The role of mycobiota-genotype association in inflammatory bowel diseases: a narrative review

Inflammatory bowel disease (IBD) is a chronic inflammatory disease affecting various parts of the gastrointestinal tract. A majority of the current evidence points out the involvement of intestinal dysbiosis in the IBD pathogenesis. Recently, the association of intestinal fungal composition With IBD susceptibility and severity has been reported. These studies suggested gene polymorphisms in the front line of host defense against intestinal microorganisms are considered to play a role in IBD pathogenesis. The studies have also detected increased susceptibility to fungal infections in patients carrying IBD-related mutations. Therefore, a literature search was conducted in related databases to review articles addressing the mycobiota-genotype association in IBD.


Inflammatory bowel disease pathogenesis
Inflammatory bowel disease (IBD) is a chronic relapsing disease affecting various parts of the gastrointestinal tract and encompasses two common disorders: Crohn's disease (CD) and Ulcerative Colitis (UC). IBD is a worldwide issue, especially in urban and westernized countries among young individuals [1], assumed to result from an improper and continuous inflammatory response to commensal microbes in a genetically susceptible host [2]. So far, the pathogenesis of the disease is considered to be a combination of genetic predisposition and environmental factors. The majority of current evidence emphasizes the involvement of intestinal dysbiosis in IBD pathogenesis [3]. While intestinal epithelial cells (IECs) are constantly exposed to microbial components; they are regarded not only as a structural but also a functional barrier in the front line of host defense against intestinal microorganisms. The functional alteration of these cells is hypothesized to be associated with IBD [4]. Bacteria as the predominant organisms of the gastrointestinal tract gained the greatest attention in IBD microbial studies [5][6][7]. Nonetheless, the association of intestinal fungal composition with mucosal inflammation in both CD and UC has recently become into consideration [8][9][10][11]. Of note, increased IBD flares were associated with increased global fungal load accompanied by alteration of certain fungal species in the microbiota [12][13][14].
To date, numerous gene polymorphisms are found to be connected to IBD susceptibility [15] and severity; for instance, an increased colitis severity was driven by activation of Leucine-rich repeat kinase 2 (LRRK2), an important enzyme that regulates innate immunity through nuclear factor kappa B (NF-κB) signaling pathway [16]. Some articles studied the association of specific intestinal bacterial microbiota with gene polymorphisms [17,18]. However, few have focused on the role of fungal subsets in the intestine. The purpose of this study was to discuss the association of fungal flora with IBD and review the articles connecting the gene polymorphisms with intestinal mycobiota in IBD cases.

Anti-Saccharomyces cerevisiae antibody
The first sparks of fungi role in IBD pathogenesis flared by detecting elevated levels of anti-Saccharomyces cerevisiae mannan antibodies (ASCA) in the sera of IBDaffected patients since the early 90 s [19,20]. A twin study in 2005 has detected ASCA in CD cases more frequently compared with healthy controls [21]. ASCA was also found commonly in CD patients with a positive family history of IBD [22] and even in unaffected relatives of CD patients [23]. ASCA was not only detected in answer to Saccharomyces antigens but also in response to Candida albicans or the presence of anti-β2 glycoprotein I antibodies in CD patients [24,25]. Marrakchi et al. revealed a positive correlation of caspase recruitment domaincontaining protein 15 (CARD15)/nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene mutation, an important intracellular pattern recognition receptor (PRR) that is expressed by dendritic cells (DCs), macrophages, and IECs [26], with ASCA expression in IBD-affected patients [27].

IBD affecting intestinal mycobiota
In addition to animal studies, some articles are conveying the alteration of intestinal mycobiome in human subjects with IBD. Ott et al. first described significantly higher fungal diversity in patients with CD in comparison with healthy controls, albeit no disease-specific fungal species were present in the CD and UC group [28]. Ever since, many studies have consistently shown an elevated abundance of Candida sp. in IBD fecal samples [29][30][31]. Lewis et al. have reported an increased amount of S. cerevisiae [29], whereas Hoarau et al. reported a reduction in intestinal S. cerevisiae abundance in IBD patients [31]. Another study in 2009 reported a significantly elevated C. albicans population obtained from fecal samples of CD patients (44%) and their healthy relatives (38%) compared to healthy controls [22]. Li et al. assessed 19 patients with active CD and 7 healthy individuals and discovered increased fecal fungal richness and diversity in C. albicans, Aspergillus clavatus, Cryptococcus neoformans, and a decrease in S. cerevisiae in CD patients. The diversity of the fecal fungal community was also positively correlated with serum C-reactive protein level and the CD activity index [13]. Another study in 2016, revealed a significant increase in global fungal load in both inflamed and noninflamed mucosa compared with healthy subjects (HS). However, no significant differences in fungal diversity were observed between the groups [12].
Unlike most similar articles, Chehoud et al. demonstrated pediatric IBD to be associated with reduced fungal diversity in the host gut microbiota. Specific Candida taxa were also found to have increased abundance in the IBD samples [30]. An additional study with de-novo pediatric IBD cases revealed a shift from the Ascomycota-predominant mycobiota in HS to a different fungal spectrum with a predominance of Basidomycetes in patients with de-novo IBD without the conflicting impact of antibiotics or immunosuppression [32]. Later, another study investigated the possible fungal dysbiosis index in IBD; the fecal fungal composition of 235 patients with IBD and 38 HS showed an increased Basidiomycota-to-Ascomycota ratio that was dramatically higher in patients with IBD flares compared to patients in remission and HS [8]. There was also a negative correlation between the abundance of S. cerevisiae and C. albicans in fecal samples of IBD subjects, suggesting a competitive environment between these two species in the gut [8,33]. The study also described a complex fungal-bacterial interaction in the fecal composition of subjects [8].
As opposed to Sokol and Mukhopadhya et al., Qiu and colleagues did not detect any significant difference in the abundance of Ascomycota, Basidiomycota, and the ratio of Ascomycota-to-Basidiomycota between the HS and UC patients. However, there was a prominent variation in the abundance of Aspergilli between the groups [11]. A recent report studied the cultivable intestinal mycobiota presented in feces obtained from 34 pediatric CD patients, 27 pediatric UC patients, and 32 healthy children. The authors observed increased load of S. cerevisiae and Candida sp. in IBD patients, which was in line with previous studies. Likewise, Di Paola et al. concluded that the presence of S. cerevisiae was associated with a favorable intestinal environment for beneficial bacterial genera, such as Faecalibacterium; whereas the absence of normal fungal flora or presence of unusual fungal species were conjugated with the presence of potential pathogenic bacteria that might lead to IBD [34]. The latest article by Nelson et al. reported an increased abundance of Candida sp. and a decreased Basidiomycota-to-Ascomycota ratio, in contrast to the previous literature, in CD cases [35]. Of note, the discrepancies between these studies might stem from different fungal extraction methods. In this regard, we provided additional information for these studies, including the fungal extraction method and the sample source, in Table 1.
Gene polymorphisms targeting innate immunity may play an important role in IBD. Although studies aiming at the role of intestinal fungi pathogenesis in IBD are scarce, studies focusing on innate immune pathways against intestinal bacteria and their inflammatory consequences have successfully revealed important roles for innate immunity in IBD. Similarly, fungal recognition in the gut may be also regulated by innate immunity [49]. Four main types of innate immune receptors that can recognize fungi through fungal Pathogen-associated molecular patterns (PAMPs) are TLRs, C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and galectin 3 on antigen-presenting cells [50]. The most studied class are the CLRs which include Dectin-1, Dectin-2, Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin receptor (DC-SIGN), Macrophage inducible Ca 2+ -dependent lectin receptor (MINCLE), and the Mannose Receptor (MR). Additionally, some CLRs can interact with TLRs to recognize fungi [51]. The β-glucan is the main PAMP that can be recognized by Dectin-1, although Dectin-1 can also recognize unidentified bacterial and endogenous ligands [52]. Dectin-2 has been recently shown to be the functional receptor for α-mannans and to be implicated in anti-bacterial immunity [53]. The α-mannose is also strongly suggested to be Mincle's ligand, which has been implied in antimycobacterial immune activity [54]. The fractalkine receptor (CX3CR-1) expressed by intestinal-resident mononuclear phagocytes (MNPs), were also characterized to have a role in initiating immune responses against fungi [55]. Through fungal recognition, these pathways initiate the inflammatory cascade by predominantly driving the immune responses through spleen tyrosine kinase (SYK)-dependent, SYK-independent, and eventually NF-κB signaling pathway towards T helper 1 (TH1) and/or T helper 17 (TH17) immunophenotypes [56]. The brief signaling cascade leading to intestinal inflammation is available in Fig. 1.

Intestinal mycobiota-genotype association
As Table 2 represents, here, we concentrated on articles reporting the mutations of innate immunity components and resulted in the gut mycobiome alteration. In a recent article, Limon et al. expressed that colonization of the colonic mucosa with Malassezia restricta, a commensal fungus typically found on the skin, might increase IBD severity in patients with CARD9 S12N risk allele. They found out that the CARD9 S12N variant induces a potent pro-inflammatory cytokine response against M. restricta in IBD [57]. By examining the SYK-CARD9 signaling axis and gut fungi, Malik et al. also demonstrated the decreased occurrence of Ascomycetes along with elevation of Saccharomycetes in Card9 −/− mice. They implied that a normal inflammasome assembly in an unperturbed SYK-CARD9 signaling axis led to protection against colitis and colon cancer and also promoted T cell-mediated anti-tumorigenic responses; thereby indicating that a healthy gut mycobiota could prevent the development of IBD [58]. According to Lamas et al., the fungal microbiota of wild type and Card9 −/− mice with induced-colitis mainly were members of the Ascomycota, Basidiomycota, and Zygomycota phyla. However, there were different measurements at the days 0, 7, and 12, and both groups reached a peak at day 7 that was higher in Card9 −/− mice. On day 7, Card9 −/− mice showed decreased fecal Ascomycota, increased fecal Basidiomycota, and Zygomycota communities [59]. CX3CR-1 T280M (rs3732378) is a common polymorphism that has been previously detected in extra-intestinal inflammatory diseases [60,61]. In 2018, Leonardi et al. described that CX3CR1 + MNPs not only modifies adaptive immune responses to intestinal fungi and controls the mycobiota during experimental colitis in animal models (without changing bacterial communities), but is also connected with a decrease in antifungal antibody responses in CD patients. They concluded that intestinal mycobiota and CX3CR1-dependent immune responses might provoke extra-intestinal manifestations of inflammatory diseases [62]. Elevated antifungal antibodies detected in patients with alcoholic liver disease, Graves' disease, spondyloarthritis, and systemic lupus erythematous corroborate this hypothesis [63]. Finally, the article provided evidence for CX3CR1 + MNPs as a mediator between gut mycobiome and both local and systemic immunity [55].
The impact of NOD2 variants on the intestinal bacterial community in CD patients has previously been described [65]. Thus, Nelson et al. investigated the presence of NOD2 polymorphisms in CD patients and its relation with fecal fungal diversity but did not find any significant correlation between NOD2 variants and specific intestinal fungi community [35].
Dectin-1 is the most important fungal PRR expressed by innate immune cells, such as macrophages, dendritic cells, and neutrophils. C-Type Lectin domain containing 7A (CLEC7A) is the gene that encodes Dectin-1. Clec7 −/− mice with induced colitis had increased proportions of opportunistic pathogenic fungi including Candida sp. and Trichosporon sp. along with a decreased frequency of nonpathogenic Saccharomyces. Iliev et al. identified a significant association between CLEC7A SNP (rs2078178) and patients suffering from medically refractory UC and delineated the role of Dectin-1 as a fungal receptor during severe forms of colitis [66]. Other gene polymorphisms were also described to influence Dectin-1-associated immunity in IBD [16,67]. Among these genes, LRRK2 has also been described as the familial Parkinson's disease genetic risk factor. Multiple variations in LRRK2 comprising N2081D, rs11175593 LRRK2/MUC19, and rs11564258 LRRK2/MUC19 were associated with IBD as well [68]. Takagawa et al. suggested an increase in severity of colitis, mediated by increased Dectin-1induced immunity, in (rs11564258) LRRK2/MUC19 polymorphism carriers [16]. Noteworthily, this variance (rs11564258) had the second-highest odds ratio in IBD patients of the European population [69]. Further studies are required to identify the intestinal mycobiota in the patients carrying this mutation.

Conclusion
In summary, the role of intestinal fungal mycobiota in IBD pathogenesis and severity index have been quite underrated. This review emphasizes that a majority of IBD-affected patients had increased diversity and richness of intestinal mycobiome, higher abundance of C. albicans and Basidiomycota-to-Ascomycota ratio, and a decreased proportion of S. cerevisiae despite a few contradictory results in other studies.
It is widely known that innate immunity takes part in intestinal fungal recognition and mutations in innate immunity mediators are linked to IBD pathogenesis. Even so, few articles aimed to examine the connection between gene polymorphisms and intestinal fungal dysbiosis in IBD.
Although DSS-induced colitis is a well-established experimental murine model with much resemblance to human IBD [70], we were able to find only three non-murine studies containing mycobiota-genotype data related to IBD patients. Additional evidence is needed to determine whether different gene polymorphisms can alter intestinal mycobiome or whether this information would be of use in providing novel insight into IBD pathogenesis. Therefore, our purpose was to highlight the importance of the matter and draw attention to this underappreciated aspect of IBD-associated research.