Gut Cells Research Articles

Functional genomics implicates natural killer cells in the pathogenesis of ankylosing spondylitis

Multiple lines of evidence indicate that ankylosing spondylitis (AS) is a lymphocyte-driven disease. However, which lymphocyte populations are critical in AS pathogenesis is not known. In this study, we aimed to identify the key cell types mediating the genetic risk in AS using an unbiased functional genomics approach. We integrated genome-wide association study (GWAS) data with epigenomic and transcriptomic datasets of human immune cells. To quantify enrichment of cell type-specific open chromatin or gene expression in AS risk loci, we used three published methods - LDSC-SEG, SNPsea, scDRS - that have successfully identified relevant cell types in other diseases. Natural killer (NK) cell-specific open chromatin regions are significantly enriched in heritability for AS, compared to other immune cell types such as T cells, B cells, and monocytes. This finding was consistent between two AS GWAS. Using RNA-seq data, we validated that genes in AS risk loci are enriched in NK cell-specific gene expression. Using the human Space-Time Gut Cell Atlas, we also found significant upregulation of AS-associated genes predominantly in NK cells. We performed co-localization analyses between GWAS risk loci and genetic variants associated with gene expression (eQTL) to find putative target genes. This revealed four AS risk loci affecting regulation of candidate target genes in NK cells: two known loci, ERAP1 and TNFRSF1A, and two under-studied loci, ENTR1 (aka SDCCAG3) and B3GNT2. Our findings suggest that NK cells may play a crucial role in AS development and highlight four putative target genes for functional follow-up in NK cells.

Identification and functional analysis of gut dsRNases in the beet armyworm Spodoptera exigua

RNA interference (RNAi)-based products have the potential to significantly contribute to insect pest control. However, RNAi efficiency varies widely among different insect orders, particularly in Lepidoptera, where it is often low. One key factor affecting RNAi efficiency is the presence of double-stranded ribonuclease (dsRNase) in the digestive tract, which can degrade dsRNA prior to uptake by gut cells. In this study, four dsRNases were identified in the beet armyworm, Spdoptera exigua, of which two were highly expressed gut dsRNases, SedsRNase1 and SedsRNase2. To assess their effect on dsRNA degradation activity via the oral route, CRISPR/Cas9-based gene editing was employed to knock out these gut dsRNases. The results indicate that all mutant strains, including SeKO1 (knockout SedsRNase1), SeKO2 (knockout SedsRNase2), and SeKO1KO2 (knockout SedsRNase1 and SedsRNase2), showed significantly decreased dsRNA degradation activity, particularly in the SeKO1KO2 mutant strain, where the weakest degradation occurred in both the gut and whole body. Additionally, we noticed that the lack of gut SedsRNases led to a slight extended developmental period and reduced reproductive capacity in S. exigua. Collectively, these findings deepen our understanding of gut SedsRNases and how they can impact the biology of the beet armyworm and can support the exploration dsRNA-based approaches for pest control.

Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia Muciniphila with Bioorthogonal Chemical Reporters.

Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota-host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin-degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non-toxic tetrazine click-compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.

Identification of Key Genes in Fetal Gut Development at Single-Cell Level by Exploiting Machine Learning Techniques.

The study of fetal gut development is critical due to its substantial influence on immediate neonatal and long-term adult health. Current research largely focuses on microbiome colonization, gut immunity, and barrier function, alongside the impact of external factors on these phenomena. Limited research has been dedicated to the categorization of developing fetal gut cells. Our study aimed to enhance our understanding of fetal gut development by employing advanced machine-learning techniques on single-cell sequencing data. This dataset consisted of 62,849 samples, each characterized by 33,694 distinct gene features. Four feature ranking algorithms were utilized to sort features according to their significance, resulting in four feature lists. Then, these lists were fed into an incremental feature selection method to extract essential genes, classification rules, and build efficient classifiers. Several important genes were recognized by multiple feature ranking algorithms, such as FGG, MDK, RBP1, RBP2, IGFBP7, and SPON2. These features were key in differentiating specific developing intestinal cells, including epithelial, immune, mesenchymal, and vasculature cells of the colon, duo jejunum, and ileum cells. The classification rules showed special gene expression patterns on some intestinal cell types and the efficient classifiers can be useful tools for identifying intestinal cells.

Effects of parenteral nutrition supplemented with beta-hydroxy-beta-methylbutyrate on gut-associated lymphoid tissue and morphology in mice.

Parenteral nutrition (PN) without enteral nutrition (EN) leads to marked atrophy of gut-associated lymphoid tissue (GALT), causing mucosal defense failure in both the gut and the extraintestinal mucosal system. We evaluated the effects of beta-hydroxy-beta-methylbutyrate (HMB) on GALT and gut morphology in PN-fed mice. Experiment 1: male Institute of Cancer Research mice were assigned to the Chow (n = 12), Control (standard PN: n = 10), or H600 and H2000 (PN containing 600 mg/kg or H2000 mg/kg body weight of Ca-HMB: n = 12 and 10, respectively) groups. After 5 days of dietary manipulation, all mice were killed and the whole small intestine was harvested. GALT lymphocyte cell numbers and phenotypes of Peyer patch (PP), intraepithelial space, and lamina propria lymphocytes were evaluated. Experiment 2: 47 mice (Chow: n = 12; Control: n = 14; H600: n = 11; and H2000: n = 10) were fed for 5 days as in experiment 1. Proliferation and apoptosis of gut immune cells and mucosa, and protein expressions (mammalian target of rapamycin [mTOR], caspase-3, and Bcl2) were evaluated in the small intestine. Compared with the Controls, the Chow and HMB groups showed significantly higher PP cell numbers, prevented gut mucosal atrophy, inhibited apoptosis of gut cells, and increased their proliferation in association with increased mTOR activity and Bcl2 expression. HMB-supplemented PN is a potentially novel method of preserving GALT mass and gut morphology in the absence of EN.

Size-dependent toxicity of microplastics induced gut cell apoptosis in brine shrimp Artemia parthenogenetica through p53 signaling pathway

Size-dependent toxicity of microplastics induced gut cell apoptosis in brine shrimp Artemia parthenogenetica through p53 signaling pathway

Spatio-temporally controlled all-in-one hydrogel microspheres for ulcerative colitis treatment based on cocktail strategy

Due to the complex pathogenesis of ulcerative colitis (UC), particularly excessive reactive oxygen species (ROS), gut microbiota dysbiosis, and hyperpolarization of M1 macrophages, the cocktail strategy is urgently needed to obtain a sufficient therapeutic outcome. Meanwhile, the accurate lesion location targeting of these combined therapeutic agents is still crucial. Herein, based on NPs-in-MPs approach, a novel integrated “all-in-one” oral delivery system (BMM/MPs), containing the natural gut microbiota regulator berberine, MnO2 nanoenzymes, and cell penetrating peptides-decorated magnolol liposomes to regulate macrophage polarization, was constructed to address these above issues in sequence. BMM/MPs could non-destructively cross the gastrointestinal tract, enduringly detain, and dissociate in the colon site due to the pH-sensitive properties. Conceivably, the released MnO2 nanoenzymes depleted the excess ROS at the colitis location into O2, sequentially promoting the acceleration of hydrogel microspheres rupture and oxidative damage remission. Accordingly, the oral administration of BMM/MPs exhibited remarkably satisfactory therapeutic effects against DSS-induced UC mice, by ameliorating the representative symptoms, scavenging ROS, relieving inflammation, and inhibiting M1 macrophage polarization at colon sites. Meanwhile, the dysbiosis of microbes was effectively regulated by facilitating SCFAs production. This study provides a feasible strategy for administration in the treatment of UC.

Scalable Bioreactor-based Suspension Approach to Generate Stem Cell-derived Islets From Healthy Donor-derived iPSCs.

Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. Differentiation was capable of generating 90.4% PDX1+/NKX6.1+ pancreatic progenitors and 100% C-peptide+/NKX6.1+ iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19+ ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 104 cells/cm2 (total 8.3 × 106 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 106 cells/mL (total 105.0 × 106 cells), reducing costs by 88.8%. This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Variations in cell plasticity and proliferation underlie distinct modes of regeneration along the antero-posterior axis in the annelid Platynereis.

The capacity to regenerate lost tissues varies significantly among animals. Some phyla, such as the annelids, display substantial regenerating abilities, although little is known about the cellular mechanisms underlying the process. To precisely determine the origin, plasticity and fate of the cells participating in blastema formation and posterior end regeneration after amputation in the annelid Platynereis dumerilii, we developed specific tools to track different cell populations. Using these tools, we find that regeneration is partly promoted by a population of proliferative gut cells whose regenerative potential varies as a function of their position along the antero-posterior axis of the worm. Gut progenitors from anterior differentiated tissues are lineage restricted, whereas gut progenitors from the less differentiated and more proliferative posterior tissues are much more plastic. However, they are unable to regenerate the stem cells responsible for the growth of the worms. Those stem cells are of local origin, deriving from the cells present in the segment abutting the amputation plane, as are most of the blastema cells. Our results favour a hybrid and flexible cellular model for posterior regeneration in Platynereis relying on different degrees of cell plasticity.

In vitro investigations on interference of selected probiotic candidates with Campylobacter jejuni adhesion and invasion of primary chicken derived cecal and Caco-2 cells

BackgroundCampylobacter (C.) jejuni is one of the most important bacterial foodborne pathogens worldwide. Probiotics such as Lactobacillus or Bacillus species are considered one option for reducing the colonization rate and magnitude in poultry, the most frequent source of human infections. Due to the lack of suitable avian in vitro models such as chicken intestinal cell lines, especially those derived from the cecum, most in vitro studies on C. jejuni host interaction have been conducted with human intestinal cell lines. In this study, we compared C. jejuni-cell interactions between primary chicken cecal cells and the human intestinal cell line Caco-2, which is derived from colorectal adenocarcinoma, and investigated possible interfering effects of selected probiotic candidates.ResultsWe detected differences in adhesion and invasion between the two tested gut cell types and between different C. jejuni strains. The probiotic inhibition of C. jejuni adhesion and invasion of human and avian gut cells was affected by host cell type, investigated C. jejuni strain and time points of probiotic treatment. Additionally, our results suggest a possible correlation between C. jejuni invasion and the detected increase in IL-6 mRNA expression.ConclusionsOur results indicate distinct differences between avian and human gut cells in their interaction with C. jejuni. Therefore, data obtained in one host species on C. jejuni-host interaction may not easily be transferrable to another one. The factors influencing the variable efficacy of probiotic intervention in chicken and human derived cells should be investigated further.

Single-cell transcriptome analysis reveals secretin as a hallmark of human enteroendocrine cell maturation

The traditional nomenclature of enteroendocrine cells (EECs), established in 1977, applied the “one cell - one hormone” dogma, which distinguishes subpopulations based on the secretion of a specific hormone. These hormone-specific subpopulations included S cells for secretin (SCT), K cells for glucose-dependent insulinotropic polypeptide (GIP), N cells producing neurotensin (NTS), I cells producing cholecystokinin (CCK), D cells producing somatostatin (SST), and others. In the past 15 years, reinvestigations into murine and human organoid-derived EECs, however, strongly questioned this dogma and established that certain EECs coexpress multiple hormones. Using the Gut Cell Atlas, the largest available single-cell transcriptome dataset of human intestinal cells, this study consolidates that the original dogma is outdated not only for murine and human organoid-derived EECs, but also for primary human EECs, showing that the expression of certain hormones is not restricted to their designated cell type. Moreover, specific analyses into SCT-expressing cells reject the presence of any cell population that exhibits significantly elevated secretin expression compared to other cell populations, previously referred to as S cells. Instead, this investigation indicates that secretin production is realized jointly by other enteroendocrine subpopulations, validating corresponding observations in murine EECs also for human EECs. Furthermore, our findings corroborate that SCT expression peaks in mature EECs, in contrast, progenitor EECs exhibit markedly lower expression levels, supporting the hypothesis that SCT expression is a hallmark of EEC maturation.

Extraembryonic gut endoderm cells undergo programmed cell death during development

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.

Transcriptional remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm.

The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut- derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications.

Allergen-Encapsulating Nanoparticles Reprogram Pathogenic Allergen-Specific Th2 Cells to Suppress Food Allergy.

Food allergy is a prevalent, potentially deadly disease caused by inadvertent sensitization to benign food antigens. Pathogenic Th2 cells are a major driver for disease, and allergen-specific immunotherapies (AIT) aim to increase the allergen threshold required to elicit severe allergic symptoms. However, the majority of AIT approaches require lengthy treatments and convey transient disease suppression, likely due to insufficient targeting of pathogenic Th2 responses. Here, the ability of allergen-encapsulating nanoparticles to directly suppress pathogenic Th2 responses and reactivity is investigated in a mouse model of food allergy. NPs associate with pro-tolerogenic antigen presenting cells, provoking accumulation of antigen-specific, functionally suppressive regulatory T cells in the small intestine lamina propria. Two intravenous doses of allergen encapsulated in poly(lactide-co-glycolide) nanoparticles (NPs) significantly reduces oral food challenge (OFC)-induced anaphylaxis. Importantly, NP treatment alters the fates of pathogenic allergen-specific Th2 cells, reprogramming these cells toward CD25+FoxP3+ regulatory and CD73+FR4+ anergic phenotypes. NP-mediated reductions in the frequency of effector cells in the gut and mast cell degranulation following OFC are also demonstrated. These studies reveal mechanisms by which an allergen-encapsulating NP therapy and, more broadly, allergen-specific immunotherapies, can rapidly attenuate allergic responses by targeting pathogenic Th2 cells.

Dietary phytochemicals as regulators of gut inflammation in the context of type 2 diabetes

Numerous disorders, including type 2 diabetes and even COVID-19, are linked to poor gut health and inflammation(1,2). In addition to impacting food digestion and absorption, gut inflammation worsens diabetes outcomes by causing gut microbial dysbiosis, disrupting tight junctions (allowing microbial metabolites to freely enter into circulation), and altering glucose absorption(3). ACE2 is a crucial regulator of gut health and has received much attention during the COVID-19 pandemic due to its role as a major viral entry protease. Studies have shown that the ACE2/Ang-(1-7)/Mas axis is important in managing inflammation and maintaining normal glucose metabolism(3). Dietary phytochemicals are plant bioactive compounds, with promising anti-inflammatory and anti-diabetic properties, and may affect these processes. In this work, we aimed to look at the link between inflammation, ACE2 and the glucose transporters, SGLT1 and GLUT2, and how phytochemicals could be used to normalise the changes brought about by inflammation in Caco-2/TC7 human intestinal epithelium cells. We first examined how gut inflammation, ACE2 and glucose transporters are related and proceeded to look at the effect of some chosen phytochemicals on regulating glucose transport via modulation of the ACE2/Ang(1-7)/Mas axis. This included genistein (an isoflavone from soyabeans), sulforaphane (an isothiocyanate found in Brassica, especially broccoli), apigenin (a flavone found in vegetables and herbs), and artemisinin (a sesquiterpene lactone used as a drug). The impact of phytochemicals on the SARS-CoV-2 viral entry receptors, ACE2 and TMPRSS2, was also examined as a secondary outcome. To induce inflammation, the Caco2/TC7 cells were co-stimulated with IL-1β (25 ng/mL) and TNF-α (50 ng/mL) for varying durations (24 h, 48 h, 72 h, 168 h) and changes in target gene expression (ACE2, SGLT1, GLUT2, TMPRSS2) were assessed by droplet digital PCR. IL-6 and IL-8 were assessed as markers of inflammation in the cell culture media by multiplex ELISA. Inflammation increased ACE2, TMPRSS2 and SGLT1 mRNA. ACE2 increased with cytokine exposure duration, coupled with an obvious decrease in IL-8, SGLT1 and TMPRSS2. Pearson correlation analysis revealed that the increase in ACE2 was strongly associated with decreases in SGLT1 (r = −0.99, p<0.01) and IL-8 (r = −0.959, p<0.05), implying ACE2 to play a crucial role in gut inflammation and postprandial glycaemia. After establishing the gut cell inflammation model, we compared the effect of the phytochemicals on our target genes in cells cultured in normal and pro-inflammatory environments. None of the tested phytochemicals were effective in reducing IL-8 secretion, while phytochemicals showed varying effects on the target genes. Genistein normalised the effects of inflammation on the target genes with less effect from the other tested phytochemicals. However, further research is required to assess the importance of genistein in vivo in the context of gut inflammation and type 2 diabetes.

Stage-specific exposure of Caenorhabditis elegans to cadmium identifies unique transcriptomic response cascades and an uncharacterized cadmium responsive transcript.

Age/stage sensitivity is considered a significant factor in toxicity assessments. Previous studies investigated cadmium (Cd) toxicosis in Caenorhabditis elegans, and a plethora of metal-responsive genes/proteins have been identified and characterized in fine detail; however, most of these studies neglected age sensitivity and stage-specific response to toxicants at the molecular level. This present study compared the transcriptome response between C. elegans L3 vs L4 larvae exposed to 20 µM Cd to explore the transcriptional hallmarks of stage sensitivity. The results showed that the transcriptome of the L3 stage, despite being exposed to Cd for a shorter period, was more affected than the L4 stage, as demonstrated by differences in transcriptional changes and magnitude of induction. Additionally, T08G5.1, a hitherto uncharacterized gene located upstream of metallothionein (mtl-2), was transcriptionally hyperresponsive to Cd exposure. Deletion of one or both metallothioneins (mtl-1 and/or mtl-2) increased T08G5.1 expression, suggesting that its expression is linked to the loss of metallothionein. The generation of an extrachromosomal transgene (PT08G5.1:: GFP) revealed that T08G5.1 is constitutively expressed in the head neurons and induced in gut cells upon Cd exposure, not unlike mtl-1 and mtl-2. The low abundance of cysteine residues in T08G5.1 suggests, however, that it may not be involved directly in Cd sequestration to limit its toxicity like metallothionein, but might be associated with a parallel pathway, possibly an oxidative stress response.

Crosstalk between hypoxia-inducible factor-1α and short-chain fatty acids in inflammatory bowel disease: key clues toward unraveling the mystery.

The human intestinal tract constitutes a complex ecosystem, made up of countless gut microbiota, metabolites, and immune cells, with hypoxia being a fundamental environmental characteristic of this ecology. Under normal physiological conditions, a delicate balance exists among these complex "residents", with disruptions potentially leading to inflammatory bowel disease (IBD). The core pathology of IBD features a disrupted intestinal epithelial barrier, alongside evident immune and microecological disturbances. Central to these interconnected networks is hypoxia-inducible factor-1α (HIF-1α), which is a key regulator in gut cells for adapting to hypoxic conditions and maintaining gut homeostasis. Short-chain fatty acids (SCFAs), as pivotal gut metabolites, serve as vital mediators between the host and microbiota, and significantly influence intestinal ecosystem. Recent years have seen a surge in research on the roles and therapeutic potential of HIF-1α and SCFAs in IBD independently, yet reviews on HIF-1α-mediated SCFAs regulation of IBD under hypoxic conditions are scarce. This article summarizes evidence of the interplay and regulatory relationship between SCFAs and HIF-1α in IBD, pivotal for elucidating the disease's pathogenesis and offering promising therapeutic strategies.

A High-Fat-Diet-Induced Microbiota Imbalance Correlates with Oxidative Stress and the Inflammatory Response in the Gut of Freshwater Drum (Aplodinotus grunniens).

Lipids are critical nutrients for aquatic animals, and excessive or insufficient lipid intake can lead to physiological disorders, which further affect fish growth and health. In aquatic animals, the gut microbiota has an important regulatory role in lipid metabolism. However, the effects of a high-fat diet on physical health and microbiota diversity in the gut of freshwater drum (Aplodinotus grunniens) are unclear. Therefore, in the present study, a control group (Con, 6%) and a high-fat diet group (HFD, 12%) were established for a 16-week feeding experiment in freshwater drum to explore the physiological changes in the gut and the potential regulatory mechanisms of bacteria. The results indicated that a high-fat diet inhibited antioxidant and immune capacity while increasing inflammation, apoptosis and autophagy in gut cells. Transcriptome analysis revealed significant enrichment in immune-related, apoptosis-related and disease-related pathways. Through 16S rRNA analysis, a total of 31 genus-level differentially abundant bacterial taxa were identified. In addition, a high-fat diet reduced gut microbial diversity and disrupted the ecological balance of the gut microbiota (Ace, Chao, Shannon and Simpson indices). Integrated analysis of the gut microbiota combined with physiological indicators and the transcriptome revealed that gut microbial disorders were associated with intestinal antioxidants, immune and inflammatory responses, cell apoptosis and autophagy. Specifically, genus-level bacterial taxa in Proteobacteria (Plesiomonas, Arenimonas, Erythrobacter and Aquabacteriumb) could serve as potential targets controlling the response to high-fat-diet stimulation.

Gut bacteria-derived serotonin promotes immune tolerance in early life.

The gut microbiota promotes immune system development in early life, but the interactions between the gut metabolome and immune cells in the neonatal gut remain largely undefined. Here, we demonstrate that the neonatal gut is uniquely enriched with neurotransmitters, including serotonin, and that specific gut bacteria directly produce serotonin while down-regulating monoamine oxidase A to limit serotonin breakdown. We found that serotonin directly signals to T cells to increase intracellular indole-3-acetaldehdye and inhibit mTOR activation, thereby promoting the differentiation of regulatory T cells, both ex vivo and in vivo in the neonatal intestine. Oral gavage of serotonin into neonatal mice resulted in long-term T cell-mediated antigen-specific immune tolerance toward both dietary antigens and commensal bacteria. Together, our study has uncovered an important role for specific gut bacteria to increase serotonin availability in the neonatal gut and identified a function of gut serotonin in shaping T cell response to dietary antigens and commensal bacteria to promote immune tolerance in early life.

The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2′s high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2′s role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.