Advances in the research of intestinal fungi in Crohn's disease

Abstract

This article reviews of the original research published by Wu et al in the World Journal of Gastroenterology, delving into the pivotal role of the gut microbiota in the pathogenesis of Crohn's disease (CD). Insights were gained from fecal microbiota transplantation (FMT) in mouse models, revealing the intricate interplay between the gut microbiota, mesenteric adipose tissue (MAT), and creeping fat. The study uncovered the characteristics of inflammation and fibrosis in the MAT and intestinal tissues of patients with CD; moreover, through the FMT mouse model, it observed the impact of samples from healthy patients and those with CD on symptoms. The pathogenesis of CD is complex, and its etiology remains unclear; however, it is widely believed that gut microbiota dysbiosis plays a significant role. Recently, with the development and application of next-generation sequencing technology, research on the role of fungi in the pathogenesis and chronicity of CD has deepened. This editorial serves as a supplement to the research by Wu et al who discussed advances related to the study of fungi in CD.

Key Words: Intestinal microbiota; Crohn's disease pathogenesis; Fecal microbiota transplantation; Mesenteric adipose tissue; Gut fungal dynamics

Core Tip: This article offers an in-depth look at the original research by Wu et al in the World Journal of Gastroenterology, focusing on the critical role of gut microbiota in Crohn's disease (CD) pathogenesis. This study utilized fecal microbiota transplantation in mouse models to shed light on the complex relationship between the gut microbiota, mesenteric adipose tissue, and creeping fat, highlighting inflammation and fibrosis in CD-affected tissues. This underscores the influence of microbiota dysbiosis in CD and complements the findings with a discussion of emerging research on the role of fungi in CD pathogenesis and chronicity, propelled by advancements in next-generation sequencing technology.

TO THE EDITOR

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is characterized by a complex pathogenesis and etiology that remains largely unknown. The original research by Wu et al[1], published in the World Journal of Gastroenterology, focused on the pivotal role of the gut microbiota in the pathogenesis of CD. Utilizing fecal microbiota transplantation in a mouse model, this study elucidated the intricate interplay between the gut microbiota, mesenteric adipose tissue, and creeping fat, highlighting inflammation and fibrosis in affected tissues. This emphasizes the impact of microbial dysbiosis in celiac disease and sheds light on emerging research on the role of fungi in the pathogenesis and chronicity of celiac disease, driven by advancements in next-generation sequencing technology.

It is widely accepted that environmental and microbial factors induce an abnormal immune response in genetically susceptible individuals, leading to chronic inflammation associated with the onset of IBD. Genetically susceptible, germ-free animals do not exhibit symptoms of colitis, indicating that microbes are necessary for IBD development[2]. With the advancement of next-generation sequencing technology, a plethora of recent studies have utilized 16S metagenomic sequencing to analyze the complex microbial composition and dysbiosis in the digestive tracts of patients with CD[3]. Concurrently, there are an increasing number of reports on the role of opportunistic pathogenic fungi in the pathogenesis and chronicity of IBD in eukaryotes. Compared to patients without CD, patients with CD exhibit higher levels of antifungal antibodies, differences that are evident even before the disease is clinically diagnosed[4], supporting the notion of fungal microbiota dysbiosis in this population. This editorial focuses on summarizing recent advances in the study of fungi in adult patients with CD from the perspectives of pathogenicity and metagenomics.

Association of gut fungi with the host immune system

The variety of gut fungi is influenced by various factors including the host's genetic background, hygiene status, lifestyle, and use of antimicrobial drugs[5]. The most common fungi found in the human gut are from the genera Aspergillus, Candida, and Fusarium[4]. Studies have shown that food sources significantly affect the composition of gut fungal microbiota, with a marked difference between populations that primarily consume animal-based diets and those that consume plant-based diets[6,7]. Although fungi constitute only approximately 0.1% to 1% of the gut microbiota at the DNA level, their average volume is more than 100 times that of bacteria, which may greatly underestimate the total biomass of gut fungi and their impact on gut microbial ecology[6].

The interaction between fungi colonizing the gut and the host immune system is crucial for maintaining the health of the host intestines. Innate immune receptors such as C-type lectin receptors (dectin-1, dectin-2, mincle) and Toll-like receptors (TLR2, TLR4, etc.) can recognize β-1,3-glucans in the fungal cell wall[8]. Dectin-1 can activate intracellular signaling through CARD9, inducing the differentiation of T cells into Th1 and Th17 cells, as well as the production of related inflammatory cytokines[7]. It also regulates the differentiation of regulatory T cells (Treg cells) by interacting with the gut microbiota, thereby regulating intestinal immune homeostasis[9].

Research on the role of fungi in IBD through C-type lectin receptors has been common[10]. Mice lacking dectin-1 are more susceptible to colitis induced by dextran sodium sulfate (DSS) and genetic polymorphisms of dectin-1 (CLEC7A) are significantly associated with the severity of UC[11]. CX3CR1+ mononuclear phagocytes, which highly express dectin-1 and other C-type lectin receptors, respond to most commensal fungi by initiating an adaptive antifungal immune response through the Syk signaling pathway[12]. Mice with impaired function of these cells are more prone to intestinal diseases induced by Candida species. Patients with CD have a missense mutation in the gene encoding CX3CR1, which is associated with an impaired antifungal response[10]. Therefore, the stability of gut fungal microbiota is closely related to the balance of mucosal immunity in the intestine.

The advancement of gut fungal microecology research driven by the development of next-generation sequencing technology

Over the past two decades, the development of next-generation sequencing technology that does not rely on culture methods has made it possible to study microorganisms in the digestive tract that are difficult to cultivate. Fungal analysis focuses on their highly conserved internal transcribed spacer regions (ITS1 or ITS2)[13,14]. Different sequencing platforms employ strategies with their own advantages. The Illumina sequencing platform uses a cyclic reversible termination sequencing strategy that has a low homopolymer error rate; the 454 pyrosequencing method can sequence long fragments of 1000 bp, lifting the length limitation of sequencing; and the SOLiD (Sequencing by Oligonucleotide Ligation and Detection) method makes large-scale parallel sequencing possible[15]. The accuracy of Illumina sequencing is greater than 99.5%, and other ligase-based sequencing methods, such as SOLiD, have an accuracy greater than 99.95%[11]. Researchers can choose an appropriate sequencing platform based on their experimental requirements.

The continuous development of computational software has made it possible to calculate operational taxonomic units with differences of less than 1%. Minimum entropy decomposition can also distinguish real differences between closely related species and sequencing errors[12]. Ecological classification concepts are used to differentiate microbial populations. For example, the Shannon and Simpson indices are used in alpha diversity analysis to calculate species diversity within a sample, whereas beta diversity analysis can assess the diversity between samples[16]. The integration of 16S and ITS sequencing strategies in the future, coupled with the development of data analysis methods for complex bacterial and fungal mixed communities, will promote the assessment of the role of fungi in health and disease[13].

The role of fungi in the pathogenesis of CD

The application of next-generation sequencing technology to examine the gut microbiota composition during the onset of CD revealed a correlation between the diversity of intestinal bacterial and fungal communities in patients as well as a dysbiosis between bacteria and fungi[17]. Compared to healthy controls, patients with CD exhibit a greater biodiversity of fungi in their colonic biopsy specimens, with a more pronounced increase in the abundance and diversity of fungi in inflamed mucosal tissue[18]. The fecal fungal microbiota of patients showed a significant increase in the quantities of Candida, Aspergillus, and Cryptococcus species, and there was a significant positive correlation between fecal fungal diversity, serum C-reactive protein levels, and CD activity index[19]. Hoarau et al[20] found that the abundance of Candida tropicalis in patients with CD was significantly higher than that in their unaffected first-degree relatives and was positively correlated with the levels of anti-Saccharomyces cerevisiae antibodies, a biological marker for CD, although the overall biodiversity of fungi was reduced. Danne[21] analyzed fecal samples from patients with IBD and found an increased ratio of Basidiomycota to Ascomycota compared to that seen in healthy individuals, with a decrease in Saccharomyces and an increase in Candida abundance. Danne et al[22] demonstrated that in patients with CD, the mucosal-associated microbiota had an excess of the Tremellales family, Cystofilobasidiaceae, and smooth Candida, with an overall increase in the number of fungi in inflamed mucosal tissues, the presence of Herpotrichiellaceae in the inflamed mucosa, and Saccharomyces and Trichosporon in the non-inflamed mucosa. Whether these fungi merely pass through or are related to intestinal mucosal immunity requires further in-depth studies. However, the aforementioned studies support the view that adult patients with CD have a greater abundance of gut fungal biological composition than healthy individuals.

The relationship between gut fungal dysbiosis in patients with CD and current treatment regimens

The current effective early treatment strategy for CD involves the use of antibodies targeting specific inflammatory molecules to alleviate chronic inflammation, with TNF-α, IL-12/23, and leukocyte adhesion molecules being common therapeutic targets[23,24]. Antibodies targeting TNF-α mainly include infliximab, adalimumab, and certolizumab, while ustekinumab is the first antibody targeting the p40 subunit of IL-12/23[25]. The immune response to opportunistic pathogenic fungi such as Candida is involved in cytokines such as TNF-α, IL-17, and IL-23[26,27]. Therefore, the aforementioned CD treatment regimens are associated with fungal infections (including histoplasmosis, yeast infections, coccidioidomycosis, and other opportunistic fungal infections)[28]. In clinical trials, compared to a placebo, the group treated with an anti-IL-17A monoclonal antibody (secukinumab) had an increased amount of fecal inflammatory mediators and an increased risk of mucocutaneous candidiasis[29]. A systematic review including 14 Literature sources with a total of 1524 patients with IBD showed that the most common fungal infection in patients with IBD was Candida infection (903 cases), with most infections occurring within 6 months of anti-TNF-α treatment[30].

To date, no clinical trials of antifungal treatments for IBD have been conducted. Theoretically, targeting the cytokine pathways of IL-17, IL-23, IL-22, and TNF-α can reduce pro-inflammatory responses and acute mucosal damage, but it may also lead to a reduction in commensal fungi and an increase in the abundance of opportunistic pathogenic fungi, providing a reasonable explanation for the clinical detection of pathogenic fungi in patients with IBD[31]. In particular, Th17 cytokines, such as IL-22, can directly regulate the expression of antimicrobial-related genes in epithelial cells, including antimicrobial products of intestinal epithelial cells Reg3γ and Paneth cells[32]. A reduction in these mucosal secretions can promote the growth and colonization of fungi on the mucosal surface[9]. Although the above phenomena have not been routinely detected, research data show that some immune-targeted treatments can increase the fungal load. Based on this, the following hypothesis can be proposed: Part of the immune response related to IBD may be driven by pathogenic fungi, and specific treatment regimens may alleviate the immune response of the digestive tract to pathogenic fungi[6].

Application of probiotic fungi in the treatment of IBD

Commensal fungi play a significant role in modulating host immune functions. In clinical treatment for CD, Saccharomyces boulardii has been utilized as an adjunct therapy to alleviate intestinal inflammation[33]. Recent years have witnessed advancements in the study of its mechanism of action. As a probiotic fungus, Saccharomyces boulardii can enhance the host intestinal immune response to pathogenic bacteria, limiting pathological inflammatory responses such as CD, UC, and Clostridium difficile colitis[34]. The supernatant from a culture of Saccharomyces boulardii can directly inhibit the secretion of pro-inflammatory cytokines by myeloid dendritic cells (DCs) induced by LPS in patients with CD[35]. Studies have shown that Saccharomyces boulardii can increase the overall level of IgA in the gut[6,36]. In a mouse model of colitis, the administration of Saccharomyces boulardii reduced histological inflammatory responses and inhibited Candida colonization in the intestine[37].

Another probiotic fungus is Candida kefyr[38]. Oral administration of Candida kefyr alleviated experimental colitis, accompanied by an increase in Treg cells and regulatory DCs in the mesenteric lymph nodes and a decrease in Th17 cells in the lamina propria[35]. With the development of high-throughput next-generation sequencing technology, future research should focus on the identification of probiotic fungi in mucosal or fecal sample microbiome analyses and their clinical applications.

CONCLUSION

Research on the role of gut fungi in CD has shed new light on the complex dynamics of IBD. Advancements in next-generation sequencing have increased our understanding of the impact of the gut mycobiome on host immunity and its association with CD. Studies have indicated a correlation between fungal dysbiosis and inflammatory markers in CD, with certain fungi showing an increased prevalence in affected individuals. The modulation of immune responses by fungal cell wall components, particularly through receptors such as dectin-1, is a key area of focus with direct implications for disease management. While current treatments targeting inflammatory cytokines show promise, they also pose risks of altering gut fungal balance, as evidenced by increased fungal infections with anti-TNF-α therapies. The emerging use of probiotic fungi as therapeutic agents indicates a novel approach for the treatment of intestinal inflammation. However, the precise mechanisms underlying the effect of fungi on CD require further elucidation. Future research must clarify the causal links between fungal dysbiosis and disease progression to pave the way for more effective microbiome-informed treatment strategies.