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 Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes. 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 c…

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). 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

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