Gut virome: New key players in the pathogenesis of inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory illness of the intestine. While the mechanism underlying the pathogenesis of IBD is not fully understood, it is believed that a complex combination of host immunological response, environmental exposure, particularly the gut microbiota, and genetic susceptibility represents the major determinants. The gut virome is a group of viruses found in great frequency in the gastrointestinal tract of humans. The gut virome varies greatly among individuals and is influenced by factors including lifestyle, diet, health and disease conditions, geography, and urbanization. The majority of research has focused on the significance of gut bacteria in the progression of IBD, although viral populations represent an important component of the microbiome. We conducted this review to highlight the viral communities in the gut and their expected roles in the etiopathogenesis of IBD regarding published research to date.

Key Words: Inflammatory bowel disease; Pathogenesis; Gut virome; Bacteriophage; Eukaryotic viruses

Core Tip: Inflammatory bowel disease (IBD) is a chronic multifactorial inflammatory disease involving the gastrointestinal tract. The exact etiopathogenesis is unknown, but it’s believed that gut microbiome dysbiosis is a cornerstone in triggering disease progression. The gut virome forms a significant part of the gut microbiome and participate in health and disease conditions. Until 2015, researchers paid little attention to their role in IBD. Subsequently, numerous studies have followed this line of inquiry, using advanced techniques to clarify this role. Herein, we emphasize the viral populations in the gut and their predicted roles in the etiopathogenesis of IBD based on current studies.

INTRODUCTION

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic condition characterized by chronic inflammation of the gastrointestinal tract (GIT)[1]. The precise etiology of IBD is complex and still not fully understood. However, studies have revealed that the onset and course of IBD are controlled by a variety of factors, including the interaction between environmental factors (e.g., intestinal microbiota) and the host immune response in genetically susceptible people[2-6]. After birth and in the early days of life, the gut microbiota begins to colonize the GIT, where they coexist in an equilibrium process and actively interact with the host[7]. In healthy settings, the composition of microbiota changes until adulthood, when it becomes more stable[8]. In particular, the gut microbiota maintains the integrity of the gut barrier, promotes the generation of nutrients (e.g., short-chain fatty acids [SCFAs] and vitamins), regulates the immunological response, and participates in the metabolism of drugs and nondigestible food, and defense against pathogenic organisms[9,10]. Typically, gut microbiota consists of bacteria, viruses, fungi, and archaea. Bacteria have received the most attention from these microbes and have been associated with developing mucosal immunity and reducing mucosal inflammation[11-14]. An abnormality in one of these immunological pathways can have a negative impact on IBD development. For instance, alterations in the function of the bacterial microbiome or a decrease in Bacteroidetes and Firmicutes levels and an increase in less prevalent bacterial species (spp.) have all been linked to IBD[15]. Nonbacterial elements of the gut microbiota have been neglected in previous studies for a variety of reasons, including their low absolute prevalence in the intestinal microbiota of humans and a scarcity of competent and specific diagnostic methods for nonbacterial genome analysis[16].

Early studies defining the gut microbiota focused on culturing bacteria, which had little success since only a tiny fraction of gut microbes can be cultivated[17]. Following that, in the early 2000s, next-generation sequencing technology emerged and allowed scientists to investigate the diversity of gut microbiota. This scientific advancement led to the emergence of the “microbiome” era, which aims to study the whole microbial genomes, paving the way for the development of the subfield of “virome research”[18]. The gut virome is still a little-studied subsection of the whole microbiome despite this significant development[19]. Regardless of the lack of representation, several publications have demonstrated that a disturbed gut virome is linked to several illnesses including type 1 and type 2 diabetes[20,21], cystic fibrosis[22], obesity[23,24], graft vs host disease[25], acquired immunodeficiency syndrome[26], colorectal cancer[27], malnutrition[28], liver diseases[29], severe acute respiratory syndrome coronavirus 2[30], as well as IBD[31].

This review provides deep insights into gut virome dysbiosis and its role in the etiopathogenesis of IBD.

AN OVERVIEW OF THE GUT VIROME

The GI system has a complex ecosystem, including bacteria, viruses, fungi, and protozoans. The overall GI microorganism communities and their constituent genes are known as the gut microbiome[32]. The gut microbiome plays a key role in developing and maintaining homeostasis and a balanced immune system through interactions with epithelial and immune cells and regulating metabolic processes (such as SCFAs and bile acids)[33-35]. Viruses form a significant part of the gut microbiome and participate in maintaining homeostasis[36].

The two main forms of viruses in the gut microbiome are phages, which infect bacteria, and viruses, which infect eukaryotic cells (such as human cells). Although both kinds have been observed in the human GIT, phages account for the vast majority of viral spp.[37] Both forms either contain DNA or RNA (single or double strand) as genetic material[38]. A phage enters its cellular host and uses its machinery to start its own reproduction process. There are two major lifecycles that characterize this process: lytic or lysogenic cycle[39]. The lytic cycle comprises attachment, entry, replication, and creation of virions, which are mainly completed through lysis of the host cell (Figure 1)[40]. The lysogenic cycle involves the formation of extrachromosomal plasmids in the cytoplasm or the integration of phage genetic material into the host genome[40]. The lysogenic cycle allows viruses to remain latent (as prophages), which ensures that the genetic material will be passed on to cellular progeny during cell division[37,41]. Induction of prophage happens either naturally at a low rate or is activated by outside stresses, initiating the DNA damage response or SOS response (Figure 1)[41,42]. Moreover, Erez et al[40] discovered the “arbitrium system,” a phage-specific communication mechanism that enables the phages to detect their levels in the surroundings and choose whether to start the lytic or lysogenic cycle. Using this approach, phages lysogenize the host at high arbitrium concentrations and lyse the host at low arbitrium concentrations[40]. Prophage serves as a reservoir for phage-encoded genes that the bacterial host may acquire[43]. This genetic reservoir may have genes that increase stress tolerance and immunity, promote virulence and biofilm production, and provide metabolic and antibiotic resistance[44-48]. As a result, these acquired bacterial activities might be advantageous (as boosting immunity) or damaging (as virulence factors) to the human host[49]. For instance, prophages can protect bacterial host cells from subsequent infection from closely related phages[49]. Moreover, prophages have the ability to disseminate virulence components that turn some commensal gut microbiota into pathogenic ones[46,50]. For instance, phages from the Inoviridae family that encode cholera toxin can incorporate with the bacterial host, transport toxin genes, and produce dangerous organisms[50,51].

Figure 1 Two major lifecycles of bacteriophage (lytic and lysogenic).

GUT VIROME IN NORMAL HEALTHY CONDITIONS

Each human has a large number of viruses, approximately 10¹³ particles per person, the majority of which are located in the gut[52-54]. According to growing data, the gut microbiome is initially quite basic, changes quickly during the first days after childbirth, and eventually becomes more diversified and stable over time[55-58]. Breitbart et al[59] conducted the first investigation documenting the gut phage population in fresh fecal samples in newborns. According to their investigation, the meconium, a newborn’s initial fecal excretion, failed to include any virus-like particles (VLPs) when examined by a direct epifluorescence microscope. On the other hand, towards the end of the first week, 10⁸ VLPs per gram of moist feces were found[59].

Furthermore, in 2015, two additional studies revealed that the gut phage population exhibited considerable changes throughout the first 2 and 2.5 years of life, respectively[60,61]. Lim et al[60] documented the existence of the Microviridae family in the dominant phages in addition to the Caudovirales class, as well as a shift from Caudovirales to Microviridae at the first 24 mo of age. Additionally, they discovered that the abundance as well as diversity of intestinal phages were maximized in the first 4 d of life and subsequently declined as individuals aged[60].

Members of the phage classes Malgrandaviricetes (spherical single-strand DNA [ssDNA]) and Caudovirales (tailed double-strand DNA) make up the biggest known populations of viruses living in a healthy human GIT[57,62]. Caudovirales are believed to infect Bacteroidetes, Firmicutes, Verrucomicrobia, Actinobacteria, and Proteobacteria, while Malgrandaviricetes are believed to infect Enterobacteria or intracellular microorganisms (such as Spiroplasma, Chlamydia, and bdellovibrio)[63,64]. The crAss-like phage, a newly identified monophyletic clade within the Caudovirales class, is thought to be the most abundant phage in the healthy human gut[62,65,66]. After the identification of crAss-like phages, several other prevalent and common viral clades, including Flandersviridae, Gubaphage, giant Lak phages, and LoVEphage, were discovered[67-69]. Small circular ssDNA viruses, such as Anelloviridae and Caudovirales, are among the most common eukaryotic viruses[62,70]. It was reported that Anelloviruses are not particularly common, but they form a very diverse and common eukaryotic viral class at the beginning of life and are reduced gradually as the microbiome matures[63]. Plant viruses are another type of eukaryotic virus that is typically observed in elevated concentrations in the healthy human GIT[71]. They are often gained by food and passed to the GIT[62,72].

Viral complexity is defined by substantial interindividual variations or notable uniqueness of viral contigs[62,73]. While people significantly differ from one another, a person’s virome can remain quite constant over time, as demonstrated by low intraindividual variance[74,75]. Generally, it is believed that the healthy gut microbiota is a diverse ecosystem, and any dysbiosis or imbalance in this ecosystem is frequently linked to the progression of diseases[76] such as IBD[6,77,78], irritable bowel syndrome[79-81], and colorectal cancer[82-84]. Although the patterns of diversity in the healthy gut virome are still out of reach, it is believed that they have a positive impact on the diversity of the microbiome[85].

ENVIRONMENTAL AND HOST FACTORS THAT AFFECT THE GUT VIROME

Many factors can affect and shape the gut virome. One of these is anthropometric factors that measure the physical properties of the host such as height, weight, age, and body mass index, among others[62,86,87]. Other factors can be divided into a number of main groups, including nutrition and its relationship to stool uniformity, lifestyle, and physical activity, diseases and medications, as well as geographical location[24,74,86,88-90]. As mentioned above, the diversity of the gut virome reaches its maximum level in the first days after birth and decreases with age[60]. Also, it is thought that people’s dietary habits impact the type of viruses in their GIT[74,91,92]. For example, consuming coffee and dairy products and consuming fruit have a positive association with the diversity of the gut virome and affect the Shannon diversity index and Bristol Stool Score[74,86,93,94]. In addition, consumption of high quantities of fats is linked to a low proportion of Caudovirales phages and a large proportion of Malgrandaviricetes phages, as well as reduced lysogenic capacity[92]. Several investigations have shown that the medications might activate prophages inside their hosts, modifying the virome of the gut, and subsequently, the life cycle[95,96]. A complex equilibrium between lytic and lysogenic phages is therefore believed to exist in a healthy condition[57]. For instance, a lysogenic lifestyle results in a higher bacterial cell number, but activation of lytic activity results in a lower bacterial cell number[57,97]. Higher microbial cell numbers probably give phages an easier means to replicate their incorporated genomes via bacterial replication instead of lysing the bacterial host[97]. Caudovirales phages, which are mainly lysogenic, are abundant in the early stages of the development of the neonatal gut virome, but as time goes on, Malgrandaviricetes phages, which are obligatorily lytic, become more prevalent[60,62,98]. This means that a reduced lysogenic and greater lytic capacity of the intestinal virome occur toward adulthood[63].

In conclusion, lysogenic and lytic phages are in a dynamic equilibrium in a healthy adult gut, and some factors can disrupt this equilibrium and encourage the induction of prophages.

PATHOPHYSIOLOGY OF IBD WITH THE ROLE OF THE GUT VIROME IN IBD

Research conducted over the past 30 years on human intestinal tissue and in vivo mouse models has revealed that intestinal homeostasis, which governs the host-microbiome interaction, is largely dependent on the integrity of the epithelial barrier, host defense mechanisms, immunological modulation, and tissue repair. Any disruption of these pathways or the cytokine networks that regulate them can result in IBD[5,99,100]. IBD is a multifactorial disease. The pathophysiology may be initiated by dysbiosis in the gut ecosystem as a result of some environmental or genetic factors[101]. Subsequently, dysbiosis triggers several inflammatory pathways[33,102,103] (Figure 2)[104]. However, the exact association between dysbiosis and inflammation in IBD is yet to be understood. Whether dysbiosis is the cause of inflammation or the inflammation leads to dysbiosis, the final result is the coexistence of dysbiosis and inflammation and the progression of IBD[104].

Figure 2 Role of gut microbiome in health condition and inflammatory bowel disease progression. In a healthy state, a balanced microbiome helps maintain homeostasis and the production of beneficial metabolites that build tight junctions and keep gut epithelium barrier integrity. On the other hand, gut microbiome dysbiosis participates in damaging tight junctions as well as dysregulating the gut barrier. This enhances the interaction of pathogens with gut epithelium and increases their penetration to the gut lumen with subsequent stimulation to immunological response and immune cells and overproduction of proinflammatory cytokines and progression of inflammatory bowel disease. PBAs: Primary bile acids; SBAs: Secondary bile acid; SCFAs: Short-chain fatty acids; IBD: Inflammatory bowel disease.

The onset and progression of IBD are influenced by several critical risk factors including genetics[105,106], diet[107,108], smoking[109], medicines, stress, mental health, and others[110]. Up to 12% of cases indicate a family history of illness, making genetics the most significant known risk factor. Additionally, diet and dietary habits have significant effects on the initiation of IBD[107]. More precisely, a low-fiber diet is thought to switch the gut microbiome from digesting fiber-derived glycans to digesting mucus-derived glycans, destroying the mucous protective layer of the gut and enhancing pathogen penetration with subsequent activation of inflammatory cascades[111]. These inflammatory responses are characterized by gut microbiome dysbiosis, including virome dysbiosis. Gut virome dysbiosis includes the reduction of Microviridae and crAss-like phages as well as the propagation of Caudovirales and perhaps other phages with lysogenic potential[31,112,113]. Regarding eukaryotic viruses, it has been revealed that patients with IBD have higher prevalence rates of particular viral families (e.g., Herpesviridae and Anelloviridae) than normal controls[31,112,114,115].

IMPLEMENTATION OF PHAGE IN IBD

After reviewing the literature, we found that gut phages may influence IBD pathogenesis by three mechanisms: (1) Change of gut phage community; (2) Modulation of gut microbial population; and (3) Modification of the local immune response.

Change of gut phage community

Regarding the alteration of the gut phage community, the majority of investigations depend on metagenomic sequencing of stool samples and intestinal biopsies. Variations among normal people and patients with IBD or experimental models have been discovered (Table 1).

Table 1 Overview of the alteration in the gut phage ecosystem in patients with inflammatory bowel disease and animal models.

Disease	No. of patients included in the study	Sample type	Interpretation of result	Ref.
CD	19	Biopsies	CD patients had considerably more VLPs than normal controls	[116]
CD	6	Ileal biopsies, colonic biopsies, gut wash samples	A significant excess of phages in biopsies and gut washes, Bacteroides phages (B10-8 and B124-14) were most predominant, and the composition of Mycobacterium phage differed between CD patients and controls in ileum tissue samples	[117]
CD	20	Stool samples, biopsies	Phage counts in stools were three times greater than in biopsies, CD patients had higher levels of Alteromonadales and Clostridiales phages	[114]
IBD	10	Colonic biopsies	Phages make up the bulk of the DNA viruses within the virome, about 50% of the phages were connected to the bacterial strains found in the colon specimens	[118]
UC and CD	(42 for UC) and (18 for CD)	Stool samples	Patients with IBD had a considerable increase in Caudovirales phages, and virome community in UC and CD patients were disease and cohort-specific	[31]
UC and CD	(5 pt. for UC) and (7 pt. for CD)	Stool samples	Caudovirales phage proportions in patients with IBD and normal controls were greater than Microviridae phage proportions. However, the Caudovirales phages were more prominent in CD than UC but not in controls. On the other hand, control persons had a larger diversity of Microviridae phages than CD patients, but not UC patients	[119]
UC	97	Rectal mucosa	Caudovirales phages were more abundant in UC cases compared to normal controls, but with lower richness, diversity, and balance, and UC patients’ mucosa had much more Enterobacteria and Escherichia phages than healthy controls	[113]
UC and CD	(42 pt. for UC) and (27 pt. for CD)	Stool samples	A stable virulent core virome is associated with a healthy gut and switched from a lysogenic to lytic cycle in temperate phages may be related to CD	[112]
CD	5	proximal and distal colonic wash samples	Considerable interpatient diversity and little, but significant, intrapatient variations between various regions	[120]
(VEO) IB	45	Stool samples	No detectable difference in the overall number of VLPs among VEO-IBD patients and normal controls, but the Caudovirales vs Microviridae ratio is larger in the VEO-IBD patients than in the controls	[121]
UC and CD	(38 pt. for UC) and (65 pt. for CD)	Stool samples	The prevalence of phages varied among patients with IBD and normal controls as well as the components of the temperate phage population were extremely distinctive to each individual. Moreover, compared to normal controls, active UC patients had a higher prevalence of temperate phages infecting Bacteroides thetaiotaomicron and Bacteroides uniformis	[122]
IBD	455	Stool samples	crAss-like phageome of the human gut has remained largely stable for 4 yr and individuals with IBD had lower levels of gut crAss-like phages	[65]
CD	19	Stool samples	CD patients had a considerably higher prevalence of crAss-like phages as well as no difference in the richness and evenness of the gut virome among CD patients and controls, but there was a substantial difference in the virome’s overall structure	[123]
Colitis	3 from C57BL/6 mice	Stool samples	The intestinal phage populations were altered and shifted to dysbiosis in the mice model, and a decrease in the variety of the phage community, such as Clostridiales phages during colitis	[124]

CD: Crohn’s disease; IBD: Inflammatory bowel disease; UC: Ulcerative colitis; VEO: Very early onset; VLPs: Virus-like particles.

In 2008, Lepage et al[116] published the first study connecting phages to IBD. They used epifluorescence and electron microscopy to compare populations of VLP in biopsies from patients with CD and healthy controls. They discovered that patients with CD had considerably more VLPs compared to healthy people[116]. This opened the door for further research to demonstrate the gut virome in different IBD subtypes and shed light on the role of the virome in the progression of IBD. Wagner et al[117] conducted a study on pediatric patients with CD to compare the alteration in phage population in GI biopsies from different sites and gut wash between patients and control individuals. They collected tissue biopsies from the ileum and colon as well as gut wash and analyzed them through metagenomic analysis. They found a significant excess of phages in biopsies and gut washes of pediatric CD patients compared to healthy controls. Furthermore, they discovered that the Bacteroides phages (B10-8 and B124-14) were the most predominant, and the composition of Mycobacterium phage differed between CD patients and controls in ileum tissue samples[117]. Further metagenomics examination of colonic specimens revealed that about 50% of phages were connected to the bacterial strains found in the colon specimens[118]. Subsequently in 2015, Pérez-Brocal et al[114] demonstrated variations in the gut bacteriome and virome communities in various types of specimens from adult patients with CD at various stages. They discovered that the phage counts in stools were three times greater than in biopsies and that the bacterial community rather than the viral populations are a better predictor of an individual’s illness status. Also, they discovered that individuals with CD had higher levels of phages infecting the bacterial orders Alteromonadales and Clostridiales, including Clostridium acetobutylicum spp. as well as Retroviridae family[114]. In the same year, Norman et al[31] established a metagenomic analysis to demonstrate the differences in gut phage populations in stool samples among UC and CD patients vs healthy controls. They showed that, compared to healthy groups, patients with IBD had a considerable increase in Caudovirales phages. Additionally, the CD and UC patients’ gut phage community was disease and cohort-specific[31]. Later, in 2019, Fernandes et al[119] examined the virome of fecal samples in children with CD, UC, and healthy controls of the same age. The result showed that Caudovirales phage proportions in both patients with IBD and normal controls were greater than Microviridae phage proportions. However, the Caudovirales phages were more prominent in CD than UC but not in controls. On the other hand, the control group showed a larger diversity of Microviridae phages than patients with CD, but not those with UC[119]. Moreover, another study by Zuo et al[113] identified the virome communities of the mucosa of patients with UC. According to their investigation, Caudovirales phages were more abundant in UC cases compared to normal controls but had lower richness, diversity, and balance. They also discovered that the mucosa of patients with UC had much more Enterobacteria and Escherichia phages than healthy controls[113]. Interestingly, Clooney et al[112] employed a whole-virome sequencing technique to re-analyze previously published data and provide extensive insights into the activity of the gut virome and its possible involvement in IBD[112]. They found that a stable virulent core virome is associated with a healthy gut, and switching from a lysogenic to lytic cycle in temperate phages may be related to CD[112]. A new virome sequencing analysis using pediatric CD patients’ proximal and distal colonic wash samples revealed considerable inter-patient diversity and little but significant intra-patient variations among different regions[120]. In another study, Liang et al[121] evaluated the dynamics of the virome in stool samples collected from children classified with very early onset (VEO) IBD, defined as IBD with onset prior to the child’s sixth birthday. They found that there is no detectable difference in the overall number of VLPs among VEO-IBD patients and normal controls but that the Caudovirales vs Microviridae ratio is larger in VEO-IBD patients than in the controls[121]. By utilizing whole-metagenome shotgun sequencing data, Nishiyama et al[122] showed the ecological composition of the temperate phage population in the human gut. They discovered that the prevalence of phages varied among patients with IBD and normal controls, and the components of the temperate phage population were extremely distinctive to each individual. Moreover, compared to normal controls, patients with active UC had a higher prevalence of temperate phages infecting B. thetaiotaomicron and B. uniformis[122]. In the most recent study, Gulyaeva et al[65] performed metagenomic sequencing on feces specimens collected from 1950 individuals, to investigate the vital function and diversity of crAss-like phages in clinical cohorts and human populations. They found that the crAss-like phageome of the human gut remained stable for 4 years and that individuals with IBD had lower levels of gut crAss-like phages[65]. Furthermore, Imai et al[123] analyzed stool samples collected from Japanese patients with CD using shotgun metagenomic sequencing. In contrast, they found that patients with CD had a considerably higher prevalence of crAss-like phages as well as no difference in the richness and evenness of the gut virome among CD patients and controls, but there was a substantial difference in the overall structure of the virome[123].

Animal studies additionally represent an essential tool for investigating the functions of intestinal phages in the pathophysiology of IBD. Duerkop et al[124] reported that in an animal model of colitis, the intestinal phage populations were altered and shifted to dysbiosis[124]. Also, they noticed a decrease in the variety of the phage community, such as Clostridiales phages, during colitis.

In summary, recent studies employed metagenomic sequencing and bioinformatic analysis to describe fecal and mucosal phage ecosystems. Most studies found that Caudovirales phages were more frequent and less diverse in patients with CD and UC than normal controls. However, different results were found in a recent study that showed no substantial variation in the number of intestinal phages between patients with IBD and normal controls[121]. Additionally, the abundance of crAss-like phage and Microviridae was decreased. Some investigations also revealed changes in specific phages, such as elevated levels of Alteromonadales and Clostridial phages in CD patients, elevated levels of Escherichia and Enterobacteria phages in UC mucosa, and decreased levels of Clostridial phages during colitis.

Modulation of the gut microbial population through bacteriophages

Virulent phages, which have the ability to lyse the bacterial host cell, are frequently identified in the GIT of patients with IBD[112]. It has been demonstrated that the invasion of phage to its bacterial host leads to modification and alteration in bacterial community with subsequent change in the abundance of certain spp.[125]. Researchers have shown that individuals with CD and UC exhibit dysbiosis in their gut microbiome, which is characterized by reduced diversity, increased hazardous proteobacteria (e.g., E. coli and Fusobacteria), and decreased beneficial Firmicutes (e.g., Clostridium clusters IV and XIVa, Faecalibacterium prausnitzii, and Rumininococci)[126-128]. Additionally, Nishiyama et al[122] found a significant increase in the prevalence of phages infecting B. thetaiotaomicron and B. uniformis, as well as a reduction in the population of their bacterial host. Considering the aforementioned studies, we conclude that there is a link between the abundance of phage and its bacterial host population. Germ-free (GF) animals are the ideal model for investigations related to the gut microbiome since they don’t have any microbial colonization in their guts[129,130]. In GF murine models, a recent investigation revealed that phage invasion directly affects vulnerable bacteria, with subsequent cascade affecting other bacterial spp. and gut metabolome[131,132].

Other than virulent phages, temperate phages can affect the viability and diversity of gut bacteriome[133]. For instance, temperate phages significantly increase the genetic variation of bacteria via horizontal gene transfer and increase the mutation rates[46,133-135]. Moreover, the induction of latent prophage through environmental stressors may activate its lytic cycle and decrease the number of bacterial hosts. In a metagenomic study conducted by Cornuault et al[136], they found a greater abundance or quantity of phages infecting F. prausnitzii in feces samples from patients with IBD in comparison to normal controls[136]. While less F. prausnitzii abundance has been demonstrated in patients with IBD, they concluded that phages might exacerbate this reduction of F. prausnitzii[136].

In summary, the relevant information is still inadequate, and theories about how phages directly or indirectly affect bacterial populations are still out of reach. So further investigations are required to fully understand the complex phage-bacteria interactions in IBD.

Modification of the local immune response through bacteriophages

After prophage induction, a process known as phage-mediated lysis describes the positive feedback inflammatory response between phage induction and gut inflammation-begins[133,137]. In this situation, intestinal inflammation induces the production of stressors by enterocytes, such as reactive oxygen species and reactive nitrogen species, which cause the host bacteria to respond to stress (SOS response)[138]. Increased bacterial host cell lysis is followed by a rise in pathogen-associated molecular patterns (PAMPs) (such as lipopolysaccharides and bacterial DNA) that activate more enterocyte receptors[133,137]. This leads to activation of a positive feedback inflammatory response and dysregulation of the immune system (Figure 3). Additionally, in the presence of a thin lining mucous layer and disrupted tight junction, large amounts of PAMPs can penetrate gut epithelium and activate Toll-like receptors (TLRs) and other immune cells located on gut epithelium[31,139-141]. As a result, inflammatory pathways are activated, resulting in increased generation of pro-inflammatory cytokines and decreased generation of anti-inflammatory cytokines[142-145] (Figure 4). A recent in vivo investigation conducted in GF mice showed upregulation in both innate and acquired immunity after administration of a phage cocktail. The results demonstrated a considerable increase in overall CD8+ and CD4+ T cells, in addition to interferon gamma (IFN-γ)-producing T helper 1 cells[146]. Furthermore, an in vitro study indicated that the detection of phage DNA by dendritic cells triggers the generation of IFN-γ through a TLR9-dependent mechanism[147].

Figure 3 Activation of positive feedback inflammatory response through induction of latent prophage and lysis of bacterial host cell. (1) Intestinal inflammation induces gut mucosa to generate stressors (like reactive oxygen species [ROS] and reactive nitrogen species [RNS]); (2) Production of stressors aggregates the stressor response (SOS) in the bacterial host cell (SOS reaction) and redox imbalance; (3) This imbalance leads to damage of bacterial DNA and induction of latent phage; (4) Switch to lytic cycle with subsequent bacterial cell rupture; (5) Accumulation of pathogen-associated molecular patterns (PAMPs) as lipopolysaccharide (LPS) and DNA, that results from bacterial lysis; (6) PAMPs activate receptors in the gut mucosa and stimulate the production of more stressors.

Figure 4 Modification of local immune response through bacteriophage. An inflamed gut is characterized by microbiome imbalance including virome. An imbalanced virome is distinguished by an expansion of Caudovirales phages and lysogenic lifecycle as well as a decline in the relative abundance of the Microviridae family and crAss-like phage. Additionally, in the presence of a thin mucus lining or broken tight junction, microbial antigens (like viral antigens) can penetrate the intestinal epithelium, activate Toll-like receptor (TLR), upregulate pro-inflammatory cytokines production, and dysregulate anti-inflammatory cytokines production. IL: Interleukin; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TGF-β: Transforming growth factor beta; TNF-α: Tumor necrosis factor alpha.

On the other hand, phages may play a significant role in protecting the intestinal barrier against bacteria and provide non-host-derived immunity[148]. Phages can stick to the mucus layer of the gut and reduce the colonization of pathogens. This adhesion was controlled by interactions between immunoglobulin-like domains, which are present on phage capsid proteins, and glycan residues, which are found in mucin glycoprotein[148].

In summary, little information is currently known about gut phages’ impact on IBD through immunological modulation. Therefore, there is an urgent need for more research on how gut phages and the immune system interact in IBD.

IMPLEMENTATION OF EUKARYOTIC VIRUSES

Early in life, eukaryotic viruses begin to colonize the gut mucosa. These viruses are members of the Anelloviridae, Adenoviridae, Picornaviridae, Picobirnaviridae, Astroviridae, and Parvoviridae families, and they become more diverse with age[60]. Such viruses may cause pathological changes or may remain dormant in healthy persons for many years, exerting significant benefits[141,149-151]. Eukaryotic virome dysbiosis, such as phage dysbiosis, has been linked to IBD pathophysiology[152-154] as eukaryotic-targeting viruses incorporate their genetic element into the human genome and can affect the physiological condition of enterocytes[141,151,154]. Patients with UC had greater levels of the eukaryotic Pneumoviridae family than controls, according to a metagenomic study involving a large cohort of UC patients. However, control individuals had higher levels of the eukaryotic Anelloviridae family[113]. On the other hand, another investigation demonstrated that patients with UC and CD had greater levels of the Herpesviridae family than normal controls[118].

Epstein-Barr virus and cytomegalovirus are the most studied eukaryotic viruses that may cause intestinal inflammation[150]. However, their role in the pathophysiology of IBD has yet to be fully understood, as their reactivation may be brought on by immunosuppressive or stressful situations that are prominent in patients with IBD, making them more likely to serve as bystanders than true disease-causing factors.

Norovirus infection was found to be a significant colitogenic factor, significantly dependent on the presence of gut microbiome, in the interleukin 10-deficient mouse model of spontaneous colitis[155]. Likewise, IBD-susceptibility gene Atg16L1^HM mouse models have shown that Norovirus infection leads to the progression of intestinal inflammation[156]. Hence, it appears that the development of colitis is accelerated by a synergistic interaction between genetic makeup and Norovirus infection as a trigger of intestinal inflammation.

These aforementioned investigations specifically focused on enterotropic viruses, which are often restricted to the GI system. On the other hand, a recent investigation using metatranscriptomic processes revealed that some eukaryotic RNA viruses with a physiological hepatic tropism were found in the gut mucosa of patients with IBD[151]. In a recent study, Massimino et al[157] discovered how the hepatitis B virus X protein, a virome-associated protein encoded by the Orthohepadnavirus genus, contributes to the pathogenesis of UC.

In summary, previous findings have indicated a link between these eukaryotic virus families and IBD pathogenesis, and more research is urgently required to demonstrate their roles in producing chronic intestinal inflammation.

CHALLENGES AND LIMITATIONS AND FUTURE TRENDS IN VIROME DESCRIPTION

Analysis of the gut virome has been neglected due to the difficulty in producing an in vitro composite culture environment that would support the simultaneous development of different microorganisms[158]. It is still challenging to recognize and categorize viral DNA in microbiological samples. Viral spp. are difficult to classify into closely related spp. due to their extraordinarily high diversity, low gene content, and quick acquisition of mutations[159]. Moreover, it is impracticable to sequence viral DNA using a targeted amplicon-like strategy as there are non-common genes that might be utilized as markers for identification[160]. Unfortunately, it can be challenging to cultivate viruses as well. Because viruses are parasitic and depend on host cells for energy and multiplication, these hosts must also be discovered and cultivated. Additionally, many GIT microorganisms cannot be cultivated, making it problematic to culture their related viruses[115].

Metagenomic analysis of the virome may appear to be a difficult process, but various approaches might help with this issue. For instance, before sequencing, viral particles from a microbiome sample can be separated and purified using size selection by centrifugation, filtration (using 0.2-μm to 0.45-μm filters), and particle precipitation using polyethylene glycol[161]. Despite these helping approaches, the metagenomic technique possesses great limitations, such as dependence of the result on the degree of fragmentation of viral genome as well as analysis of DNA sequence only and ignoring RNA[162]. Although revolution in -omics approaches, such as metagenomics, metataxonomics, and metatranscriptomics, share common limitations[152]. (1) They must depend on reliable databases that provide information on the various genomes and their explanation; otherwise, the analysis will be challenging and may miss some crucial information[158]; (2) Studies must be carried out on purified RNA and DNA specimens, and occasionally, the yields are insufficient to cover poorly represented communities. In addition, residual host DNA and RNA molecules may persist in the sample after purification, leading to false outcomes[158]; (3) The sequencing depth must be very high to obtain accurate outcomes, particularly for metatranscriptomics, which may be expensive[163]; and (4) The present statistical method is constrained by the concept that the predictor variables are independent of one another and do not consider the complexity of the biological ecosystem[158].

The use of more modern computational techniques, such as VIP and VirFinder, which offer workflows to map, filter, and detect viruses from metagenomic sequences[164,165], as well as METAVIR, an online library for identifying viral genes from metagenomic data[166], can make the understanding of human virome easier. Future gut virome investigations should include approaches like tracking viral protein exacerbation[167] or host DNA reduction, as well as high-throughput sequencing of the microbiome in patient samples[159]

COMMUNITY TYPING AS A NEW APPROACH TO DESCRIBING GUT VIROME

The idea of community typing, also known as “enterotyping” was first developed in bacterial studies to simplify the complexity and categorize the diversity of the gut microbiome[168]. The Dirichlet Multinomial Mixture approach is used for community typing, which is based on probability-based modeling and takes into account particular microbiome data properties such as relative scarcity[169]. With this technique, samples from the same community (those with comparable bacterial abundance patterns) are classified into microbial configurations without stating any assumption about the underlying separate character of the strata[169]. These techniques reliably divided the gut microbiome into the 4 enterotypes Ruminococcus, Bacteroides 1, Bacteroides 2 (Bact2), and Prevotella and found several connections to the abovementioned risk factors, including diet and illnesses[170-173].

Due to the massive insights and enterotyping helping in the understanding of the microbiome of the human gut[171,174,175], it has been hypothesized that viral community typing might be a valuable method to pursue knowledge of the gut virome as well. Regarding this idea, Song et al[176] analyzed many published data and found that most people could be sorted into two viral community types depending on their gut virome; however, they were unable to identify their taxonomical makeup because of the elevated incidence of viral dark matter. Additionally, it has been demonstrated that the gut virome of patients with IBD existed in two viral community types: community type CrM, which includes either crAss-like phage and Malgrandaviricetes, or community type CA, which includes Caudovirales phages[177]. Moreover, the community type CA was linked to reduced virome diversity, dysbiosis in the Bact2-enterotype, and active illness, demonstrating the clinical potential of these community types[177,178].

In summary, viral community typing has great promise as a future strategy for discovering alterations in viral composition in health and illness.

CONCLUSION

IBD is a multifactorial chronic inflammatory disease involved in GIT. IBD is divided into two subtypes: UC and CD. The exact etiopathogenesis is still unknown, but the researchers are doing their best to remove this ambiguity. Most researchers focus on the role of gut bacteriome in the etiogenesis of IBD and ignore other microorganism communities as viruses. In 2015, Norman and partners conducted the first investigation revealing the role of gut virome dysbiosis in IBD. Since then, many studies have been conducted with evolution in novel approaches to describe virome dysbiosis and its role in disease and health conditions. In this review, we give an overview of gut virome and its role in normal health conditions. Further, we give deep insights into the implementation of gut virome in IBD pathogenesis regarding the role of both bacteriophages and eukaryotic viruses. Finally, we describe the challenges and limitations in describing gut virome and how the appearance of novel approaches as community typing opens the door for further research to understand the role of gut virome in disease states, including IBD.