Microbiota Therapy in Inflammatory Bowel Disease

Abstract

Background

In both Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD) the immune reaction is – at least partially – directed against components of the luminal microbiota of the gut. These immune responses as well as other factors contribute to a phenomenon frequently described as “dysbiosis” meaning an alteration of the composition of the colonic microbiota. To improve the dysbiosis and to restore the normal composition of the colonic microbiota, fecal microbiota transplantation (FMT) has been tested as a therapeutic option to induce and maintain remission in IBD patients.

Summary

This review will first discuss changes in the composition of the intestinal microbiota found in IBD patients and second the therapeutic potential of microbiological interventions for the treatment of these patients. FMT has been studied in several clinical trials in both, CD and UC. Reported results and subsequent meta-analyses indicate that FMT may be effective to induce remission in UC. However, the optimal route of FMT, the necessary number of administrations and the question whether life bacteria of freshly prepared stool is more effective than frozen are still unclear. Concepts associated with an optimization of FMT such as the “super donor concept” or the “consortia-approach” will be discussed to illustrate open questions and difficulties associated with microbiota therapy in IBD.

Key Messages

The microbiota composition in IBD patients shows significant alterations compared to healthy individuals termed as “dysbiosis”. FMT and other therapeutic approaches to modify the microbiota composition have been studied in clinical trials in recent years. Efficacy has been shown in UC; however, many questions with respect to the optimization of microbiota therapy remain to be answered.

Introduction

The pathophysiology of inflammatory bowel disease (IBD) is still not completely understood. It is evident that in both Crohn’s disease (CD) and ulcerative colitis (UC) an activation of the adaptive immune system with an infiltration of T-cells into the intestinal mucosa occurs. Nevertheless, IBD is not regarded to be a classical “autoimmune disease.” The immune reaction of T-cells in IBD is – at least partially – directed against components of the luminal microbiota (and not against body’s own tissue antigens). In addition, many so-called risks genes or single nucleotide polymorphisms that have been associated with the pathogenesis of IBD are located in components of the innate immune system recognizing the invasion of bacteria or viruses across the epithelial barrier. Usually, those variants are loss of function variants which may indicate that potentially dangerous or invasive components of the luminal microbiota cannot be recognized properly (Fig. 1).

Fig. 1.

The mucosal “fire wall concept”: there are several lines of defense (“fire walls”) that prevent the invasion of bacteria into the mucosa. Left panel: the mucus layer, the intact epithelial barrier, the mucosal innate immune system, and finally the adaptive immune system prevent the invasion of the microbiota into the mucosa in healthy individuals. Right panel: in patients with IBD, the barrier functions are impaired on several levels leading to an invasion of microbiota compounds and to an activation of inflammation.

Another group of risks genes or disease-associated single nucleotide polymorphisms is essential for the integrity and function of the epithelial barrier (Fig. 1). Those variants may cause a leakiness of the epithelial barrier and the penetration of physiologically apathogenic commensal microbes, fragments and antigens of those microbes or secreted bacterial, fungal or viral proteins and lipids into the intestinal wall. Besides alterations of the cell-cell contacts (such as tight junctions or adherence junctions), a reduction of the thickness of the mucus layer above the epithelial cells has been reported. The mucus layer contains a protein called MUC-2 which is known to interact with microbial species. Intestinal epithelial cells prevent invasion of bacteria also by the secretion of small antimicrobial proteins such as α-defensins and β-defensins. The major producer of these defensins are Paneth cells. Their morphology is altered in patients with CD; subsequently CD has been termed to represent a “Paneth cell disease” by some authors [1]. These data indicate that antibacterial mechanisms are impaired in IBD further disrupting the epithelial barrier defense leading to an increased permeability for commensal bacteria of the intestinal lumen.

The protective mechanisms that in healthy subjects interfere with an invasion of microbiota components into the mucosa and the body have been termed “mucosal firewalls” [2] (Fig. 1). As mentioned, these “protective firewalls” consist of the epithelial cell layer and its cell-cell contacts, the subepithelial macrophages and other innate immune cells, the dendritic cell commensal sampling as well as the mesenteric lymph node system [2] (Fig. 1).

It is not surprising that the composition of the intestinal microbiota has gained interest with respect to the pathogenesis of IBD and the activation of the intestinal innate and adaptive immune system. First description of the disturbance of the composition of the luminal microbiota, so-called dysbiosis, was already published in 2004 [3, 4]. Here, the most prominent and reproduced changes in the composition of the intestinal microbiota will be described first and then the potential of therapeutic microbiological interventions for the treatment of IBD will be discussed.

There are several “microbiota intervention concepts” that have been introduced into therapeutic considerations in both CD and UC. In view of the undoubtedly present alterations of the intestinal (both luminal/fecal and mucosa-associated) microbial composition, the most obvious concept is to restore the “normal” microbiological composition by fecal microbiota transplantation (FMT). Clinical trials with FMT have been performed in patients suffering from CD as well as UC. Currently, it appears that there are fewer clinical trials in CD and their results are considerably less promising as compared to those in UC. A remarkable number of clinical trials provide evidence that FMT is more effective than placebo to induce and maintain remission in patients with active UC.

However, many questions so far remain unanswered. The optimal route of FMT, the necessary number of administrations, the question whether life bacteria of freshly prepared stool is more effective than frozen are still unclear. In addition, there are concerns of transmission of infectious diseases via fecal stool samples, evidently culminating during the COVID-19 pandemic. But not only infections may be transduced. The lack of standardized transplant production and the fact that the huge aggregates of even non-pathogenetic microbial organisms may also associate with other sequela aside from infectious disease also gave raise to concerns of transmission of antibiotic resistance genes and even susceptibility for other disease states, including ischemic heart disease, multiple sclerosis, rheumatoid arthritis, obesity/metabolic syndrome, or colonic cancer.

Subsequently, new approaches have been discussed and tested. On one hand, there was the concept of a so-called “super donor” for FMT samples that would be more effective at least in certain situations as compared to the standard “average donor.” To avoid the potential risks of FMT, it has been discussed that well-defined consortia of bacteria may be produced under GMP conditions and then used instead of stool samples for the treatment of patients. A further concept is to use specific bacterial strains such as Faecalibacterium prausnitzi as there seems to be a frequent reduction of this species in many patients with IBD. Advantages and disadvantages of those concepts will also be discussed further below.

Microbiota Composition in Patients with IBD

As mentioned before the composition of the “intestinal microbiota” has been found to be altered in patients with IBD [5, 6]. The normal human intestinal microbiota is a complex ecosystem with many crosstalks and interactions. Besides its dominant component of “bacteria,” it consists of fungi (the “mycobiome”), viruses and phages (the “virome”), and some archaea, which predominantly colonize the colon. Fungal commensalism in the gut is influenced by the formation of antifungal antibodies in IBD [7]. Changes in the gut virome have been shown to impact intestinal barrier function and consecutive immune reactions leading to inflammatory responses [8–10]. There is evidence that also the phage community structure in IBD is altered having a direct influence on the gut bacterial ecosystem [11]. The interactions between the host and the microbiota are bidirectional [12] adding a further level of complexity when analyzing the impact of the microbiota in the pathogenesis of IBD. While studies on the mycobiome and the virome are still somewhat impeded by methodological difficulties, the vast majority of information has been achieved with respect to the bacterial composition of the microbiota in health and IBD [13].

For the analysis of the bacterial composition in healthy subjects and patients with IBD in most studies, 16S rRNA sequencing has been used. 16S rRNA amplicon sequencing for many years has been the standard for taxonomic identification of bacteria since current microbial taxonomies based on genomes have still some limitations [14]. However, analyses of the intestinal bacterial colonization based on 16S rRNA sequencing can only provide information on bacterial community structure down to a certain phylogenetic level without reliable species information. Furthermore, it does not provide information on metabolic activities of those bacteria and consecutively no information on functional aspects. A metagenomic approach may be more informative. However, the same bacterial strain can change its metabolic preferences and activities depending on the ecosystem it is introduced and the crosstalk with other species [15, 16].

Patients with IBD have a reduced bacterial diversity when compared to the healthy population without intestinal diseases. In general, they show fewer species of bacteria with anti-inflammatory properties and more species that are believed to have pro-inflammatory effects. Frequently, a decrease in anaerobic bacterial species has been reported which also could reduce anti-inflammatory properties of the intestinal microbiota. However, these changes are not specific for IBD as similar signs of dysbiosis also have been found in a large and constantly growing number of other diseases, including celiac [17–19] or liver disease [20–25].

The dysbiosis in IBD seems to be most severe in patients with active mucosal inflammation [26–29]. However, frequently changes in the abundance of specific bacterial species that were found in one cohort could not be reproduced in other cohorts. In an own study, we analyzed the microbial community of IBD patients in biopsy samples from the Swiss IBD cohort study (SIBDCS) biobank as well as a second independent cohort of patient-biopsies for validation [13]. In pre-studies for these analyses, we found that 16S sequencing from biopsy samples mirroring the composition of the mucosa-associated intestinal microbiota translated into better reproducibility and more reliable results than the analysis of stool samples. It appeared that the mucosa-associated microbiota is more stable and less affected by nutritional influences. The dominant bacterial phylotypes in the gastrointestinal lumen of IBD patients as well as healthy controls were Bacteroidetes, Firmicutes, and Proteobacteria [13]. Confirmatory to other studies we found that alpha diversity as measured by the Shannon and Simpson indices was significantly lower in patients with CD, as compared to patients with UC and healthy controls [13]. We also found significant differences at phylum and genus level that we compared in an unsupervised meta-analysis with previously published data.

This unsupervised meta-analysis confirmed that CD and UC patients have an altered mucosa-associated bacterial composition. CD patients clearly show a lower bacterial diversity compared to controls and UC patients. Differences in bacterial richness and diversity between healthy controls and UC patients were less obvious. Specific differences in CD were a reduced abundance of Faecalibacterium, Lachnospiraceae, and Ruminococcaceae, which produce short-chain fatty acids [13]. Furthermore, in CD a reduced abundance of the secondary bile acid producer Oscillospira and of Bifidobacteria was found. Besides producing short-chain fatty acids the genus Ruminococcus degrades mucus. Alterations that were consistently found in several other studies in CD patients include a relative reduction of Faecalibacterium prausnitzii and of Roseburia hominis as well [30–40].

These changes in the composition of the intestinal microbiota are dependent on the diet and have functional and metabolic consequences [41–43] (Fig. 2). Complex carbohydrates are degraded and fermented by the microbiota in the large intestine [41] (Fig. 2). Through the produced metabolites (e.g., succinate, lactate, propionate, formate, acetate, butyrate) this fermentation process can influence the metabolism of intestinal epithelial cells and subsequently the mucosal barrier function (“mucosal fire walls”). These bacterial metabolites (i.e., succinate) can also impact gut health by the modulation of inflammation [44] (Fig. 2).

Fig. 2.

Dietary fibers, starches, sugars, and proteins are metabolized and fermented by the intestinal microbiota. In the healthy gut, end metabolites such as acetate, butyrate, or propionate are most abundant. In IBD patients, the dysbiotic microbiota leads to an accumulation of intermediate metabolites such as formate, lactate, or succinate.

It has to be kept in mind that the mentioned analyses are far from a longitudinal picture of compositional developments, but rather a one time point snap-shot in the disease course of a chronic, lifelong disease. Microbiota changes may occur permanently during the disease course. Interestingly, looking into the subset of patients having provided several biopsy samples over a period of up to 9 years, we observed the individual compositional changes to be rather stable over time [13]. As mentioned, disease activity itself might interfere with and directly affect the composition of the intestinal microbiota. We found that active disease had a significant influence on Enterobacteriaceae and Klebsiella in CD patients and Ruminococcus and Prevotella in UC patients [13].

In a subgroup of these patients with detailed microbiota analysis, we studied prospectively collected standardized and validated outcome parameters on psychological functioning, comorbidities, and quality of life. In patients with a higher perceived level of stress, we found a significantly lower alpha diversity [45]. Anxiety and depressive symptoms were significantly associated with a decreased beta-diversity [45]. Furthermore, a negative correlation between the abundance of Clostridia, Bacilli, Bacteroidia, as well as Beta- and Gamma-Proteobacteria, and psychological distress was found [45]. Interestingly, our results indicated that the relative abundance of Bifidobacterium in CD patients and Desulfovibrio in UC patients correlated with the severity of depression [45]. These data indicate that in patients with IBD the microbial composition in the gut may influence the psychological well-being and contribute to IBD symptoms such as fatigue or anxiety.

Evidently, many confounders could have influenced these results. Patients with active inflammation usually do not eat normally and try different diets to improve the symptoms. It is well known that a change in nutrition and diet is followed by changes in the composition of the intestinal microbiota. In line with this, we observed in another study from the SIBDCS that a considerable fraction of IBD patients follow a rather restrictive dietary pattern (4% vegetarian and 5% gluten-free diet), which was not associated with a favorable course of IBD but distinctive alterations in intestinal microbial composition on the one hand and lower psychological well-being on the other [46].

Concepts of Microbiota Interventions in IBD

Besides FMT several other concepts for microbiota interventions in IBD have been developed. FMT is impossible to standardize and therefore always at risk for variable effects even when a specific set of donors is used. It has been demonstrated that nutritional changes alter the colonic microbiota composition and subsequently each FMT from one donor will have significant variations over time. There have been attempts by the pharmaceutical industry to reduce batch-to-batch variability and infectious risks. One of those products was SER-287, a donor-based stool preparation with mainly spores initially showed positive results in an induction phase-I study in UC patients [47], however, failed to reach the endpoint in a subsequent phase 2b trial.

In contrast, the most minimalistic concept of a microbiota intervention is the supplementation of just one bacterial strain, i.e., a probiotic bacteria preparation. A number of probiotics have been tested in IBD patients in clinical trials of varying quality. Only studies with E. coli Nissle in UC patients have been shown to maintain remission similar to 5-amino-salicylic acid.

In between these two extremes is the concept of microbiota consortia. Defined bacterial consortia can have a composition based on certain selection criteria and can be well characterized. Subsequently, these consortia may represent a safer and more controlled alternative to FMT [16].

Clinical Trials with FMT in UC

In 1989, 2 case reports were published on FMT in UC patients. Justin Bennett and Mark Brinkman reported in Lancet about a self-experiment with fecal transplantation. Justin Bennett, MD, had continuously active, severe UC for a period of 7 years as confirmed by endoscopy and histology [48]. His disease did not sufficiently respond to standard treatment with sulfasalazine and corticosteroids. He had a steroid dependent disease course and experienced bloody diarrhea and cramping when reducing the steroid dose below 30 mg/day. 6 months before the report was published in Lancet in January 1989, the authors performed a fecal microbiota transplant with stool from a healthy donor via an enema application. Justin Bennett experienced a rapid improvement and was clinically symptom free without medication. 3 months after the fecal microbiota, transplantation histology showed no active inflammation confirming the treatment success [48]. The authors suggested that a pre-treatment with antibiotics might be necessary so that the new microbiota can be maintained [48]. In the same year, Thomas Borody, a strong supporter of the approach of FMT raised the question: “Bowel-flora alteration: a potential cure for inflammatory bowel disease and irritable bowel syndrome?” [49].

However, the treatment strategy of FMT was not followed up in clinical trials for the next decades. It took until 2013 when van Nood and colleagues [50] published a successful trial on the duodenal infusion of donor feces for recurrent Clostridioides difficile infection in the New England Journal of Medicine to regain interest in FMT in patients with IBD.

Since then a number of studies have been published that were well designed. Moayyedi et al. performed a study in 78 patients with mild to severe UC in which 38 patients received a FMT product and 37 received placebo [51]. The donor feces were introduced by enema. The endpoint of clinical remission was achieved in 24% of FMT patients at week 7 but only in 5% of the placebo group (p = 0.03) [51]. In this study, 6 healthy donors were used. Interestingly, the majority of clinical remissions was induced by only one donor (donor B) whereas feces from all other donors did not show an effect different from placebo. This raised the concept of so-called “super donors” that had consequences for all subsequent clinical trials [51].

A further large clinical study on fecal transplantation in UC patients was published by Paramsothy and coworkers 2 years later [52]. Due to the concept of a super-donor, the authors did not use one healthy donor for a fecal transplantation but used pooled fecal samples from 3 to 7 donors to maximize the chance of having included such super-donor in the preparation. Patients with active UC with a Mayo score of 4–10 were randomized to receive FMT or placebo. The first preparation was given by colonoscopy followed by five administrations of FMT per week by enema over 8 weeks. This indicates that it was quite an intensive FMT protocol. The endpoint of this trial also was very rigorous. The authors evaluated steroid-free clinical remission with endoscopic remission or response at week 8. Forty-two patients received the FMT and 43 patients received placebo. 27% of the verum patients reached the trial endpoint in contrast to only 8% of the placebo patients (p = 0.021) [52].

In both studies, there was no difference between treatment groups and placebo groups with respect to adverse events. In the meantime, a number of additional clinical trials have been published and over the years several meta-analysis came to the conclusion that there is a significant positive effect of fecal microbiota transplantation in UC. A recent Cochrane meta-analysis looked into 12 studies on FMT in 550 UC patients [53]. In the 12 studies analyzed there was no homogeneous route of administration of FMT. The FMT preparation was either administrated in the form of capsules or delivered as suspension by naso-duodenal tube from the oral route. In other studies, rectal administration was chosen via an enema or colonoscopy. Induction of clinical remission was the endpoint and 10 of those studies. The meta-analysis indicates that FMT increases the rates of induction of clinical remission in UC patients as compared to placebo or sham-control with an odds ratio of 1.79 (CI: 1.13–2.84) [53]. Five studies also evaluated the endpoint of endoscopic remission and demonstrated an odds-ratio of endoscopic remission of 1.45 as compared to controls (CI = 0.64–3.29). The risk of any adverse events was not increased in the FMT groups [53]. The authors conclude that “FMT may increase the proportion of people with active UC who achieve clinical and endoscopic remission” [53]. A small study (prematurely terminated due to COVID 19) revealed promising results in 35 patients with active UC using an oral lyophilized microbiota preparation and indicated that such as preparation may be a feasible way to achieve induction of remission in UC subsequent to antibiotic treatment [54].

Clinical Trials with FMT in CD

The data on the effect of microbiota transplantation in CD are substantially less convincing as compared to UC. So far, only one RCT has been published in abstract form to evaluate the effect of FMT in CD. Sokol and colleagues published a clinical trial in which 21 CD patients that achieved clinical remission upon treatment with systemic steroids that were randomized to receive either FMT or a placebo preparation [55]. Evidently, the clinical endpoint was maintenance of remission and not induction of remission. No statistical significant difference was found; however, this clinical trial was most likely underpowered.

Clinical Trials with Microbiota Interventions Other than FMT in IBD

Microbiota intervention other than FMT (in direct or lyophilized form) is increasingly investigated aiming to enable a more standardized and targeted approach to impact the recipient’s microbial composition and/or metabolic function. Encouraging results were obtained in a phase 1b study in UC patients who received (double-blinded) an oral formulation of Firmicutes spores for 8 weeks after 6 days of vancomycin pretreatment. Clinical remission was significantly higher (up to 40%) in the group receiving verum [47]. However, as mentioned above, in phase 2 the predefined endpoint could not be reached.

In a mouse model of colitis, severity of intestinal inflammation promoted by an abnormal expansion of Enterobacteriaceae could be reduced by administration of tungstate [56]. The authors referred to this approach as “precision editing of the gut microbiota” and their work undermines that not exclusively microbes or their metabolic products may induce microbial changes that ultimately could translate into a therapeutic benefit. In contrast, also a variety of small molecules or anorganic substances and even subtle manipulations achieved by small molecules might represent a microbial therapeutic intervention.

Challenges and Conceptual Questions in Microbiota Therapy in IBD

One of the major challenges for the interpretation of published clinical trials on microbiota therapy in IBD as well as for the design of future clinical trials is the lack of standardized protocols for a donor feces preparation, the route of administration, the number of administrations, and the observation time. It is not clear whether at all and which antibiotic pretreatment is necessary to optimize the conditions for FMT. As mentioned above dysbiosis has consistently been observed in IBD patients. Despite the fact that dysbiosis appears to be more pronounced in CD patients, the efficacy of FMT counterintuitively seems to be higher in UC. It is assumed that the primary goal of FMT is to reverse dysbiosis, implicating to have a causative role in the emergence of IBD. However, it might plausibly be assumed that those bacteria having induced immunogenicity need to be replaced (or at least diminished) by a less or even non-immunogenic microbial consortium. In other words, the agent that causes the autoimmune inflammation needs to be replaced by “non-immunostimulatory material.” There is still a debate, which of those concepts is supported by better evidence.

Furthermore, it is unclear whether one single FMT is able to provide a sufficient and sufficiently sustained microbiota (ex-)change or whether several FMTs are required, perhaps even in considerably longer intervals over years, compared to those having been used in trials with multiple FMT interventions. The study by Moayyedi and coworkers seems to indicate that one transplantation might be sufficient. Manuscripts reporting on single transplantations provide conflicting results. In a meta-analysis, Mocano and coworkers [57] provided evidence that repeated FMT is better than single transplantation (43 vs. 30%) and that antibiotic pretreatment also improves FMT efficacy [57].

Many routes of administration have been used in the aforementioned clinical trials, among them were nasal duodenal tube, capsules as upper GI route and enemas and colonoscopies for the rectal route. Meta-analyses provided conflicting results [57–66].

The treatment for maintenance of remission after FMT remains also to be elucidated. It is unclear, how a long-term efficacy of FMT in IBD patients can be achieved. A repetition of FMT every 8 weeks has been discussed. Other authors suggested monthly transplantation.

Another open question surrounding the context of FMT for IBD is the selection of fecal donors and the preparation of the fecal samples. There is a potential for transmitting infections, pathogens, and antibiotic resistance genes up to susceptibility for other disease states. There are different strategies for the screening of fecal donors that all lack sufficient basis of evidence. Fecal testing usually includes C. difficile toxin A/B, Campylobacter, Salmonella, Shigella, Escherichia coli, Rotavirus, Norovirus, Adenovirus, COVID-19, and Monkey pox. As this testing may take time and as exposure to pathogens changes over time the question is crucial whether efficacy is associated with fresh, frozen, or lyophilized faeces. Especially when searching for viruses that might not be detectable at the time of infection it becomes evident that the stool needs to be stored for some time. Therefore, a recently published consensus statement might encourage and provide guidance to design studies on FMT in a more homogeneous fashion [67].

Several important statements have been made in this consensus manner prescription: With respect to donor selection and testing the authors refer to national and international guidelines currently available for CD featureless fecal transplantation [67]. However, those national and international guidelines are also heterogeneous and do not provide a unique standard. Some of the suggested testing lacks evidence, which again will cause problems for the planning of respective clinical trials. The participants of the consensus conference did not find agreement on the definition of donor characteristics with respect to optimization of treatment results. This most important question remains to be answered and will require significant further research.

The consensus participants stated that it is still too early to use FMT for the induction of remission and mild to moderate UC and that patients which have an interest in this procedure should be included into clinical trials [67]. They further suggest repeated applications in contrast to single applications.

With respect to other open questions such as the route of administration, antibiotic pretreatment, testing of engraftment and others, the authors do not give a recommendation but point to further studies that need to answer those questions [67]. Subsequently, it is questionable whether this consensus statement will homogenize the trial landscape in microbiota therapy in IBD.

Instead of using fresh/frozen microbiota (FMT, consortia, probiotics) as therapeutics, there is further effort to understand the complex, bi-directional crosstalk between the host and its microbiota, e.g., through metabolites produced by bacteria or fungi. Utilizing immunomodulatory microbial metabolites could lead to novel treatments for IBD patients [68].

Finally, in view of the assumption that impaired intestinal barrier function might represent the initial trigger for subsequent microbial invasion and dysbiosis, one could speculate, whether any attempts to manipulate microbial composition could be most promising to prevent the occurrence of IBD in well-selected subjects with increased risk of IBD. For instance, intestinal permeability was prospectively assessed using the urinary fractional excretion of lactulose-to-mannitol ratio in a large prospective study of first-degree relatives from CD patients over almost 8 years and the investigators identified an abnormal lactulose-to-mannitol ratio to antecede a diagnosis of CD [69]. Theoretically, microbial manipulation could successfully halt the development in CD (or at least reveal to be a more successful approach than treating an already established perhaps long-standing form of the disease).

Conflict of Interest Statement

Gerhard Rogler is co-founder and head of the Scientific Advisory Committee of PharmaBiome, a microbiota therapy company.

Author Contributions

G.R., L.B., and A.K. contributed to the writing of the manuscript.