Intestinal Epithelial Response Research Articles

Pig Milk Exosome Packaging ssc-miR-22-3p Alleviates Pig Intestinal Epithelial Cell Injury and Inflammatory Response by Targeting MAPK14

Inflammatory diseases of the intestinal tract in piglets severely impair the economic performance of pig farms. Pig milk exosomes can encapsulate miRNAs which can then enter the piglet intestine to play an immunomodulatory role. Previously, we comparatively analyzed and identified exosomal miRNAs in the colostrum and mature milk of Bamei and Landrace pigs, and we screened for ssc-miR-22-3p, which is associated with inflammation and immune response; however, the role played by ssc-miR-22-3p in the immune response in IPEC-J2 cells is not yet clear. In this study, we first constructed a pig intestinal inflammatory response model using Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly (I:C)), and we investigated the role of ssc-miR-22-3p targeting MAPK14 in the regulation of LPS and Poly (I:C)-induced inflammatory injury in IPEC-J2 cells by RT-qPCR, cell counting kit-8 (CCK-8), EdU staining, lactate dehydrogenase (LDH) activity assay, and dual luciferase reporter gene assay. We successfully established LPS and Poly (I:C)-induced cell damage models in IPEC-J2 cells. The immune response of IPEC-J2 cells was stimulated by induction of IPEC-J2 cells at 10 μg/mL LPS and 20 μg/mL Poly (I:C) for 24 h. Overexpression of ssc-miR-22-3p decreased cytokine expression and promoted cell viability and proliferation. The functional enrichment analysis revealed that ssc-miR-22-3p targets genes enriched in the pathways of negative regulation of inflammatory response and bacterial invasion of epithelial cells. The validity of the binding site of ssc-miR-22-3p to MAPK14 was tested by a dual luciferase reporter gene. Pig milk exosome ssc-miR-22-3p promotes cell viability and proliferation by targeting MAPK14, and it alleviates LPS and Poly (I:C)-induced inflammatory responses in IPEC-J2 cells.

324 Use of organoids as in vitro model to study intestinal function

Abstract Organoids are tridimensional structures cultured in the lab from stem cells. Organoids have been developed for many different organs, particularly those with Lgr5+ stem cells. The organs of the digestive system belong to this group of organs and to date, organoids have been established from all the digestive tract tissues and organs including liver, gall bladder and pancreas. Although most protocols to generate organoids have focused on human and mouse organs, organoids from domestic and production animals like dogs, cats, rabbits, pigs, chickens, turkeys, and horses are actively used in research. In this presentation, the principles of developing intestinal organoids will be covered, along with a summary of how they have been used to address intestinal function and disease in our lab and by others. In our lab, we use swine enteroids (intestinal organoids) to study the intestinal epithelial responses to nutrients and pathogens. We have explored differential responses of the intestinal epithelium to organic and inorganic sources of dietary zinc, and we are currently investigating the mechanism of pathogenesis of Lawsonia intracellularis, the causative agent of porcine intestinal adenopathy. We will also discuss methods for modeling intestinal physiology using enteroids, and the advantages and challenges of working with swine enteroids.

Network Pharmacology Analysis and Retrospective Cohort Study Revealing the Effect of Qingyi Decoction on Intestinal Paralysis in Patients with Severe Acute Pancreatitis.

Intestinal dysfunction plays an important role in the clinical progress and prognosis of severe acute pancreatitis (SAP). Qingyi decoction (QYD) has shown beneficial effects on intestinal function recovery, but the prevention actions of the QYD on intestinal paralysis and its mechanism have not been fully explored. The possible molecular mechanism was unraveled by network pharmacology, including active ingredients and potential target prediction, as well as GO, KEGG, and REATCOME pathway enrichment analyses. The potential interactions between the main active ingredients of the QYD and core genes were explored by molecular docking. A retrospective cohort study on 137 patients with SAP from Tianjin Nankai Hospital was conducted to evaluate the preventive effect of QYD on intestinal paralysis. A total of 110 active ingredients in QYD were screened out, and 37 key targets were predicted by network pharmacology. GO, KEGG, and REATCOME enrichment analyses showed that bioinformatics annotation of the hub genes was mainly involved in intestinal epithelial functions and inflammatory response pathways. The main components of QYD possessed good affinity with IL-6, TNF, CASP3, CXCL8, and CRP by molecular docking. Patients who used QYD plus usual care seemed to have fewer intestinal paralysis rates, lower risk of renal insufficiency, ARDS and blood purification therapy, and shorter hospital and ICU stays. The multivariable regression analyses indicated that the mode of nasogastric and enemas administration of QYD (P = 0.010) and timely intervention with QYD (P = 0.045) were the independent protective factors for intestinal paralysis prevention in patients with SAP. In conclusion, QYD can be used as an effective adjuvant procedure to prevent the occurrence and development of intestinal paralysis in patients with SAP. The mechanisms may be involved in the anti-inflammatory response and maintenance of intestinal epithelial function.

Parthanatos initiated by ROS-induced DNA damage is involved in intestinal epithelial injury during necrotizing enterocolitis

Necrotizing enterocolitis (NEC) involves intestinal epithelial damage and inflammatory response and is associated with high morbidity and mortality in infants. To improve therapeutic prospects, elucidating underlying molecular mechanisms of intestinal epithelial damage during NEC is of the essence. Poly (ADP-ribose) polymerase 1 (PARP1)-dependent parthanatos is a programmed inflammatory cell death. In the present study, the presence of parthanatos-associated proteins PARP1 and poly (ADP-ribose) (PAR), along with high expression of DNA damage-associated biomarkers, 8-hydroxy-2’-deoxyguanosine (8-OHdG) and phosphorylation of histone H2AX (γH2AX), were discovered in the intestinal tissues of NEC infants. Additionally, the upregulated expression of PARP1 and PAR in NEC intestinal tissues correlated distinctly with clinical indices indicative of NEC incidence and severity. Furthermore, we demonstrated that inhibiting the expression of parthanatos-associated proteins, by either pharmacological blockage using 3-aminobenzamide (3-AB), an inhibitor of PARP1, or genetic knockout using Parp1-deficient mice, resulted in substantial improvements in both histopathological severity scores associated with intestinal injury and inflammatory reactions. Moreover, in an in vitro NEC model, reactive oxygen species (ROS)-induced DNA damage promoted the formation of PAR and nuclear translocation of apoptosis-inducing factor (AIF), thus activating PARP1-dependent parthanatos in Caco-2 cells and human intestinal organoids. Our work verifies a previously unexplored role for parthanatos in intestinal epithelial damage during NEC and suggests that inhibition of parthanatos may serve as a potential therapeutic strategy for intervention of NEC.

Role of long non-coding RNA in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a group of recurrent chronic inflammatory diseases, including Crohn's disease (CD) and ulcerative colitis (UC). Although IBD has been extensively studied for decades, its cause and pathogenesis remain unclear. Existing research suggests that IBD may be the result of an interaction between genetic factors, environmental factors and the gut microbiome. IBD is closely related to non-coding RNAs (ncRNAs). NcRNAs are composed of microRNA(miRNA), long non-coding RNA(lnc RNA) and circular RNA(circ RNA). Compared with miRNA, the role of lnc RNA in IBD has been little studied. Lnc RNA is an RNA molecule that regulates gene expression and regulates a variety of molecular pathways involved in the pathbiology of IBD. Targeting IBD-associated lnc RNAs may promote personalized treatment of IBD and have therapeutic value for IBD patients. Therefore, this review summarized the effects of lnc RNA on the intestinal epithelial barrier, inflammatory response and immune homeostasis in IBD, and summarized the potential of lnc RNA as a biomarker of IBD and as a predictor of therapeutic response to IBD in the future.

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

Ferulic Acid Derivatives Ameliorate Intestine Barrier Destruction by Alleviating Inflammatory Responses in Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), a chronic disorder affecting the colon and rectum, involves the overproduction of pro-inflammatory cytokines causing damage to tight junctions (TJ) in the intestinal epithelial cells and chronic inflammation. The current mainstay of treatment, sulfasalazine, often causes adverse effects, thereby necessitating the exploration of alternative herbal medicines with fewer side effects. Portulaca oleracea L. (P. oleracea), a traditional medicinal herb, contains feruloyl amide compounds. We synthesized new compounds by conjugating ferulic acid (FA) with (±)-octopamine. Our study focused on novel FA derivatives that demonstrate protective effects against the intestinal epithelial barrier and inflammatory responses. In lipopolysaccharide-induced cells, C1 and C1a inhibited the production of inflammatory mediators. In Caco-2 cells, these compounds maintained the TJ protein expression, thereby demonstrating their protective effects on the epithelial barrier. In a mouse model of dextran sulfate sodium-induced IBD, a treatment with these compounds ameliorated features including a body weight reduction, colon shortening, an increased disease activity index, and histopathological changes. Furthermore, C1a demonstrated greater efficacy than C1 at the same concentration. These findings suggest that the novel FA derivative (C1a) effectively alleviates clinical signs and inflammatory mediators in IBD, making these compounds potential candidates as natural medicines for the treatment of IBD.

The Role of Ferroptosis in Inflammatory Bowel Disease: Mechanisms and Therapeutic Implications

Inflammatory Bowel Disease (IBD), encompassing Crohn's disease and ulcerative colitis, is characterized by chronic inflammation of the gastrointestinal tract, with an increasing incidence worldwide. Recent advancements in cellular biology have identified ferroptosis, a form of programmed cell death driven by iron-dependent lipid peroxidation, as a critical player in the pathology of IBD. This article reviews the current understanding of ferroptosis and its distinctive mechanisms, including the role of GPx4, Nrf2-HO-1 pathways, and iron metabolism in the context of IBD. It also examines the dual nature of iron in intestinal health and disease, contributing to both physiological functions and pathological processes through oxidative stress and inflammation. The implications of ferroptosis in the intestinal epithelial cell death, barrier function, and immune response are discussed, highlighting its potential as a novel therapeutic target. Despite the promising insights, the article underscores the need for further research to elucidate the complex mechanisms of ferroptosis in IBD and to translate these findings into effective therapeutic strategies. The emerging evidence positions ferroptosis at the crossroads of metabolic, inflammatory, and cell death pathways, offering a unique perspective on the interplay between nutrition, genetics, and immunity in intestinal health and disease.

Development and application of a multi-step porcine in vitro system to evaluate feedstuffs and feed additives for their efficacy in nutrient digestion, digesta characteristics, and intestinal immune responses

In vitro model provides alternatives to the use of live animals in research. In pig nutrition, there has been a tremendous increase in in vivo research over the decades. Proper utilization of in vitro models could provide a screening tool to reduce the needs of in vivo studies, research duration, cost, and the use of animals and feeds. This study aimed to develop a multi-step porcine in vitro system to simulate nutrient digestion and intestinal epithelial immune responses affected by feedstuffs and feed additives. Seven feedstuffs (corn, corn distillers dried grains with solubles [corn DDGS], barley, wheat, soybean meal, soy protein concentrates, and Corynebacterium glutamicum cell mass [CGCM]), feed enzymes (xylanase and phytase), and supplemental amino acids (arginine, methionine, and tryptophan), were used in this in vitro evaluation for their efficacy on digestibility, digesta characteristics, and intestinal health compared with the results from previously published in vivo studies. All in vitro evaluations were triplicated. Data were analyzed using Mixed procedure of SAS9.4. Evaluations included (1) nutrient digestibility of feedstuffs, (2) the effects of feed enzymes, xylanase and phytase, on digestibility of feedstuffs and specific substrates, and (3) the effects of amino acids, arginine, tryptophan, and methionine, on anti-inflammatory, anti-oxidative, and anti-heat stress statuses showing their effects (P < 0.05) on the measured items. Differences in dry matter and crude protein digestibility among the feedstuffs as well as effects of xylanase and phytase were detected (P < 0.05), including xylo-oligosaccharide profiles and phosphorus release from phytate. Supplementation of arginine, tryptophan, and methionine modulated (P < 0.05) cellular inflammatory and oxidative stress responses. The use of this in vitro model allowed the use of 3 experimental replications providing sufficient statistical power at P < 0.05. This indicates in vitro models can have increased precision and consistency compared with in vivo animal studies.

A30 MICROBIOTA AND NOD2 REGULATE SMALL INTESTINAL RESTITUTION THROUGH FETAL-LIKE INTESTINAL STEM CELLS

Abstract Background Inflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract, resulting in recurrent injury to the intestinal epithelium. Restitution of the small intestinal epithelium is a coordinated response that involves the dedifferentiation of epithelial cell lineages, proliferation of Lgr5+ intestinal stem cells, and fetal-like stem cell reversion. While gut microbiota are critical mediators of intestinal inflammation, their impact on epithelial restitution remains unclear. Aims We aim to identify the how microbes regulate small intestinal epithelial restitution following damage. Our hypothesis is that gut microbiota accelerate restitution through pattern recognition receptor-driven signals following fetal-like stem cell reversion. Methods Irradiation (IR, 12Gγ) was used to induce a synchronized small intestinal epithelial restitution response in mice. Intestinal restitution kinetics were assessed transcriptionally (scRNA-Seq, qPCR) and histologically. Small intestinal organoids were used to assess epithelial restitution kinetics in vitro. Results ScRNA-Seq of small intestinal epithelial cells following IR from germ-free mice (GF), and specific pathogen-free mice (SPF) mice revealed that microbiota induced greater expression of fetal-like stem cell reversion markers, Ly6a and Clu, and greater expression of the proliferation marker, Pcna. These results were supported histologically as irradiated SPF mice observed an increase in fetal-like stem cells marked by Ly6a and Clu and an increase BrdU+ proliferating cells. ScRNA-seq and in situ hybridization highlighted that fetal-like intestinal stem cells upregulate expression of Nod2, a bacterial pattern recognition receptor. Using intestinal organoids to assess the function of Nod2, we observed that muramyl dipeptide driven Nod2-signlaing potentiates an interferon gene signature following IFNγ and TNFα co-stimulation. Further supporting the role for Nod2 in intestinal restitution, intestinal epithelium specific Nod2KO mice decreased BrdU+ proliferating cells post-IR compared to littermate controls. Conclusions Microbiota promote small intestinal restitution following IR through Nod2-signaling in fetal-like intestinal stem cells. Funding Agencies CIHR

GRANZYME A-PRODUCING CD4 T CELLS ARE DRIVERS OF IMMUNE CHECKPOINT BLOCKADE-INDUCED COLITIS

Abstract Immune checkpoint blockade (ICB) (i.e. anti-CTLA4 and/or anti-PD-1) is a type of cancer immunotherapy that is broadly and effectively used across multiple cancer types. Despite this efficacy, ~60% of individuals of ICB recipients develop autoimmune-like inflammation in the skin, lungs and intestines. Of these barrier sites, an inflamed intestine is the most common form of severe disease that requires cessation of immunotherapy and potential tumor rebound. ICB-induced colitis therefore represents a significant health concern in an increasing population of cancer patients receiving this otherwise effective immunotherapy. Previous work has shown evidence of a dysregulated intestinal CD4 T cell response in ICB colitis patients, however, how CD4 T cells might contribute to disease has remained unclear. Here, we identify a unique subset of T helper (Th) 1-like CD4 T cells that express the serine protease granzyme A (GZMA) that are specifically elevated in ICB-induced colitis patients, suggesting a pathogenic role for GZMA-expressing Th cells in disease. As mouse models for ICB-induced colitis are virtually non-existent, we have profiled existing mouse models of intestinal inflammation for the presence of colonic GZMA-expressing CD4 T cells for more in-depth functional studies. Using this approach, we have identified the T cell transfer model of colitis as a model that mimics the GZMA CD4 T cell phenotype observed in human ICB colitis patients. Within this model, GZMA-expressing CD4 T cells arise early in the intestines after transfer and only minimally express other CD4 T helper (Th) cell lineage cytokines (i.e. IFN-γ or IL-17) indicating that these cells may represent a unique Th cell subtype that are associated with intestinal disease. Indeed, transfer of GZMA-deficient CD4 T cells into colitis-prone mice resulted in reduced intestinal inflammation and damage, despite efficient trafficking of these cells to the intestines. Mechanistically, CD4 T cell-derived GZMA promoted an exaggerated intestinal epithelial cell response to the microbiota, but was not required for immune-editing of the microbiota in this setting. Taken together, our data indicate that GZMA-producing Th cells are likely major contributors to ICB-induced colitis and may represent a valuable therapeutic target for limiting intestinal disease during ICB therapy.

P087 IFN-γ-Induced Intestinal Epithelial Cell Type-Specific Cytotoxic Responses of Human Enteroids: PANoptosis and The Protective Role of Selective JAK1 Inhibitors

Abstract Background Interferon-gamma (IFN-γ) plays an important role in the development of intestinal injury and inflammatory bowel disease (IBD). Using human intestinal organoid (enteorid) models, this study was investigated 1) what is the major response of intestinal epithelial cells (IECs) induced by IFN-γ to elucidate; 2) which programmed cell death (PCD) pathways cause IFN-γ-induced cell death on IECs in the absence of intestinal microbiota and immune cells; 3) how the expression of cell-cell adhesion molecules in epithelial cells is altered by IFN-γ; 4) how epithelial cell differentiation is altered by IFN-γ; 5) whether there is a difference in IFN-γ-induced PCD between different epithelial cell types; and 6) which molecules can block IFN-γ-induced PCD in IECs. Methods Using human enteroids, the major response of IECs induced by IFN-γ was evaluated, focusing on the IFN-γ-induced PCD pathway. The intestinal epithelial cell type-specific response to IFN-γ was assessed by bulk and single-cell RNA sequencing (RNA-seq). Furthermore, the molecules to block IFN-γ-induced PCD in IECs were evaluated. Results As the concentration of IFN-γ in the culture media increased, disruption of organoid structure, growth arrest, and cell death were observed in IFN-γ-treated enteroids on organoid reconstitution, MTT, and EdU assay. Bulk RNA-seq and western blot were identified the activation of pyroptosis, apoptosis, and necroptosis to form the collective inflammatory cell death pathway of PANoptosis. Single-cell RNA-seq indicated that IFN-γ altered epithelial cell differentiation in human enteroids, including expansion of the intstinal stem cell (ISC) population and depletion of the enterocyte population. PCD-associated gene expression was upregulated in enterocytes and goblet cells, but not in ISCs and paneth cells. Individual PCD inhibitors may be insufficient to prevent IFN-γ-induced cytotoxicity, whereas upadacitinib interferes with the downstream signaling of IFN-γ by inhibiting the activity of JAK1 kinase, resulting in effective blocking of PANoptosis. Conclusion PANoptosis is the major mechanism of IFN-γ-induced IEC damage, which was blocked by a selective JAK1 inhibitor. ECs were particularly susceptible to IFN-γ-induced PANoptosis. * This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2022R1A2B5B02002092 and 2021R1C1C2095192) and Future Medicine 2030 Project of the Samsung Medical Center (SMO1230011).

Targeted modulation of intestinal epithelial regeneration and immune response in ulcerative colitis using dual-targeting bilirubin nanoparticles.

Rationale: The therapeutic benefits of bilirubin in the treatment of ulcerative colitis (UC) are considerable, whereas the underlying mechanism of bilirubin on UC remains unclear remains unexplored. In addition, the weak hydrophilicity and toxicity have limited its translational applications. Methods: We have developed a colon dual-targeting nanoparticle, for orally delivering bilirubin through hydrogel encapsulation of hyaluronic acid (HA)-modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles (HA-PLGABilirubin). Confocal microscopy and in vivo imaging were used to evaluate the uptake and the targeted property of HA-PLGABilirubin in UC. Immunohistochemistry, immunofluorescence, and transcriptomic analyses were applied to examine the therapeutic effect and potential mechanism of HA-PLGABilirubin in UC. Results: Our results indicated that HA-PLGAbilirubin can significantly enhance the release of bilirubin at simulated intestinal pH and demonstrate higher cellular uptake in inflammatory macrophages. Moreover, in vivo biodistribution studies revealed high uptake and retention of HA-PLGAbilirubin in inflamed colon tissue of UC mouse model, resulting in effective recovery of intestinal morphology and barrier function. Importantly, HA-PLGAbilirubin exerted potent therapeutic efficacy against ulcerative colitis through modulating the intestinal epithelial/stem cells regeneration, and the improvement of angiogenesis and inflammation. Furthermore, genome-wide RNA-seq analysis revealed transcriptional reprogramming of immune response genes in colon tissue upon HA-PLGAbilirubin treatment in UC mouse model. Conclusion: Overall, our work provides an efficient colon targeted drug delivery system to potentiate the treatment of ulcerative colitis via modulating intestinal epithelium regeneration and immune response in ulcerative colitis.

5-Methoxytryptophan Alleviates Dextran Sulfate Sodium-Induced Colitis by Inhibiting the Intestinal Epithelial Damage and Inflammatory Response.

Colitis is a refractory intestinal inflammatory disease significantly affecting the quality of a patient's life and increasing the risk of exacerbation. The primary factors leading to colitis encompass infections, insufficient blood flow, and the buildup of collagen as well as white blood cells. Among various available therapeutics, 5-methoxytryptophan (5-MTP) has emerged as one of the protectants by inhibiting inflammatory damage. Nonetheless, there is no report on the role of 5-MTP in the treatment of colitis. To verify the anti-inflammatory effect of 5-MTP in vivo, we first constructed mouse model with dextran sulfate sodium-induced colitis. Furthermore, the macrophage infiltration and release of inflammatory factors through western blot (WB) and hematoxylin-eosin staining analyses were examined. Intestinal epithelial cell tight junction damage and apoptosis were investigated by WB analysis, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Finally, we examined the generation of cellular inflammation and analyzed the influence of 5-MTP on M1 polarization at the cellular level. This study initially confirmed that 5-MTP possessed an excellent therapeutic effect on colitis. 5-MTP inhibits macrophage infiltration and the generation of inflammatory factors. In addition to its effects on immune cells, 5-MTP significantly inhibits intestinal epithelial cell tight junction damage and apoptosis in vivo. Moreover, it inhibits inflammation and M1 polarization response in vitro. 5-MTP counteracts excessive inflammation, thereby preventing intestinal epithelial tight junction damage. In addition, inhibition of apoptosis suggests that 5-MTP may be a potential therapeutic agent for colitis.

Segmented filamentous bacteria-induced epithelial MHCII regulates cognate CD4+ IELs and epithelial turnover.

Intestinal epithelial cells have the capacity to upregulate MHCII molecules in response to certain epithelial-adhesive microbes, such as segmented filamentous bacteria (SFB). However, the mechanism regulating MHCII expression as well as the impact of epithelial MHCII-mediated antigen presentation on T cell responses targeting those microbes remains elusive. Here, we identify the cellular network that regulates MHCII expression on the intestinal epithelium in response to SFB. Since MHCII on the intestinal epithelium is dispensable for SFB-induced Th17 response, we explored other CD4+ T cell-based responses induced by SFB. We found that SFB drive the conversion of cognate CD4+ T cells to granzyme+ CD8α+ intraepithelial lymphocytes. These cells accumulate in small intestinal intraepithelial space in response to SFB. Yet, their accumulation is abrogated by the ablation of MHCII on the intestinal epithelium. Finally, we show that this mechanism is indispensable for the SFB-driven increase in the turnover of epithelial cells in the ileum. This study identifies a previously uncharacterized immune response to SFB, which is dependent on the epithelial MHCII function.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumour development in mice fed a high fat diet.

High fat diets, and inflammation are risk factors for colon cancer, however the underlying mechanisms remain to be fully elucidated. The transcriptional co-repressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing lipid oxidation genes and the rate of lipid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis, however whether this is driven by the intestinal metabolic reprogramming, and whether this predisposes these mice to intestinal tumourigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumourigenesis in mice fed a standard (STD) or HF diet (HFD). Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by a HFD. Hdac3deletion also accelerated intestinal tumour development, specifically when fed a HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed a HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of a HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing lipid oxidation, DNA damage and intestinal epithelial cell turnover.

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

ABSTRACT 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 transit, the anti-inflammatory immune system, wound healing, normal social behavior in mice, and prevents bone reabsorption. Oxytocin impacts these functions and oxytocin signaling is required for L. reuteri-mediated wound healing and social behavior; however, the events in the gut leading to oxytocin stimulation and beneficial effects are unknown. Here we report 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 find that L. reuteri facilitates oxytocin secretion 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 cells, 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.

Maturation of the medaka immune system depends on reciprocal interactions between the microbiota and the intestinal tract.

The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.

TET3-mediated DNA oxidation is essential for intestinal epithelial cell response to stressors

DNA methylation functions as a repressive epigenetic mark that can be reversed by the Ten-eleven translocation (TET) family of DNA dioxygenases that sequentially oxidize 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised by DNA base-excision repair factors leading to unmodified cytosines. TET enzymes were recently implicated as potential risk factors for inflammatory bowel disease (IBD), but the contribution of TET-mediated DNA oxidation to intestinal homeostasis and response to environmental stressors are unknown. Here, we show prominent roles of TET3 in regulating mouse intestinal epithelial differentiation and response to luminal stressors. Compared with wild-type littermates, mice with intestinal epithelial cell-specific ablation of Tet3 (Tet3ΔIEC) demonstrated a decreased transcriptome involved in innate immune response, Paneth cell differentiation, and epithelial regeneration. Tet3IEC mice exhibited an elevated susceptibility to enteric pathogen infection that is correlated with a decreased epithelial 5hmC abundance. Infection of human enterocytes or mice with the pathogenic bacteria acutely increased 5hmC abundance. Genome-wide 5hmC profiling revealed a shift of genomic enrichment of 5hmC toward genes involved in activating Notch, Wnt, and autophagy pathways. Furthermore, chemical stressor dextran sulfate sodium (DSS) represses epithelial 5hmC abundance in a temporal fashion, and Tet3IEC mice exhibited increased susceptibility to DSS experimental colitis with reduced regenerative capacity. TET3 is a critical regulator of gut epithelial DNA methylome and transcriptome, especially in response to luminal stressors, for the maintenance of tissue homeostasis.