What do we already know about this subject?

The intestinal microbiota of neonates is characterised by lower bacterial diversity and a higher proportion of pathogenic bacteria. Moreover, their intestinal immune system is immature. These two factors modify the intestinal epithelial barrier and enhance the production of pro-inflammatory mediators.

Necrotising enterocolitis (NEC) is an ischemic and inflammatory disorder of the gastro- intestinal tract which affects premature neonates. The physiopathology of NEC remains poorly understood but intestinal dysbiosis is present, and also an inflammatory process. The use of antibiotics and antacids promote dysbiosis and also increases the risk of onset of NEC.

Neonates can also suffer from other ischaemic and inflammatory disorders, such as small bowel volvulus and localised gastro- intestinal perforations.

What are the main insights from this study?

This single centre pilot study profiled the composition of the FM and MM and also the mononuclear cells of the lamina propria and the pro-inflammatory cytokines of two groups of neonates: full-term or premature. Seven infants had extensive intestinal ischaemia (EII) (5 NEC, 1 small bowel volvulus and 1 total colonic ischaemia) and 7 infants had localised intestinal ischaemia (LII) (4 isolated perforations and 3 cases of intestinal atresia). The FM of 9 full-term infants was used as control. The MM of neonates with EII, compared with those with LII, contained: more Proteobacteria (p = 0.049) and fewer Bacteroidetes (p = 0.007) and Verrucomicrobia (p = 0.01) (Figure 1); fewer Bacteroides, Lachnospiracee, Ruminococcaceae and Akkermansia muciniphila (p < 0.05).

The FM was less diverse (Shannon index) in EII than in LII (p = 0.01). The relative abundance for the MM was similar between EII and LII for Proteobacteria and Firmicutes (p < 0.05). Similarly, a bacterial distribution with more Enterobacteriaceae in EII and more Ruminococcaceae, Bacteroides, Lachnospiracee and Staphylococcaceae in cases of LII.

The EII group exhibited increased numbers of lymphocytes B, T and NK (Natural Killer), in particular T CD3+ lymphocytes, TH17 (Figure 2A) and reduced numbers of T lymphocyte regulators (Tregs), with increased numbers of cells expressing TNFa (Figure 2B) and INFg (Figure 2C).

What are the consequences in practice?

This pilot study confirms that lack of bacterial diversity and the predominance of Enterobacteriaceae are risk factors for NEC, as is a reduction in numbers of Akkermansia muciniphila. Correction of this dysbiosis could modify the TH17/Tregs imbalance and reduce the production of mediators of inflammation (TNFa and INFg).

What do we already know about this subject?

Cholesterol is a key biological molecule that functions as a structural component of all animal cell membranes and is a pre-cursor of steroid hormones, vitamin D, and bile acids. Two main sources of cholesterol are thought to influence concentrations of this metabolite in serum: endogenous cholesterol synthesised in the liver and exogenous cholesterol derived from dietary components of animal origin (Figure 1). The cholesterol synthesised in hepatocytes is transported to the gallbladder and is then secreted into the small intestine along with other bile salts. In the intestine, biliary cholesterol (~1–2 g/day) mixes with dietary cholesterol (~0.2–0.4 g/day in the average American diet), and both sources are eventually transported into enterocytes for packaging into lipoprotein particles and secretion into the plasma. Hypercholesterolaemia is a risk factor for cardiovascular disease (CVD), which is the cause of one-fourth of all deaths in industrialised countries.

Reducing cholesterol transport in the intestine is a clinically validated strategy for lowering serum cholesterol levels. A range of gut microbes metabolise and modify dietary and host-derived molecules in the small intestine. Because both sources of cholesterol pass through this environment, the gut microbiota may influence serum cholesterol levels. Indeed, microbiota transfer from human donors with elevated serum cholesterol levels can impart this hypercholesterolaemia phenotype to mice. [2, 3] Other studies have reported that administering specific bacterial species can have cholesterol-lowering effects. [4] However, the precise mechanisms underlying these observations are currently unknown. The gut microbiota may exert cholesterol-lowering effects by metabolising intestinal cholesterol to coprostanol (Figure 1), which would reduce the amount of cholesterol absorbed from the intestine.

This microbiota-dependent transformation has been known to occur in humans since the early 1900s. Several coprostanol-generating gut bacteria with similar physical and biochemical characteristics have been reported from a variety of different sources including rats, baboons, and humans. However, most of these strains are not currently available and were never sequenced. Early work showed that coprostanol formation by this group of gut bacteria proceeds through an indirect reduction pathway involving the initial oxidation of cholesterol (1) to cholestenone (2), followed by reduction of the D4,5 double bond to form coprostanone (3), and subsequent re-reduction of ketone to generate coprostanol (4) (Figure 1). The bacterial enzymes responsible for this metabolism were never identified. More recently, other reports have implicated additional phylogenetically diverse gut bacteria in coprostanol formation. [5] While efforts to elucidate how gut microbial metabolism of cholesterol affects human serum cholesterol levels span over 100 years, mechanistic support for this connection has remained elusive due to a limited understanding of the gut microbes, genes, and enzymes responsible for coprostanol formation.

What are the main insights from this study?

The authors used a multi-disciplinary strategy to discover gut bacterial enzymes. This strategy, based on correlations between metagenomics and metabolomics data from existing human cohorts, identified and characterised an extensive family of cholesterol dehydrogenase enzymes from a clade of uncultured intestinal bacteria implicated in the metabolism of cholesterol to coprostanol. Firstly, the enzyme responsible for the first step in cholesterol transformation, called ismA, was identified in Eubacterium coprostanoligenes, a bacteria already known for this function. Analysis of sequencing data from human cohorts then identified homologous enzymes in a group of uncultured anaerobic bacteria.

The presence of these ismA genes in the microbiome was associated with the presence of coprostanol in stools and lower faecal cholesterol levels. Finally, to demonstrate the potential for these cholesterol- metabolising bacteria to influence human health, the authors showed that presence of ismA genes in human metagenomes is associated with a decrease in total cholesterol concentrations in serum that is on par with the effects observed from variants in human genes involved in lipid homoeostasis

What are the consequences in practice?

Overall, these findings confirm the role of gut-bacterial metabolism in modulating host cholesterol levels in the intestine and also, more importantly, on a systemic level. This work paves the way for the use of the gut microbiota as a predictive biomarker of high cholesterol and establishes the foundations for microbiota-targeted therapeutic interventions.

THE ONGOING THREAT OF RESPIRATORY VIRAL INFECTIONS

Viral respiratory tract infections are still a major concern worldwide as they lead to considerable socio-economic and health issues. Despite vaccine campaigns and antiviral medications, influenza - the commonly called “flu” - remains the most impacting in terms of infected individuals (5-10% of the global population) and deaths (> 600,000 per year). Along with annual epidemics, influenza is also responsible for pandemics every 10-20 years, the most famous being the 1918-1919 Spanish flu (50 million deaths) and the most recent, the 2009 H1N1 influenza pandemic (400,000 excess deaths) [1]. Pandemics are not restricted to influenza, as exemplified by the COVID-19 [2]. Of great concern is the increasing frequency of pandemics occurring over the last few decades, a phenomenon explained in part by climate changes and human practises, in general.

THE GUT MICROBIOTA IN HEALTH AND DISEASES

The gut microbiota has a crucial role in the maintenance of human health and is critical in the control of (respiratory) infections [3, 4]. Many factors can alter the diversity and composition of the gut microbiota, leading to dysbiosis. Among them, disease situations, such as infections and chronic inflammatory or metabolic disorders, can trigger profound alteration of the gut microbiota’s composition and function. Changes in intestinal bacterial communities can influence disease outcomes even in distant organs (including the lungs) [3, 4], as demonstrated by transfer experiments with dysbiotic microbiota. Below, we summarize the effects of acute viral respiratory infections on the gut microbiota.

VIRAL RESPIRATORY TRACT INFECTIONS LEAD TO GUT DYSBIOSIS

Clinical evidences suggest a gut microbiota dysbiosis during influenza infection. The relative abundance of Actinobacteria, Erysipelotrichea, Clostridia and beneficial butyrate producers (Lachnospiraceae and Ruminococcaceae families) is decreased in patients H1N1. On the other hand, opportunistic pathogens such as Escherichia- Shigella and Prevotella develop [5]. In experimental (mouse) models, transient gut dysbiosis also occurs with a peak at 5-7 days post-infection [6-9]. Changes occurred at taxonomic levels with no change in alpha diversity. Infection blunts the growth of health-promoting bacteria such as Lactobacilli, Bifidobacteria, and segmented filamentous bacteria. Many species capable of fermenting dietary fibres into short-chain fatty acids (SCFAs) are affected. In line, the production of SCFAs dramatically drops during influenza infection [9]. The reduced levels of beneficial commensals associate with the overgrowth of deleterious bacteria including Gammaproteobacteria (Escherichia coli) and mucus-degrading bacteria such as Verrucomicrobia (Akkermansia genus) and Ruminococcus. SARS-CoV-2 infection also triggers gut microbiota alterations in patients, including lower abundance of butyrate producers such as several genera from the Ruminococcaceae and Lachnospiraceae (Roseburia) families [5, 10]. On the other hand, a significantly higher relative abundance of opportunistic bacterial pathogens including Streptococcus (class Bacilli), Rothia and Actinomyces was observed. Of note, overgrowth of opportunistic fungal pathogens (Aspergillus and Candida spps) was also described in COVID-19 patients [11]. Collectively, viral respiratory infections lead to depletion of beneficial commensals and enrichment of opportunistic harmful pathogens. Putative changes in the gut microbiota’s structure, composition and functional activity might be biomarkers of disease severity.

MECHANISMS LEADING TO GUT DYSBIOSIS

There are probably several causes of gut dysbiosis during viral respiratory infections; these may include the release of inflammatory cytokines and the reduced food intake (Figure 1). Infection induces substantial weight loss due to a loss of appetite. Pair-feeding experiments in mice clearly indicate that a rapid fall in food intake mimics the changes in the gut microbiota observed during influenza infection [8]. Recent evidence suggests a role for TNFa in inappetence-associated dysbiosis during viral respiratory infection [12]. Type I and II interferons, which are essential for the host antiviral response, also play a part in gut dysbiosis [5,6].

Hypoxia (a feature of acute viral respiratory infection), alterations of the enteric nervous system and dysregulated local immune response are also likely to participate in gut dysbiosis [13] (Figure 1). In the case of COVID-19, along with these mechanisms, local viral replication is likely to play a role in gut dysbiosis. Angiotensin- converting enzyme II (ACE2), the receptor of SARS-CoV-2, is instrumental to maintain the gut’s microbial ecology. Considering the lack of available ACE2 during SARS-CoV-2 infection, one expects that this might influence the composition and functions of the gut microbiota [13].

CONSEQUENCES OF GUT DYSBIOSIS ON SECONDARY OUTCOMES

Gut dysbiosis during viral respiratory infection has local and distal consequences and might be an important contributor of disease severity and fatal outcomes (Figure 2). Patients experiencing viral respiratory infection can develop gastroenteritis- like symptoms such as abdominal pain, nausea, vomiting and diarrhoea. Alterations of the gut microbiota may explain these disorders. It is also likely that altered gut microbiota including the emergence of pathobionts and mucus degrading bacteria play a role in intestinal inflammation and disruption of the gut barrier integrity [6]. In turn, intestinal barrier leakage might increase endotoxin concentrations in the blood, ultimately triggering inflammation, cytokine overproduction and lung dysfunctions [14]. Acute viral respiratory infections can lead to secondary enteric infections and sepsis. Gut dysbiosis (and drop of SCFAs) might be important in this setting. Indeed, SCFAs play a key role in intestinal homeostasis, barrier integrity and control of enteric pathogens [15]. Along with local disorders, our recent data show that gut dysbiosis can remotely hamper host defense in the lungs [9] (Figure 2). In healthy conditions, the gut microbiota remotely arms the lungs against bacterial infection, in part by reinforcing the bactericidal activity of pulmonary macrophages [16].

During influenza, this axis is altered and opportunistic bacteria invade the lungs leading to bacterial superinfection, a major cause of death during influenza epidemics and pandemics [1]. We have shown that the reduced production of acetate (the major SCFAs) by the gut microbiota is partly responsible for this effect [9]. Collectively, dysbiosis might influence the gastrointestinal and pulmonary signs and symptoms (and overall mortality) of viral respiratory infections. Can we use the gut-lung pathway as a basis to better control the severity and mortality rate of viral respiratory infections?

INTERVENTIONAL PERSPECTIVES

The gut microbiota is vital in the lung’s defences against respiratory infection and interventional strategies that target intestinal commensals to preventively arm the lungs against viral pathogens and to protect the microbiota against the perturbations associated with viral infections are of major interest (Figure 3). This is particularly true in individuals with a general imbalance of the gut microbiota such as the elderly and individuals with co-morbidities, well known to be more susceptible to infections.

Approaches like (i) dietary ingredients intended to nourish our beneficial microorganisms (like prebiotic fibres) and (ii) indigenous bacteria (known as probiotics) to replenish our gut with missing beneficial microorganisms and to optimize their metabolic functions, might be relevant. These strategies, especially personalized (i.e. based on the analysis of the gut microbiota in the “at risk” population) might improve clinical outcomes and accelerate the recovery of patients that experience acute viral respiratory tract infections. Probiotics, including some Bifidobacteria and Lactobacillus spp, can decrease the severity of influenza infection, through still undetermined mechanisms [17]. Of highly topical concern, a recent study showed that oral bacteriotherapy in addition to the standard drug therapy showed promising clues in the management of COVID-19 patients [18].

The trillions of microorganisms that inhabit the human gut possess a greatly expanded set of genes compared to the host genome. Many of these genes encode protein-based catalysts, or enzymes, that enable gut microbes to perform a wide range of chemical reactions, expanding the chemistry associated with the human body. A hallmark of gut microbial metabolism is its variability; just as the composition of the microbiota differs between individuals, so too can the metabolic capabilities of this community. As we continue to identify associations between the gut microbiota and health and disease outcomes, it is becoming increasingly important to characterize microbial metabolic transformations at a molecular level.

One prominent activity associated with the gut microbiota is the ability to chemically modify the structures of small molecule drugs. [1] Orally administered drugs encounter gut microbes either prior to absorption in the small intestine or in the large intestine if they are poorly orally bioavailable. Orally administered or injected drugs, or drug metabolites, also reach the microbiota if they undergo biliary excretion into the intestine. Because a drug’s pharmacological activity directly arises from its chemical structure, microbial metabolism can have a large effect on drug action.

Effects of gut microbial drug metabolism

Gut microbial metabolism has various downstream consequences for drug action and efficacy (Figure 1). As the early examples of azo drugs illustrate, microbial metabolism of ‘prodrugs’ (inactive precursors) may be required to generate the active pharmacological agent. This knowledge has inspired the rational design of additional strategies for targeted drug release in the large intestine that rely on microbial metabolic activities Metabolism by the gut microbiota can also have negative effects on drug activity by disrupting interactions with intended host targets. One example is the natural product- based cardiac medication digoxin. In 5-10% of patients, the gut microbiota reduces the a, b-unsaturated lactone ring of digoxin to give dihydrodigoxin. This subtle modification, which is performed by the gut bacterium Eggerthella lenta, greatly reduces the binding affinity for digoxin’s target Na+/K+ ATPase, resulting in a loss of efficacy. [2] Another prominent example is the front-line Parkinson’s disease treatment L-dopa. Metabolism of L-dopa to dopamine by host enzymes in the brain is critical for alleviation of symptoms. Gut microbial metabolism of L-dopa also produces dopamine. [3,4] Because dopamine generated in the periphery cannot cross the blood brain barrier, this activity may reduce the amount of L-dopa that reaches the brain.

Finally, in addition to reducing activity, the chemical modifications installed by gut microbes can produce unwanted toxicity. For example, gut microbial metabolism was implicated in the lethality of co-administering the antiviral medication sorivudine with fluoropyrimidine chemotherapeutics. This outcome was traced to gut microbial metabolism of sorivudine to bromovinyluracil. This metabolite inhibits a key host enzyme involved in detoxifying 5-fluorouracil, increasing its concentration to lethal levels.

An important characteristic of gut microbial drug metabolism is its variability across patients. This phenomenon has its origins in the variability of the gut microbiota. Though some metabolic activities are found in many organisms, others are carried out by a small, low abundant subset of the gut community. Metabolism can vary between individual strains of the same species, as even closely related bacteria can have large differences in their genomes. It is therefore perhaps unsurprising that community composition is often a poor predictor of metabolism, and metabolism of individual drugs can be extensive in some individuals and absent in others. This variation likely has important but incompletely understood consequences for patients taking a range of small molecule drugs.

Understanding drug metabolism at a molecular level

In order to fully understand gut microbial drug metabolism, it is necessary to link individual activities with microbes, genes, and enzymes. Identifying specific drug-metabolizing microbes is typically needed to enable downstream mechanistic studies. This may be accomplished through screening available gut microbial isolates or isolating metabolizing organisms directly from complex gut microbiota samples. An important next step is connecting transformations of interest to genes and enzymes. This is crucial for studying metabolism in complex gut communities, as the genes encoding metabolic enzymes allow detection and prediction of individual activities in microbial genomes and microbiome sequencing data. Linking drug metabolism to microbial genes can be accomplished in multiple ways, including rationally searching genomes for enzymes with the requisite catalytic capabilities, using RNA-Seq to identify genes that are specifically upregulated in response to a drug, and using comparative genomics to associate genes with metabolic capabilities.

Identifying new metabolic activities

Until 2019, approximately 60 examples of gut microbial drug metabolism were reported. Two recent studies leveraged approaches from high-throughput screening and experimentation to perform large scale surveys of gut microbial drug metabolism, greatly expanding the scope of known transformations. Goodman and co-workers screened 76 human gut bacterial isolates for their ability to metabolize 271 small molecule drugs and found that two thirds of the drugs were depleted by at least one organism. [8] The Donia group performed an analogous screen of 575 drugs using a patient gut microbiome sample ex vivo and uncovered 45 new transformations. [6] These efforts suggest the scope of drugs subject to metabolism may be larger than previously known; however, the vast majority of these newly reported activities have not yet been confirmed in vivo, so their relevance for patients is unknown.

Manipulating gut microbial drug metabolism

Once the gut microbiota has been found to transform a small molecule drug, a logical next step is to ask how this activity may be controlled, both to assess the consequences of metabolism for drug action and to improve patient therapy should metabolism prove detrimental. Various methods have been employed to achieve this goal. Using gnotobiotic animal models (germfree animals colonized in a controlled manner with a defined microbiota), one can compare communities containing either drug metabolizing gut strains or deletion mutants missing specific activities. The utility of this approach was nicely illustrated by the Goodman lab’s studies of brivudine. [5]

However, genetic manipulation is challenging in native, complex microbial communities, prompting evaluation of alternative approaches. One potential strategy is to leverage knowledge of gut bacterial physiology to guide manipulation of the gut environment via dietary interventions. For example, digoxin Turnbaugh and co-workers noted that the presence of L-arginine downregulates drug metabolism by E. lenta. [2] They then showed that administering protein-rich diets to gnotobiotic mice colonized with E. lenta reduced drug inactivation in vivo.

Another exciting strategy is to identify small molecules that inhibit the activity of gut microbial drug metabolizing enzymes, as pioneered by the Redinbo lab in their studies of irinotecan metabolism. Irinotecan is a prodrug that is metabolized by host cells to the active topoisomerase inhibitor SN- 38. SN-38 is metabolized by the host via glucuronidation, which produces an inactive conjugate (SN-38G). This metabolite is excreted into the intestine, where the glucuronide is removed by gut bacterial b-glucoronidase (GUS) enzymes. This reactivation causes dose-limiting gastrointestinal tract toxicity. The Redinbo group used highthroughput screening to identify selective inhibitors of gut bacterial GUS enzymes, and found they prevented the severe side effects caused by irinotecan in a mouse model. [9] Subsequent work revealed that these compounds increase the efficacy of irinotecan by limiting its toxicity. [10] Together, this work has provided exciting proof-of-concept for therapeutically targeting gut bacterial metabolism and has prompted additional inhibitor discovery efforts.

Future frontiers

The successful development of GUS inhibitors as therapeutic candidates highlights one way in which gaining a molecular understanding of gut microbial drug metabolism could benefit patients. Another area that could be transformed by this knowledge is precision medicine. With an understanding of how specific therapeutics are metabolized by gut microbes, physicians could one day use microbiome sequencing data or microbiota-based diagnostic assays in deciding whether and how to prescribe particular medications.

Our growing appreciation of gut microbial drug metabolism may also influence the drug discovery process itself. Due to past associations with toxicity and side effects, many functional groups known to be transformed by gut bacteria are typically avoided by medicinal chemists. One could imagine uncovering new, unanticipated transformations early in drug development by screening individual gut microbes or complex patient communities for metabolism ex vivo, similarly to how drug candidates are typically tested for metabolism by host enzymes. Differences in gut microbiota composition and functions between animal models and humans should be taken into account in preclinical and clinical studies. Finally, it may be advisable to incorporate microbiome sample collection and analysis for drug metabolism into clinical trials. Correlating metabolism with differences in toxicity or efficacy might help in interpreting the results of such trials and defining target patient populations.

GUT MICROBIOTA AND THE INTESTINAL BARRIER

The gut microbiota is an initial barrier protecting the intestinal mucosa from pathogens. This complex ecosystem inhabits the gastrointestinal tract where it remains stable and limits access to the intestinal niches and to the nutrients required for the multiplication of exogenous bacteria by the phenomenon called “colonisation resistance” [1] (Figure 1). The enterocytes, which provide a physical barrier between the intestinal lumen and the host, absorb water and nutrients and secrete antimicrobial peptides, AMPs (RegIIIg, b-defensins and cathelicidin) [2]. By the recognition of microbe-associated molecular patterns, (MAMPs) by specific receptors (including the Toll-Like-Receptors, TLR), these cells will be able to transduce the signal to cytokines and chemokines thus signalling infection and recruiting immune cells (Figure 2). Paneth cells also participate in colonisation resistance by secreting AMPs (lysosyme, a-defensins, RegIIIg) [2]. The goblet cells – mucus-secreting – and the M cells have gatekeeping action, transporting antigens, intact and captured at random in the intestinal lumen arising from commensal bacteria or pathogens or dietary antigens. These will then be prepared by the dendritic cells (DC) and presented to the adaptive immune system. This function is vital to intestinal tolerance and the induction of mucosal immune responses [2]: there therefore is a constant balance between pro- and anti- inflammatory responses (Figure 2). In particular, this was demonstrated in mice models of induced colitis and in TLR receptor-deficient mice: the absence of microbiota or recognition of this reduces the proliferation of intestinal epithelial cells or barrier repair [2]. Lastly, the mucus also provides protection by capturing AMPs, which act to prevent the pathogens from reaching the epithelium. In the model of Muc2-deficient mice (Muc2 is the gene coding for one of the proteins making up the mucus), an increase in the translocation of commensal bacteria is observed and these animals develop intestinal inflammatory diseases [3].

CROSSTALK BETWEEN THE GUT MICROBIOTA AND THE INNATE IMMUNE SYSTEM

Among the players of the innate immune system which participate in intestinal homeostasis, antigen-presenting cells (APC), such as the macrophages (Mj) and the DCs have a major role. The Mj and the DCs synthesise IL-10 and thus promote differentiation of Treg [4] and the maturation of the Th17 lymphocytes via the implication of commensal bacteria: the segmented filamentous bacteria (SFB). These have the particular ability to adhere to the intestinal epithelial cells causing active stimulation of the immune system [5] (Figure 3). A study shows that colonisation of mice by these SFB, induces the differentiation of Th17 thus resulting in protection from Citrobacter rodentium (the murine equivalent of EPEC and EHEC). It has been suggested that this protection is due to the capacity of the SFB to cause Th17 to stimulate the synthesis of IL-22, a cytokine known to stimulate the synthesis of AMPs [6]. To come back to the DCs, these, by extending their dendrites between the epithelial cells, are able to phagocyte the bacteria present in the intestinal lumen. These commensal bacteria are then transported to the mesenteric lymph nodes to induce the production of IgA secreted by the plasma cells [1]. The innate lymphoid cells (ILC) also play an important role in intestinal homoeostasis; this is related to their capacity to initiate and direct intestinal immune responses. More specifically, the type 3 ILCs (ILC3) have a unique place in the interaction with the gut microbiota. By synthesising IL-22, these cells stimulate the production of mucus, AMPs and the secretion of chemokines and recruitment of polymorphonuclear (PMN) cells (Figure 2) [1].

CROSSTALK BETWEEN THE MICROBIOTA AND THE ADAPTIVE IMMUNE SYSTEM

The final maturation of the adaptive immune system is characterised by the colonisation of the intestinal mucosa by mature effector T-lymphocytes with inflammatory properties (Th17), T-lymphocytes with antiinflammatory properties (Treg) and B-lymphocytes (Figure 2). Besides effects on the macrophages and the differentiation of the Th17 cells, the SFB also stimulate the development of the lymphoid follicles and participate in the differentiation of the B-lymphocytes to IgA-producing plasma cells the action of which is the containment of pathogenic bacteria in the mucus [5]. Other commensal bacteria can stimulate adaptive immune responses: a mixture of 17 Clostridia strains isolated from a human faecal sample and introduced in mice induced an anti-inflammatory response by stimulating the Treg [7]. Faecalibacterium prausnitzii has also been identified for its anti-inflammatory action in vitro and in vivo by acting on the NF-kB factor, DCs and Mj which secrete IL-10 and enhance differentiation of Treg to the detriment of Th17 [8]. Of the Bacteroidetes, Bacteroides fragilis and B. thetaiotaomicron have also been described as exerting anti-inflammatory activity. B fragilis synthetises a polysaccharide A (PSA) that prevents pro-inflammatory IL-17 production and stimulates the anti-inflammatory of secretion IL-10 (Figure 3). In a specific model of Helicobacter hepaticus-induced colitis, PSA stimulated the development of lymphoid follicles, stimulated Treg lymphocyte cells and protected the mice [9].

MICROBIAL METABOLITES: IMPORTANT MEDIATORS IN THE CROSSTALK BETWEEN THE MICROBIOTA AND ADAPTIVE IMMUNITY

Short-chain fatty acids (SCFAs), tryptophan metabolites and bile salts are the principal metabolites produced by the gut microbiota which exert a protective effect against infections [9, 10]. Butyrate, propionate and succinate are known to act on intestinal homoeostasis, on mucus secretion, but also on the various cells of the immune system. Among other effects, butyrate has anti-inflammatory and anti- microbial effects. This action is exerted via the G-coupled protein receptors (GPR) found on the epithelial cells and the macrophages [9]. F prausnitzii produces large quantities of butyrate, which may partly explain its anti-inflammatory effect. It inactivates NF-kB and thus suppresses synthesis of the pro-inflammatory cytokines IFN-g, TNF-a, IL-1b, IL-8 by the enterocytes [8] (Figure 3) . It also induces metabolic and epigenetic modifications (via histone deacetylases, HDACs) macrophages in mice, thus amplifying their anti-microbial activities in vitro and in vivo [11]. Commensal bacteria can also metabolise tryptophan and produce antimicrobial substances. An example is the Lactobacilli, which utilise it as an energy source to synthesise an indole that binds to aryl hydrocarbon receptors (AhR) present on the ILC3. AhR triggers IL-22 secretion by the ILCs and this further drives the secretion of AMPs and protects against infections [9].

MICROBIOTA – INTESTINAL IMMUNE SYSTEM CROSSTALK FOR PROTECTION AGAINST VIRAL INFECTIONS

Among the enteric viruses, norovirus and rotavirus are the main causes of gastroenteritis [12]. The enteric viruses infect various cell types: enterovirus 71 specifically infect the goblet cells, whereas the rotavirus has a preferential tropism for the enterocytes [13] (Figure 4A). The gut microbiota acts as a barrier against enteric viral infections. The viruses have evolved and become adapted to their host, implementing mechanisms that enable them to cross the intestinal barrier and escape barrier immunity: it is in fact difficult to infect mice effectively with human enteric viruses by the oral route [13]. Virus penetration into the enterocyte triggers the secretion of type III interferon (IFN). Detection of a virus can induce IL-la, which activates the ILC3 to produce IL-22. This IL protects against enteric viral infections and acts synergistically with type III IFN to induce the expression of antiviral effectors and IL-15. Recognition of a virus by TLR-3 leads to the activation of the NF-kB pathway and to the production of IL-15 also. IL-15 activates the cytotoxic lymphocytes (NK cells). Those viruses, which have traversed the intestinal barrier, trigger the production of type I IFN by the macrophages of the lamina propria (Figure 4B). Some enteric viruses (rotavirus, reovirus, enterovirus) are able to adhere to the intestinal bacteria, enhancing penetration into the intestinal epithelial cells [13]. The SFB, which accelerate epithelial cell turnover produce protection against rotavirus infection in mice by expulsing infected cells [14]. The bile acids metabolised by the gut microbiota also act to protect the small intestine (but not the colon) from acute infection by norovirus in mice by enhancing the production of type III IFN in the small intestine [15].

Covid-19 is a respiratory illness caused by a novel coronavirus (SARS-CoV-2) and is still affecting tens of millions of people worldwide today. Although most of Covid-19 patients present respiratory symptoms, up to 20% of them have gastrointestinal (GI) symptoms including diarrhea [1], suggesting that the digestive tract is an extrapulmonary site of disease expression and SARS-CoV-2 infection. In addition, Covid-19 presents a wide spectrum of disease severity, varying from asymptomatic, mild, severe, and up to critical resulting in respiratory failure or even death [2].

The GI tract is the largest immune organ in humans, playing critical roles in host defense against pathogens infections. Trillions of microorganisms live and colonize the human gut – bacteria, fungi, viruses, and other life forms that are collectively known as the microbiota – regulating the host immunity. Therefore, it is of paramount importance to understand if the gut microbiota modulates the host susceptibility to and severity of SARS-CoV-2 infection, as well as the impact of SARS-CoV-2 infection on the host GM and its downstream long-term effect on human health.

The gut bacterial microbiota and Covid-19

Covid-19 patients had significant alterations in the gut bacterial microbiome compared with healthy individuals, characterized by depletion of beneficial commensals and enrichment of opportunistic pathogens in the gut (Figure 1) [3]. Depletion of gut symbionts persisted even after the resolution of Covid-19. The baseline (at hospitalization) abundance of the bacteria Coprobacillus, Clostridium ramosum and Clostridium hathewayi showed positive correlation with Covid-19 severity, whereas there was an inverse correlation between the abundance of Faecalibacterium prausnitzii (known as an anti-inflammatory bacteria) and the disease severity.

SARS-CoV-2 uses the angiotensin converting enzyme 2 (ACE2) receptor to enter the host and this receptor is highly expressed in both the respiratory and gastrointestinal tracts [4]. ACE2 is important in controlling intestinal inflammation and gut microbial ecology [5]. Four Bacteroides species : B. dorei, B. thetaiotaomicron, B. massiliensis, and B. ovatus, were reported to inversely associate with ACE2 expression in murine gut [6]. Interestingly, their abundances in faecal microbiome also showed inverse correlation with faecal SARS-CoV-2 viral load in Covid-19 patients during the disease course. These findings suggest that the human bacterial GM is affected by Covid-19 and might calibrate the host defense against SARS-CoV-2 infection.

The fungal microbiome and Covid-19 

The GI tract also harbors a large number of fungi, collectively known as the mycobiome (fungal microbiome), which have been shown to be causally implicated in GM assembly and immune development [7]. Patients with Covid-19 also had altered gut mycobiomes, characterized by enrichment of Candida albicans and highly heterogeneous mycobiome configurations (Figure 1) [8]. The diversity of the fecal mycobiome in patients with Covid-19 at discharge was 2.5-fold higher than that in healthy individuals. Opportunistic fungal pathogens, Candida albicans, C. auris, and Aspergillus flavus were highly present in faeces of Covid-19 patients during the disease course. Two respiratory symptom- associated fungal pathogens, A.flavus and A. niger, were detected in faecal samples from a subset of patients with Covid-19, even after disease resolution. Unstable gut mycobiomes and prolonged dysbiosis persisted in approximately 30% of patients with Covid-19.

The gut virome and Covid-19 

Through shotgun RNA viral sequencing, a signature of active gut viral infection were found in 47% of patients with Covid-19, even in the absence of gastrointestinal symptoms and after respiratory clearance of SARS-CoV-2 [9], suggesting “quiescent” SARS-CoV-2 infection in the GI tract and potential faecal-oral transmission risk. Patients with such gastrointestinal SARSCoV- 2 activity harboured abnormal GM compositions and functions, featured by high abundances of opportunistic pathogens and enhanced capacity for biosynthesis of nucleotide and amino acid and carbohydrate metabolism (glycolysis) [9].

The human GI tract also harbours abundant viral/phage members collectively known as the gut virome. Covid-19 patients had under-representation of Pepper mild mottle virus (RNA virus) and multiple bacteriophage lineages (DNA viruses) and enrichment of environment-derived eukaryotic DNA viruses in faecal samples, compared to non-Covid-19 subjects (Figure 1) [10]. Faecal virome in SARSCoV- 2 infection showed more stress-, inflammation- and virulence-associated gene encoding capacities. At patient baseline, faecal abundances of the RNA virus, Pepper chlorotic spot virus, and multiple bacteriophage species inversely correlated with Covid-19 severity. These viruses were also inversely associated with blood levels of pro-inflammatory proteins, white cells and neutrophils, indicating gut resident viruses might tune host immune response to SARS-CoV-2 infection. Among Covid-19 severity-associated DNA virus species, 40% species showed inverse correlation with age, which may underlie the observation that elderly subjects are at a higher risk for a more severe Covid-19.

SEVERE PRE-ECLAMPSIA AND MICROBIOTA

^{Lin CY, Lin CY, Yeh YM, et al. Severe preeclampsia is associated with a higher relative abundance of Prevotella bivia in the vaginal microbiota. Sci Rep 2020; 10: 18249.}

Severe preeclampsia (SPE) is a hypertensive disorder of pregnancy that can have serious consequences for both mother and child. It is characterized by hypertension and manifestations of a multisystem disorders. The role the vaginal microbiota may play in the pathogenesis of SPE remains unknown. The present study revealed that women with SPE had increased relative abundance of vaginal Prevotella bivia (Pb). Pb is an anaerobic gram-negative bacterial species that was previously associated with pelvic inflammatory disease and bacterial vaginosis. Previous studies have shown that obesity was a risk factor for SPE and that vaginal microbiota of obese women was characterized by increased diversity and predominance of Prevotella spp. In this study, the Body Mass Index (BMI) was the strongest SPE predictor and the authors suggest that the higher relative abundance of Pb in the vaginal microbial, which is tightly regulate by BMI, may be involved in the pathogenesis of SPE.

CERVICAL MUCUS ESSENTIAL FOR FEMALE REPRODUCTIVE HEALTH

^{Lacroix G, Gouyer V, Gottrand F, Desseyn JL. The cervicovaginal mucus barrier. Int J Mol Sci 2020; 21: 8266.}

Cervical mucus (CM) is key for women health: it protects vaginal epithelium, helps to maintain fertility and fecundity. In this study, the authors assessed its role in both the physiological state and in bacterial vaginosis. Normal vaginal microbiota is characterized by Lactobacillus spp dominance. Undisturbed cooperation between vaginal microbiota, CM and host cells is necessary for vaginal health. This includes acidification by lactic acid, production of reactive oxygen species, interaction between mucins and cells, and diffusion of signaling cells. Bacterial vaginosis, which may cause preterm birth, is characterized by depletion of Lactobacilli leading to impaired vaginal barrier function. During pregnancy, a cervical mucus plug (CMP) is formed to prevent the vaginal microbes to ascend into the uterus, which protects the fetus from pathogens. CMP contains mucus, antimicrobial compounds and immune cells. A shorter, more permeable and less mucoadhesive CMP has been found in women at high risk for preterm birth compared with those at low risk. In addition to oral or vaginally administered antibiotics, bacterial vaginosis may be treated by restoring vaginal Lactobacillus flora. In conclusion, CM is essential for the fertility and protects from bacterial vaginosis and sexually transmitted infections, which increase the risk of infertility and preterm births.

AGE-RELATED SKIN MICROBIOTA PROFILES

^{Li Z, Bai X, Peng T, et al. New insights into the skin microbial communities and skin aging. Front Microbiol 2020: 11: 565549.}

Intrinsic skin aging is a natural aging process determined by internal factors while photoaging is the accelerated aging of the skin due to repeated exposure to ultraviolet radiation (UV). Little is known about how the skin microbiota influences the aging process (either natural or photoaging) and on the effects of age-related skin microbes. To answer this question, the authors analyzed 160 skin samples from the cheek and the abdomen of 80 individuals of varying ages to develop age-related microbiota profiles. They found that abundance of Cyanobacteria was higher in the children group and was associated with decreased UV-induced skin damage and pigmentation. In young and middle-aged, Staphycococcus, Cutibacterium and Lactobacillus improved skin barrier and protected from photoaging. Cutibacterium may modulate immune responses and suppress inflammation and slow aging processes. In young and middle-aged people, Staphylococcus may protect from intrinsic skin aging and maintain skin microbiota homeostasis. The authors suggest that these findings may have great innovation and clinical value, and that the development and use of microbial skin homeostasis regulators may reduce the incidence of age-related skin diseases.

SACCHARIDE ISOMERATE MODULATES SKIN MICROBIOTA

^{Sfriso R, Claypool J. Microbial reference frames reveal distinct shifts in the skin microbiota after cleansing. Microorganisms 2020; 8: 1634.}

Many intrinsic, extrinsic and host-related factors modulate skin microbiota. Skin cleansing products such as bar or liquids soaps and deterge have impact on the skin microbiota. Saccharide isomerate (SI) is a plant-derived moisturizer that resembles the natural carbohydrate fraction of the upper layer of the skin. SI binds to the skin stronger than other moisturizer ingredients and keeps skin hydrated longer than usual. The investigators performed a placebo- controlled, single-blind, and randomized clinical study to investigate how skin cleansing with liquid soap containing SI affects skin microbiota over time. Of potentially beneficial organisms, Paracoccus marcusii was positively associated with the active formulation. This bacteria naturally produces astaxanthin, a potent antioxidant carotenoid having potential positive effects on health. P. mercusii is also a potentially carcinogenic polyaromatic hydrocarbons degrader and a biosurfactants producer and may have a key role in maintaining healthy skin. SI wash also reduced the abundance of “coryneforms” (Brevibacterium casei and Rothia mucilaginosa) linked to skin infections and represents uncharacterized benefit of the active wash formulation. These results suggest that skin wash with SI may have beneficial effects on skin microbiota.

FECAL MICROBIOTA TRANSPLANTION (FMT) FOR CESAREANSECTION- DELIVERED INFANTS TO RESTORE NORMAL GUT MICROBIOTA

^{Korpela K, Helve O, Kolho K-L, et al. Maternal fecal microbiota transplantation in cesarean-born infants rapidly restores normal gut microbial development: a proof-of concept study. Cell 2020; 183: 324-34.}

The gut microbiota of infants born vaginally differs from that of CS born infants since they are not exposed to maternal microbes during delivery. Several studies reported that CS may be associated to short- and long-term consequences, including an increased risk of chronic immune diseases. In this study, the efficacy and safety of fecal microbiota transplant (FMT) has been evaluated as a means of restoring the gut microbiota of babies born by CSD. Seven CSD infants received a stool-transplant from their own mother at the first milk feeding, and the composition of their gut microbiota was compared to that of 82 babies born vaginally or by CS without FMT. During the 3-month follow-up, no adverse effects was reported. One week post-FMT, the gut microbiota of CSD infants was similar to that of vaginally delivered infants while CSD-infants without FMT had lower microbial diversity. FMT corrected the bacterial signature of CSD delivered infants by rapid normalization of Bacteroidales which was lower in CSD group and also reduced potential pathogens typical for CSD infants. This proof-of-concept study showed that FMT normalizes gut microbiota development in CSD infants.

CESAREAN SECTION AND CHILDHOOD ASTHMA RISK

^{Stokholm J, Thorsen J, Blaser MJ, et al. Delivery mode and gut microbial changes correlate with an increased risk of childhood asthma. Sci Transl Med 2020; 12, eaax9929.}

The authors analyzed the effects of cesarean section (CS) delivery on gut microbiota composition during the first year of life and examined if the perturbations were associated with a risk of developing asthma in the first 6 years of life. They included 700 children from the COPSAC₂₀₁₀ (Copenhagen Prospective Studies on Asthma in Childhood₂₀₁₀) cohort, of whom 22% (151) were born by CS and 78% (549) by vaginal delivery. Gut microbiota composition varied with delivery mode: CS born babies had lower abundance of Bacteroidetes and Actinobacteria at 1 week of age, but the abundance of Firmicutes and Proteobacteria were higher compared with vaginally born children. At genus level, only 3 genera were different at age 1 year and CS delivery was associated with higher relative abundance of a genus belonging to the family Enterobacteriaceae and Escherichia/Shigella. A microbial profile was identified that predicted the birth mode at one week, one month, and one year of age. CS delivered children who retained a CS gut microbiota signature at age 1 year had a three times increased risk of developing asthma by age 6. This increased asthma risk was ameliorated in CS-born children whose gut microbiota at the age of 1 year resembled that of vaginally born children. It indicates that healthy maturation of a dysbiotic CS gut microbiota could ameliorate some of the risk of asthma associated with CS delivery.

MICROBIOTA, ENVIRONMENTAL AND HOST FACTORS IN HEALTH AND DISEASE

The gut microbiome has been associated with a large number of diseases, but it is still not clear how a healthy or unhealthy microbiome should be defined. A large Dutch population-based study (OP178 R Gacesa et al.) demonstrated common microbial patterns across several diseases (including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), asthma, diabetes and mental disorders), making it possible to define clusters of health- and disease-linked gut microbes and functions. Specifically, the microbiome associated with diseases was found to be characterized by a significant increase in prevalence and abundance of opportunistic pathogens of genera Clostridium, Gordonibacter and Eggerthella, by a reduction in carbohydrate catabolism, synthesis of amino-acid and vitamins, and by an increase in synthesis of long-chain fatty acids. On the other hand, the healthy microbiome showed high abundances of butyrate-producing commensals from genera Alistipes, Roseburia, Faecalibacterium and Butyrivibrio. The authors also showed that the microbiome was primarily shaped by the environment and lifestyle, and therefore concluded that alterations through improving diet, lifestyle and the environment, and use of probiotics can be advocated to improve general health. Furthermore, a longitudinal follow-up study (OP201 L Chen et al.) highlighted that microbial changes over time seem to be driven by environmental exposures and can affect the metabolic health of the host.

MICROBIOTA IN INTESTINAL DISEASES

Lactose restriction is the cornerstone of treating gastrointestinal (GI) complaints in subjects with lactose malabsorption due to lactase deficiency. However, the severity of gut symptoms, such as flatulence, bloating and diarrhea, after lactose intake in these subjects varies substantially, and the reason for this remains unclear. Via analyses from the Dutch Microbiome project (OP177 MDF Brandao Gois et al.), a plausible mediating role of the gut microbiome between dairy intake and the occurrence of gut symptoms in subjects with lactase deficiency was demonstrated, and in particular the Bifidobacterium genus was found to be of potential relevance. Hence, modulating the gut microbiota composition may influence the sensitivity to dairy products in subjects with lactose malabsorption.

Even though the exact mechanisms that explains food-related GI symptoms in patients with IBS remains unclear, different dietary adjustments improves GI symptoms in subsets of patients. A posthoc analysis of a previously published clinical trial (P0786 E Colomier et al.) revealed patterns of psychological, nutritional, and microbial factors that can predict treatment response to both the traditional NICE (National Institute for Health and Care Excellence) diet for IBS and the low fermentable oligo-, di-, monosaccharides, and polyols (FODMAP) diet for specific symptoms. This indicates that individual tailoring of dietary treatment advice in IBS will be possible in the near future.

Gut microbes and their metabolites are involved in the pathophysiology of a number of intestinal diseases, including IBS and IBD, with several abstracts at UEG week 2020 highlighting this. In IBD, a large cohort study nicely confirmed the presence of gut dysbiosis in both ulcerative colitis (UC) and Crohn’s disease (CD) (OP002 A Vich Vila et al.), and that this was translated into the fecal metabolite profile, which could be used as a potential biomarker to distinguish between IBD and non-IBD and between UC and CD. Specifically, metabolites related to sphingolipid synthesis were increased in IBD, whereas fatty acid metabolites were decreased. Furthermore, in a proof-of-concept study (OP045 L Oliver et al.), a combination of four microbiome markers (Faecalibacterium prausnitzii and one of its phylogroups (PHG-II), Ruminococcus sp., and Methanobrevibacter smithii) could predict the treatment response to anti-TNF treatment with a positive predictive value of 100% and negative predictive value of 75%. This indicates that microbiome analyses can be used to personalize treatment in IBD in the near future. The role of gut microbiota in IBS was highlighted in several abstracts, including a study supporting good long-term effects of fecal microbial transplantation in IBS (OP059 M El-Salhy et al.), which was associated with changes in the faecal bacterial and short chain fatty acid profile and increase in enteroendocrine cells (P0783 M El Salhy et al.). Moreover, another study demonstrated a distinct intestinal microenvironmental profile in IBS with a link to the predominant bowel habit of the patient (P0651 C Iribarren et al.), with the separation between IBS and health and among IBS subtypes (IBS with diarrhea versus IBS with constipation) being mostly driven by metabolites involved in e.g. amino acid metabolism and certain cellular and molecular functions. Hence, it seems to be more important what the microbes do than the composition per se. There were also abstracts focusing on animal models of relevance for IBS pathophysiology. These studies highlighted the importance of gut microbiota for the development of abnormal gutbrain interactions (P0052 M Constante et al.), as well as the role of stress in inducing gut dysbiosis and visceral hypersensitivity (OP056 C Petitfils et al.). These studies are of great relevance for our understanding of gut-brain interactions in IBS and the role of gut microbes and their metabolites in these interactions, and fits well into the concept that IBS and other functional GI disorders are now called disorders of gut-brain interactions.

MICROBIOTA IN EXTRAINTESTINAL DISEASES

Finally, there were also studies focusing on the gut microbiome in extraintestinal diseases. Gut microbiome alterations were demonstrated in both renal and liver transplant recipients (OP180 JC Swarte et al., and OP112 y Li et al.). Patients with end stage renal disease were characterized by low gut microbial diversity, increased richness of virulence factors, and antibiotic resistance genes. The microbial diversity decreased further post-renal transplantation and gut microbiota composition was not restored. Furthermore, immunosuppressive agents had a profound effect on gut microbiota composition. The authors concluded that these changes could have far-reaching implications for the outcome of renal transplantation. Similar findings regarding microbial diversity, gut microbiota composition and effect of immunosuppressive agents were noted also in liver transplant recipients, and intriguingly microbial diversity was associated with survival post liver transplantation, therefore revealing a new potential biomarker or therapeutic target.