Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans.

Background and aims: WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results: Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion: WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement: All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Perspectives of Rare Disease Experts on Newborn Genome Sequencing.

Importance: Newborn genome sequencing (NBSeq) can detect infants at risk for treatable disorders currently undetected by conventional newborn screening. Despite broad stakeholder support for NBSeq, the perspectives of rare disease experts regarding which diseases should be screened have not been ascertained. Objective: To query rare disease experts about their perspectives on NBSeq and which gene-disease pairs they consider appropriate to evaluate in apparently healthy newborns. Design, Setting, and Participants: This survey study, designed between November 2, 2021, and February 11, 2022, assessed experts' perspectives on 6 statements related to NBSeq. Experts were also asked to indicate whether they would recommend including each of 649 gene-disease pairs associated with potentially treatable conditions in NBSeq. The survey was administered between February 11 and September 23, 2022, to 386 experts, including all 144 directors of accredited medical and laboratory genetics training programs in the US. Exposures: Expert perspectives on newborn screening using genome sequencing. Main Outcomes and Measures: The proportion of experts indicating agreement or disagreement with each survey statement and those who selected inclusion of each gene-disease pair were tabulated. Exploratory analyses of responses by gender and age were conducted using t and χ2 tests. Results: Of 386 experts invited, 238 (61.7%) responded (mean [SD] age, 52.6 [12.8] years [range 27-93 years]; 126 [52.9%] women and 112 [47.1%] men). Among the experts who responded, 161 (87.9%) agreed that NBSeq for monogenic treatable disorders should be made available to all newborns; 107 (58.5%) agreed that NBSeq should include genes associated with treatable disorders, even if those conditions were low penetrance; 68 (37.2%) agreed that actionable adult-onset conditions should be sequenced in newborns to facilitate cascade testing in parents, and 51 (27.9%) agreed that NBSeq should include screening for conditions with no established therapies or management guidelines. The following 25 genes were recommended by 85% or more of the experts: OTC, G6PC, SLC37A4, CYP11B1, ARSB, F8, F9, SLC2A1, CYP17A1, RB1, IDS, GUSB, DMD, GLUD1, CYP11A1, GALNS, CPS1, PLPBP, ALDH7A1, SLC26A3, SLC25A15, SMPD1, GATM, SLC7A7, and NAGS. Including these, 42 gene-disease pairs were endorsed by at least 80% of experts, and 432 genes were endorsed by at least 50% of experts. Conclusions and Relevance: In this survey study, rare disease experts broadly supported NBSeq for treatable conditions and demonstrated substantial concordance regarding the inclusion of a specific subset of genes in NBSeq.

A Roadmap for the Human Gut Cell Atlas.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

Therapy Development for Microvillus Inclusion Disease using Patient-derived Enteroids.

Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na + /H + exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Inhibition of Notch signaling with the γ-secretase inhibitor, DAPT, recovered apical brush border structure and functional Na + /H + exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID. Conflict-of-interest statement: The authors have declared that no conflict of interest exists.

Gasdermin D pore-forming activity is redox-sensitive.

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.

The genetics of monogenic intestinal epithelial disorders.

Monogenic intestinal epithelial disorders, also known as congenital diarrheas and enteropathies (CoDEs), are a group of rare diseases that result from mutations in genes that primarily affect intestinal epithelial cell function. Patients with CoDE disorders generally present with infantile-onset diarrhea and poor growth, and often require intensive fluid and nutritional management. CoDE disorders can be classified into several categories that relate to broad areas of epithelial function, structure, and development. The advent of accessible and low-cost genetic sequencing has accelerated discovery in the field with over 45 different genes now associated with CoDE disorders. Despite this increasing knowledge in the causal genetics of disease, the underlying cellular pathophysiology remains incompletely understood for many disorders. Consequently, clinical management options for CoDE disorders are currently limited and there is an urgent need for new and disorder-specific therapies. In this review, we provide a general overview of CoDE disorders, including a historical perspective of the field and relationship to other monogenic disorders of the intestine. We describe the genetics, clinical presentation, and known pathophysiology for specific disorders. Lastly, we describe the major challenges relating to CoDE disorders, briefly outline key areas that need further study, and provide a perspective on the future genetic and therapeutic landscape.

Nociceptor neurons direct goblet cells via a CGRP-RAMP1 axis to drive mucus production and gut barrier protection.

Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.

Membrane Lipids in Epithelial Polarity: Sorting out the PIPs.

The development of cell polarity in epithelia, is critical for tissue morphogenesis and vectorial transport between the environment and the underlying tissue. Epithelial polarity is defined by the development of distinct plasma membrane domains: the apical membrane interfacing with the exterior lumen compartment, and the basolateral membrane directly contacting the underlying tissue. The de novo generation of polarity is a tightly regulated process, both spatially and temporally, involving changes in the distribution of plasma membrane lipids, localization of apical and basolateral membrane proteins, and vesicular trafficking. Historically, the process of epithelial polarity has been primarily described in relation to the localization and function of protein 'polarity complexes.' However, a critical and foundational role is emerging for plasma membrane lipids, and in particular phosphoinositide species. Here, we broadly review the evidence for a primary role for membrane lipids in the generation of epithelial polarity and highlight key areas requiring further research. We discuss the complex interchange that exists between lipid species and briefly examine how major membrane lipid constituents are generated and intersect with vesicular trafficking to be preferentially localized to different membrane domains with a focus on some of the key protein-enzyme complexes involved in these processes.

The epithelial-specific ER stress sensor ERN2/IRE1ß enables host-microbiota crosstalk to affect colon goblet cell development.

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1ß, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor, ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free WT mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis.

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein's putative involvement in multiple developmental and stem cell maintenance pathways.

Minimally Invasive Image-Guided Gut Transport Function Measurement Probe.

Introduction: Diseases such as celiac disease, environmental enteric dysfunction, infectious gastroenteritis, type II diabetes and inflammatory bowel disease are associated with increased gut permeability. Dual sugar absorption tests, such as the lactulose to rhamnose ratio (L:R) test, are the current standard for measuring gut permeability. Although easy to administer in adults, the L:R test has a number of drawbacks. These include an inability to assess for spatial heterogeneity in gut permeability that may distinguish different disease severity or pathology, additional sample collection for immunoassays, and challenges in carrying out the test in certain populations such as infants and small children. Here, we demonstrate a minimally invasive probe for real-time localized gut permeability evaluation through gut potential difference (GPD) measurement. Materials and Methods: The probe has an outer diameter of 1.2 mm diameter and can be deployed in the gut of unsedated subjects via a transnasal introduction tube (TNIT) that is akin to an intestinal feeding tube. The GPD probe consists of an Ag/AgCl electrode, an optical probe and a perfusion channel all housed within a transparent sheath. Lactated Ringer's (LR) solution is pumped through the perfusion channel to provide ionic contact between the electrodes and the gut lining. The optical probe captures non-scanning (M-mode) OCT images to confirm electrode contact with the gut lining. A separate skin patch probe is placed over an abraded skin area to provide reference for the GPD measurements. Swine studies were conducted to validate the GPD probe. GPD in the duodenum was modulated by perfusing 45 ml of 45 mM glucose. Results: GPD values of -13.1 ± 2.8 mV were measured in the duodenum across four swine studies. The change in GPD in the duodenum with the addition of glucose was -10.5 ± 2.4 mV (p < 0.001). M-mode OCT images provided electrode-tissue contact information, which was vital in ascertaining the probe's proximity to the gut mucosa. Conclusion: We developed and demonstrated a minimally invasive method for investigating gastrointestinal permeability consisting of an image guided GPD probe that can be used in unsedated subjects.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling.

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNß expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.

BACKGROUND: Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1. METHODS: Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples. RESULTS: Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions. CONCLUSIONS: Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.