How Gut Bacteria Influence Intestinal Immunity Through Tryptophan Breakdown

As the rate-limiting enzyme of KP, IDO1 also plays an important role in regulating the adaptive immunity of vertebrate hosts (Zelante et al.,). When attempting to counterbalance tissue damage, high expression of IDO1 by intestinal mononuclear cells can mediate anti-inflammatory and immunosuppressive effects of IDO1 on the intestinal mucosa (Wolf et al.,) by regulating host immunomodulatory activity via kynurenine production, mucosal amino acid nutrition, mucosal immune reactivity, and gut microbial community metabolism (Dai and Zhu,). Moreover, through the influence of IDO1 on T-cells, KP may also serve as the basis of the sensitive balance between pro-inflammatory (excitotoxic quinolinic acid) and anti-inflammatory (neuroprotective KA) states in the GI tract (Kaszaki et al.,). This balance can affect intestinal motor or sensory function through a profound influence on the excitability of enteric neurons. In turn, inflammatory mediators tightly regulate KP (Campbell et al.,). Specifically, inflammatory cytokines and interferon (IFN)-γ induce expression of IDO1 in the GI tract and other tissues. In addition, the severity of the inflammation induced is linked to the translocation of β-catenin from the cell membrane to the cytoplasm/nucleus (Cooper et al.,). Impairment of IDO1 activity, which is detected in inflamed or neoplastic intestinal epithelial cells, can reduce nuclear β-catenin and cell proliferation (Thaker et al.,). ^{2013 2004 2010 2008 2014 2000 2013}

In addition, IDO1 is an important activating enzyme in host-microbiota symbiotic relationships via regulation of Trp metabolism (Niño-Castro et al.,; Romani et al.,). The gut microbiota may influence host Trp degradation and circulating Trp concentrations through KP (O'mahony et al.,). In GF animals, KP metabolism (kynurenine:Trp ratio) is decreased due to the microbiota deficiency (Clarke et al.,), and colonization by a normal microbiota in GF animals increases KP metabolism (kynurenine:Trp ratio) and reduces plasma Trp. In microbiota-deficient animals, KP cannot be detected in the central nervous system (CNS), but mice infected with Toxoplasma gondii have increased levels of kynurenine, KA, 3-hydroxykynurenine and quinolinic acid in brain tissue (Notarangelo et al.,). In contrast, colonization byin rodents increases Trp levels, circulating KA and the KA:kynurenine ratio and decreases the kynurenine:Trp ratio, suggesting reduced activity of IDO and Trp metabolism through KP, with no effect on kynurenine concentrations (Desbonnet et al.,). This increase in KA and decrease in IDO activity appear to conflicting, and other factors need to be taken into account when considering the influence ofcolonization on host Trp metabolism. Colonization byin rats also decreases ileum IDO mRNA levels and serum kynurenine concentrations, consistent with the effect ofculture cell-free supernatant in reducing IDO1 activity in HT-29 intestinal epithelial cells (47% reduction) (Freewan et al.,; Valladares et al.,). As an explanation,feeding was proven to alter the distribution of ileum and colon IDO1 in rats, and increased ileum lumen HOproduced bywas found to be a strong inhibitor of IDO1 activity. The signaling molecule HOpossibly mediated host-microbiota symbiotic interactions. Moreover, the impact of altered IDO1 activity on the degradation of 5-HT is also important, as lower IDO activity leads to both decreased kynurenine and increased 5-HT concentrations. ^{2014 2014 2015 2013 2014 2008 2013 2013} Bifidobacteria infantis B. infantis Lactobacillus johnsonii L. johnsonii L. johnsonii L. johnsonii 2 2 2 2

There is also a correlation between IDO and inducible nitric oxide synthase (iNOS). On the one hand, the NO produced by iNOS inhibits IDO activity by direct interaction or by stimulating IDO degradation. On the other hand, 3-hydroxyanthranilic acid, a kynurenine metabolite, inhibits the expression and catalytic activity of iNOS. Other kynurenine metabolites, quinolinic, and picolinic acids, can also enhance IFN-γ-dependent iNOS expression (Xu et al.,). iNOS is involved in the immune response after gut microbiota exposure and helps to limit inflammation. During the inflammatory response, leukocyte recruitment, and adhesion are regulated by iNOS. iNOS-derived NO is maintained at high levels, which is considered a host-protective effect. Similarly, commensal bacterial exposure promotes iNOS expression, which further enhances IgA (a major class of immunoglobulin) secretion by intestinal B cells, which is beneficial for promoting the intestinal barrier function. Conversely, iNOS acts as a microbicidal mediator, reducing microbial growth, and indirect antimicrobial effects are suggested to be caused by local arginine depletion after induction of iNOS or NO-dependent induction of IFN-γ (Bogdan,). In another report, increased iNOS stimulated by quinolinic and picolinic acids together with 3-hydroxykynurenine and 3-hydroxyanthranilic acids enhanced lipid peroxidation and activated an arachidonic acid cascade, resulting in the production of inflammatory factors such as prostaglandins and leukotrienes (Oxenkrug,). Overall, the iNOS pathway acts as a mediator between the gut microbiota and host immune system, and it is also related to Trp metabolism. Nonetheless it remains unclear how the iNOS pathway and Trp metabolism simultaneously participate in this mutual interaction, and further elucidation is required. ^{2017 2001 2010}

The microbiota influences host IDO and Trp metabolism through KP, though there are conflicting results for the same or different experiments. For example, IDO and KP metabolism decrease due to the deficiency in the microbiota in GF animals, whereas GF animals colonized with a normal microbiota orhave increased IDO and KP metabolism. However, colonization of probiotics (and) in conventional rodents reduced IDO activity and KP metabolism. The reason for these differences may be variation in the original gut microbial background in the transplanted animals, with one being GF and others conventional. Another reason may be the different types of colonized microbes. In relation to both experimentation and therapy via IDO activity regulation, these factors should be further investigated. T. gondii B. infantis L. johnsonii

Kynurenines possess antimicrobial activities, which can directly impact proliferation of the gut microbiota (Niño-Castro et al.,). The influence of the gut microbiota on host Trp metabolism in KP is associated with the immune system. GF animals that lack a microbiota have an immature immune system, which is associated with reduced Trp metabolism in KP (Clarke et al.,). After intestinal microbiota colonization in GF animals, immune system function is reinstated, and aberrant KP metabolism is normalized (Clarke et al.,). GI Toll-like receptors (TLRs), which recognize microbial components in the GI tract, act as crucial junctions (Kawai and Akira,; Wang et al.,). In the GF state, TLR expression is reduced, which is associated with increased KP metabolism that may be mediated by IFN-γ-dependent or -independent IDO1 induction (Clarke et al.,). ^{2014 2013 2013 2010 2010 2012}

In KP, kynurenine, KA, CA, and XA act as direct ligands of AhR, stimulating AhR and AhR-dependent gene expression in a concentration-dependent manner, and simultaneously modulate intestinal homeostasis. Additionally, AhR itself plays a role in regulating levels of IDO1 and TDO1 expression (Bessede et al.,). The absence of AhR causes an increase in endogenous KA levels in mice (García-Lara et al.,). AhR may be an important mediator in the complex crosstalk between the gut microbiota, KP and the immune response. ^{2014 2015}

In addition, transmembrane G protein-coupled receptors (GPCRs) sense metabolic intermediates to activate signaling pathways and play roles in regulating GI homeostasis and intestinal immunity. GPCRs, including GPR43, GPR109A, and GPR120, exert anti-inflammatory effects (Tilg and Moschen,). For instance, GPR43 deficiency induces severe inflammatory reactions in the mouse intestine (Maslowski et al.,). GPR35 is predominantly expressed in immune cells and in the GI tract, suggesting it may play an important role in immunological regulation. Trp metabolites, such as serotonin, melatonin, KA and niacin, are known GPCR ligands, and KA acts as a ligand for GPR35. Based on elevated KA levels, anti-inflammatory effects of KA during inflammation, and increased expression of GPR35 in immune cells, several studies have suggested that KA may have important functions in immunological regulation via GPR35 activation and subsequent signaling (Wang et al.,). Several bacteria can also catabolize Trp through KP (Genestet et al.,). Most strains ofisolated from cystic fibrosis patients can produce a high level of kynurenine, which can promote bacterial survival and allow bacteria to circumvent the innate immune response by scavenging neutrophil reactive oxygen species (ROS) production (Genestet et al.,). Additionally, inKP, kynurenine acts as the main precursor of thequinolone signal, which is another virulence factor of these bacteria (Genestet et al.,). ^{2015 2009 2006 2014 2014 2014} P. aeruginosa P. aeruginosa Pseudomonas

In summary, the interface for the gut microbiota, KP and immune response is tightly controlled and complex. Gut microbial composition plays an important role in regulating KP to subsequently influence host immunity, and variations in the composition of the gut microbiota can influence an individual's immunity and health through adjusted Trp metabolism in KP.

The serotonin pathway is one of the core signaling pathways in the gut (Gershon and Tack,; Lesurtel et al.,). Serotonin plays a role in regulating the permeability of the intestine and mucosal inflammation. In the murine intestine, serotonin is associated with inflammation during chemically induced colitis. Suppressing the production of mucosal serotonin is beneficial for relieving inflammation (Margolis et al.,), and studies have found that GI-selective TPH (Trp hydroxylase) inhibitors may act as a cure for several GI diseases caused by serotonin pathway dysregulation (Shi et al.,). Changes in serotonin concentrations induced by the gut microbiota can regulate the host immune response and subsequently influence the coping strategy by which the host defends against pathogens or disease. Microbial SCFA metabolites can active GPCRs on intestinal epithelial cells and thus have a major role in regulating epithelial barrier integrity and intestinal immunity. At the same time, SCFAs promotetranscription and colonic serotonin production from enterochromaffin cells and stimulate colonic transit, steps that are essential for serotonin homeostasis (Reigstad et al.,). Plasma serotonin levels were found to be decreased by 2.8-fold and levels of Trp increased in GF male Swiss Webster mice compared with conventional mice (Wikoff et al.,). At the same time, GF mice have decreased gut motility compared to normal animals, and decreased serotonin levels are one possible reason for this defect (Ridaura and Belkaid,). In another experiment involving GF Swiss Webster mice, the concentration of hippocampal serotonin was significantly increased in GF and colonized GF animals compared with conventional mice (Clarke et al.,). The possible factors responsible for differences in serotonin levels in GF mice is worth further study. When discussing the effect of microbial colonization on host 5-HT levels, it is also important to consider the influence of altered IDO1 activity and KP metabolism induced by colonization, as lower IDO activity may result in decreased kynurenine and increased 5-HT concentrations. ^{2007 2008 2013 2008 2015 2009 2015 2013} TPH1

Colonization of GF animals with the gut microbiota from humans or other mice can significantly increase gut motility, and this can be partially blocked by a pharmacologic antagonist of serotonin receptors (Kashyap et al.,). Indeed, serotonin can promote immunity and inflammation in various models of mucosal infections. Combining its effects on intestinal physiology and the gut microbiota, serotonin has been suggested to directly and indirectly influence GI motility and the immune system, which in turn shapes the composition and localization of the microbiota. ²⁰¹³

Melatonin acts as a powerful anti-inflammatory molecule, and the role of melatonin in the gut, especially its role in gut permeability, has recently been explored (Anderson and Maes,). Melatonin release in the gut is 400-fold higher than that in the pineal gland (Bubenik et al.,), with a peak after food intake. Melatonin has positive impacts on gut disorders, including inflammatory bowel disease (IBD) (Eliasson,) and GI cancer (Glenister et al.,). ^{2015 1977 2014 2013}

The impact of melatonin on the gut microbiota has been examined. Specific gut bacteria determine the availability of Trp to the host and then regulate serotonin and subsequent melatonin synthesis (Wikoff et al.,). Moreover, melatonin can alleviate the increase in gut permeability and immune activation induced by(Sun et al.,). ^{2009 2013} Escherichia coli

Inflammasomes, such as NOD-like receptor 3 (NLRP3) and pyrin domain-containing 6 (NLRP6), are known to be important effectors of gut permeability and interactions with gut bacteria. These inflammasomes are important for maintaining gut homeostasis (Zambetti and Mortellaro,), including regulating gut permeability, and are crucial for IL-1b and IL-18 release. Activation of the alpha 7 nicotinic acetylcholine receptor (α7nAChr), an inflammasome activator in lipopolysaccharide (LPS) models (Kim et al.,), or melatonin (Galley et al.,) can reduce the incidence of sepsis by decreasing NLRP3 activation. Melatonin is a significant positive regulator of α7nAChr (Markus et al.,), suggesting that several regulatory effects of melatonin may be mediated by α7nAChr induction in the gut. For example, the protective effects of melatonin against induced gut permeability is mediated, at least in part, by α7nAChr (Sommansson et al.,). Several potential mediators that connect melatonin, gut bacteria, and gut immunity have been discovered, but the specific mechanisms of these connections remain to be determined. ^{2014 2014 2014 2010 2013}

Indole is a major bacterial Trp metabolite. The generation of indole is catabolized by tryptophanase, which can be induced by Trp or repressed by glucose in most bacteria (Figure). Bacterial species including, andspp are capable of producing indole (Keszthelyi et al.,). Recently, indole has been recognized as a signaling molecule that can regulate bacterial motility, biofilm formation, antibiotic resistance, persister cell formation, and virulence (Li and Young,) and that plays a role in affecting host cell invasion by other non-indole-producing species, such asandand even the yeast(Li and Young,). In the porcine gut with a low-non-starch polysaccharide diet, the maximum concentration of indole (~0.12 mM) was found in the distal part of the cecum, where the majority of gut bacteria had settled, whereas the amount of indole in the hind intestine was lower in animals fed a high-non-starch polysaccharide diet (Knarreborg et al.,). An explanation is that easily fermented carbohydrates such as non-starch polysaccharides, but not protein, are preferentially fermented by the intestinal microbiota, decreasing the production of indole from Trp degradation. Indole can also be detected in human feces, between 0.25 and 1.1 mM (consistent with the levels that are readily produced bywhen cultured in a rich medium), which suggests that intestinal epithelial cells are exposed to indole when the fermented substrate is available for Trp degradation in gut (Bansal et al.,). 1C ^{2009 2013 2013 2002 2010} E. coli, Proteus vulgaris, Paracolobactrum coliforme, Achromobacter liquefaciens Bacteroides Salmonella enterica P. aeruginosa C. albicans E. coli

As a specific bacterial signal, indole is abundant in the healthy mammalian gut and positively influences intestinal health. Indole has also been recognized as a beneficial signal in intestinal epithelial cells that can ameliorate intestinal inflammation in mammals (Bansal et al.,). Moreover, compared to other bacterial Trp metabolites, indole is the most effective molecule (Davis,). Indole administration can attenuate damage of the GI tract induced by non-steroidal anti-inflammatory drugs (NSAIDs), modulating inflammation mediated by innate immune responses and alterations in the gut microbiota composition (Whitfield-Cargile et al.,). At millimolar concentrations, indole can weaken the invasion and colonization capabilities of enteric bacteria by reducing expression ofPathogenicity Island-1 (SPI-1) genes, which facilitate bacterial invasion into host cells. After exposure to indole, the expression level of genes associated with strengthening the mucosal barrier and mucin production is increased, which is usually positively correlated with improvement in the resistance of human enterocyte HCT-8 cells. Indole exposure can also reduce TNF-α-mediated activation of NF-κB, expression of the proinflammatory chemokine IL-8, and the adherence of pathogenicto HCT-8 cells, though it does increase production of the anti-inflammatory cytokine IL-10. Variations in NF-κB activation and cellular resistance are highly specific to indole, as exposure to other indole-like molecules does not induce a similar response (Bansal et al.,). ^{2010 2014 2016 2010} Salmonella E. coli

Indole has been shown to promote the epithelial barrier functions of intestinal cells by fortifying epithelial tight junctions between cells through the pregnane X receptor (PXR) (Bansal et al.,; Shimada et al.,; Thaiss et al.,), which might contribute to resistance to inflammation. Indole can also enhance secretion of glucagon-like peptide-1 (GLP-1), an incretin with profound influences on host metabolism (Chimerel et al.,; Thaiss et al.,). As an important Trp microbiota-derived metabolite, indole can activate AhR signaling and subsequently promote local IL-22 production, which is important for intestinal homeostasis and further drives the secretion of antimicrobial peptides and protects against pathogenic infection (Levy et al.,). Considering theseandresults, it can be concluded that indole has beneficial effect on intestinal health. For normal cells, indole exposure can strengthen the mucosal barrier and mucin production by inducing expression of associated genes, thereby increasing resistance to pathogen invasion; for inflammatory cells, indole exposure can suppress activation of NF-κB and proinflammatory chemokine production and simultaneously increase anti-inflammatory cytokine production, thus ameliorating inflammation and damage. ^{2010 2013 2016 2014 2016 2016} in vitro in vivo

As Trp can induce the enzyme tryptophanase, exogenous Trp levels influence the production of indole inin the gut (Li and Young,). High-protein diets have also been reported to induce bacterial tryptophanase activity, which can result in overproduction of indole in the colon. Because indole affects bacterial physiology in a concentration-dependent manner, the level of indole that can be produced by microbiota is noteworthy. For example, 0.5 mM indole affects the motility of bacteria, the formation of biofilm, and the secretion of several virulence factors. Higher indole concentrations (1–2 mM) influence expression of multidrug exporters and several virulence factors; even higher indole concentrations (3–5 mM) can inhibit cell division and affect plasmid stability (Li and Young,). Regarding the relationship among bacterial physiology, intestinal homeostasis and intestinal inflammation, we suggest that several nutritional factors, such as a high-protein or high-Trp diet, may impact intestinal homeostasis and intestinal inflammation via indole as an intermediary. This may constitute an effective approach to increasing the production of indole to modulate intestinal immunity through nutritional regulation. E. coli ^{2013 2013}

In the gut, a portion of Trp can be catabolized to indolic acid derivatives by bacteria, including indole-3-acetic acid (IAA), indole-3-aldehyde (IAld), indole acryloyl glycine (IAcrGly), indole lactic acid, and indole acrylic acid (IAcrA) (Figure) (Keszthelyi et al.,). Several intestinal bacteria, such as, and, can catabolize Trp to tryptamine and indole pyruvic acid, which are then converted to indole-3-acetic acid, indole propionic acid, and indole lactic acid (Smith and Macfarlane,). Indole-3-acetic acid can be further combined with glutamine to produce indolyl acetyl glutamine in the liver or oxidized to indole-3-aldehyde (IAld) through peroxidase-catalyzed aerobic oxidation (De Mello et al.,). Indolyl propionic acid can also be further converted to indolyl acrylic acid (IAcrA) and combined with glycine to yield indolyl acryloyl glycine (IAcrGly) in the liver or kidney (Figure) (Keszthelyi et al.,). 1 ^{2009 1997 1980} 1 ²⁰⁰⁹ Bacteroides, Clostridia E. coli

The effects of several indolic acid derivatives on the gut microbiota and intestinal homeostasis have been reported. Severalspecies are capable of producing IAcrA, which can suppress inflammation by promoting intestinal epithelial barrier function and mitigating inflammatory responses. After LPS stimulation, IAcrA enhances both IL-10 production and mucin gene expression. Mucins can be utilized as an energy source by commensal bacteria, and IL-10 acts as an anti-inflammatory cytokine (Hasnain et al.,). Therefore, IAcrA is suggested to have an important anti-inflammatory function in the intestine, and stimulating IAcrA production to promote anti-inflammatory responses has therapeutic benefits (Wlodarska et al.,).can metabolize Trp into indolyl propionic acid, which protects mice from DSS-induced colitis (Venkatesh et al.,). Indolyl propionic acid significantly enhances IL-10 production (an anti-inflammatory cytokine) after LPS stimulation and reduces TNF production (a proinflammatory cytokine). Indole-3-aldehyde (IAld), which is able to activate ILC3s to produce IL-22 via AhR, is abundantly produced byin the presence of Trp in the gut. Additionally, mutation ofcaused the loss of its capacity to produce IAld and induce IL-22 in the presence of Trp (Romani et al.,). Several indolic acid derivatives are toxic to the microbiota. For instance, certain indolic compounds are known to have bacteriostatic effects on gram-negative enterobacteria, especially the generaand, Furthermore, indolyl acetic acid has been reported to inhibit the growth and survival of, specifically(Nowak and Libudzisz,). Indole-3-aldehyde (IAld), a metabolite produced from Trp by commensal lactobacilli, can act as a ligand of AhR and subsequently activate AhR-dependenttranscription (Zelante et al.,). IL-22 mediates pivotal innate antifungal resistance in mice (De Luca et al.,) and humans (Puel et al.,) and provides colonization resistance against the fungusand mucosal protection from inflammation. Several authors have suggested that indolyl acryloyl glycine (IAcrGly) can increase intestinal epithelial permeability, and it has been hypothesized that increased intestinal permeability is caused by membrane damage induced by the precursor of IAcrGly: indolyl acrylic acid. Such membrane damage results in increased permeability and permeation of compounds that can disrupt normal intestinal homeostasis (Keszthelyi et al.,). Peptostreptococcus Clostridium sporogenes L. reuteri L. reuteri Salmonella Shigella Lactobacillus L. paracasei IL-22 C. albicans ^{2013 2017 2014 2014 2006 2013 2010 2010 2009}

What can influence the production of these derivatives? Increased and prolonged excretion of urinary indolic acid derivatives has been detected in a number of diseases, such as Hartnup disorder, celiac disease and other malabsorption syndromes (Haverback et al.,). However, the effects of increased indolic acid derivatives on the immune system, especially on intestinal immunity, have not been evaluated in these diseases, and doing so may be useful for interpreting pathology and improving available therapies. Additionally, excessive Trp overload in the colon and concomitant gut microbiota alteration has been hypothesized to increase the production of Trp microbiota-derived metabolites, though this is merely a hypothesis. The types of metabolites that can be accelerated and whether other factors can influence production need to be validated. ¹⁹⁶⁰

Skatole is another intestinal Trp microbiota-derived metabolite (Jensen et al.,), the precursor of which is indole-3-acetic acid (IAA) (Figure) (Yokoyama and Carlson,). Intestinal microbiota convert Trp to indole and IAA, and skatole is then synthesized from IAA via decarboxylation (Figure). However, the level of skatole produced is usually low. Compared to other Trp bacterial-derived metabolites, skatole concentrations in the intestine are highly variable, which may be due to the two-step production process mediated by at least two different bacterial species; the concentration of the intermediate IAA may be another rate-limiting factor (Yokoyama and Carlson,)., andcan convert IAA to skatole (Cook et al.,; Whitehead et al.,). The actual site of skatole production in the intestine is likely the small intestine and colon, and skatole can be efficiently absorbed by both. In pigs, the concentration of skatole in the colon is in excess of 30 μg/g (Yoshihara and Maruta,). After oral supplementation with L-Trp, the concentration of skatole in bovine ruminal fluid is ~36 μg/ml. Skatole can influence the growth and reproduction of certain intestinal bacteria and has bacteriostatic effects on gram-negative enterobacteria. The generaandare slightly more sensitive to the bacteriostatic effects of skatole than areandspecies. In dilute solutions, skatole can also inhibit the growth and fermentation of. In this regard, skatole may determine the composition of intestinal microbes and the intestinal microbial ecosystem and protect the ecological niches of the bacteria that produce it (Yokoyama and Carlson,). Regardless, the influence of skatole on host diseases through effects on intestinal microbes is not well-characterized. ¹⁹⁹⁵ 1B ¹⁹⁷⁹ 1B ^{1979 2007 2008 1977 1979} Lactobacillus, Clostridium Bacteroides Salmonella Shigella Escherichia Aerobacter Lactobacillus acidophilus

The production of skatole is associated with both healthy and disease states. The fecal skatole concentration in humans varies considerably and may indicate different health statuses. Fecal skatole levels in healthy individuals are usually ~5 μg/g feces, whereas fecal skatole levels may be as high as 80 to 100 μg/g feces in persons who suffer from disturbed intestinal digestion (Yokoyama and Carlson,). Researchers have suggested that after absorption but before detoxification, skatole may have a damaging effect on the activity and function of intestinal epithelial mucosa (Yokoyama and Carlson,). In cattle, intraruminal and intravenous administration of skatole induces clinical features and lung lesions similar to Trp-induced disease. Skatole has been recognized as a primary cause for Trp-induced disease, which manifests as acute pulmonary edema and emphysema, generally resulting in death (Yokoyama and Carlson,). ^{1979 1979 1979}

With an efficacy equivalent to that of indole, skatole exhibits modest dose-dependent activation to stimulate murine and human AhR. Within the GI tract, abundant generation of skatole may underlie the establishment of an axis to regulate intestinal physiology, which may involve microbiota-indole-AhR-mediated maintenance of intestinal homeostasis throughout a longer lifespan (Hubbard et al.,). In another report, skatole has been described as an inhibitory factor for, leading to decreased formation of pregnenolone, which is the precursor of mineralocorticoids, glucocorticoids, and sex steroids (Mosa et al.,). In the gut, synthesis of endogenous steroid hormones, such as the anti-inflammatory glucocorticoid cortisol, is critical for the maintenance of intestinal homeostasis (Bouguen et al.,). Along with reducedexpression (Coste et al.,) and decreased glucocorticoid production (Huang et al.,), disorders of intestinal steroidogenesis have been associated with IBD. Skatole has also been suggested to play a role in the disturbance of intestinal homeostasis and in the development of IBD via inhibition ofexpression and glucocorticoid production. [2015a] ^{2016 2015 2007 2014} CYP11A1 CYP11A1 CYP11A1

Nutrients are important factors that influence the concentrations of skatole in the intestine and other tissues. As indicated above, oral supplementation with Trp can induce high concentrations of skatole in bovine ruminal fluid. Sugar concentrations in the intestine are another factor. Decarboxylase is a key enzyme in the second step of skatole production, and under induction-repression regulation, sugar concentrations in the intestine have a significant impact on decarboxylase activity and subsequently influence skatole production (Yokoyama and Carlson,). Antibiotic use is another important factor influencing skatole levels. The most sensitive regulator of the microbial population and conversions mediated by bacteria in the gut is the application of antibiotics (Engberg et al.,). For instance, pigs fed zinc bacitracin have reduced skatole concentrations in the blood and backfat compared with non-supplemented control pigs (Hansen et al.,). In addition, significant gender differences in both indole and skatole concentrations in the blood and backfat were observed (≤ 0·001) (Hansen et al.,). As skatole stimulates physiological responses in a dose-dependent manner, maintaining appropriate concentrations in the gut is beneficial for maintaining the intestinal microbial ecosystem, intestinal homeostasis and intestinal health. ^{1979 2000 2000 2000} P

Tryptamine is produced by the decarboxylation of Trp, which is common in the plant kingdom but rare in bacteria (Figure) (Williams et al.,). The common gut Firmicutesandare capable of decarboxylating Trp to tryptamine (Williams et al.,). Although Trp decarboxylation is rare in bacteria, the Human Microbiome Project demonstrated that at least 10% of the human population possesses at least one bacterium encoding a Trp decarboxylase among the intestinal microbiota (Williams et al.,). Therefore, the generation and physiological role of tryptamine in the gut is non-negligible. 1A ^{2014 2014 2014} C. sporogenes Ruminococcus gnavus

Tryptamine, a β-arylamine, is a neurotransmitter with documented effects on intestinal motility that acts on the enteric nervous system to modulate intestinal homeostasis (Wlodarska et al.,). However, tryptamine can induce ion secretion by intestinal epithelial cells. In an experiment using an Using chamber, 3 mM tryptamine induced a marked change in short-circuit currents, indicating that it can influence colonic ion secretion, which subsequently regulates GI motility. It is hypothesized that tryptamine-mediated signaling might affect the transit of food particles and bacteria through the gut lumen (Williams et al.,). Tryptamine can also reduce the invasion and colonization capabilities of enteric pathogens, such asserovar Typhimurium (Davis,). In addition, tryptamine exerts inhibitory activity against IDO1, which then influences immune surveillance. Upregulation of IDO1 is reported to be associated with the escape of malignant cells from immune surveillance (Muller and Scherle,; Katz et al.,), and inhibition of IDO1 activity is regarded as an important target in interventions related to immune escape (Whiteside,). Therefore, effects of tryptamine on IDO1 inhibition may contribute to a more effective tumor-reactive response by immune cells, which is considered part of a viable strategy for anticancer therapies (Tourino et al.,). Similar to other Trp metabolites, tryptamine acts as a ligand for AhR and activates AhR to regulate intestinal immunity (Islam et al.,). In turn, AhR plays a role in regulating tryptamine production when the intestinal balance is disturbed. For instance, in wild-type (WT) and-knockout (KO) mice, the concentrations of tryptamine in feces were similar in both Trp diet groups before DSS treatment. However, after DSS treatment, colonic tryptamine levels were markedly lower inKO mice compared to WT mice, which might be due to dysbacteriosis induction in the former (Islam et al.,). In addition, tryptamine is a ligand for trace amine-associated receptors (TAARs) and potentiates the inhibitory response of cells to serotonin (Zucchi et al.,). Tryptamine can also induce the release of serotonin (Takaki et al.,). Fluctuation in intestinal serotonin levels can modulate GI motility (Lundgren,; Turvill et al.,) and is also involved in the pathology of IBD (Linden et al.,,; Bischoff et al.,). ^{2015 2014 2014 2006 2008 2006 2013 2017 2017 2006 1985 1998 2000 2003 2005 2009} Salmonella enterica Ahr Ahr

Alteration of tryptamine production is mediated by Trp-microbial metabolism. The concentration of tryptamine in feces increases ~3-fold in conventional vs. GF mice (Marcobal et al.,). Although only a small fraction of Trp is converted to tryptamine by the intestinal microbiota, levels of tryptamine can be drastically increased after Trp supplementation (Vikström Bergander et al.,). Indeed, a Trp-supplemented diet can increase tryptamine levels compared with a control diet. For instance, the concentrations of tryptamine in the colon and serum were found to be increased in Trp-supplemented diet mice compared to control diet mice (Islam et al.,). On the basis of these studies, Trp can be absorbed from the diet by microbes and then converted to tryptamine; the type and distribution of Trp metabolites are altered and influence the normal physiological function of the host intestine. ^{2013 2012 2017}

Spondyloarthritis (SpA) occurs in approximately one percent of the population in the United States (Lawrence et al.,). Half of SpA patients can have intestinal inflammation but not overt gastrointestinal symptoms (also called subclinical inflammation) (Vereecke and Elewaut,). Based on the close relationship observed between SpA and intestinal inflammation, the microbiota has been suggested to be a potential factor in influencing immune responsiveness in SpA patients (Fantini,; Scher et al.,). In pediatric or adult SpA patient feces, taxonomic differences in bacteria have been identified compared to healthy individuals (Costello et al.,; Stoll et al.,). ^{2008 2017 2009 2015 2014 2014}

The influence of the gut microbiota in SpA has been studied (Lawrence et al.,; Costello et al.,; Stoll et al.,). With the decreased gut microbial diversity in pediatric SpA, relatively reduced Trp metabolism and lower levels of Trp metabolites in patients have been reported. The decrease in Trp metabolites in SpA patients is consistent with a similar finding in the synovial fluid of rheumatoid arthritis patients (Kang et al.,). Changes in fecal metabolomics and gut microbiota differences in Trp metabolism in pediatric SpA have been ascribed to alteration in the gut microbiota (Stoll et al.,). In the Trp metabolism pathway, 21 unique Trp metabolites were found to be reduced in the feces of SpA patients, as detected by charged ions, including the following: three bacterial Trp metabolites (indole-3-acetate, indole-3-acetaldehyde, and methyl indole-3-acetate); eight metabolites of kynurenine (such as 3-hydroxy-L-kynurenine, anthranilate, glutaryl-CoA, kynurenate); and six metabolites of 5-hydroxy-Trp. In addition, 16S sequence data of the fecal microbiome verify the relative decrease in Trp metabolism in SpA patients compared with controls (Stoll et al.,). Based on the above-described reports, the gut microbiota in enthesitis-related arthritis (one type of SpA) patients potentially acts as a proinflammatory factor due to altered Trp catabolism. As reported above, quite a lot of these Trp metabolites are associated with intestinal immune and intestinal inflammation, but it remains to be investigated which ones play dominant roles in causing subclinical inflammation in SpA. At the same time, which genes and gut microbes that target the specific lesser Trp metabolites should be further analyzed. ^{2008 2014 2014 2015 2016 2016}

IBS is one of the most commonly diagnosed chronic functional GI disorders, with a prevalence of ~15% in western populations (Soares,). The pathophysiology of IBS is related to intestinal epithelial cells, enteroendocrine cells, neuronal cells, and immune cells. Alterations in gut microbiota diversity and composition have been implicated in IBS. Focusing on the symptomatology of IBS, changes in the intestinal microflora balance are an important factor (Nakai et al.,; Spiller,; Bhattarai et al.,). Increases in thetoratio (Clarke et al.,) and the abundance ofandspecies are observed in IBS patients (Hong and Rhee,), whereasandpopulations (Balsari et al.,) are decreased. ^{2014 2003 2008 2017 2009 2014 1982} Firmicutes Bacteroidetes Streptococcus Ruminococcus Lactobacillus Bifidobacterium

In addition, IBS is associated with increased Trp metabolism via KP (Jenkins et al.,). The kynurenine:Trp ratio is positively related to symptom severity in IBS (Fitzgerald et al.,), and IFN-γ activation and subsequent IDO1 oxidation of Trp may be a pathogenic mechanism of IBS (Fitzgerald et al.,). In addition, dysfunction of the serotonergic system is associated with the pathophysiology of IBS. Serotonergic modulation through acute Trp increase treatment in IBS patients induced more severe GI symptoms compared with acute Trp depletion treatment (Kilkens et al.,; Shufflebotham et al.,). In IBS, the severity of symptoms and alterations of both gut and brain serotonin concentrations are associated with changes in the microbiota balance (Jenkins et al.,). IBS patients have lower serotonin concentrations in the small intestine, but colonic serotonin production can be promoted by effects of bacterial products on enterochromaffin cells, such as SCFAs (Reigstad et al.,). At the genetic level, microbiota from humanized and conventional mice increased colonic Trp hydroxylase 1 () (a rate-limiting enzyme for mucosal 5-HT synthesis) expression through the stimulatory activities of SCFAs. For instance, butyrate, a SCFA, can activeexpression in mice through the inducible zinc finger transcription factor ZBP-89 (Essien et al.,). In the intestinal tract, nearly all SCFAs are produced through bacterial fermentation. Concentrations of SCFAs in the cecum of GF mice were reported to be ~1 mmol/kg, whereas average concentrations in the conventional mouse cecum are ~125 mmol/kg. Moreover, the variety and concentrations of SCFAs in human feces vary widely among individuals, which may be attributable to the complex microbial community or dynamic diet composition (Høverstad and Midtvedt,). ^{2016 2008 2008 2004 2006 2016 2015 2013 1986} Tph1 Tph1

Microbial-origin stimuli and the gut serotonergic system may act as key factors influencing the symptomatology of IBS. To alleviate GI symptoms through serotonergic modulation in IBS patients, theeffects of local concentrations and proportions of different colonic SCFAs on mucosal 5-HT homeostasis should first be investigated, as SCFAs affectexpression in a concentration-dependent manner; how to balance mucosal 5-HT homeostasis by manipulating the complex microbial community is challenging for this strategy. In addition, more data about the coadjustment between SCFAs and gut serotonergic system are required. in vivo Tph1

IBD, defined as a chronic, remitting, and relapsing inflammatory disorder of the gut, includes Crohn's disease (CD), and ulcerative colitis (UC) (Matsuoka and Kanai,; Lamas et al.,). Multifaceted factors, including dysregulation of the mucosal immune system, an unbalanced gut microbial community, and disruption of the mucosal barrier, are related to the pathogenesis of IBD (Flint et al.,; Pillai,; Kostic et al.,; Matsuoka and Kanai,; Lamas et al.,; Liu et al.,). Of these factors, the gut microbial community is gaining more attention due to its influence on intestinal health. ^{2015 2017 2012 2013 2014 2015 2017 2017}

The pathogenesis of IBD is associated with alterations in the composition of the intestinal microbiome, but whether these alterations are causal or a result of inflammation in IBD is still under dispute (Gkouskou et al.,). In several reports, the gut microbiota has been considered to trigger inflammation in IBD (Sartor,), as short-term treatment with antibiotics can markedly alleviate intestinal inflammation (Sartor,; Kostic et al.,). In addition, an altered microbiome and altered interactions between intestinal microbes and mucosal immunity have been suggested to cause increased intestinal permeability and inflammation in IBD (Sartor,; Hold et al.,). ^{2014 2008 2004 2014 2004 2014}

Reductions in bacterial diversity have been reported in IBD patients (Martin-Subero et al.,), and these are accompanied by increased intestinal permeability and enhanced intestinal bacteria infiltration, which can induce immune responses and ultimately systemic inflammation (Xavier and Podolsky,; Martin-Subero et al.,). IBD patients exhibit reduced intestinal mucosal barriers and decreased mucin glycosylation, and the disproportionate increase in several mucolytic microbes in IBD is suggested to be partly due to bacterial adaptation to the altered mucin glycosylation (Png et al.,). Several specific bacteria have been associated with IBD. For example, a decrease in(Frank et al.,) and(Nemoto et al.,) levels are reported in IBD patients, as were increases inandlevels (Rowan et al.,; Jia et al.,). A positive correlation between the invasive potential ofand the severity of IBD in the host was also found (Strauss et al.,), suggesting that invasive strains ofmay influence IBD pathology (Kostic et al.,). In contrast, several specific species of gut bacteria may have protective effects against IBD (Kostic et al.,). For instance, species of, andmay protect the host from mucosal inflammation by downregulating inflammatory cytokines or stimulating expression of IL-10, an anti-inflammatory cytokine (Sokol et al.,; Llopis et al.,). ^{2015 2007 2015 2010 2007 2012 2010 2012 2011 2014 2014 2008 2009} Bacteroidetes Bacteroides Desulfovibrio Bilophila Fusobacterium Fusobacterium Bifidobacterium, Lactobacillus Faecalibacterium

IBD is also associated with altered host and gut bacterial Trp metabolites. Plasma levels of kynurenine and KA are increased (Forrest et al.,) and concentrations of plasma Trp are decreased in IBD patients, and increased IDO1 activity has been reported in the peripheral blood and colon cells of IBD patients (Munn and Mellor,; Martin-Subero et al.,). In IBD, increased pro-inflammatory cytokines, including IFN-γ, IL-1, and IL-6, have been suggested to induce Trp catabolic pathways to reduce plasma Trp levels and increase Trp catabolite levels (Martin-Subero et al.,). In CD patients and IBD animal models, increased 5-HT levels are found in inflamed mucosa, suggesting the important role of 5-HT in driving intestinal inflammation in IBD (Levin and Van Den Brink,). Several specific gut bacterial Trp metabolites are also involved in the pathophysiology of IBD (Matsuoka and Kanai,). In dogs with IBD, bacterial Trp metabolites (indole acetate and indole propionate) that are suggested to have anti-inflammatory functions in the intestine are significantly decreased (Honneffer et al.,). In IBD patients, levels of IAA (anti-inflammatory function in the intestine) are reduced in feces, suggesting that reduced bacterial Trp metabolism may contribute to the etiology of IBD (Lamas et al.,). Furthermore, in IBD patients, the abundance of bacteria that can cleave terminal fucose residues from intestinal mucins by utilizing α-L-fucosidases is significantly reduced, correlating with the reduced production of indole acrylic acid and indole-3-propionic acid from Trp (Wlodarska et al.,). Studies have also suggested a strategy of increasing indole acrylic acid production by restoring bacterial Trp metabolism in the intestine to promote anti-inflammatory responses in IBD patients, which may have beneficial therapeutic effects. ^{2003 2007 2015 2015 2014 2015 2015 2016 2017}

CRC is the third-most prevalent cancer that causes mortality. The effects of the gut microbiota on CRC progression have been examined. GF animals have lower tumor burdens compared to conventionally raised counterparts. Similarly, depletion of the gut microbiota through antibiotics has also been shown to reduce CRC occurrence (Grivennikov et al.,). Alterations in gut microbiota composition have been found in murine tumorigenicity studies. In CRC patients, the fecal microbiota demonstrate decreased temporal stability but increased diversity ofandsubgroups compared with the control group (Grivennikov et al.,). ^{2012 2012} Clostridium leptum Clostridium coccoides

Trp metabolism via KP has proven to be a potent target for immunotherapy. KP is activated in multiple tumor types; IDO1 initiates KP and is expressed in the primary tumor and in infiltrating myeloid-derived cells in CRC (Uyttenhove et al.,; Ferdinande et al.,; Théate et al.,). IDO1 appears to be chronically activated in cancer patients (Huang et al.,; Weinlich et al.,). IDO1 is commonly overexpressed in CRC and often accompanied by reduced Trp levels and increased levels of KP metabolites (Liu et al.,; Walczak et al.,; Engin et al.,). As introduced in above, IDO1 plays an essential role in maintaining microbiota diversity, and IDO1 activation can reduce microbial proliferation. Hence, we suggest that overexpression of IDO1 in CRC may be the cause of alterations and decreased temporal stability of the gut microbiota. As depletion of the gut microbiota can reduce CRC occurrence, decreasing the microbial proliferation induced by IDO1 activation may constitute self-regulation as a defense against CRC. In fact, research has proven that blockage of IDO1 activity can directly enhance the immune capacity of tumor-bearing mice against the tumors (Uyttenhove et al.,; Muller et al.,). However, the influence of the blockage of IDO1 activity on the mouse gut microbiota was not discussed, and it remains unclear whether the mouse gut microbiota played a role in this IDO1-blockage treatment. Regardless, selective inhibition of IDO1 is proposed to upregulate cellular immunity, with therapeutic potential in cancer, including CRC, and the influence of IDO1 on the gut microbiota is a factor that must be taken into account. ^{2003 2012 2015 2002 2007 2010 2011 2015 2003 2005}

IDO1 also functions as a mediator in host-microbe crosstalk, which has effects on the pathology of GI cancer. When mediating host-microbe interactions, expression of IDO1 can be induced by activation of several binding pattern recognition receptors (PRRs), especially TLR4 and TLR9 (Ciorba et al.,; Santhanam et al.,). For example, TLR4 can be activated by LPS, a primary bacterial toxin. The ability of LPS to activate PRRs and then IDO1, reducing the generation of LPS and other similar toxins by controlling gut microbes, may be important for reducing the risk of GI cancer. Furthermore, new antagonists for PRRs to reduce expression of IDO1 may be beneficial against tumors. Indoles, as Trp microbial metabolites, have particular relevance for Trp metabolism and CRC (Raman et al.,), and several indoles derived from dietary Trp can activate AhR, which has important roles in intestinal homeostasis and controlling GI cancer. The influence of the gut microbiota and Trp metabolism on CRC pathogenesis will guide the development of targets and new approaches for the prevention and treatment of CRC. ^{2010 2016 2013}