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

Ying Xiao; Xiaoying S Zhong; Xiaowei Liu; Qingjie Li

doi:10.3791/63350

JoVE Journal > Immunology and Infection

Imunologia e Infecção

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

Published: April 06, 2022

doi:

10.3791/63350

Ying Xiao^1,2, Xiaoying S Zhong¹, Xiaowei Liu², Qingjie Li¹

¹Division of Gastroenterology, Department of Internal Medicine,University of Texas Medical Branch at Galveston, ²Department of Gastroenterology,Xiangya Hospital of Central South University

Summary

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.

Abstract

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.

Introduction

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 'healthy' 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'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'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. IL-10^−/− 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-TNFα therapy¹⁶. Aging IL10^−/− mice (>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.

Protocol

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

Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes.
1. Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 °C in dry heat for 30 min. Use sterile conical tubes as well. Weigh the conical tubes and write down their weight on the tubes.
Turn on the biosafety cabinet in the animal room.
Take an autoclaved clean mouse cage without any bedding and place it in the biosafety cabinet. Remove the cover and the food rack and place them inside the cabinet.
Place some sterile paper towels on the bottom of the cage and place the metal rack back on top of the cage.
Identify age-matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse (C57BL/6J) by the tail and place it on the metal rack on top of the clean cage.
Place the cage cover on top of the rack and wait for the animal(s) to defecate.
NOTE: Put a few mouse littermates on the rack simultaneously.
Collect the fecal pellets and put them in a sterile 50 mL conical tube. Pool the pellets by sex. Do not mix fecal pellets collected from males and females.
Weight the tube again and calculate the weight of the fecal pellets.

2. Preparation of fecal suspension

Prepare a sterile solution (10% glycerol in normal saline).
Add 10 mL of 10% glycerol/normal saline to the conical tube for each gram of fecal pellets.
NOTE: Increase the solution volume to 20 mL if necessary. This study used 1 mL of solution for each pellet (5-10 mg) as well.
Homogenize the mixture at a low speed with a benchtop homogenizer or a blender inside a fume hood to resuspend the feces (3 X 30 s).
Filter the fecal suspension through 2 layers of sterile cotton gauze (10.2 cm x 10.2 cm). Store the filtrate temporarily in a refrigerator for up to 6 h or package it into sterile cryogenic vials and store it in a -80 °C freezer.
Thoroughly clean the homogenizer or blender following a standard procedure.

3. Administration of fecal suspension by oral gavage

Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing.
Transfer the fresh or thawed fecal suspension to 1 mL syringes.
NOTE: Each mouse will receive a total of 200 µL of fecal suspension, and each IL-10^-/- mouse in the control group will get 200 µL of 10% glycerol/normal saline¹⁷.
Weigh the mice and choose the right gavage needle size and maximum dosage volume.
NOTE: For mice with a bodyweight between 20-25 grams, use a 20 G 3.81 cm curved gavage needle with a 2.25 mm ball. Please check gavageneedle.com for more information.
Test the gavage needle by measuring the length from the tip of the mouse's nose to the xiphoid process (bottom of the sternum). Fill the syringe with 10% glycerol/saline or fecal suspension and remove air bubbles inside the syringe and the needle.
NOTE: If the needle is longer than the length, put a mark on the needle shaft/tubing at the level of the nose. Do not pass the needle/tubing through the animal past that point to prevent gastric perforation.
Place one mouse cage in the biosafety cabinet, remove the plastic cage cover, and leave the metal rack in place.
Grab one mouse by the tail and put it on the metal rack. Hold the mouse by the tail with one hand and use the thumb and middle fingers of another hand to restrain the animal by grasping the skin over the shoulders. This way, the forelegs are stretched out to the side, which will prevent the front feet from pushing the needle out. Gently extend the animal's head backward and hold the head in place with one hand.
NOTE: Practice mouse handling until the experimenter has full confidence before proceeding to the experiment.
Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper palate until the needle reaches the esophagus. Pass the needle smoothly in one motion. Do not force the needle if any resistance is felt. Take the needle out and try again.
Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. Do not rotate the needle or push the needle forward, which may rupture the esophagus. After dosing, gently pull the needle out.
Return the mouse to its home cage. Monitor the animal for 5-10 min by looking for signs of labored breathing or distress. Monitor the mice again between 12-24 h after the FMT.

4. Disease monitoring and euthanasia

Monitor the mice longitudinally for IBD onset by occult fecal blood and/or colonoscopy²¹. Evaluate heart function by trans-thoracic echocardiography²²^,²³.
Euthanize the animals by decapitation under a deep plane of anesthesia with isoflurane (1%-4%).
Collect the blood in microcentrifuge tubes with anticoagulant and centrifuge at 1000-2000 x g for 10 min in a refrigerated centrifuge (4 °C). Save the supernatant, designated plasma in a -80°C freezer.
Upon euthanization, prepare the mouse colon using the Swiss-roll technique²⁴ for histopathologic analysis (H&E staining)²⁵.
Measure B-type natriuretic peptide (BNP) in plasma using an enzyme immunoassay (EIA) kit²³.

Representative Results

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 <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 < 0.05). Echocardiography detected a significant decrease in left ventricle ejection fraction (LVEF) in the IL-10^-/- mice, compared to the WT mice; the decrease was significantly abrogated by FMT (Figure 1B, n = 5, p < 0.05). These findings suggest that healthy donor FMT mitigated colitis-induced cardiac impairment.

Figure 1. BNP up-regulation and LVEF down-regulation were mitigated by fecal microbiota transplantation (FMT) in IL-10 knockout mice. (A) Plasma BNP concentration (pg/mL) in wild type (WT) and IL-10 knockout (KO) mice treated with vehicle (Veh) or FMT. (B) LVEF of WT and IL-10 KO mice treated with/without FMT. Results were presented as mean ± SD (n = 5). * p < 0.05 vs. WT mice treated with vehicle (Veh). # p < 0.05 vs. IL-10 KO mice treated with Veh. Please click here to view a larger version of this figure.

Discussion

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 adopted oral gavage as the delivery route, which has been proven effective²⁷. We chose the oral route because oral administration is the most convenient and economical and is preferred by most patients. In addition, FMT by oral capsules has been found to be an effective approach to treating recurrent CDI in clinical trials²⁸. Other common upper GI delivery routes are nasogastric tube, nasojejunal tube, jejunostomy tube, and esophagogastroduodenoscopy. The common lower GI delivery routes include colonoscopy, colonic transendoscopic enteral tubing (TET), and rectal enema. However, the optimal route of FMT remains uncertain²⁰. Currently, upper GI administration is considered most appropriate²⁹, but there is no ideal route that suits all patients.

The tools and containers used in the fecal pellets collection should be sterile to prevent any cross-contamination. Fecal pellets collected from males and females should not be mixed because of sex differences in immunity. In animal models and humans, there are differences in microbiota between the sexes³⁰. These sex differences often result in sex-dependent alterations in local GI inflammation, host immunity, and susceptibility to a series of inflammatory disorders³¹. The fecal pellets can be homogenized in sterile phosphate-buffered saline or 10-50% glycerol/normal saline, and 10% glycerol has been widely adopted²⁹. In this study, 10%, 20%, and 50% glycerol/saline were used, and they all offered good bacterial preservation under freezing conditions. Homogenization should be done with a low speed and inside a fume hood to minimize exposure to respirable aerosols produced in this process. The fecal suspension should be filtered through two layers of sterile gauze or a 20 µm nylon filter to get rid of large particles which could block gavage needles. The filtered fecal suspension for immediate use should be placed in a refrigerator, and the rest can be aliquoted and stored in a -80 °C freezer. Freezing is particularly necessary if the fecal matter is obtained from human donors. Meta-analysis has found that frozen FMT is as effective as fresh FMT in patients with recurrent CDI³²^,³³^,³⁴. However, Cui et al. also observed that at 6 months after FMT, the response rate was 26.7% higher in CD patients in the fresh fecal bacteria group than in the frozen fecal bacteria group³⁵, suggesting that fresh FMT is a better choice than frozen FMT in some cases.

The optimal dose and frequency of FMT infusion remain unknown at this stage. Studies have found that the duration of clinical response to single FMT treatment is transient and insufficient to induce fundamental changes in the recipient intestinal flora, and sequential FMT therapy is needed to maintain CD remission³⁶^,³⁷. We performed FMT once a month for 3 consecutive months, and our data showed good therapeutic efficacy. In an ongoing study, we are performing monthly FMT in a group of IL-10-deficient mice for 12 months and will evaluate gut microbiota, intestinal inflammation, and heart function at the end of the study. We expect sequential FMT to show better therapeutic efficacy than single FMT.

While FMT offers enormous potential to mend disturbed gut microbiota and safety data are emerging³⁸^,³⁹, there is still a lack of medical evidence on the safety, mode of administration, bacterial dose, frequency of administration, and long-term prognosis of FMT for the treatment of human diseases. On March 12, 2020, the FDA issued a safety alert that FMT is associated with a potential risk of serious or life-threatening infections⁴⁰. The infections were caused by enteropathogenic E. coli and Shiga toxin-producing E. coli, and the FMT product was supplied by a stool bank company based in the United States. Thus, further mechanistic studies and long-term observations in animals are still required to truly comprehend the use of FMT as a treatment modality in patients. FMT in rodents will remain a powerful tool in microbiome research, which could eventually make the FMT procedure an easily accessible treatment with lower toxicity than other synthetic drugs.

Declarações

The authors have nothing to disclose.

Acknowledgements

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).

Materials

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

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Xiao, Y., Zhong, X. S., Liu, X., Li, Q. Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model. J. Vis. Exp. (182), e63350, doi:10.3791/63350 (2022).