Biology subjects

Danhof, H. A.

Publications and source records attributed to Danhof, H. A..

7 recordsLinked to original sources

bioRxiv2026

Infant gut microbiomes contribute to metabolic states that impact brain function

Alterations in the gut microbiome are associated with neurodevelopmental disorders, but causal mechanisms and therapeutic strategies remain undefined. Here, we demonstrate that human infant microbiomes isolated during the first six months of life drive behavioral impairments in mice and that microbiota-based interventions restore mice to normal behavior. Early-life microbiomes from twelve infants who later exhibited cognitive deficits at 2 years old (low-scoring) transferred adverse metabolic, brain, and behavioral phenotypes to mice, in contrast to microbiomes from twenty-three cognitively typical or high-scoring infants. Deficits in mice were rescued by fecal microbiota transplant from high-scoring infants or a rationally designed consortium that promoted amino acid levels. We confirmed lower fecal amino acid concentrations in low-scoring infants and replicated the association between early-life microbiome composition and cognitive outcomes in a second geographically independent infant cohort. Altogether, we discovered an early-life microbiome-mediated metabolic state causally linked to cognitive deficits and amenable to microbial intervention.

microbiology↗

bioRxiv2024

Limosilactobacillus reuteri promotes the expression and secretion of enteroendocrine- and enterocytederived hormones

Observations that intestinal microbes can beneficially impact host physiology have prompted investigations into the therapeutic usage of such microbes in a range of diseases. For example, the human intestinal microbe Limosilactobacillus reuteri strains ATCC PTA 6475 and DSM 17938 are being considered for use for intestinal ailments including colic, infection, and inflammation as well as non- intestinal ailments including osteoporosis, wound healing, and autism spectrum disorder. While many of their beneficial properties are attributed to suppressing inflammatory responses in the gut, we postulated that L. reuteri may also regulate hormones of the gastrointestinal tract to affect physiology within and outside of the gut. To determine if L. reuteri secreted factors impact the secretion of enteric hormones, we treated an engineered jejunal organoid line, NGN3-HIO, which can be induced to be enriched in enteroendocrine cells, with L. reuteri 6475 or 17938 conditioned medium and performed transcriptomics. Our data suggest that these L. reuteri strains affect the transcription of many gut hormones, including vasopressin and luteinizing hormone subunit beta, which have not been previously recognized as being produced in the gut epithelium. Moreover, we find that these hormones appear to be produced in enterocytes, in contrast to canonical gut hormones which are produced in enteroendocrine cells. Finally, we show that L. reuteri conditioned media promotes the secretion of several enteric hormones including serotonin, GIP, PYY, vasopressin, and luteinizing hormone subunit beta. These results support L. reuteri affecting host physiology through intestinal hormone secretion, thereby expanding our understanding of the mechanistic actions of this microbe.

microbiology↗

bioRxiv2024

Emerging Clostridioides difficile ribotypes have divergent metabolic phenotypes

Clostridioides difficile is a gram-positive spore-forming pathogen that commonly causes diarrheal infections in the developed world. Although C. difficile is a genetically diverse species, certain ribotypes are overrepresented in human infections. It is unknown if metabolic adaptations are essential for the emergence of these epidemic ribotypes. Here, we tested carbon substrate utilization by 88 C. difficile isolates and looked for differences in growth between 22 ribotypes. By profiling clinical isolates, we assert that C. difficile is a generalist species capable of growing on a variety of carbon substrates. Further, C. difficile strains clustered by phylogenetic relationship and displayed ribotype-specific and clade-specific metabolic capabilities. Surprisingly, we observed that two emerging lineages, ribotypes 023 and 255, have divergent metabolic phenotypes. In addition, although C. difficile Clade 5 is the most evolutionary distant clade and often detected in animals, it displayed more robust growth on simple dietary sugars than Clades 1-4. Altogether, our results corroborate the generalist metabolic strategy of C. difficile and demonstrate lineage-specific metabolic capabilities. In addition, our approach can be adapted to the study of additional pathogens to ascertain their metabolic niches in the gut. IMPORTANCEThe gut pathogen Clostridioides difficile utilizes a wide range of carbon sources. Microbial communities can be rationally designed to combat C. difficile by depleting its preferred nutrients in the gut. However, C. difficile is genetically diverse with hundreds of identified ribotypes and most of its metabolic studies were performed with lab-adapted strains. Here, we profiled carbon metabolism by a myriad of C. difficile clinical isolates. While the metabolic capabilities of these isolates clustered by their genetic lineage, we observed surprising metabolic divergence between two emerging lineages. We also found that the most genetically distant clade grew robustly on simple dietary sugars, posing intriguing questions about the adaptation of C. difficile to the human gut. Altogether, our results underscore the importance of considering the metabolic diversity of pathogens in the study of their evolution and the rational design of therapeutic interventions.

microbiology↗

bioRxiv2023

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit1, the anti-inflammatory immune system2-4, wound healing5-7, normal social behavior in mice8-10, and prevents bone reabsorption11-17. Each of these functions is impacted by oxytocin18-22, and oxytocin signaling is required for L. reuteri-mediated wound healing5 and social behavior9; however, the initiating events in the gut that lead to oxytocin stimulation and related beneficial functions remain unknown. Here we found evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We subsequently found that L. reuteri facilitates oxytocin secretion directly from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells23, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

microbiology↗

bioRxiv2022

Separation of the effects of small intestinal microbiome-diet interactions on human gut hormone secretion

Microbial regulation of gut hormones is a potential mechanism by which the gut microbiome acts on systemic physiology. However, there are limited systems that permit study of how small intestinal microbes and diet modulate gut hormone secretion. Here we present the platform Culturing and Application of Microbes on Intestinal Organoids (CAMIO) and demonstrate its usage in studying the effects of diet and microbes on gut hormones. We validate that CAMIO supports long-term cultivation of a small intestinal microbiome in different dietary sugars and show that CAMIO permits measurement of gut hormones released from jejunal organoids in response to products of the small intestinal communities. In doing so, we observe differential secretion of ghrelin, PP, and PYY according to whether the microbial communities were grown in glucose-fructose versus sucrose or trehalose. We expect CAMIO to be useful in mechanistically understanding how diet and microbes collectively regulate gut hormones.

microbiology↗

bioRxiv2020

ROTAVIRUS INDUCES INTERCELLULAR CALCIUM WAVES THROUGH ADP SIGNALING

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains incompletely characterized. Here we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids. Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 antagonists or CRISPR/Cas9 knockout of P2Y1. Blocking the paracrine ADP signal reduces rotavirus replication, inhibits rotavirus-induced serotonin release and fluid secretion, and reduces diarrhea severity in neonatal mice. This is the first evidence that viruses exploit ICWs to amplify diarrheal signaling; a finding which has broad implications for gastrointestinal physiology. SummaryRotavirus triggers the extracellular release of ADP from infected cells to dysregulate nearby uninfected cells and activate pro-disease pathways.

microbiology↗

bioRxiv2019

Human intestinal enteroids with inducible neurogenin-3 expression as a novel model of gut hormone secretion

BackgroundEnteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but due to their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are a biologically relevant in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, like the intestine, HIEs spontaneously produce few EECs, which limits their study.\n\nMethodsTo increase the number of EECs in HIEs, we used lentivirus transduction to stably engineer jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIEs). We examined the impact of NGN3 induction on EECs by quantifying the increase in the enterochromaffin cells and other EEC subtypes. We functionally assessed secretion of serotonin and EEC hormones in response to norepinephrine and rotavirus infection.\n\nResultsTreating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, demonstrating increased enterochromaffin cell differentiation. Despite increased ChgA-positive cells, other differentiated cell types of the epithelium remained largely unchanged by gene expression and immunostaining. RNA sequencing of doxycycline-induced tetNGN3- HIEs identified increased expression of key hormones and enzymes associated with several other EEC subtypes. Doxycycline-induced tetNGN3-HIEs secreted serotonin, monocyte chemoattractant protein-1, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin in response to norepinephrine and rotavirus infection, further supporting the presence of multiple EEC types.\n\nConclusionsWe have combined HIEs and inducible-NGN3 expression to establish a flexible in vitro model system for functional studies of EECs in enteroids and advance the molecular and physiological investigation of EECs.\n\nSynopsisEnteroendocrine cells have low abundance but exert widespread effects on gastrointestinal physiology. We engineered human intestinal enteroids with inducible expression of neurogenin-3, resulting in increased enteroendocrine cells and facilitating investigations of host responses to the dynamic intestinal environment.

microbiology↗