 (2003).</li><li>Kline, K. T., 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=Neonatal+injury+increases+gut+permeability+by+epigenetically+suppressing+E-Cadherin+in+adulthood.">Neonatal injury increases gut permeability by epigenetically suppressing E-Cadherin in adulthood.</a> The Journal of Immunology. 204 (4), 980-989 (2020).</li><li>DeGruttola, A. K., Low, D., Mizoguchi, A., Mizoguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+dysbiosis+in+disease+in+human+and+animal+models.">Current understanding of dysbiosis in disease in human and animal models.</a> Inflammatory Bowel Diseases. 22 (5), 1137-1150 (2016).</li><li>Chevalier, G., 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=Effect+of+gut+microbiota+on+depressive-like+behaviors+in+mice+is+mediated+by+the+endocannabinoid+system.">Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.</a> Nature Communications. 11 (1), 6363 (2020).</li><li>Kao, 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=Effect+of+oral+capsule-+vs+colonoscopy-delivered+fecal+microbiota+transplantation+on+recurrent+clostridium+difficile+infection:+A+randomized+clinical+trial.">Effect of oral capsule- vs colonoscopy-delivered fecal microbiota transplantation on recurrent clostridium difficile infection: A randomized clinical trial.</a> JAMA. 318 (20), 1985-1993 (2017).</li><li>Cammarota, G., 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=International+consensus+conference+on+stool+banking+for+faecal+microbiota+transplantation+in+clinical+practice.">International consensus conference on stool banking for faecal microbiota transplantation in clinical practice.</a> Gut. 68 (12), 2111-2121 (2019).</li><li>Vemuri, R., 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+microgenderome+revealed:+sex+differences+in+bidirectional+interactions+between+the+microbiota,+hormones,+immunity+and+disease+susceptibility.">The microgenderome revealed: sex differences in bidirectional interactions between the microbiota, hormones, immunity and disease susceptibility.</a> Seminar Immunopathology. 41 (2), 265-275 (2019).</li><li>Wilkinson, N. M., Chen, H. -. C., Lechner, M. G., Su, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+immunity.">Sex differences in immunity.</a> Annual Review of Immunology. , (2022).</li><li>Lee, C. H., 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=Frozen+vs+fresh+fecal+microbiota+transplantation+and+clinical+resolution+of+diarrhea+in+patients+with+recurrent+clostridium+difficile+infection:+A+randomized+clinical+trial.">Frozen vs fresh fecal microbiota transplantation and clinical resolution of diarrhea in patients with recurrent clostridium difficile infection: A randomized clinical trial.</a> JAMA. 315 (2), 142-149 (2016).</li><li>Hamilton, M. J., Weingarden, A. R., Sadowsky, M. J., Khoruts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+frozen+preparation+for+transplantation+of+fecal+microbiota+for+recurrent+clostridium+difficile+infection.">Standardized frozen preparation for transplantation of fecal microbiota for recurrent clostridium difficile infection.</a> American Journal of Gastroenterology. 107 (5), 761-767 (2012).</li><li>Tang, G., Yin, W., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+frozen+fecal+microbiota+transplantation+as+effective+as+fresh+fecal+microbiota+transplantation+in+patients+with+recurrent+or+refractory+Clostridium+difficile+infection:+A+meta-analysis.">Is frozen fecal microbiota transplantation as effective as fresh fecal microbiota transplantation in patients with recurrent or refractory Clostridium difficile infection: A meta-analysis.</a> Diagnostic Microbiology and Infectious Disease. 88 (4), 322-329 (2017).</li><li>Cui, B., 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=Fecal+microbiota+transplantation+through+mid-gut+for+refractory+Crohn's+disease:+safety,+feasibility,+and+efficacy+trial+results.">Fecal microbiota transplantation through mid-gut for refractory Crohn's disease: safety, feasibility, and efficacy trial results.</a> Journal of Gastroenterology and Hepatology. 30 (1), 51-58 (2015).</li><li>Li, 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=Timing+for+the+second+fecal+microbiota+transplantation+to+maintain+the+long-term+benefit+from+the+first+treatment+for+Crohn's+disease.">Timing for the second fecal microbiota transplantation to maintain the long-term benefit from the first treatment for Crohn's disease.</a> Applied Microbiology and Biotechnology. 103 (1), 349-360 (2019).</li><li>Moayyedi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+fecal+microbiota+transplantation+in+patients+with+inflammatory+bowel+disease.">Update on fecal microbiota transplantation in patients with inflammatory bowel disease.</a> Gastroenterology and Hepatology. 14 (5), 319 (2018).</li><li>Saha, S., Mara, K., Pardi, D. S., Khanna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+safety+of+fecal+microbiota+transplantation+for+recurrent+clostridioides+difficile+infection.">Long-term safety of fecal microbiota transplantation for recurrent clostridioides difficile infection.</a> Gastroenterology. 160 (6), 1961-1969 (2021).</li><li>Perler, B. K., 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=Long-term+efficacy+and+safety+of+fecal+microbiota+transplantation+for+treatment+of+recurrent+clostridioides+difficile+infection.">Long-term efficacy and safety of fecal microbiota transplantation for treatment of recurrent clostridioides difficile infection.</a> Journal of Clinical Gastroenterology. 54 (8), 701-706 (2020).</li><li>Safety alert regarding use of fecal microbiota for transplantation and risk of serious adverse events likely due to transmission of pathogenic organisms. FDA Available from: <a target="_blank" href="https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely">https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely</a> (2020)</li></ol>

As an innovative investigational treatment, FMT has become a hot topic in the treatment of various disorders in recent years since dysbiosis of the commensal microbiota is implicated in the pathogenesis of multiple human diseases, including IBD, obesity, diabetes mellitus, autism, heart disease, and cancer26. Although the mechanism has not been determined, it is believed that FMT works by building a new biological flora and preventing the loss of residual bacteria. The method presented herein adop...

The human intestinal micro-ecosystem is extremely complex, with more than 1000 species of bacteria in the intestine of a healthy person1. The intestinal flora is involved in maintaining the normal physiological functions of the intestine and the immune response and has an inseparable relationship with the human body. Accumulating evidence suggests that the intestinal microbiome constitutes the last human organ, which is part of the human body, not just a group of parasites2. A &#39;healthy&#39; symbiotic relationship between the gut microbiota, their metabolites, and the host immune system established in early life is critical to maintaining gut homeostasis. In some abnormal conditions such as chronic inflammation, changes in the internal and external environment of the body seriously disrupt the gut homeostasis, resulting in a persistent imbalance of the gut&#39;s microbial community, named dysbiosis3. In fact, exposure to multiple environmental factors, including diet, drugs, and pathogens, can lead to changes in the microbiota.
Dysbiosis is associated with the pathogenesis of a variety of intestinal diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and pseudomembranous enteritis, as well as a growing list of extra-intestinal disorders, including cardiovascular disease, obesity, and allergy4. Microbiota profiling revealed that patients with IBD have a dramatic decrease in bacterial diversity, as well as marked alterations in the populations of some specific bacterial strains5,6. These studies demonstrated less Lachnospiraceae and Bacteroidetes but more Proteobacteria and Actinobacteria in IBD patients. It is believed that the pathogenesis of IBD is related to various pathogenic factors, including abnormal intestinal flora, dysregulated immune response, environmental challenges, and genetic variants7.Abundant evidence suggests that intestinal bacteria play a role in the initiation and application phases of IBD8,9, indicating that correcting gut dysbiosis may represent a novel approach for the therapy and/or maintenance treatment of IBD.
The prototype of fecal microbiota transplantation (FMT) began in ancient China10. In 1958, Dr. Eiseman and his colleagues successfully treated four cases of severe pseudomembranous enteritis with fecal matter from healthy donors via enema, opening a new chapter in modern Western medicine using human feces to treat human diseases11. Clostridium difficile infection (CDI) has been found to be the main cause of pseudomembranous enteritis12 and FMT is highly effective in the treatment of CDI. In the last eight years, FMT has become a standard-of-care therapy for the treatment of recurrent CDI13, prompting further studies investigating the role of FMT in other disorders, such as IBD. Over the past twenty years, numerous case reports and cohort studies have documented the use of FMT in patients with IBD14. A meta-analysis, including 12 trials, showed that 62% of patients with Crohn&#39;s disease (CD) achieved clinical remission after FMT, and 69% of CD patients had clinical response15. Despite these encouraging findings, the role of FMT in the management of IBD remains uncertain, and the mechanisms by which FMT ameliorates intestinal inflammation are poorly understood. Further investigation is necessary before FMT can join the current armamentarium of treatment options for IBD in the clinics.
In this protocol, we applied FMT on IL-10-/- mice, which develop colitis spontaneously after weaning and have served as a gold standard to mirror the multifactorial nature of IBD16,17,18.&#160;IL-10&#8722;/&#8722; mice have been extensively used to dissect IBD etiology because they present similar molecular and histological features to IBD patients, and, like patients, the disease can be ameliorated with anti-TNF&#945; therapy16. Aging IL10&#8722;/&#8722; mice (&#62;9 months of age) have an increased heart size and impaired cardiac function compared to age-matched wild-type mice19, making it an excellent model for studying colitis-induced heart diseases. However, other murine models of colitis, such as the dextran sodium sulfate model and the T-cell-induced colitis model, can be used as well. We administered fecal suspension via oral gavage, proven to be an effective and better route than enema in humans20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Syringe, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-829-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Knipex</td>
			<td>926443</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain natriuretic peptide EIA kit</td>
			<td>Sigma</td>
			<td>RAB0386</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Lab</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved feeding Needles</td>
			<td>Kent Scientific</td>
			<td>FNC-20-1.5-2</td>
			<td></td>
		</tr>
		<tr>
			<td>GLH-115 homogenizer</td>
			<td>Omni International</td>
			<td>GLH-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>MilliporeSigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-10 knockout mice</td>
			<td>Jackson Lab</td>
			<td>004366</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical care</td>
			<td>NDC66794-017-10</td>
			<td></td>
		</tr>
		<tr>
			<td>USP normal saline</td>
			<td>Grainger</td>
			<td>6280</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Euthanex Corp.</td>
			<td>EZ-108SA</td>
			<td></td>
		</tr>
	</tbody>
</table>

therapeutic evaluation of fecal microbiota transplantation in an interleukin 10-deficient mouse model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

We performed healthy donor FMT 3 times (once a month for 3 months) on 2-month old C57BL/6J wild type (WT) and IL-10 knockout mice. Age-matched C57BL/6J mice (age difference should be &#60;2 months) served as the fecal donors and fresh fecal pellets were used each time. EIA assays revealed that BNP was markedly elevated in the plasma of IL-10-deficiency mice and that healthy donor FMT significantly mitigated the increase in BNP levels (Figure 1A, n = 5, p &#60; 0.05). Echocardiograph...

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Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Research

JoVE Journal

Immunology and Infection

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

评估介入疗法对人体粪便微生物群影响的体内批处理培养模型

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

科研

免疫与感染

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

大鼠异位心脏和心肌细胞移植的免疫模型

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

穆林心脏移植袖口技术的优化

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

实验性自身免疫性葡萄膜炎：眼内炎症小鼠模型

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Partial Heterotopic Hindlimb Transplantation Model in Rats

大鼠部分异位欣德利姆移植模型

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

分析宿主-微生物群相互作用的肠道肠道器官培养系统

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

在盲肠结扎和穿刺诱导的脓毒症小鼠模型中评估可靠的生物标志物

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

用于过敏性接触性皮炎评估的小鼠耳模型

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...