Host–microbiome interactions: the aryl hydrocarbon receptor as a critical node in tryptophan metabolites to brain signaling

ABSTRACT

Tryptophan (Trp) is not only a nutrient enhancer but also has systemic effects. Trp metabolites signaling through the well-known aryl hydrocarbon receptor (AhR) constitute the interface of microbiome-gut-brain axis. However, the pathway through which Trp metabolites affect central nervous system (CNS) function have not been fully elucidated. AhR participates in a broad variety of physiological and pathological processes that also highly relevant to intestinal homeostasis and CNS diseases. Via the AhR-dependent mechanism, Trp metabolites connect bidirectional signaling between the gut microbiome and the brain, mediated via immune, metabolic, and neural (vagal) signaling mechanisms, with downstream effects on behavior and CNS function. These findings shed light on the complex Trp regulation of microbiome-gut-brain axis and add another facet to our understanding that dietary Trp is expected to be a promising noninvasive approach for alleviating systemic diseases.

Introduction

Tryptophan (Trp) is not only a nutritional enhancer but also serves as a key prerequisite that aligns gastrointestinal physiology and central nervous system (CNS) function. This process is achieved through the regulation of indole pathway, kynurenine (Kyn) pathway and serotonin (5-hydroxytryptamine, 5-HT) synthesis.^1,2 Being metabolized into numerous bioactive metabolites³ (Table 1), Trp metabolism has become part of the cellular and organismal communication strategies. These intermediates can serve as ligands for aryl hydrocarbon receptor (AhR)^12,13 (Table 2). In recent years, the focus on AhR has shifted to its mode of action in response to physiological ligands. After activation, AhR has been shown to participate in a broad variety of physiological and pathological processes, which not only focus on intestinal homeostasis, but are also highly to the autoimmune and neoplastic diseases of CNS.²⁸ Recent research has shown that AhR can suppress proinflammatory cytokines in astrocytes²⁹ and microglia³⁰ that has potential to be a novel factor of interest for several brain diseases such as plasticity,³¹ alzheimer’s disease (AD)³² and epilepsy³³ in “gut-brain” axis.

Table 1.

AhR ligands derived from endogenous tryptophan (Trp) metabolism and roles in intestinal homeostasis.^a

Trp metabolites	Relation to AhR	Roles in intestinal homeostasis	References
Kynurenine	Human, mouse, rat and pig AhR activator	Serous kynurenine levels are elevated in a CD mouse model and IBS patients	4,5
Kynurenic acid	Human and mouse AhR activator	Kynurenic acid levels are decreased in the duodenal mucosa but enhanced in plasma from IBS patients	6,7
Cinnabarinic acid	human and mouse AhR activator	Modulates the gut immune response by inducing AhR-dependent IL-22 production; protects against oxidative stress-induced apoptosis by activating AhR	8,9
Xanthurenic acid	Human AhR agonist	Promotes tumor cell migration; a decrease in parasite development caused by IDO1 inhibition is entirely rescued by xanthurenic acid; elevated xanthurenic acid urinary levels are detected in a CD mouse model	6,10,11
Serotonin	Human and mouse AhR activator	Mucosal serotonin can facilitate intestinal inflammation; reduction of serotonin levels in the intestine is related to intestinal homeostasis disturbances	7

^aAhR, aryl hydrocarbon receptor; CD; Cohn’s disease; IBS, irritable bowel syndrome; IDO1, indoleamine 2,3-dioxygenase 1; IL-22, interleukin-22

Table 2.

AhR ligands derived from microbial tryptophan (Trp) metabolism and roles in intestinal homeostasis.

Trp metabolites	Relation to AhR	Roles in intestinal homeostasis	References
Indole	Human, mouse and rat AhR ligand	Strengthens the gut mucosal barrier and mucin generation; increases IL-10 expression and reduces inflammatory indicators	14
FICZ	Human and mouse AhR agonist	Plays a protective role in IL-6-induced intestinal epithelial barrier injury; improves DSS-induced colitis in mice	15-17
TrA	Human and mouse AhR agonist	Reduces pro-inflammatory cytokine-induced permeability in a T84 cell monolayer	18
IAA	Human and mouse AhR activator	A uremic toxin that can serve as a predictor of CKD; induces endothelial inflammation and oxidative stress in vitro	19-21
IAld	Mouse AhR ligand	Induces IL-22 production, resists fungal infections and balances mucosal reactivity in mice	20
3-methylindole (skatole)	Human AhR activator	A pulmonary toxin; is also responsible for the fecal odor in pig carcasses; the roles of skatole in intestinal functions remain unclear	22
IS	Human and mouse AhR agonist	This uremic toxin correlates with the development of CKD and cardiovascular disease; urinary IS level is a common marker of intestinal dysbiosis	23,24
Indigo	Human, mouse and rat AhR activators	Ameliorates chemically induced murine colitis; activates the IL-22 pathway via AhR	25
Indirubin	Human, mouse, and rat AhR ligand	Indirubin-activated AhR accelerates IL-22 production by mucosal type 3 innate lymphoid cells, thus inducing the generation of antimicrobial peptides and tight junction proteins	26,27

^aAhR, aryl hydrocarbon receptor; CKD, chronic kidney disease; DSS, dextran sulfate sodium; FICZ, 6-formylindolo[3,2-b]carbazole; IAA, indole-3-acetic acid; IAld, indole-3-aldehyde; IL, interleukin; IS, indoxyl sulfate (indicant); TrA, tryptamine.

Gut microbiota and CNS are connected via multiple bidirectional pathways, including neural (vagus), metabolism, and immune signaling.³⁴ Microbe-derived neuroactive metabolites include Trp precursors and metabolites, which are secreted into the circulation and traffic to the CNS.³⁵ In turn, CNS is capable of shaping microbial function and composition by regulating neurotransmitters to achieve bidirectional communication.

In this article, we review the most recent insights regarding the Trp metabolism in “microbiome-gut-brain” axis, in which, AhR serves as a critical node in microbiota to brain signaling. In the gut, under the direct or indirect regulation of the microbiota, the three major Trp metabolism pathways lead to serotonin, Kyn, and indole derivatives, some of which are ligands for AhR.^3,36,37 These derivatives involved in immune, metabolic, and neural (vagal) communication mechanisms in “microbiome-gut-brain” axis under AhR regulation is reinforced discussed, with a focus on the consequences on both physiology and diseases.

The basic recognition of AhR

AhR belongs to xenobiotic receptors (XRs). It has functionally evolved into cellular sensor for both endogenous and exogenous stimuli³⁸ to regulate the clearance and detoxification of xenobiotics.³⁹ Unlike membrane-bound receptors, most XRs shuttle between the cytoplasm and nucleus. They modulate the expression of target genes involved in cell proliferation, metabolism, and immune responses.³⁸ XR transcriptional activity is regulated by binding to diverse and low-affinity small molecules through a highly conserved ligand-binding domain (LBD).⁴⁰ AhR individually and/or interactively influences cellular metabolism and their activation also play an important role in “gut-brain” axis. Thus, understanding its basic feature is a key prerequisite for exploring the role of AhR in regulating host homeostasis via “microbiota-gut-brian” connection (Figure 1).

Figure 1.

The role of AhR activated by Trp metabolites in host homeostasis. Tryptophan (Trp) can be catabolized into a number of bioactive molecules through microbial metabolism, enzymatic catalysis, and exposure to sunlight, most of which are identified as aryl hydrocarbon receptor (AhR) ligands that bind directly to AhR. Gut bacteria can utilize tryptophan (Trp) as a nitrogen source and certain microbes also have the ability to synthesize Trp and influence Trp metabolism to generate ligands. Upon activation by ligands, AhR translocated from the cytoplasm into the nucleus and dimerized with AhR nuclear translocator (ARNT). This heterodimer then localized to the promoters of AhR target genes to induce gene expression. Meanwhile, AhR activation is linked to the expression of inflammatory cytokines, including IL-6, IL-10, and IL-22, and production of CYP genes such as CYP1 and CYP3A, all of which play important roles in modulating host homeostasis.

Structures and main features of AhR

AhR is a ligand-controlled transcription factor with a basic helix-loop-helix (bHLH) and per-AhR nuclear translocator (ARNT)-Sim domains, which are highly conserved during vertebrate evolution. A wide range of compounds, such as endogenous amino acid derivatives have been shown to regulate the expression of target genes by acting as AhR agonists or inhibitors.^25,41 As a nucleocytoplasmic shuttling protein, AhR is mainly located in the cytoplasm as a complex in the absence of AhR ligands. This complex contains a 90-kDa heat shock protein (Hsp90), the Hsp90-interacting protein p23 and AIP (also known as XAP2 and ARA9).⁴² When exposed to ligands, AhR immediately binds to these chemicals. The translocation of AhR from the cytoplasm into the nucleus is then initiated. In the nucleus, AhR dissociates from Hsp90, p23, and AIP and forms a heterodimer with ARNT. The AhR-ARNT dimer then binds to the promoter of target genes and activates the transcription of genes such as cytochrome P450 (CYP)1A1, CYP1A2 and CYP1B1,⁴³ which in turn promote feedback to regulate AhR activity by affecting AhR receptor metabolism⁴⁴ (Figure 1).

AhR involved in the signaling from gut to brain

AhR: AhR participates in a broad variety of physiological and pathological processes that are highly relevant to intestinal homeostasis, autoimmune, and neoplastic diseases of CNS. The reduced level of AhR agonists that derived from intestinal microbiota has been reported in multiple conditions, including IBD,⁴⁵ metabolic syndrome,¹² and CNS diseases.^29,46 AhR has involved in the regulation of multiple organ functions but its mRNA expression differs between tissues,⁴⁷ its abundant expression in the gut indicates its important roles in regulating intestinal function. As reported, AhR regulates intestinal epithelial cell (IEC) regeneration, preventing malignant outgrowth,^28,48 while deficits in the levels of AhR or its ligands significantly decrease the number of intestinal IELs and increases epithelial damage.⁴⁹ With a special focus on the immune system, AhR activation has multiple effects in dendritic cells (DCs) and T cells, which inhibits induction of cytokines that promote polarization of pathogenic T cell subsets and reduces the expression of MHC class II in DCs^28,50.

AhR has been regarded as a key inducer of CYPs.⁵¹ CYP1 gene expression can be induced by AhR upon exposure to intra- and/or extra-cellular ligands.⁴³ The microbiota-derived metabolite indoxyl sulfate (IS) is a potent endogenous AhR ligand that is reported to regulate the transcription of various genes, including CYP1A1, CYP1B1, CYP1A2, and IL-6.²³ Meanwhile, CYP1 enzymes also play key roles in attenuating intracellular AhR activation by oxidizing AhR ligands such as 6-formylindolo[3,2-b]carbazole (FICZ).⁴⁴ However, excessive CYP1A1-induced metabolic clearance of AhR agonists resulted in an impaired AhR-dependent intestinal immune system, such as the loss of type 3 innate lymphocytes in the small intestine and colon as well as increased susceptibility to gut pathologies.⁴³ In CYP1 enzyme-deficiency (Cyp1a1/1a2/1b1-/-) Th17 cells, AhR-dependent IL-22 production was increased, which suggested that disruption of CYP1 function may modify AhR-orchestrated immune response.⁴³

As the cognition of “microbiome-gut-brain” axis grows, AhR activation offers a therapeutic avenue for the regulation of CNS inflammation. In astrocytes and microglia, AhR suppresses pro-inflammatory nuclear factor-κB (NF-κB) signaling.^28,52 On the basis of the potential function of some microbial metabolites on CNS through AhR-dependent mechanisms, the role of AhR in the modulation of inflammation-promoting activities of microglia and astrocytes by the commensal microbiota has been investigated recently.

Trp metabolites act as ligands of AhR

AhR ligands generated in the kynurenine pathway

The Kyn pathway is the dominant Trp metabolic pathway. Approximately 90% of ingested Trp is degraded along this pathway in both immune and epithelial cells.^53,54 N-formylkynurenine, which is generated through Trp degradation by Trp 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2,⁵⁵ is further catabolized into Kyn by Kyn formamidase. Kyn and one of its downstream products, kynurenic acid (KA), have been demonstrated to play an important role in cancer and the immune system by serving as an AhR agonist.^6,56 To date, only one experimental study has shown that xanthurenic acid, a Kyn catabolite, can activate human AhR in a TDO-dependent manner. Excess xanthurenic acid and over-expressed TDO can facilitate the migration of tumor cells, but these cells are inoperative when treated with AhR inhibitors.¹⁰ Another downstream metabolite of Kyn, 3-hydroxyanthranilic acid, which is oxidized into cinnabarinic acid, is also an endogenous AhR ligand and can protect cells against apoptosis that is induced by oxidative stress in an AhR-dependent manner.⁸ However, in addition to Kyn and KA, there are no published studies investigating the role of other AhR ligands derived from the Kyn pathway on CNS function (Figure 2).

Figure 2.

Key Trp metabolites and enzymes in the kynurenine (Kyn) pathway. Tryptophan (Trp) is first catabolized into N-formylkynurenine, a precursor of kynurenine, by tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase 1/2 (IDO1/2). Gut bacteria (such as Ruminococcus gnavus) and inflammatory stimuli (such as IFN-γ) can induce IDO expression, thus impacting Trp metabolism along the Kyn pathway. So far, the identified aryl hydrocarbon receptor (AhR) ligands derived from the Kyn metabolic pathway, including Kyn, kynurenic acid, xanthurenic acid, and cinnabarinic acid, are highlighted with a red outline.

AhR ligands produced in the serotonin pathway

Serotonin is a pivotal neurotransmitter that is present in the gut (~95%) and CNS (~5%).⁵⁷ It is produced in enterochromaffin (EC) cells via Trp hydroxylase 1 (TpH1).⁵⁸ Moreover, it has also been reported that indigenous spore-forming bacteria present in mouse and human microbiota could accelerate serotonin generation in colonic EC cells.^58,59 These cells carry TpH1, an enzyme that is responsible for the degradation of Trp into 5-hydroxytryptophan, a short-lived metabolite that is further decarboxylated to serotonin by aromatic amino acid decarboxylase (AAAD). Moreover, certain microbiota can directly utilize Trp to synthesize serotonin in vitro.⁶⁰ The serotonin can be further degraded along two metabolic pathways by different enzymes, whereby monoamine oxidase (MAO) is responsible for the metabolic conversion of most serotonin to 5-hydroxyindoleacetic acid (5-HIAA). In another serotonin catabolism route, melatonin can be produced through the decomposition of serotonin in the pineal gland (Figure 3).

Figure 3.

Major Trp catabolites generated in the serotonin pathway. Both gut microbes and enterochromaffin cells can impact the synthesis of serotonin (5-hydroxytryptamine, 5-HT) by regulating the rate-limiting enzyme tryptophan (Trp) hydroxylase (TPH), which catabolizes Trp into the serotonin precursor 5-hydroxytryptophan. Serotonin is then converted into melatonin or 5-hydroxyindoleacetic acid by different enzymes. In the serotonin pathway, serotonin is the only reported aryl hydrocarbon receptor (AhR) ligand. AADC, aromatic amino acid decarboxylase and MAO, monoamine oxidase.

Serotonin is an endogenous agonist of human AhR.⁶¹ Abnormal serotonin metabolism within the gut correlates to gastrointestinal diseases, such as irritable bowel syndrome.⁶² In addition to serotonin itself, its catabolites, such as 5-HIAA, are potential AhR ligands.⁶³ Melatonin has been reported to modulate a number of physiological functions, notably circadian rhythm, free radical scavenging, and oxidation resistance.⁶⁴ However, whether melatonin can directly act as an AhR ligand remains to be elucidated.

AhR ligands derived from the indole pathway

Indole and its derivatives generated by gut microorganisms

Gut microbiota expressing tryptophanase can catabolize Trp into indoles, which are important signaling molecules that modulate intestinal health.¹⁴ Numerous studies have shown that indole and indole-containing chemicals can activate AhR.^25,65 IAA is a natural AhR ligand derived from Trp fermentation by gut bacteria; its generation can be divided into the following three major pathways: the indole-3-pyruvic acid (IPyA), tryptamine, and indole-3-acetamide (IAM) pathways.⁶⁶ In the context of Trp, aminotransferases can increase AhR activity via the metabolic formation of IPyA, a compound that can be converted into a number of AhR activators, such as FICZ, IAA, 3-methylindole (skatole), and IAld. Tryptamine generated through the degradation of Trp by decarboxylase is also a bacteria-derived AhR ligand.⁶⁷ Moreover, an uremic toxin, IS is produced by the microbial metabolism of indoles and is also a potent endogenous ligand that can activate human AhR directly ²³ (Figure 4).

Figure 4.

The indole-containing chemicals produced by the microbial metabolism of Trp. In addition to the above endogenous metabolic pathways, tryptophan (Trp) can be decomposed into a wide range of indole-containing metabolites by commensal bacteria. Most of these microbial metabolites are recognized as aryl hydrocarbon receptor (AhR)-selective agonists, including the renal toxins indoxyl sulfate, indigo indole-3-acetic acid, indole-3-aldehyde, and 3-methylindole, all of which are highlighted in the red outline. The high-affinity AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ) can be generated by exposing Trp to visible and ultraviolet (UV) light or through an enzymatic formation pathway independent of UV irradiation. ArAT, aromatic amino acid aminotransferases; TMO, tryptophan 2-monooxygenase; TNA, tryptophanase; and TrD, tryptophan decarboxylase.

Trp photoproducts

Trp is the most powerful near-ultraviolet (UV) absorbing amino acid. Upon exposure to visible or UV light, Trp can be converted to photoproducts, including FICZ and 1-(1 H-indol-3-yl)-9 H-pyrido[3,4-b]indole (IPI), both of which have been reported for their AhR-inducing properties.⁶⁸ AhR activated by FICZ could protect the intestinal epithelial barrier from the destruction caused by tumor necrosis factor-alpha (TNF-α)/IFN-γ,^15,69 demonstrating that FICZ may be developed as a potent drug for resistance to intestinal diseases. Recent studies also show that FICZ can be formed through novel light-independent pathways¹⁶ (Figure 4). In the absence of light, the oxidant hydrogen peroxide (H₂O₂) is capable of converting Trp to FICZ. During the enzymatic hydrolysis of Trp, one of the Trp catabolites, indole-3-pyruvate (I3P), is spontaneously decarboxylated to generate indole-3-acetaldehyde (I3A), which subsequently dimerized to form FICZ and its oxidation product, indolo[3,2-b]carbazole-6-carboxylic acid (CICZ).¹⁶ Moreover, another Trp downstream compound, tryptamine, can also be converted into I3A by monoamine oxidase.¹⁶ This suggests that an endogenous FICZ production pathway exists in the absence of light. Cellular FICZ can be cleared by CYP1A enzymes, which is an important part of the negative feedback loop to inhibit excessive and/or prolonged AhR activation.⁴⁴ Thus, FICZ is a transient AhR activator in the cell system. However, some I3P derivatives produced during FICZ formation process can inhibit CYP1A1, thereby enhancing the potency of FICZ and thereby increased its power as an AhR activator.¹⁶

Additionally, IPI has an equivalent CYP1A-inducing efficacy to FICZ.⁶⁸ IPI-induced 7-ethoxyresorufin demethylase (EROD) activity notably decreased in AhR-defective c35 cells, supporting that the IPI response depends on AhR.⁶⁸ However, the AhR-activating efficacy of IPI in vivo and its mechanism are unclear. Additionally, the function of the IPI-AhR pathway remains unclear.

The AhR as a critical node in Trp metabolites to brain signaling

It is well established that gut-brain axis is a fundamental component underlying health and diseases.⁷⁰ The effects of the microbiota are not limited to the intestine but also signal to the brain influencing CNS inflammation and being involved in neuropsychiatric disorders.⁷¹ The maintenance of a mutualistic state between them is actually in the action of Trp metabolites through AhR.

Trp metabolites in the intestine regulates “microbiome-brain” axis via AhR-dependent immunity pathway

The three major pathways of Trp metabolism in the gut produces serotonin, Kyn, and indole derivatives are under the direct or indirect regulation of the microbiota,³ which is involved in the regulation of host immunity by activating AhR.⁷² AhR expressed in many host cell types including leukocytes and brain-resident cell types.²⁹ Stimulation of AhR by Trp metabolites has become a focal point in this regard, with dual emphasis on promoting anti-inflammatory responses, ameliorating CNS inflammation and maintaining host homeostasis^72,73 (Figure 5).

Figure 5.

The AhR as a critical node in Trp metabolites to brain signaling. The effects of Trp metabolites via gut microbes are not limited to the intestine but may also influence the central nervous system (CNS) inflammation and be involved in neuropsychiatric disorders, which is partly regulated through AhR. Trp metabolites, such as indole derivatives, active microglia, which transfer signals (activating TGF-α or suppressing VEGF-β) through AhR in astrocytes to inhibit CNS inflammation. Meanwhile, the IFN-I-AhR-SOCS2-NF-κB pathway can also mediate responses to CNS inflammation.

The connection of Kynurenine-TDO/IDO-AhR mitigates inflammation and autoimmune diseases

Kyn and its metabolites 3-hydroxykynurenine (3HK) can cross the blood-brain barrier whereas their high selection and significant implications for pathogenic activities of neurological disorders and the metabolism of neurotransmitters.^74,75 After taken up by astrocytes, microglia, and neurons, the neuroprotective KA can be generated by astrocytes whereas neurotoxic KP metabolites such as quinolinic acid (QA) is produced by microglia.^75-77

The realization of this neuroactive function not only depends on downstream products of the Kyn pathway via AhR, but is also associated with the coevolution of IDO1, TDO2, and AhR.³ Trp catabolism via the Kyn pathway is mediated by IDO and TDO, which are considered as rate-limiting enzymes and produce Kyn as an AhR agonist.^78,79 Considering the close interaction between AhR and IDO/TDO, their coevolution is indeed vital in immunological regulation.

The expression of IDO1 is regulated by AhR⁸⁰ via an autocrine AhR-IL6-STAT3 signaling loop.⁸¹ As reported, IDO1 contributes to intestinal homeostasis.⁸² In the absence of AhR ligands, IDO1 activity is inhibited, together with stimulating immune tolerance⁸³ and suppressing antigen-specific T lymphocyte activation.⁸⁴ The stimulating effect of intestinal bacteria in IDO1 activity has been clearly demonstrated.⁸⁵ Additionally, the c-SRC-dependent phosphorylation of IDO1 regulated by Kyn is realized by AhR activation, which further inhibits immunopathology triggered by gut microbes, including Salmonella typhimurium and group B Streptococcus,⁸⁶ and elicits the generation of transforming growth factor (TGF)-β1 by dendritic cells.⁸⁵ Recently, emerging evidence implicates the AhR-IDO1 pathway in autism spectrum disorder (ASD) as well. As reported, the pathogenesis of ASD is related to high IDO1 activity.⁸⁷ Considering the facilitating effect of ARNT on AhR, the association between ARNT and ASD severity⁸⁸ may partially prove the involvement of AhR in this cerebral process.⁸⁹

In addition to IDO1, the expression of TDO2 can also be detected in the brain^85,90 and activated in gliomas.⁹¹ Recently, lipopolysaccharide has been shown to stimulate the expression of TDO2, which subsequently produces Kyn. Via activating AhR-dependent pathway, the promotion of tumor cell motility and survival, the prevention of endotoxin tolerance,⁸⁶ and the inhibition of anti-tumor immune responses⁹¹ are demonstrated.

Indole derivatives-AhR pathway regulates CNS inflammation in “microbiome-gut-brain” axis

In the action of certain microbiota, metabolite molecules, such as IAA, IS, IPA, I3A, and IAld, derived from dietary Trp activate microglia, which transfer signaling through the AhR in astrocyte cells to mediate responses to CNS inflammation and reduce CNS autoimmunity.⁷²

Glia, consisting of not only microglia but also astrocytes, serves as the switch that participates in regulating the immune microenvironment of the brain.⁹² In addition to the abundance of cell populations, diverse functions ensure astrocytes to exert vital roles in CNS during health and diseases.^93-95 As reported, their involved functions ranging from regulating synaptic and neuronal transmission to regulating CNS development, repair, cell metabolism, and immunoregulation.^96-98 IFN-I signaling in astrocytes in combination with Trp microbial metabolites activate AhR.²⁹ The activated AhR subsequently inhibits NF-κB activation via inducing the expression of suppressor of cytokine signaling 2 (Socs2).⁹⁹ Moreover, effective anti-inflammatory and neurodegeneration-arresting characters of interferon-alpha receptor-1 (IFNAR-1) are demonstrated to be mediated by AhR.²⁹ Thus, this IFN-I-AhR-Socs2-NF-κB pathway suggests that targeting IFNAR1 signaling may be a therapeutic approach of CNS inflammation and also demonstrates a molecular mechanism for the protective effect of AhR ligands against CNS autoimmunity.

In addition to astrocytes, microglia is also a kind of immune cells of CNS and is reported to express AhR.^92,100,101 Some certain astrocytes are instructed by microglia and these two cell types communicate on a molecular level to mediate responses to CNS inflammation.¹⁰² Microbial metabolites of Trp regulate the activation of microglia, which is accompanied by the generation of TGFα and Vascular endothelial growth factor B (VEGF-B), regulating CNS associated diseases and the transcriptional program of astrocytes via AhR.³⁰ In-depth research has shown that microglia-derived TGFα acts via the ErbB1 receptor in astrocytes to exert neuroprotective functions and promote beneficial astrocyte activities.^103,104 Conversely, the production of VEGF-B triggers Vascular endothelial growth factor receptor 1 (FLT-1) signaling in astrocytes exacerbates their pathogenic activities and worsens experimental allergicerlcephalomyelitis (EAE) development.¹⁰⁵ Additionally, VEGF-B and TGFα also participate in the formation of multiple sclerosis (MS) lesion stage³⁰ in CD14+ cells and involve in microglial control of astrocytes in humans, suggesting but not enough to prove implications of TGF-α and VEGF-B for humans.

Gut microbial Trp metabolism connects with extended central reward network via AhR-based metabolic pathway

Gut microbiota has taken the limelight as a key regulator of brain-gut axis signaling, which influence extends beyond the gut and is involved in many aspects of human health and disease, including hedonic food intake, ingestive behavior,^106,107 and obesity.^73,108 Microbiota-derived Trp metabolites are associated with connectivity in key regions of the brain’s extended reward network.⁷³ Supporting this concept is associations between increased weight and changed brain activity and connectivity, highlighting the possible role of the brain in the pathophysiology of obesity.^109,110

Microbe-derived Trp metabolites are associated with connectivity of key regions of the brain’s extended reward network, in which the amygdala-nucleus accumbens (NAcc) circuit and the amygdala-anterior insula (aINS) circuit are vital to microbial-gut-brain signaling influencing non-homeostatic food intake.¹¹¹ Based on IAA signal, the activated AhR may explain the positive association between indoles, the amygdala-aINS circuit, and food addiction scores.⁷³ After activated, AhR can act as a transcription factor and regulate the rate-limiting enzymes in Trp metabolism along the Kyn pathway.⁸⁵

Meanwhile, with the involvement of AhR, microbial-derived indole derivatives bi-directionally regulate the release of the anorectic hormone glucagon-likepeptide1 (GLP-1).¹¹² Specifically, GLP-1 secretion is increased in colonic enteroendocrine L cells when being exposured to physiological levels of indole, whereas prolonged exposure causes adverse effect.¹¹² It is noteworthy that interacting with the intestinal enteroendocrine L cells and GLP-1 is a potential mechanism that indole affects the reward circuit.⁷³ As reported, GLP-1 is speculated to act locally on vagal afferent nerve terminals considering most GLP-1 is rapidly inactivated by dipeptidyl peptidase 4 prior to leaving the gut.¹¹³ Based on the vagal afferent, GLP-1 then signals to the brain circuits and nucleus tractus solitarius (NTS). The completion of this process plays an important role in regulating ingestive behavior.¹¹³ Consistently, another research on the GLP-1 agonist Exenatide also indicates that Exenatide can affect appetite control by regulating the functional connectivity of NTS-related reward regions.¹¹⁴ Considering AhR is involved in glucose and insulin-regulated metabolism,¹² AhR activation results in reduced fasting glucose levels, increased glucose and insulin dysmetabolism, and improved GLP-1 secretion,^12,72 which highlights how Trp metabolites target ingestive behavior via “gut-brain” axis.

Trp metabolism regulates “microbiome-brain” axis via AhR-based neural (vagal) pathway

Although the microbiome-gut-brain communication can be mediated by metabolic and immune pathways, hijacking vagus nerve signaling may still be the most direct and fastest way that gut microbiota regulates brain function.¹¹⁵ The vagus nerve, which contains a paired afferent and efferent fiber, links the viscera with the brain and innervates most of the gastrointestinal tract.¹¹⁶ The vagal afferent nerves transmit signals from the gastrointestinal tract to the CNS.³⁴ Instead of directly contacting with gut microbiota, vagal afferents sense luminal signals of microbial metabolites or products through their diffusion across gut barrier.¹¹⁷ These microbiota-derived neuromodulatory metabolites include short-chain fatty acids (SCFA),¹¹⁶ branched-chain amino acids,¹¹⁸ peptidoglycans,¹¹⁹ GABA,¹²⁰ catecholamines,¹²¹ and Trp precursors and metabolites, such as serotonin.¹²² The vagus nerve is involved in maintaining corporeal homeostasis by regulating hunger, satiety, neurotransmitter levels, and inflammation in the brain.¹²³ As reported, oleoylethanolamide belongs to a kind of fatty-acid derivates. Its synthesis after fat digestion activates intestinal PPAR-α receptors,¹²⁴ which in turn activates the vagal nerve and transfers signals from the gut to the brain to promote satiety.¹²⁵ In the involvement of gut microbiota, Trp is actively metabolized into indole or serotonin^58,126 that significantly affect host biosynthesis and modify host neurotransmitter pools.⁵⁸ Considering c-Fos protein expression in the dorsal vagal complex can be an indicator of vagus nerve activation,¹²⁷ the overexpressed c-Fos after indole treatment indicates an activation of the vagal afferent fibers in the intestinal mucosa induced by indole.¹²⁸ Moreover, the production of serotonin by the intestinal EC cells can also be stimulated by SCFA accumulation.¹²⁹ EC cells possess receptors for several bacteria metabolites, such as GPR35 and its ligand kynurenic acid.¹²⁹ On adequate stimulation of these receptors, serotonin was released in a calcium-dependent fashion.¹²⁹ In turn, the intimate, synapse-like contact of vagal afferent nerve establishes a pathway by which this local serotonin can regulate the activity of gut vagal afferents via serotonin type 3 receptors (5-HT3R) on unmyelinated vagal afferents in the gut mucosa resulting in altered vagal afferent input.⁷³

Conclusions

Trp is not only a nutritional enhancer but also plays a key role in modulating CNS homeostasis by being metabolized into numerous bioactive chemicals, most of which have far been identified as AhR ligands. These Trp metabolites activated AhR serve as chemical messengers that mediate the bidirectional crosstalk between the gut microbe and CNS and can regulate host homeostasis via different routes of immune, metabolic, and neural (vagal) communication.

However, there are still some existing questions that need to be addressed. First, AhR activation by certain Trp metabolites may be the result of the action of other compounds generated in Trp metabolic pathways. Second, dose–response experiments are required in future studies, as the optimal dosage of Trp metabolites in the human or animal diet remains to be elucidated, and this is pivotal for maintaining a healthy nervous system. Third, ligands and function of AhR are different between various species. Thus, many effects of Trp metabolites on AhR in mouse models need to be further confirmed in human cells. Finally, the exact mechanism of how Trp metabolites transmit signals to the brain through AhR still needs to be elucidated.

These metabolites serve as a kind of chemical language communicating in “gut-brain” axis and ultimately affect the outcomes of many disorders, including IBD, cancer, metabolic syndrome, autoimmune diseases, and neurodegenerative diseases. Efforts to identify the molecular mechanisms of how these Trp metabolites regulate host physiology will markedly provide new insights toward successful translation of microbiome-gut-brain axis research from bench to bedside and increase our understanding of developing therapeutic intervention that may alleviate the associated CNS diseases.

Funding Statement

This work was supported by the National Key R&D Program of China (2018YFD0500601 and 2017YFD0500501), the National Natural Science Foundation of China (31930106, 31829004 and 31722054), the National Ten-thousand Talents Program of China (23070201) and the 111 project (B16044).

Abbreviations

AAAD aromatic amino acid decarboxylase

Aβ beta amyloid

AD alzheimer’s disease

AhR aryl hydrocarbon receptor

aINS amygdala-anterior insula

ARNT AhR nuclear translocator

ASD autism spectrum disorder

BBB blood–brain barrier

BBDP BioBreeding diabetes-prone

bHLH basic helix-loop-helix

CAR constitutive androstane receptor

CICZ indolo[3,2-b]carbazole-6-carboxylic acid

CNS central nervous system

C. sporogenes Clostridium sporogenes

CYP cytochrome P450

DCs dendritic cells

DSS dextran sulfate sodium

EAE experimental allergicerlcephalomyelitis

EC enterochromaffin

E. coli Escherichia coli

EROD 7-ethoxyresorufin deethylase

FICZ 6-formylindolo[3,2-b]carbazole

FITC fluorescein isothiocyanate

FLT-1 Vascular endothelial growth factor receptor 1

GLP-1 glucagon-likepeptide1

hPXR human nuclear pregnane X receptor

Hsp90 90-kDa heat shock protein

H₂O₂ hydrogen peroxide

IAA indole-3-acetic acid

IAld indole-3-aldehyde

IAM indole-3-acetamide

IBD inflammatory bowel disease

IDO Indoleamine 2,3-Dioxygenase

IEC intestinal epithelial cell

IELs intestinal intraepithelial lymphocytes

IFNAR-1 interferon-alpha receptor-1

IFN-γ interferon-γ

IL-10R IL-10 receptor

IL-22 interleukin-22

IPA indole-3-propionic acid

IPI 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole

IPyA indole-3-pyruvic acid

IS indoxyl sulfate

I3A indole-3-acetaldehyde

I3P indole-3-pyruvate

KA kynurenic acid

Kyn kynurenine

LAT1 large neutral amino acid transporter

LBD ligand-binding domain

L. johnsonii Lactobacillus johnsonii

MAO monoamine oxidase

MS multiple sclerosis

NAcc amygdala-nucleus accumbens

NF-κB nuclear factor-kappa B

NTS nucleus tractus solitarius

PXR pregnane X receptor (NR1I2)

QA quinolinic acid

gnavus Ruminococcus gnavus

RXR retinoic acid receptor (NR2B1)

SCFA short-chain fatty acids

Socs2 suppressor of cytokine signaling 2

TDO Trp 2,3-dioxygenase

TGF transforming growth factor

Th17 T helper 17 cells

TNBS trinitrobenzene sulfonic acid

TNF-α tumor necrosis factor alpha

TPH1 Trp hydroxylase

Trp Tryptophan

UGT UDP-glucuronosyltransferase

UV near-ultraviolet

VEGF-B Vascular endothelial growth factor B

XR Xenobiotic receptors

3HK 3-hydroxykynurenine

5-HIAA 5-hydroxyindoleacetic acid

5-HT serotonin (5-hydroxytryptamine)

5-HT3R serotonin type 3 receptors.

Disclosure of potential conflicts of interest

The authors declare that they have no competing interests.

Authors’ contributions

The review was mainly conceived and designed by XM. Literature was collected by NM. The manuscript was mainly written by NM and edited by TH, Lee J. J and XM. XM resourced the project. All authors contributed to, read, and approved the final manuscript.