Exploring gut microbiota as a novel therapeutic target in Crohn’s disease: Insights and emerging strategies

Abstract

Extensive research has investigated the etiology of Crohn’s disease (CD), encompassing genetic predisposition, lifestyle factors, and environmental triggers. Recently, the gut microbiome, recognized as the human body’s second-largest gene pool, has garnered significant attention for its crucial role in the pathogenesis of CD. This paper investigates the mechanisms underlying CD, focusing on the role of ‘creeping fat’ in disease progression and exploring emerging therapeutic strategies, including fecal microbiota transplantation, enteral nutrition, and therapeutic diets. Creeping fat has been identified as a unique pathological feature of CD and has recently been found to be associated with dysbiosis of the gut microbiome. We characterize this dysbiotic state by identifying key microbiome-bacteria, fungi, viruses, and archaea, and their contributions to CD pathogenesis. Additionally, this paper reviews contemporary therapies, emphasizing the potential of biological therapies like fecal microbiota transplantation and dietary interventions. By elucidating the complex interactions between host-microbiome dynamics and CD pathology, this article aims to advance our understanding of the disease and guide the development of more effective therapeutic strategies for managing CD.

Key Words: Crohn’s disease; Gut microbiome; Dysbiosis; Creeping fat; Fecal microbiota transplantation

Core Tip: Crohn’s disease (CD) represents a complex and challenging medical condition, characterized by the intricate interplay between genetic predisposition, gut microbiota dysbiosis, and immune dysregulation. This paper discusses the key effects of the gut microbiota on CD, as well as how they affect creeping fat. Furthermore, the paper highlights the urgent need to address gut microbiota imbalances, particularly focusing on pathogenic species and their mechanisms, as a critical therapeutic target. The findings underscore the importance of a personalized approach to CD management, emphasizing the need for continued research and innovation to address this complex disease.

TO THE EDITOR

In this article, we review a recently published article by Wu et al[1] in the World Journal of Gastroenterology, titled “Role of gut microbiota in Crohn’s disease pathogenesis: Insights from fecal microbiota transplantation in mouse model”. The study delves into the disrupted characteristics of mucosal-associated lymphoid tissue (MAT) and intestinal tissues in Crohn’s disease (CD) that were directly observed through hematoxylin and eosin (HE) staining, Masson’s trichrome staining, and immunohistochemical staining. Furthermore, by establishing mouse models, they detected excessive expression of proinflammatory factors in creeping fat (CrF), which robustly validates the previous understanding of the role of CrF in the pathogenesis of CD. This paper outlines the critical contributions of gut microbiome studies to the pathogenesis of CD, with a particular focus on emerging therapies targeting the intestinal flora, notably fecal microbiota transplantation (FMT) and dietary interventions. Special emphasis is placed on the significance of gut microbiome research in elucidating disease mechanisms and providing novel perspectives and potential solutions for the treatment of CD.

CD AND ITS PATHOLOGICAL CHARACTERISTICS

CD

CD, one of the major entities within the spectrum of inflammatory bowel diseases (IBDs), commonly affecting the terminal ileum and proximal colon[2]. Most patients present with an inflammatory phenotype at diagnosis, but over time, complications such as fibrosis, strictures, fistulas, or abscesses may develop, often leading to the need for surgery[3].

Etiological analysis

Despite significant advancements in experimental techniques and models, the etiology of IBD remains unclear. Higher incidence rates of IBD observed in monozygotic and dizygotic twins, as well as among certain families, strengthen the case for a genetic influence on the disease. Additionally, researchers have identified hundreds of IBD-associated genetic polymorphisms and mutations through genome-wide association studies, further supporting the genetic underpinnings of IBD[4,5]. However, genetics alone is insufficient to account for the onset of the disease. With industrialization, changes in lifestyle, and urbanization in modern society, the incidence and prevalence of IBD have been on the rise[6]. Nonetheless, the timeframe of these developments is too short to be explained by genetic drift or natural selection. In this context, non-genetic factors such as environmental influences, higher ultra-processed food and lower unprocessed/minimally processed food intakes[7], smoking[8], and antibiotic use[9] all play a significant role in the pathogenesis of these diseases. In recent years, with the increasing depth of investigations into the gastrointestinal microbiota, more studies have indicated that dysbiosis of the gut microbiome is a significant factor in triggering and promoting the development of IBD.

CrF in CD

CrF is a hallmark of CD[10,11], characterized by the abnormal expansion of perienteric adipose tissue. This phenomenon involves the gradual encasement and infiltration of mesenteric fat around affected intestinal segments, resulting in a characteristic “creeping” appearance. A large number of cohort studies and clinical trials demonstrate that CrF exacerbates CD inflammation through various mechanisms[11-13] such as the release of pro-inflammatory molecules (leptin[14] and resistin[15]) and activation of immune cells (macrophages[16] and CD4+ T cells[17,18]). This is further supported by a few clinical trials that CrF promotes inflammation via SP/NKR1 activation and upregulating interleukin-17A (IL-17A) mRNA in preadipocytes and IL-17A receptor mRNA in colonic tissue[19,20].

It is worth noting that the gut microbiome, especially bacteria, may be a potential trigger for CrF. Emerging evidence suggests that CrF, the migration of MAT to sites of intestinal barrier dysfunction, may represent a protective response. This process aims to prevent systemic dissemination of potentially harmful bacteria that have translocated across the compromised intestinal epithelium. A study demonstrated that while bacterial translocation occurs in both normal MAT and CD-associated CrF, the characteristics and metabolic functions of the gut microbiota differ significantly[21]. For instance, a specific population of translocated enteric bacteria, i.e., Clostridium innocuum, is found in both the ileal mucosa and CrF. This bacterium promotes M2 macrophage polarization, stimulating adipose tissue remodeling and CrF formation[21]. Furthermore, adipocytes and preadipocytes within CrF express functional pattern recognition receptors, such as Toll-like receptors and nucleotide-binding oligomerization domain-containing protein 1[22]. These pattern recognition receptors recognize microbe-associated molecular patterns derived from translocated bacteria, triggering downstream signaling cascades that activate transcription factors like nuclear factor-κB (NF-κB) and induce the production of pro-inflammatory cytokines and chemokines[23]. Another study investigated whether microbial translocation in CD is a key factor in the progression of CrF[24]. The researchers identified a subset of mucosa-associated intestinal bacteria that consistently translocate during ileal resections in CD and remain viable in CrF; they pinpointed Clostridium innocuum as a marker of this population, noting strain variation between mucosal and adipose tissue isolates, which suggests a preference for lipid-rich environments[24]. Single-cell RNA sequencing revealed that CrF exhibits both pro-fibrotic and pro-adipogenic effects, along with an activated immune cell environment responsive to microbial stimuli. This was further confirmed in germ-free mice colonized with Clostridium innocuum[24].

Role and alterations of the gut microbiome in CD

The gut microbiota, including bacteria, fungi, viruses, and archaea, is closely related to the immune system and is of great importance to the normal physiological functions of the body[25,26]. Multiple studies have shown that gut microbiota dysbiosis is an important cause of the occurrence and aggravation of CD, and is closely related to the clinical manifestations of CD, such as inflammation and fibrosis. Wu et al[1] suggested a potential role of gut microbiota dysbiosis in the pathogenesis of CD, but their evidence was limited. Given the increasing focus on gut microbiota dysbiosis in CD research, we describe the impact of bacterial, fungal, viral, and archaeal dysbiosis, and their interactions, on CD development.

Intestinal bacterial microbiota in CD

The gut microbiome, especially bacteria, is the most extensively researched microbial community in patients with CD[27]. Studies using culture-independent methods (quantitative polymerase chain reaction, FISH analysis, 16S rRNA gene sequencing, shotgun-sequencing, and Illumina sequencing) on fecal samples consistently show that the observed decrease of some beneficial bacteria, such as Bacteroides and butyrate-producing Firmicutes, including Faecalibacterium prausnitzii and Roseburia spp., alongside an increase in pathogenic bacteria, may significantly contribute to pro-inflammatory effects[28,29]. Such dysbiosis can lead to the disruption of certain metabolites produced by the microbiota, subsequently increasing the inflammatory response. For instance, short-chain fatty acids, particularly butyrate, play a crucial role as a histone deacetylase inhibitor, effectively suppressing the activation of NF-κB and downregulating pro-inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor (TNF)-α[29]. Secondary bile acids, which result from microbial metabolism, are critical in immune regulation, promoting regulatory T cell differentiation, and disruptions in bile acid metabolism worsen inflammation in IBD patients[30,31]. The reduced availability of tryptophan-derived metabolites, which modulate immune responses via aryl hydrocarbon receptors, correlates with higher disease susceptibility[32]. Sphingolipids, produced by both host and microbes, also regulate inflammation, with microbial sphingolipids like those from Bacteroides shown to protect against colitis by inhibiting natural killer T cell proliferation[33]. Furthermore, some clinical trials and relevant pre-clinical studies found that Faecalibacterium prausnitzii actively suppresses NF-κB activation and attenuates IL-8 production in intestinal epithelial cells via its secretory metabolites[28,34]. In addition, CD is characterized by an increased abundance of Ruminococcus gnavus and mucosa-associated adherent-invasive Escherichia coli (AIEC), which bind to and invade intestinal epithelial cells through mechanisms involving microtubule polymerization and actin recruitment, inducing the secretion of inflammatory cytokines, particularly the transcriptional levels of interferon-γ and IL-8[35]. These cytokines are closely related to the immune response during CD, indicating the significant role of AIEC in perpetuating intestinal inflammation. When surviving and replicating in macrophages, AIEC can also induce TNF-α secretion, further contributing to the inflammatory milieu in CD[36]. A multi-center clinical study encompassing Spanish and Belgian CD cohorts investigated fecal microbiota composition using sample collection, genomic DNA extraction, high-throughput 16S rRNA gene sequencing, and bioinformatic analysis[37]. The results indicated that the depletion of beneficial bacteria, rather than an overabundance of pathogenic bacteria, is more strongly associated with CD[37]. These beneficial bacteria include butyrate-producing species such as Faecalibacterium prausnitzii, members of the Lachnospiraceae family (e.g., Roseburia and Ruminococcus), and Oscillospira[37]. These findings support and extend previous reports of dysbiosis in CD[37].

Intestinal fungal microbiota in CD

Although the fungal community is far less abundant than bacteria, constituting only 0.1% of the gut microbiota[38], many studies have shown that fungi also play an important role in the pathogenesis of CD[39,40]. The abundance of Candida albicans, Malassezia restricta, and Debaryomyces hansenii, which are the most commonly detected fungal species in stool samples of CD subjects[41-43], shows significant changes in patients with CD. Recent studies have revealed that Malassezia restricta worsens colitis in mice through a caspase-recruitment domain 9-dependent mechanism. Upon Malassezia restricta recognition by C-type lectin receptors on immune cells, caspase-recruitment domain 9 is recruited to form a complex with B-cell lymphoma/leukemia 10 and mucosa-associated lymphoid tissue lymphoma translocation protein 1, activating the NF-κB pathway. This triggers production of TNF-α, IL-8, and other pro-inflammatory mediators, amplifying immune cell recruitment and inflammation[42]. Debaryomyces, specifically Debaryomyces hansenii, has been identified as being enriched in inflamed intestinal tissues of patients with CD and impairs colonic healing by modulating the myeloid cell-interferon γ-C-C chemokine ligand 5 signaling axis[43]. Compared to healthy individuals, patients with CD exhibit an elevated proportion of Candida albicans and a reduced proportion of Saccharomyces cerevisiae in their gut. Candida albicans, originally a symbiotic bacterium in the gut, can delay intestinal mucosal healing and increase the secretion of IL-17 and IL-23 in diseases such as CD, thereby exacerbating inflammation[44]. Saccharomyces cerevisiae, a common probiotic in the gut, can strongly inhibit the adhesion of AIEC to the brush border of intestinal epithelial cells in CD. Furthermore, it restores barrier function by blocking AIEC-induced expression of Claudin-2, which forms tight junctions in pores on the plasma membrane of intestinal epithelial cells. These effects are accompanied by a reduction in the release of pro-inflammatory cytokines IL-6, IL-1β, and KC from the intestinal mucosa[45]. Additionally, several studies have indeed found that high serum titers of anti-Saccharomyces cerevisiae antibodies (ASCA) [immunoglobulin G (IgG) and IgA], which are directed against yeast cell wall-associated mannan, serve as a clinical biomarker for CD. The IgG ASCA-positive rate is 60%-70% in patients with CD[46].

Fungal-bacterial interactions

With the deepening of research on the intestinal microbiota in recent years, the interaction between different species has gradually attracted attention. The coexistence of fungi and some specific intestinal bacteria may be crucial for the development or improvement of intestinal diseases. Recently, Seelbinder et al[47] confirmed that antibiotic treatment significantly altered the composition of fungi, demonstrating that bacterial dysbiosis may lead to fungal dysbiosis. This study also showed that the use of Candida albicans aggravated the severity of the disease, while the use of Saccharomyces boulardii alleviated the symptoms of the disease. A cohort study, including patients from Northern France-Belgium, found a positive correlation between the abundance of Candida tropicalis and both Streptococcus mitis and Escherichia coli[48]. Additionally, the study showed an association between the levels of ASCA, a known biomarker for CD, and the abundance of Candida tropicalis[48].

Intestinal viruses and archaea in CD

The role of virome in CD may promote intestinal inflammation. CD is associated with an increase in temperate phages, critical for gut bacterial diversity and fitness, impacting microbial balance and CD progression. The “core phage” comprises double-stranded DNA Caudovirales (Myoviridae, Podoviridae, and Siphoviridae) and single-strand DNA Microviridae. CD patients exhibit altered viromes, with decreased Microviridae and Virgaviridae, and increased Caudovirales, Alteronomonadales, and Clostridiales phages. However, the specific virus-CD relationship remains elusive[49,50]. There is a paucity of relevant research in this area, and in vitro studies have demonstrated that bacteriophages can stimulate macrophages to induce MyD88-dependent pro-inflammatory cytokine production[51]. Besides, bacteriophages may also play a therapeutic role in CD treatment. A cocktail of three bacteriophages was demonstrated to reduce symptoms and significantly reduce AIEC in dextran sodium sulfate-induced colitis in mice[52]. Currently, research on archaea in CD is limited, with the majority of studies focusing on methanogens, which constitute approximately 10% of the intestinal anaerobic microbiota[53]. For instance, the presence of methanogens and the role of methane as a signaling molecule have been shown to prolong the contact time between the mucosa and toxic metabolic byproducts such as hydrogen sulfide. This extended exposure can enhance their absorption by the intestinal epithelium or lead to damage to the intestinal epithelial barrier, thereby increasing the risk of pathogen translocation[54]. Methanobrevibacter smithii is the most abundant species among methanogens, which accounts for approximately 94% of them, and recent studies have revealed that Methanobrevibacter smithii has been associated with increased acetate production[54,55] and acetate may exert a proinflammatory effect[56]. Additionally, the release of single-strand DNA from Methanosphaera stadtmanae, recognized as a microbe-associated molecular pattern, has been shown to stimulate the secretion of significant amounts of proinflammatory cytokines, such as IL-1β, TNF-α, and type-I and type-III interferons, through the activation of Toll-like receptor 8[57].

Virus-bacterial interactions

The trans-cohort study by Cao et al[58] describes the cross-relationship between 50 types of bacteriophages and 30 species of bacteria, revealing a significant dysbiosis in the bacteriophage-bacteria ecological network of the small intestinal mucosa in patients with CD. Specifically, the phage-bacteria interaction network, which is typically centered around Bifidobacterium and Lachnospiraceae in healthy individuals, is significantly weakened, while the phage-bacteria interaction network centered around Prevotella is markedly strengthened in CD. Another study has focused on the virus-bacteria interaction network, identifying a positive correlation between DNA bacteriophages and certain bacteria (such as Enterobacter cloacae and Escherichia coli), while showing a negative correlation with Faecalibacterium prausnitzii[59]. Furthermore, these studies have noted distinct changes in virus-bacteria associations under different disease states, including a close correlation of torquetenovirus, Bacteriophage spp., and Bacteroides phage B124-14 with fecal bacteria during active disease periods, which significantly diminishes during remission.

MICROBIOTA TRANSPLANTATION AND NUTRITIONAL THERAPY IN CD TREATMENT EFFICACY AND MECHANISMS

For the intractable chronic inflammation in CD, as well as severe complications such as intestinal stenosis and fistulas, various targeted surgical options and biological agents have been developed so far. With the updating of the concept of targeted drugs in recent years, some targeted therapies with theoretically better therapeutic effects and fewer side effects, such as FMT and dietary interventions, have gradually been developed and put into clinical use, providing more options for the treatment of CD from a new perspective.

Conventional therapy

Approximately 15%-50% of patients with CD will require surgery within ten years following the diagnosis[60], including fistula repair, colectomy and ileostomy, proctectomy, and strictureplasty[2,61,62]. Notably, there is evidence indicating that biological therapy is more effective if introduced earlier in the disease course, preparing for early intervention[2]. The selection of medical therapy is determined by the patient’s risk profile and the severity of the disease. Mild-to-moderate disease can be treated by oral mesalamine, immunomodulators like thiopurines (6-mercaptopurine and azathioprine), methotrexate, and steroids[63]. Moderate-to-severe disease (including fistulizing disease) is best treated with combined immunomodulators and biologics or biologics alone, including budesonide (corticosteroids), azathioprine, and mercaptopurine (immunomodulators)[64]. Anti-TNF agents (vedolizumab, ustekinumab, and infliximab) block the downstream effects of the TNF inflammatory cascade. These agents are efficacious in steroid-resistant or immunomodulator-refractory CD[65-67]. Notably, combination therapy with an immunomodulator and anti-TNF is more effective than monotherapy with either medication. Anti-IL-12/23 agents (ustekinumab and risankizumab) are efficacious in patients who failed prior corticosteroid, immunomodulator, or anti-TNF treatment.

Emerging biological therapies

Despite demonstrating efficacy in managing CD, conventional therapeutic interventions are correlated with a myriad of adverse reactions, encompassing metabolic disorders, serious infections, and dermatitis, etc.[68,69]. For instance, corticosteroids have the risk of causing cystoid appearance and hepatic steatosis, while anti-tumor necrosis factor therapy may cause severe bacterial infections in the lungs and a psoriatic eczema-like skin reaction. Initial analysis of early treatment regimens showed that some of these treatments disrupted the host gut microbiome, an occurrence linked to increased pathogenesis of CD[70-72]. Thus, introducing an adjuvant or supporting therapy that can modulate the gut microbiota and prevent gut microbiome dysbiosis may be a novel approach to supporting the management of CD. Not only does this approach have the potential of restoring the microbiome balance and it may also help achieve better response to the currently used medications[73,74].

Dietary therapy for maintenance of remission in CD

As the pathogenesis of CD becomes clearer, dietary factors are increasingly recognized for their role in disease management. Exclusive enteral nutrition (EEN) is well-established as an effective therapy for inducing remission in pediatric CD, with success rates of 60% to 80%[75]. EEN not only avoids the adverse effects of steroids but also improves nutritional status, promotes mucosal healing, and manages complications like strictures and fistulae[76-79], potentially reducing the need for surgery. In adult CD patients, corticosteroids and biologics are typically first-line treatments. However, a high-quality study has proposed EEN as an alternative therapy for adults, aiming to mitigate or avoid the side effects of conventional treatments and offering a standardized protocol for future research[80]. Despite its benefits, EEN has limitations, including its limited long-term effects on the gut microbiota and poor palatability, which hinder widespread adoption[81]. To address these limitations, alternative dietary strategies, such as partial enteral nutrition (PEN), low FODMAP diets, the specific carbohydrate diet, and Mediterranean diets, have been explored[82-84]. The efficacy of these interventions remains debated, and they are rarely used as standalone treatments[85].

The CD exclusion diet (CDED), characterized by low fat and animal protein and high carbohydrates and dietary fiber, has emerged as a promising alternative to EEN[86-88]. Clinical trials in pediatric CD patients have shown that CDED, combined with PEN, achieves remission rates comparable to EEN[89-91]. Recent studies indicate that CDED, alone or with PEN, effectively induces remission, reduces inflammation, and produces lasting changes in the gut microbiota[88,92]. Specifically, the combination of PEN and CDED reduces Actinobacteria and Proteobacteria while increasing beneficial Clostridia species after six weeks. Unlike EEN, which often sees microbiota reverting to baseline after remission, CDED-induced changes persist for 12 weeks post-therapy, suggesting a more sustainable approach to gut microbiota modulation and CD management[86]. It is noteworthy that the 2024 European Crohn’s and Colitis Organization Guidelines on pharmacological treatment of CD have emphasized the importance of dietary therapy, offering a highly valuable option for personalized treatment of CD.

FMT

FMT involves the processing of stool from a healthy donor into a therapeutic formulation that is then administered to individuals with gut microbiota dysbiosis, with the aim of ameliorating the imbalance in the patient’s intestinal microbiota. FMT has shown remarkable efficacy in treating refractory Clostridioides difficile infection[93-96], with a 90% success rate in the treatment of antibiotic recurrence cases. Given the critical impact of gut microbiota dysbiosis on the pathogenesis of CD, FMT is currently being explored as a therapeutic option for IBD.

A retrospective study integrated 11 high-quality CD reports involving a total of 106 CD patients and conducted a meta-analysis. The pooled clinical remission rate with a random effects model across these 11 reports was 0.62 (95% confidence interval: 0.48-0.81). Among them, seven studies reported a combined proportion of 0.79 for CD patients achieving clinical response after FMT treatment, with low heterogeneity (I² = 43%). Additionally, metagenomic analysis revealed that pre-FMT patients displayed reduced species diversity and significant disparities in microbiome composition when compared to their respective donors. Notably, following FMT, clinical responders, in contrast to non-responders, developed significantly elevated species diversity, more closely aligning with the donor’s microbiome configuration[97]. Several additional meta-analyses also support this viewpoint. Gutin et al[98] reported that in FMT for CD patients, responders had a more abundant presence of Enterobacteriaceae and Bifidobacteriaceae members, while non-responders had a higher relative abundance of Lachnospiraceae and Ruminococcaceae members[98]. Kao et al[99] reported that several groups of bacteria disappeared after FMT (Enterococcus, Lactobacillus, Streptococcus, Burkholderia group, and Erysipelothrix group), while the abundance of strains beneficial for CD (Faecalibacterium and Roseburia) increased. This indicates that the overall efficacy of FMT in CD patients is relatively good based on these studies. Additionally, the postoperative complications including abdominal pain, bloating, diarrhea and constipation typically following FMT are mild. And Gutin et al[98] and Sokol et al[100] further verified that the adverse reactions observed in patients were not associated with FMT. This validates the favorable safety characteristics of FMT as a treatment modality. Wu et al[26] have validated in mice models that FMT from healthy donors can improve CD, whereas FMT from CD patients exacerbates the condition. These findings strongly support the aforementioned viewpoint.

FUTURE PERSPECTIVES

Future research should focus on the extensive coverage of diverse populations across different regions, including germ-free animal models and genetically edited animal samples, while integrating cross-sectional and longitudinal cohort studies to ensure sample diversity and representativeness. The identification of key microbial strains and the development of related research methodologies should be expanded, emphasizing the application of metagenomic single nucleotide polymorphism/single-nucleotide variation analysis workflows, such as GT-Pro, and the utilization of AI algorithms to construct multidimensional diagnostic models linking species, genes, and single-nucleotide variations. The research should incorporate culture-based omics, bacterial gene editing technologies, and germ-free animal models to minimize and control the impact of confounding variables in experiments. By systematically summarizing research findings and validating causal relationships in animal models, further clinical trials can be conducted. Based on these results, treatment protocols for celiac disease should be optimized to achieve more precise early diagnosis, complemented by advanced imaging techniques to expedite disease assessment and promote the development of personalized therapeutic strategies. Microbiome-based therapeutic approaches, such as FMT and dietary interventions, have already been implemented in clinical settings and have shown promising therapeutic outcomes. These strategies aim to more effectively alleviate disease symptoms, reduce adverse events during treatment, and enhance the quality of life for patients.

CONCLUSION

CD, a complex chronic inflammatory bowel disorder, is characterized by its unique symptom of CrF. Dysbiosis of the gut microbiome exerts profound influence on the progression of the disease. Traditional therapies often exacerbate microbial imbalance and are accompanied by numerous adverse effects. Microbiome-based targeted therapies, with their unique efficacy and safety profiles, have paved a new avenue for personalized treatment of CD.