Species differences in specific ligand-binding affinity and activation of AHR: The biological basis for calculation of relative effective potencies and toxic equivalence factors.

In 2022 the World Health Organization (WHO) published updated 'Toxic Equivalence Factors' (TEFs) for a wide variety of chlorinated dioxins, dibenzofurans and PCBs [collectively referred to as 'dioxin-like chemicals'; DLCs) that interact with the aryl hydrocarbon receptor (AHR)]. Their update used sophisticated statistical analysis of hundreds of published studies that reported estimation of 'Relative Effective Potency' (REP) values for individual DLC congeners. The weighting scheme used in their assessment of each study favored in vivo over in vitro studies and was based largely on rodent studies. In this Commentary, we highlight the large body of published studies that demonstrate large species differences in AHR-ligand activation and provide supporting evidence for our position that the WHO 2022 TEF values intended for use in human risk assessment of DLC mixtures will provide highly misleading overestimates of 'Toxic Equivalent Quotients' (TEQs), because of well-recognized striking differences in AHR ligand affinities between rodent (rat, mouse) and human. The data reviewed in our Commentary support the position that human tissue-derived estimates of REP/TEF values for individual DLC congeners, although uncertain, will provide much better, more realistic estimates of potential activation of the human AHR, when exposure to complex DLC mixtures occurs.

Immune cell-intrinsic Ah receptor facilitates the expression of antimicrobial REG3G in the small intestine.

The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.

Induction of AHR Signaling in Response to the Indolimine Class of Microbial Stress Metabolites.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays an important role in gastrointestinal barrier function, tumorigenesis, and is an emerging drug target. The resident microbiota is capable of metabolizing tryptophan to metabolites that are AHR ligands (e.g., indole-3-acetate). Recently, a novel set of mutagenic tryptophan metabolites named indolimines have been identified that are produced by M. morganii in the gastrointestinal tract. Here, we determined that indolimine-200, -214, and -248 are direct AHR ligands that can induce Cyp1a1 transcription and subsequent CYP1A1 enzymatic activity capable of metabolizing the carcinogen benzo(a)pyrene in microsomal assays. In addition, indolimines enhance IL6 expression in a colonic tumor cell line in combination with cytokine treatment. The concentration of indolimine-248 that induces AHR transcriptional activity failed to increase DNA damage. These observations reveal an additional aspect of how indolimines may alter colonic tumorigenesis beyond mutagenic activity.

Alpha-tocopherylquinone-mediated activation of the Aryl Hydrocarbon Receptor regulates the production of inflammation-inducing cytokines and ameliorates intestinal inflammation.

This study investigated the role of Alpha-tocopherylquinone (TQ) in regulating the intestinal immune system and the underlying mechanisms. In the experimental dextran sodium sulfate and T cell-mediated colitis models, TQ significantly reduced the mRNA levels of interleukin (IL)-6, IL-1ß, IL-17A, IL-23, and tumor necrosis factor (TNF)-α and the abundance of proinflammatory macrophages, T helper (Th)17 cells, and ILC3s in the colons of wild-type mice. TQ also prevented lipopolysaccharide (LPS)-induced activation of NFκB and signal transducer and activator of transcription (Stat)-3 pathways in the human macrophage U937 cells. Pharmacological inhibition or CRISPR-Cas-9-mediated knockout of Aryl hydrocarbon Receptor (AhR) prevented the anti-inflammatory effects of TQ in the LPS-treated U937 cells. Furthermore, TQ reduced the mRNA levels of the LPS-induced pro-inflammatory cytokines in the WT but not Ahr-/- mice splenocytes. TQ also reduced IL-6R protein levels and IL-6-induced Stat-3 activation in Jurkat cells and in vitro differentiation of Th17 cells from wild-type but not Ahr-/- mice naive T cells. Additionally, TQ prevented the pro-inflammatory effects of LPS on macrophages and stimulation of T cells in human PBMCs and significantly reduced the abundance of tumor necrosis factor-α, IL-1ß, and IL-6hi inflammatory macrophages and Th17 cells in surgically resected Crohn's disease (CD) tissue. Our study shows that TQ is a naturally occurring, non-toxic, and effective immune modulator that activates AhR and suppresses the Stat-3-NFκB signaling.

Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

Molecular networking identifies an AHR-modulating benzothiazole from white button mushrooms (Agaricus bisporus).

Diet-derived aryl hydrocarbon receptor (AHR) ligands have potential to maintain gut health. However, among the myriad bioactive compounds from foods, identifying novel functional ligands which would significantly impact gastrointestinal health is a challenge. In this study, a novel AHR modulator is predicted, identified, and characterized in the white button mushroom (Agaricus bisporus). Using a molecular networking approach, a methylated analog to benzothiazole was indicated in white button mushrooms, which was subsequently isolated and identified as 2-amino-4-methyl-benzothiazole(2A4). Cell-based AHR transcriptional assays revealed that 2-amino-4-methyl-benzothiazole possesses agonistic activity and upregulated CYP1A1 expression. This contrasts with previous findings that whole white button mushroom extract has overall antagonistic activity in vivo, underscoring the importance of studying the roles each chemical component plays in a whole food. The findings suggest that 2-amino-4-methyl-benzothiazole is a previously unidentified AHR modulator from white button mushroom and demonstrate that molecular networking has potential to identify novel receptor modulators from natural products.

Endogenous Tryptophan-Derived Ah Receptor Ligands are Dissociated from CYP1A1/1B1-Dependent Negative-Feedback.

The aryl hydrocarbon receptor (AHR) exerts major roles in xenobiotic metabolism, and in immune and barrier tissue homeostasis. How AHR activity is regulated by the availability of endogenous ligands is poorly understood. Potent AHR ligands have been shown to exhibit a negative feedback loop through induction of CYP1A1, leading to metabolism of the ligand. Our recent study identified and quantified 6 tryptophan metabolites (eg, indole-3-propionic acid, and indole-3-acetic acid) in mouse and human serum, generated by the host and gut microbiome, that are present in sufficient concentrations to individually activate the AHR. Here, these metabolites are not significantly metabolized by CYP1A1/1B1 in an in vitro metabolism assay. In contrast, CYP1A1/1B metabolizes the potent endogenous AHR ligand 6-formylindolo[3,2b]carbazole. Furthermore, molecular modeling of these 6 AHR activating tryptophan metabolites within the active site of CYP1A1/1B1 reveal metabolically unfavorable docking profiles with regard to orientation with the catalytic heme center. In contrast, docking studies confirmed that 6-formylindolo[3,2b]carbazole would be a potent substrate. The lack of CYP1A1 expression in mice fails to influence serum levels of the tryptophan metabolites examined. In addition, marked induction of CYP1A1 by PCB126 exposure in mice failed to alter the serum concentrations of these tryptophan metabolites. These results suggest that certain circulating tryptophan metabolites are not susceptible to an AHR negative feedback loop and are likely important factors that mediate constitutive but low level systemic human AHR activity.

Contribution of Circulating Host and Microbial Tryptophan Metabolites Toward Ah Receptor Activation.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays an integral role in homeostatic maintenance by regulating cellular functions such as cellular differentiation, metabolism, barrier function, and immune response. An important but poorly understood class of AHR activators are compounds derived from host and bacterial metabolism of tryptophan. The commensal bacteria of the gut microbiome are major producers of tryptophan metabolites known to activate the AHR, while the host also produces AHR activators through tryptophan metabolism. We used targeted mass spectrometry-based metabolite profiling to determine the presence and metabolic source of these metabolites in the sera of conventional mice, germ-free mice, and humans. Surprisingly, sera concentrations of many tryptophan metabolites are comparable between germ-free and conventional mice. Therefore, many major AHR-activating tryptophan metabolites in mouse sera are produced by the host, despite their presence in feces and mouse cecal contents. Here we present an investigation of AHR activation using a complex mixture of tryptophan metabolites to examine the biological relevance of circulating tryptophan metabolites. AHR activation is rarely studied in the context of a mixture at relevant concentrations, as we present here. The AHR activation potentials of individual and pooled metabolites were explored using cell-based assays, while ligand binding competition assays and ligand docking simulations were used to assess the detected metabolites as AHR agonists. The physiological and biomedical relevance of the identified metabolites was investigated in the context of a cell-based model for rheumatoid arthritis. We present data that reframe AHR biology to include the presence of a mixture of ubiquitous tryptophan metabolites, improving our understanding of homeostatic AHR activity and models of AHR-linked diseases.

Complex chemical signals dictate Ah receptor activation through the gut-lung axis.

The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in rapid clearance within the intestinal tract, limiting systemic exposure and subsequent AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that functionally increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA), a gut bacterial metabolite of ellagitannins, as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). In mice, dietary exposure to UroA in a 10% broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in the liver. Thus, CYP1A1 dietary competitive substrates can lead to enhanced systemic AHR ligand distribution from the gut, likely through the lymphatic system, increasing AHR activation in key barrier tissues. Finally, this report will lead to a reassessment of the dynamics of distribution of other hydrophobic chemicals present in the diet.

Aryl Hydrocarbon Receptor Activation Coordinates Mouse Small Intestinal Epithelial Cell Programming.

In the face of mechanical, chemical, microbial, and immunologic pressure, intestinal homeostasis is maintained through balanced cellular turnover, proliferation, differentiation, and self-renewal. Here, we present evidence supporting the role of the aryl hydrocarbon receptor (AHR) in the adaptive reprogramming of small intestinal gene expression, leading to altered proliferation, lineage commitment, and remodeling of the cellular repertoire that comprises the intestinal epithelium to promote intestinal resilience. Ahr gene/protein expression and transcriptional activity exhibit marked proximalHI to distalLO and cryptHI to villiLO gradients. Genetic ablation of Ahr impairs commitment/differentiation of the secretory Paneth and goblet cell lineages and associated mucin production, restricts expression of secretory/enterocyte differentiation markers, and increases crypt-associated proliferation and villi-associated enterocyte luminal exfoliation. Ahr-/- mice display a decrease in intestinal barrier function. Ahr+/+ mice that maintain a diet devoid of AHR ligands intestinally phenocopy Ahr-/- mice. In contrast, Ahr+/+ mice exposed to AHR ligands reverse these phenotypes. Ligand-induced AHR transcriptional activity positively correlates with gene expression (Math1, Klf4, Tff3) associated with differentiation of the goblet cell secretory lineage. Math1 was identified as a direct target gene of AHR, a transcription factor critical to the development of goblet cells. These data suggest that dietary cues, relayed through the transcriptional activity of AHR, can reshape the cellular repertoire of the gastrointestinal tract.

Complex chemical signals dictate Ah receptor activation through the gut-lung axis.

The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in the rapid clearance within the intestinal tract, limiting AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA) as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). Dietary exposure to UroA in a broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in liver. Thus, CYP1A1 dietary competitive substrates can lead to intestinal escape, likely through the lymphatic system, increasing AHR activation in key barrier tissues.

The Ah Receptor from Toxicity to Therapeutics: Report from the 5th AHR Meeting at Penn State University, USA, June 2022.

The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

Aryl hydrocarbon receptor activation affects nitrergic neuronal survival and delays intestinal motility in mice.

Despite progress describing the effects of persistent organic pollutants (POPs) on the central nervous system, the effect of POPs on enteric nervous system (ENS) function remains underexplored. We studied the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a POP, and a potent aryl hydrocarbon receptor (AHR) ligand, on the ENS and intestinal motility in mice. C57Bl/6J mice treated with TCDD (2.4 µg/kg body weight) for 8 weeks (once per week) exhibited significant delay in intestinal motility as shown by reduced stool frequency, prolonged intestinal transit time, and a persistence of dye in the jejunum compared to control mice with maximal dye retention in the ileum. TCDD significantly increased Cyp1a1 expression, an AHR target gene, and reduced the total number of neurons and affected nitrergic neurons in cells isolated from WT mice, but not Ahr-/- mice. In immortalized fetal enteric neuronal cells, TCDD-induced nuclear translocation of AHR as well as increased Cyp1a1 expression. AHR activation did not affect neuronal proliferation. However, AHR activation resulted in enteric neuronal toxicity, specifically, nitrergic neurons. Our results demonstrate that TCDD adversely affects nitrergic neurons and thereby contributes to delayed intestinal motility. These findings suggest that AHR signaling in the ENS may play a role in modulating TCDD-induced gastrointestinal pathophysiology.

Contribution of circulating host and microbial tryptophan metabolites towards Ah receptor activation.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays an integral role in homeostatic maintenance by regulating cellular functions such as cellular differentiation, metabolism, barrier function, and immune response. An important but poorly understood class of AHR activators are compounds derived from host and bacterial metabolism of tryptophan. The commensal bacteria of the gut microbiome are major producers of tryptophan metabolites known to activate the AHR, while the host also produces AHR activators through tryptophan metabolism. We used targeted mass spectrometry-based metabolite profiling to determine the presence and metabolic source of these metabolites in the sera of conventional mice, germ-free mice, and humans. Surprisingly, sera concentrations of many tryptophan metabolites are comparable between germ-free and conventional mice. Therefore, many major AHR-activating tryptophan metabolites in mouse sera are produced by the host, despite their presence in feces and mouse cecal contents. AHR activation is rarely studied in the context of a mixture at relevant concentrations, as we present here. The AHR activation potentials of individual and pooled metabolites were explored using cell-based assays, while ligand binding competition assays and ligand docking simulations were used to assess the detected metabolites as AHR agonists. The physiological and biomedical relevance of the identified metabolites was investigated in the context of cell-based models for cancer and rheumatoid arthritis. We present data here that reframe AHR biology to include the presence of ubiquitous tryptophan metabolites, improving our understanding of homeostatic AHR activity and models of AHR-linked diseases.

Lead optimization of aryl hydrocarbon receptor ligands for treatment of inflammatory skin disorders.

Therapeutic aryl hydrocarbon receptor (AHR) modulating agents gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR's known ligand promiscuity, we generated novel AHR modulating agents by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. SGA388 showed the highest agonist activity with potent normalization of keratinocyte hyperproliferation, restored expression of skin barrier proteins and dampening of chemokine expression by keratinocytes upon Th2-mediated inflammation in vitro. The topical application of SGA360f and SGA388 reduced acute skin inflammation in vivo by reducing cyclooxygenase levels, resulting in less neutrophilic dermal infiltrates. The minimal induction of cytochrome P450 enzyme activity, lack of cellular toxicity and mutagenicity classifies SGA360f and SGA388 as novel potential therapeutic AHR ligands and illustrates the potential of medicinal chemistry to fine-tune AHR signaling for the development of targeted therapies in dermatology and beyond.

AHR is a master regulator of diverse pathways in endogenous metabolism.

The aryl hydrocarbon receptor (AHR) is a transcription factor with roles in detoxification, development, immune response, chronic kidney disease and other syndromes. It regulates the expression of drug transporters and drug metabolizing enzymes in a proposed Remote Sensing and Signaling Network involved in inter-organ communication via metabolites and signaling molecules. Here, we use integrated omics approaches to analyze its contributions to metabolism across multiple scales from the organ to the organelle. Global metabolomics analysis of Ahr-/- mice revealed the role of AHR in the regulation of 290 metabolites involved in many biochemical pathways affecting fatty acids, bile acids, gut microbiome products, antioxidants, choline derivatives, and uremic toxins. Chemoinformatics analysis suggest that AHR plays a role in determining the hydrophobicity of metabolites and perhaps their transporter-mediated movement into and out of tissues. Of known AHR ligands, indolepropionate was the only significantly altered molecule, and it activated AHR in both human and murine cells. To gain a deeper biological understanding of AHR, we employed genome scale metabolic reconstruction to integrate knockout transcriptomics and metabolomics data, which indicated a role for AHR in regulation of organic acids and redox state. Together, the results indicate a central role of AHR in metabolism and signaling between multiple organs and across multiple scales.

Early Life Short-Term Exposure to Polychlorinated Biphenyl 126 in Mice Leads to Metabolic Dysfunction and Microbiota Changes in Adulthood.

Early life exposure to environmental pollutants may have long-term consequences and harmful impacts on health later in life. Here, we investigated the short- and long-term impact of early life 3,3',4,4',5-pentacholorobiphenyl (PCB 126) exposure (24 µg/kg body weight for five days) in mice on the host and gut microbiota using 16S rRNA gene sequencing, metagenomics, and 1H NMR- and mass spectrometry-based metabolomics. Induction of Cyp1a1, an aryl hydrocarbon receptor (AHR)-responsive gene, was observed at 6 days and 13 weeks after PCB 126 exposure consistent with the long half-life of PCB 126. Early life, Short-Term PCB 126 exposure resulted in metabolic abnormalities in adulthood including changes in liver amino acid and nucleotide metabolism as well as bile acid metabolism and increased hepatic lipogenesis. Interestingly, early life PCB 126 exposure had a greater impact on bacteria in adulthood at the community structure, metabolic, and functional levels. This study provides evidence for an association between early life environmental pollutant exposure and increased risk of metabolic disorders later in life and suggests the microbiome is a key target of environmental chemical exposure.

Multi-Omics Strategies for Investigating the Microbiome in Toxicology Research.

Microbial communities on and within the host contact environmental pollutants, toxic compounds, and other xenobiotic compounds. These communities of bacteria, fungi, viruses, and archaea possess diverse metabolic potential to catabolize compounds and produce new metabolites. Microbes alter chemical disposition thus making the microbiome a natural subject of interest for toxicology. Sequencing and metabolomics technologies permit the study of microbiomes altered by acute or long-term exposure to xenobiotics. These investigations have already contributed to and are helping to re-interpret traditional understandings of toxicology. The purpose of this review is to provide a survey of the current methods used to characterize microbes within the context of toxicology. This will include discussion of commonly used techniques for conducting omic-based experiments, their respective strengths and deficiencies, and how forward-looking techniques may address present shortcomings. Finally, a perspective will be provided regarding common assumptions that currently impede microbiome studies from producing causal explanations of toxicologic mechanisms.

Gestational Insulin Resistance Is Mediated by the Gut Microbiome-Indoleamine 2,3-Dioxygenase Axis.

BACKGROUND & AIMS: Normal gestation involves a reprogramming of the maternal gut microbiome (GM) that contributes to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of the GM-maternal metabolism interaction. METHODS: The GM and plasma metabolome of CD1, NIH-Swiss, and C57 mice were analyzed with the use of 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry throughout gestation. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM was studied with the use of fecal microbial transplants (FMTs). RESULTS: Significant variation in GM alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time point specific, with the species enriched at gestation day 15/19 (G15/19), a point of heightened IR, being distinct from those enriched before or after pregnancy. Metabolomics revealed elevated plasma kynurenine at G15/19 in all 3 mouse strains. IDO1, the rate-limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine level effects on IR derive from the GM in both mouse and human pregnancy. CONCLUSIONS: GM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation, and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1. (Gestational Gut Microbiome-IDO1 Axis Mediates Pregnancy Insulin Resistance; EMBL-ENA ID: PRJEB45047. MetaboLights ID: MTBLS3598).