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

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 strains^5,6. 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 IBD^8,9, 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 IBD^16,17,18. 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

1. 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.
2. Turn on the biosafety cabinet in the animal room.
3. 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.
4. Place some sterile paper towels on the bottom of the cage and place the metal rack back on top of the cage.
5. 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.
6. 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.
7. 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.
8. Weight the tube again and calculate the weight of the fecal pellets.

2. Preparation of fecal suspension

1. Prepare a sterile solution (10% glycerol in normal saline).
2. 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.
3. 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).
4. 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.
5. Thoroughly clean the homogenizer or blender following a standard procedure.

3. Administration of fecal suspension by oral gavage

1. Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing.
2. 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.
3. 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.
4. 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.
5. Place one mouse cage in the biosafety cabinet, remove the plastic cage cover, and leave the metal rack in place.
6. 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.
7. 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.
8. 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.
9. 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

1. Monitor the mice longitudinally for IBD onset by occult fecal blood and/or colonoscopy²¹. Evaluate heart function by trans-thoracic echocardiography^22,23.
2. Euthanize the animals by decapitation under a deep plane of anesthesia with isoflurane (1%-4%).
3. 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.
4. Upon euthanization, prepare the mouse colon using the Swiss-roll technique²⁴ for histopathologic analysis (H&E staining)²⁵.
5. Measure B-type natriuretic peptide (BNP) in plasma using an enzyme immunoassay (EIA) kit²³.

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). Echocardiograph...

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 adop...

Materials

Name	Company	Catalog Number	Comments
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