Targeting the microbiome in Crohn’s disease

KEYWORDS:

• Anti-mycobacterial therapy
• Crohn’s disease
• Crohn’s strictures
• dysbiosis
• fecal microbiota transplantation
• gut microbiome
• inflammatory bowel disease
• Mycobacterium avium subspecies paratuberculosis (MAP)
• profound remission

1. Crohn’s disease and the microbiome

In 1932, Burrill Crohn identified the eponymous enteritis, Crohn’s disease, and separated it from tuberculosis (TB) infection of the gut. By serendipitously failing to identify an infectious cause through acid-fast staining, nor successfully cultivating any mycobacteria in animals, he was able to identify this new disease as a separate entity [1]. In an era dominated by infectious disease, Crohn would have done well to take notes from the Scottish surgeon Thomas Dalziel. Dalziel in 1913 was struck by the similarity between his patients with non-tuberculosis enteritis and the disease described in cattle by Heinrich Johne in 1895, from which another mycobacterium was isolated [2]. This organism was Mycobacterium avium subspecies paratuberculosis (or MAP).

Fast forward 130 years, the current theory on the pathogenesis of Crohn’s disease involves a complex interplay between genetics and how the host ‘handles gut microbes’ (now recognized as the gut microbiome). MAP is recognized as a dominant player within the microbiome that can be a trigger to an inappropriate and excessive immune response within affected gut mucosa. Autoimmunity was erroneously incriminated as the driving force of chronic gut inflammation, while infection never went away. Following the ‘first flush’ of the antibiotic era when classic infection was easily diagnosed and easily treated, the complexities of ‘infection’ became incorporated into the idea of the ‘host-parasite relationship.’ Here ‘handling’ of microbes by the host was a critical determinant of clinical outcome.

Outcomes in Crohn’s disease ranged from overwhelming clinical infection demanding antibiotic therapy, to ‘chronic inflammation’ where the aetiologic microbe was never identified. In the latter case, treatment was most effective when directed at suppressing the host inflammatory response. It was the character of the ‘Host’s Response,’ that influenced the position of a particular disease within this spectrum. Crohn’s disease ‘grew-up’ in this intellectual framework. Infection as an etiology was proposed by clinical markers, yet mainline therapy was based on immune suppression. Mycobacteria only reemerged as a microbe of interest in the 1980s when isolated reports of undifferentiated mycobacteria were published [3]. A further milestone in our understanding occurred when the phenotype of Crohn’s was successfully transferred. This work by Chiodini [4], transferred MAP from a patient to experimental animals which developed ileitis; fulfilling much of Koch’s postulates [5], Greenstein [6] and Hermon-Taylor [7] then brought together PCR data that had been accumulating since 1992 [8] to again implicate MAP. This time showing the presence of the DNA insertion sequence known as IS900, thought to be specific for MAP [9], being present in many individuals with Crohn’s.

However, three observations required additional resolution:

1. PCR data was positive in only 60–90% of Crohn’s patients while many normal subjects had positive PCR assays [10].

2. In some geographic areas, other intracellular microbes were linked with Crohn’s diseases, such as Yersinia enterocolitica [11].

3. Clinical benefit from surgical procedures removing inflamed tissue from the gut and response to antibiotics, best known for their effect on anaerobic bacteria, suggested a less specific role for the gut microbiome in the pathogenesis of Crohn’s disease [12].

Genetic studies have made a significant contribution to our understanding of Crohn’s as an expression of a particular host–parasite relationship. Familial clustering of Crohn’s has long been recognized and modern genetic studies transformed our understanding of the gut microbiome (specifically MAP), in the pathogenesis of Crohn’s disease. Genome-wide studies have identified over 160 susceptible genetic loci, 30 of which are specific to Crohn’s [13].

Amongst the genetic studies, nonfunctioning mutations in NOD2 were the most common genetic defects, with homozygous individuals having a 17-fold increase in ileal forms of Crohn’s disease [14]. NOD2 is an intracellular pattern recognizing receptor for DAMPS and PAMPS (Damage and Pathogen – Associated Molecular Patterns) and is expressed in Paneth cells [15]. NOD2 connects to NF-kB where it links with the secretion of cytokines [16] inside the cell, it also regulates the secretion of defensins which in turn regulates the gut microbiome [17]. Thus, a tightly woven relationship between the microbiome and mucosal protection is mediated by NOD2 [18].

The loss of NOD2 function is also associated with a disturbed gut microbiome characterized by an increase in Actinobacteria and Proteobacterial species [19]. Mycobacteria being within the phylum of Actinobacteria [20]. Furthermore, Actinobacteria is associated with high TNF-alpha secretion levels [21]. When the mechanisms were investigated further, the glycolated cell-wall peptidoglycan of mycobacteria (MDP) was found to be responsible, which is established already as a potent stimulant of NF-kB [22]. To further tie down any associations, the levels of TNF alpha secretion also correlated with MAP positive PCR results in subjects [23]. Hence, a potential mechanism explains how an individual’s genetic mutations could lead to an aberrant inflammatory response.

Altogether the evidence suggests that gut health is finely balanced with the gut microbiome. Homeostasis is maintained by host factors including: NOD2-generated protection, disturbance of this ‘balance’ leads to dysbiosis, altered cytokine expression within the mucosa, and recruitment of adaptive immunity characterized by expanded Th1 cells [24]. The driver of these mucosal events suggests intracellular bacteria, with MAP identified as dominant within most microbiome communities [25]. Finally, Crohn’s disease must also be considered alongside the concept of a ‘Crohn’s Syndrome’ which explains the numerous infections which can also mimic Crohn’s disease [26] but which occur in the absence of MAP particularly TB of the gut.

The presence of intracellular MAP and the likely involvement more broadly of a dysbiotic microbiome in promoting clinical Crohn’s disease have led to the development of therapy aimed at eradicating intracellular mycobacteria using selected combination of antibiotics, and the ‘normalising’ of the gut microbiota using Fecal Microbiome Transplantation (FMT). Though such a therapy may not be applicable to all Crohn’s cases, it is particularly interesting in the ileocolic forms of the disease where the restoration of the microbiome by FMT has already shown remarkable responses in other conditions.

2. Development of antibiotic Anti-MAP therapy in Crohn’s disease

Given successful parallel developments in the treatment of Mycobacterium tuberculosis (M. tb), Mycobacterium leprae (M. leprae) and Mycobacterium bovis (M. bovis) Hermon-Taylor’s group in 1997 reported the use of ‘double therapy’ to treat Crohn’s disease, with reasonable success, but no cure [27]. This group later used quadruple therapy but with poor results perhaps because most components had poor activity intracellularly against MAP [28]. Given double therapy was not successful in treating M. tb or M. bovis and intracellularly active antibiotics are necessary, we explored the effectiveness of a novel triple therapy with rifabutin, clarithromycin, and clofazimine as the third component which has a 70-day half-life [12,29]. This approach was supported by well-known variations in intracellular/extracellular accumulation values of different antibiotics [25], and a theory that cell-wall deficient MAP, latent within the host’s cell, could be a mechanism by which MAP could resist antibiotic eradication. M. bovis infection, which is known to infect humans, causes very similar gut lesions to that of MAP, leading to strictures and chronic inflammation and can be clinically and histologically indistinguishable from Crohn’s disease. However, M. bovis can be effectively controlled and cured with rifampicin, isoniazid and ethambutol over a 6–12-month course, in contrast, MAP could not as used by Hampson et al. 1989 [28]. Taking the analogy further, the stricture resolution observed in Crohn’s disease in response to antibiotics parallels that in TB [30]. Along these lines our antibiotic triple therapy centered on using Rifabutin, Clarithromycin, Clofazimine, and/or Metronidazole early on in our development of anti-MAP therapies [31–33]. In our small case series, we included a cohort on a ramping-up combination of rifabutin (450 mg/d), clarithromycin (750 mg/d) and clofazimine (2 mg/kg/d) with progress monitored via colonoscopy, histology, clinical response and the Harvey-Bradshaw activity index. Over 54 months of follow-up, three patients achieved complete long-term remission without any further therapy, 6 out of 12 patients achieved complete remission using combination antibiotics therapy alone (without immunosuppression), and two patients achieved partial clinical response but with mild histological inflammation [12].

In the treatment of pediatric patients, responses have been even more effective. In those not previously exposed to immunosuppressants, remission was achieved rapidly in all subjects [34–36]. Since our original study, other larger trials have been instigated, with variable results [37,38] due to insufficient dosage of antibiotics or from other confounders. Yet despite there is still data that continues to show the addition of antibiotics as superior to standard symptomatic treatment alone [39,40].

3. Brief review of fecal microbiota transplantation in Crohn’s

The first reports on prolonged remission of Crohn’s disease using FMT was by our group in 1989 [41]. The 31-year-old patient with severe Crohn’s disease remained in remission for 18 months after FMT was performed. While FMTs were superior to probiotics alone, their reported applications in Crohn’s disease are still relatively sparse. Paramsothy et al. found and reviewed only 11 Crohn’s studies in 2017 finding a 50.5% remission rate in 42 out of 83 Crohn’s disease individuals [42]. Meta-analysis within these 4 studies in this group showed clinical responses as high as 63% in 59 individuals [42]. Overall, however, the studies lacked long-term follow-up and no permanent remission by any one therapy was reported.

By 2021, there were significantly more studies [43–46]. Some groups reported quality of life improvements only [43], while others considered FMT only as a fail-over therapy for when all other approaches proved futile [44]. Caldeira et al. in 2020 reviewed 66 studies altogether measuring a remission rate of 47.6% in the FMT group versus 27% in control groups [47]. Later in 2021, Fehily et al. reviewed 15 studies between 2014 and 2020 also finding a definitively improved clinical response rates with FMT in the weeks and months following treatment [48]. Unfortunately, both studies observed a phenomenon of tapering efficacy rates over time, where treatment responses were significantly higher 0–6 months following FMT but merged slowly toward the standardized immunotherapy efficacy rates over the longer term. Few of the studies reviewed by Caldeira and Paramsothy extended beyond several years – a missing critical insight [42,47]. In spite of this, a number of ongoing registered clinical trials are still recruiting or have completed, which should shed further light on the issue [49,50].

4. Anti-MAP followed by FMT can achieve prolonged remission

Clinical observations led us to combine anti-MAP antibiotics with FMT as follow-up. Initially, the use of FMT in the single patient reported in 1989 suggested there may be a role in Crohn’s disease [40]. Later, FMTs performed on several Crohn’s disease patients with Clostridium difficile infection resulted in prolonged remission of the patient’s Crohn’s disease. Secondly, we recognized that prolonged antibiotics could permanently disrupt the gut microbiome, which could only be restored by FMT. Thus, in a subset of our patients whose calprotectin levels and mucosal histology were normalized, we formally treated this cohort with combination treatment of Anti-MAP antibiotics and FMT and followed their outcomes indefinitely. So far 30–40% of this group achieved more than three years of total absence of Crohn’s disease in the absence of all therapy, which is beyond the definitions of remission. One subject has been in remission for 23 years [35,51]. These clinical observations in an ever-growing cohort of Crohn’s disease patients resembles the prospect of a cure.

5. The future of Crohn’s disease management

There is no generally recognized cure for Crohn’s disease and the majority of patients on traditional immunosuppressive therapy will relapse. Historical data show that nearly two-thirds will require surgery within 10 years of diagnosis [52]. However, recent experience with newer therapies indicates long-term absence of Crohn’s disease can be achieved [35]. Recognizing the newly discovered genetic changes, more subjects must be followed for longer periods to determine whether recurrence occurs in the presence of persistent molecular defects or if other interactions are involved.

A future solution may lie in combining several emergent therapies. Firstly, antibiotic and immunization regimes designed to target MAP or other microbial ‘drivers’ of mucosal inflammation could be effective. Secondly, genetic mutations contributing to Crohn’s could be corrected using gene therapy which can repair specific genetic defects. In the interim, FMT offers an all-in-one approach that can be further adapted to exclude known and newly implicated microbial triggers and their metabolites.

Thus far, our most effective clinical response has been the aforementioned combination anti-MAP antibiotics with FMT as follow-up [35]. Using the current definition of ‘remission’ as a CDAI of less than 150. Our new approach offers an opportunity to terminate Crohn’s disease permanently in a subgroup of patients. Our current working criteria describing a cure of Crohn’s disease includes:

1. Clinical remission (CDAI < 150)

2. Off all active Crohn’s disease therapy >3 years

3. Colonoscopy (no inflammation) >3 years

4. Histology (no inflammation) >3 years

5. Normal calprotectin levels

Having encountered many of our own cohort fulfilling these criteria, we expect subjects in overseas cohorts to be similarly under-represented, and in need of reclassification.

6. Beyond remission

Here, we review the concept of a cure for Crohn’s disease in preference to the unsatisfactory status of ‘extended prolonged remission.’ Having observed total mucosal healing in some subjects over many years and in some cases decades, we believe we can safely designate many of these subjects cured. Researchers can no longer claim Crohn’s is not curable simply because they have not seen cured patients. Still, there is more research required to apply these new principles, especially in the majority of patients who will relapse under traditional symptomatic therapies. The parallels between Crohn’s disease treatment and Helicobacter-infected duodenal ulcers and the use of acid suppression are hard to ignore. Therefore, the recognition of ‘cured’ patients in a subgroup of Crohn’s provides the impetus for the development of a more universally robust and permanent therapy in many more.

Declaration of interest

T Borody has a pecuniary interest in the Centre for Digestive Diseases, Finch Therapeutics, Gioconda Ltd, and holds patents in the use of FMT and antibiotics for treatment of Crohn’s disease and other forms of inflammatory bowel diseases. Other authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgments

We acknowledge Dr John Ng for his kind help in editing and proofreading the document.

Additional information

Funding

This paper was not funded.