Archaea constitute a domain of single-celled organisms alongside two other domains, eukarya, and bacteria (Woese et al., 1990). Although archaea share some features with both bacteria (lack of nucleus, introns, the presence of a single circular chromosome) and eukaryotes (presence of histones for chromosomal DNA packaging) (Gaci et al., 2014), they are classified as a distinct class of organisms that comprises two major kingdoms: Euryarchaeota encompassing the methanogens and their phenotypically diverse relatives, and Crenarchaeota comprising the relatively tight clustering of extremely thermophilic archaebacteria, whose general phenotype appears to resemble the ancestral phenotype of the Archaea (Woese et al., 1990).

Archaea have the distinctive feature of colonizing a broad range of habitats, because of their evolutionary advantage in using specific pathways metabolizing a versatile panel of energy sources, ranging from the sunlight to both organic and inorganic substances (Valentine, 2007). Now we know that archaea are not only extremophile species colonizing severe environments, but they can be also found in moderate climates and can populate plants and animal intestines, representing an important constituent of the gut microbiota (Janssen and Kirs, 2008; Berg et al., 2016; Raymann et al., 2017).

Previous pieces of evidence positioned the archaeal species as ranging from 0.1 to 21.3% of the microbial entities colonizing the digestive tract (Kim et al., 2020). Recently, the development of culture-independent methods (i.e., Next-Generation Sequencing) has been opening a new horizon for the study of the composition of gut microbiota, where the methanogens have been suggested as a predominant archaeal group among gut microbial entities (Matijašić et al., 2020) and their colonization rate ranges from 25 to 95% of human stools (Stewart et al., 2006; Dridi et al., 2009; Hoffmann et al., 2013). Specifically, methanogens perform anaerobic respiration generating methane as a final product of metabolism (methanogenesis). Indeed, they decrease the gas pressure in the colon by consuming H₂ and CO₂ to produce methane (Gaci et al., 2014). Therefore, archaeal methanogens are likely to compete with sulfate-reducing bacteria for H₂ production in the human colon (Conway de Macario and Macario, 2009). Consequently, unbalance between methanogens and sulfate-reducing bacteria may alter gut mucosal homeostasis, resulting in intestinal dysbiosis.

Detailed analysis of gut archaeome revealed the predominance in the intestine of Methanobrevibacter (M.) smithii and Methanosphaera (M.) stadtmanae species, belonging to the archaeal phylum of Euryarchaeota (Conway de Macario and Macario, 2009; Dridi et al., 2009; Gaci et al., 2014) and likely harboring the strong capability to establish a syntrophic association with several bacterial species (Samuel et al., 2007). Besides these two species, haloarchaea, a non-methanogenic euryarchaeota belonging to the salt-loving family of archaea (Kim et al., 2020) (Figure 1A), and other members of several archaeal orders have been identified as components of the gut microbiota. Among these, methanogenic members of the orders Methanosarcinales, Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanopyrales were found to populate the human gut, along with the members of Desulfurococcales, Sulfolobales, Thermoproteales, Nitrososphaerales, and Halobacteriales orders, also detected in the human intestine (Gaci et al., 2014). A recent work, while confirming an increased prevalence of M. stadtmanae in the majority of human samples, failed to detect any non-methanogenic archaeal lineages (Raymann et al., 2017), unless the Haloferax massiliensis and Haloferax assiliense, demonstrating that halophilic archaea can inhabit the human gut (Khelaifia and Raoult, 2016; Khelaifia et al., 2018).

The contribution of the archaeome to the host physiology has not been completely understood, even if some relationship with the metabolism of dietary food has been highlighted, as for the M. smithii, found to trigger calories intake from the diet (Gaci et al., 2014). Moreover, methanogens exist in a syntrophic relationship with bacterial species within the gut. Indeed, short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, as well as hydrogen gas, are produced as a result of anaerobic bacterial fermentation (Samuel and Gordon, 2006). As mentioned above, by eliminating H₂ in the colon and thus affecting bacterial energy production, methanogens optimize the energy yield of the entire human microbiota (Chaudhary et al., 2018) (Figure 1A). As another example of host-microbiome interaction perpetuated by archaeal commensals, Methanomassiliicoccus luminyensis, recently isolated from human feces (Borrel et al., 2014), was found as sensitive to human-derived antimicrobial peptides and exhibited low immunogenicity toward human immune cells in vitro, thus perfectly resembling a commensal gut microbe (Bang et al., 2017).

Despite the evidence depicting the archaea as commensals, their role in IBD pathogenesis has not been fully elucidated yet (Aldars-García et al., 2021). Some indications come from studies regarding the M. stadtmanae, found to promote in vitro production of TNF and to be more abundant in IBD patient stools by comparison with the controls, suggesting a possible involvement in gut inflammation (Lecours et al., 2014) (Figure 1B). Importantly, M. stadtmanae was displayed to significantly activate human dendritic cells (Bang et al., 2014), further supporting this archaeon to participate in IBD pathogenesis (Lecours et al., 2014). Also, a recent report showed significantly lower M. smithii levels among IBD patients compared to healthy individuals, while normal M. smithii level was recovered in disease remission (Ghavami et al., 2018).

The recent IBD metatranscriptomics meta-analysis, the IBD TaMMA, analyzing a very large sample size (Massimino et al., 2021), highlighted for the first time differences in archaeome composition between colon and ileum from UC and CD patients. Indeed, such a transcriptome analysis compendium revealed colonic and ileal samples to largely differ in terms of archaeal compositions (Massimino et al., 2021). More interestingly, Nitrosophaerales, Haloferacales, Natrialbales, and Thermococcales were among the most abundant archaea orders in CD ileum, whereas the most abundant orders in UC ileum were Methanococcales, Methanobacteriales, Methanosarcinales, Methanomicrobiales, evidencing the differences between the two diseases in the ileal part, more “methanogenic” in UC. Also, Methanomicrobiales were found higher in UC colons, where it was the sole archaeal order to be statistically significant, while no differences were observed in colons from CD patients. From these insights, we can conclude that each intestinal tract may display differential abundances of archaea not only featuring the specific gut tract, but also the specific disease conditions. Such differences may contribute also to fostering diversified metabolic processes in the different intestinal segments, finally influencing the overall intestinal homeostasis. The archeaome composition in healthy conditions and the changes occurrgin during IBD pathogenesis are summarized in Table 1.

Notably, the IBD-associated bacterial dysbiosis may contribute to the archaeome composition shift, with an advantage of methylotrophic archaeal species (Figure 1B), particularly M. stadtmanae, finally increasing the inflammatory response within the human gut (Lecours et al., 2014). In this regard, a proposed leading theory explaining the possible contribution of archaea to IBD pathogenesis is that the SCFA butyric acid, produced in the colon by bacterial fermentation of dietary fibers and resistant starch, is a key regulator of syntrophism between archaea and bacteria in the gut (Matijašić et al., 2020). However, the archaeal overgrowth and the subsequent increased removal of SCFA from the biofilms are the two potential factors that cause dysbiosis, triggering bacteria to become endoparasites and enter intestinal epithelial tissues, which in turn leads to inflammatory processes in the human gut (White, 2017) (Figure 1B).

In conclusion, even if a causal link between archaeome dysbiosis and IBD pathogenesis has not been demonstrated yet, the evidence coming from previous studies suggested that archaea may play a major role in both health and IBD conditions. However, the way to the full definition of archaea-triggered mechanisms and functions in the gut is still long and deserves much more attention as a key player in regulating intestinal physiology. Moreover, its dysregulation may contribute, in combination with the other commensals, to sustain gut chronic inflammation, thus resulting important for the better comprehension of IBD pathogenesis and the development of new therapeutic lines of interventions aimed at the reconstitution of the archaeal composition within the intestinal microbiota.

Mycobiome is a general designation used for the description of fungal communities (molds and yeasts) of the microbiome (Cui et al., 2013) and is supposed to include nearly 0.1% of the total microbes in the gut (Arumugam et al., 2011; Nash et al., 2017). Mycobiome has been detected in the GI tracts of several mammals including humans, mice, rats, pigs, and many ruminants and non-ruminants (Liggenstoffer et al., 2010; Iliev et al., 2012).

Although most of the fungi-related studies were based on culture-dependent methods (Anderson, 1917), thus hampering the understanding of the fungal community of the microbiota, the recent advance in sequencing techniques (Meyer et al., 2010) has been progressively enlarging our knowledge about the mycobiome composition within the gut (Sokol et al., 2017; Qiu et al., 2020), where a lower diversity of fungal community than the bacterial was found (Nash et al., 2017).

In the human gut, many studies reported Ascomycota, Basidiomycota, and Mortierellomycota as the most dominant phyla of fungi (Qiu et al., 2020), while the most abundant genera composing the gut mycobiome are the Candida (particularly C. albicans), Saccharomyces (particularly S. cerevisiae), Penicillium, Aspergillus, Cryptococcus, Malassezia (particularly M. restricta), Cladosporium, Galactomyces, Debaryomyces, and Trichosporon (Sokol et al., 2017; Qiu et al., 2020) (Figure 1A).

Also, mycobiome entities exist in a tight equilibrium with the host and with the other actors of the intestinal microbiota, such as bacteria, thus altogether contributing to the maintenance of the overall tissue homeostasis (Santus et al., 2021). Evidence from experimental models showed C. Albicans to contribute to the recolonization of the intestine by bacterial species (Bacteroides) after antibiotic treatment (Mason et al., 2012). Interestingly, another study suggested the overall fungal and bacterial composition of the microbiota to be impacted by dietary habits in Japanese and Indian individuals. Results from this study revealed the higher abundance in Indian participants of Candida and Prevotella, maybe resulting from a higher dietary intake of vegetables used as a growth factor by the various Candida species (Pareek et al., 2019).

Some species populating the gut mycobiome were demonstrated to act as probiotics, with therapeutic potential for the host. As an example, the probiotic Saccharomyces (S.) boulardii was found to prevent antibiotic-associated diarrhea (Szajewska and Kołodziej, 2015) by regulating the immune system and by exerting an antimicrobial activity (Kelesidis and Pothoulakis, 2012). Another probiotic species is the Candida kefyr, found to reduce the severity of colitis by decreasing the abundance of Bacteroides and increasing the Lactobacillales (Takata et al., 2015) (Figure 1B). This shift in microbiota composition was associated with a decrease in interleukin (IL) 6 production (Takata et al., 2015). Moreover, it is suggested that fungi can be detected by the host immune system through Dectin-1 (CLEC7A), a c-type lectin receptor recognizing fungal wall β-glucans, resulting in a host immune response (Iliev et al., 2012) (Figure 1A).

Besides this beneficial impact on the host’s health, mycobiome dysbiosis was also associated with IBD. Higher levels of anti-S. cerevisiae antibodies raised against a component of the fungal cell wall were detected in CD patients’ sera compared to controls resulting in a reliable CD biomarker and predictors of the disease course (Quinton et al., 1998).

Reduced fungal diversity and increased abundance of Candida (C.) species were found in both pediatric and adult IBD patients (Figure 1B). More specifically, in adult IBD patients an increased Basidiomycota/Ascomycota ratio was found, coupled with the enrichment of C. albicans and the reduction of S. cerevisiae (Chehoud et al., 2015; Sokol et al., 2017). Moreover, Gibberella Moniliformis, Alternaria Brassicola, Aspergillus clavatus, and the Cystofilobasidiaceae were found to be increased in IBD patients (Liguori et al., 2016), while Malassezia sympodialis was markedly decreased (Hoarau et al., 2016). In CD, C. cropicalis and C. glabrata were found augmented by comparison with the control (Hoarau et al., 2016; Liguori et al., 2016).

Fungal dysbiosis observed in IBD patients was found to be associated also with composition shifts of other microbial compartments. As an example, the reduction of S. cerevisiae was observed in association with the reduction of several bacterial genera, such as Bifidobacterium, Blautia, Roseburia, and Ruminococcus (Qiu et al., 2020).

From a mechanistic point of view, the intestinal mycobiome was delineated also as a contributor during the inflammatory process. For example, by treating bone marrow-derived dendritic cells with two heat-killed yeast strains of S. cerevisiae and C. albicans, Sokol and colleagues found that the IL6 levels were comparable among treatments, but the anti-inflammatory cytokine IL10 was significantly higher after the stimulation with S. cerevisiae by comparison with the C. albicans (Figure 1B), suggesting an anti-inflammatory effect of the former (Sokol et al., 2017).

Additionally, C. tropicalis together with E. coli and Serratia marcescens bacteria were found to form a biofilm functioning as a commensal niche enriched in fungal hyphae, usually increased in pathogenic conditions (Hoarau et al., 2016). Interestingly, C. tropicalis was shown to positively correlate with Serratia marcescens and Escherichia coli in CD, further supporting their role in sustaining chronic inflammation as a “team” in the commensal niche (Hoarau et al., 2016).

The widely accepted mechanisms through which intestinal mycobiota may interact with the host’s mucosal physiology are prevalently based on the interaction between fungal species and innate immunity, involving specific receptors and signals driving the inflammatory response (Mahmoudi et al., 2021).

Among the main types of innate immune receptors that can recognize fungal Pathogen-Associated Molecular Patterns (PAMPs), Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and galectin 3 on antigen-presenting cells are well recognized and characterized (Bourgeois and Kuchler, 2012). The most investigated belonging to the CLRs include Dectin-1, recognizing the PAMP β-glucan (Taylor et al., 2007), Dectin-2, Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin receptor (DC-SIGN), Macrophage inducible Ca²⁺-dependent lectin receptor (MINCLE), suggested to recognize the α-mannose (Yamasaki et al., 2009), the Mannose Receptor (MR). Some of CLRs were found to directly interact with TLRs to recognize fungi (Vautier et al., 2012). Within the intestine, also, the fractalkine receptor (CX3CR-1) expressed by resident mononuclear phagocytes was recently discovered to mediate the interaction between the intestinal mycobiota and host immunity in both health conditions and inflammatory state (Leonardi et al., 2018). The proposed mechanism through which fungal recognition by immune cells triggers an innate immunity cascade occurs through the activation of the spleen tyrosine kinase (SYK) or the SYK-independent RAF-1 activation, which ultimately trigger the NF-κB signaling pathway toward T helper 1 and/or T helper 17 immunophenotypes (Richard and Sokol, 2019).

Susceptibility against mycobiome-related complications in IBD patients may rely on the genetic alteration within fungal recognition receptors-encoding genes. As some examples, polymorphisms in genes encoding for Detectin-1, TLR-1 and 3, MINCLE, Caspase recruitment domain-containing protein 9 (CARD9), Dectin-1, CD209, and CX3CR1 are the most investigated as associated with fungal dysbiosis/altered innate immunity during IBD pathogenesis (Iliev et al., 2012; Sokol et al., 2017; Leonardi et al., 2018; Limon et al., 2019).

The majority of the studies reported so far elucidated the mycobiome composition in small sample-sized IBD patient cohorts, mainly on stools and exploiting targeted DNA sequencing and PCR (Mahmoudi et al., 2021). By contrast, IBD TaMMA (Massimino et al., 2021) highlighted CD and UC ilea both featuring an increased abundance of Glomerellales, Tremellales, and Hypocreales, coupled with decreased abundance of Schizosaccharomycetales. Some orders were instead differentially abundant in the two conditions. Saccharomycetales, Ustilaginales, Malasseziales, Eurotiales, Mycosphaerellales, and Magnaporthales were found to be differentially abundant exclusively in UC ileum, while Saccharomycetales, Ustilaginales, and Sordariales were dysregulated only in CD ileum. Interestingly, differently from the ileal tract, very few orders were found to be differentially abundant in colons, perhaps resembling the different immune competence of the two tissues (Mann et al., 2016).

The mycobiome composition in healthy conditions and during IBD pathogenesis are summarized in Table 1.

Besides the long list of results coming from experimental colitis models extensively narrated by Beheshti-Maal and colleagues (Beheshti-Maal et al., 2021) and discussed later in this review, the real link between IBD pathogenesis and mycobiome dysbiosis is still missing, although some evidence lied ahead. Indeed, by combining the results from previous studies, fungal dysbiosis has been emerging to likely modulate the IBD bacteriome (Iliev and Leonardi, 2017), known to be pivotal in chronic inflammation onset and perpetuation as much as the immune system (Iliev and Leonardi, 2017). In this regard, in a study comparing samples from CD patients and their not-affected relatives, the inter- and intra-kingdom interactions between bacteriome and mycobiome were proposed to impact the host’s immune system in the setting of CD. Specifically, C. tropicalis interacts with potential bacterial pathogens, and that these interactions may play an important role in CD pathogenesis (Hoarau et al., 2016). Of note, the authors also indicated the microbiotas, specifically the mycobiomes, of familial samples as distinct from the non-familial (Hoarau et al., 2016). This may be partially explained by the consensus that members of a family share genetics, environment, diet, and bacterial microbiota being more similar to each other than they are to the unrelated individuals (Schloss et al., 2014).

Additionally, mycobiome entities were proposed to directly intervene in the release of IBD-specific proinflammatory cytokines (Beheshti-Maal et al., 2021), as suggested by the correlation found between specific gut mycobiome compositions with the expression levels of a series of pro-inflammatory cytokines in UC inflamed mucosa (Qiu et al., 2017). As a consequence, it is reasonable to indicate the mycobiome composition alterations, such as the increased abundances of Candida species often found in IBD mucosa, to be associated with inflammation and disease severity, as previously shown by Li and colleagues (Li et al., 2014). Consistently, Kowalska-Duplaga and colleagues observed that the abundance of Candida in CD patients decreased during the therapeutic intervention, particularly with anti-TNFα treatment (Kowalska-Duplaga et al., 2019), indicating a possible direct link between pro-inflammatory conditions and mycobiome composition. Besides the IBD-associated Candida species enrichment, an unbalanced Basidiomycota/Ascomycota ratio was also observed to correlate with flare-up/remission conditions in IBD (Sokol et al., 2017).

Overall, these pieces of evidence, although not specifically defining the mycobiome-triggered functional mechanisms underlying IBD pathogenesis, propose the mycobiome as important as the other microbiota compartments in orchestrating and contributing to the chronic gut inflammation onset and perpetuation, thus opening additional horizons for the investigation of IBD-associated microbiota diversity.

Whether on one hand human studies are relevant from a clinical point of view, unfortunately, they cannot provide mechanistic and functional notions that may help to determine whether gut mycobiota and archaeome dysbiosis are causal for intestinal inflammation onset and progression or this is only a consequence associated with the inflammatory process. Over the years, the established models of experimental colitis have been offering the chance to depict a more comprehensive view of the entire process involving intestinal microbiota in the aetiogenesis of gut inflammation, mainly illustrating the intestinal bacteriome roles (Zhang et al., 2017) and, to a lesser extent, the mycobiome and archaeome functions. As an example, Qiu and colleagues reported how the fungal composition of the GI tract may change during Dextran sulfate sodium (DSS)-induced colitis, finally uncovering that, while fungi were higher in the ileal tract by comparison with the colon independently of the inflammation state, the fungal Shannon diversity index of the DSS-induced colitis mice was lower than the controls in each gut segment (Qiu et al., 2015). Additionally, the inflammatory process caused a fungal translocation from the gut lumen to extra-enteric organs (such as the spleen and the mesenteric lymph node) during the experimental model of chronic colitis only in the inflamed intestinal tracts (Qiu et al., 2015), indicating that the inflammatory process itself may accelerate distal fungal invasion, likely because of the increased intestinal permeability. In line with previous evidence, the authors also showed that the fungal depletion was paralleled with a shift in mucosal bacterial composition (Qiu et al., 2015).

The direct association between fungi and bacteria during experimental intestinal inflammation was shown in another recent study highlighting Enterobacteriaceae to have a positive effect on fungal colonization of the gut, finally influencing the progression of gut inflammation. Indeed, C. albicans requires the presence of specific bacteria that trigger intestinal inflammation to increase the intensity, so that antibiotic treatment resulted as beneficial against DSS-induced colitis (Sovran et al., 2018). Also, C. albicans colonization of mouse intestines induced a strong Th17 response, suggesting that fungal composition infer specific immune changes in the host. Furthermore, Chiaro and colleagues in 2017 demonstrated that increased intestinal colonization with S. cerevisiae aggravated colitis by influencing purine metabolism, leading to extensive damage of the gut epithelia, reversible thanks to the inhibition of the purine pathway (Chiaro et al., 2017). These results further strengthened the concept that a tight interaction between mycobiota and the host’s metabolism exists, overall influencing the host’s physiology.

While some studies about fungi are existing in the context of experimental colitis, fewer pieces of evidence described the functional contribution by archaea during gut inflammation in vivo. This is mainly due to the difficulty in isolating archaeal species (Vemuri et al., 2020), and thus the functional studies in vivo manipulating the archaeome composition are still missing. Nevertheless, a very recent study reported its characterization in animals. Indeed, Mohamed and colleagues found methanogens to be increased during experimental colitis, coupled with bacterial dysbiosis, thus confirming the importance of archaeome-bacteriome equilibrium also in vivo (Mohamed et al., 2021).

Moreover, previous evidence reported that Methanobrevibacter smithii contributed to digestive health by directing Bacteroides thetaiotaomicron-mediated fermentation of dietary fructans to acetate, and in turn B. thetaiotaomicron-derived formate fostered M. smithii for methanogenesis, thus demonstrating a link between this archaeon and bacterial utilization in balancing host’s metabolism (Samuel and Gordon, 2006). Although this study did not demonstrate the effect of methanogens during intestinal inflammation, it does propose their contribution to metabolic health, suggesting their involvement in maintaining tissue physiology.

Conclusively, the paucity of studies about mycobiome and archaeome manipulation in vivo during intestinal inflammation strongly suggests that there is an urgent need to enlarge the knowledge about the mechanism directed by these two neglected components of the intestinal microbiota to make a step forward to the full comprehension of the entire microbiota-mediated mechanism in health and disease.