Intestinal IncRNAs and circRNAs Regulated by Dietary-Lipid Stimuli.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

Hepatitis E virus (HEV), a leading cause of acute viral hepatitis worldwide, is primarily transmitted via the fecal-oral route. A clinical study has reported that the intestine of a chronic hepatitis E patient is positive for HEV. However, whether the intestinal epithelium acts as a barrier for HEV transmission or whether productive enteric infection enhances transfer of the virus to the liver remains unclear. The advent of organoid technology provides a valuable platform for advancing the study of HEV-host interactions in a more physiologically relevant context. In this study, we demonstrate that primary human intestinal organoids (HIOs) efficiently support HEV replication. The infection was sustained in differentiated HIOs with specific phenotypes of intestinal cell types, namely enterocyte, goblet cell, and enteroendocrine cell lineages. Next, we constructed a gut-liver axis model using a transwell system by co-culturing HIOs with human liver-derived organoids. Importantly, infectious viral particles produced in HIOs were capable of transmission to human liver-derived organoids in this model. Bile acids are essential mediators of gut-liver crosstalk. We found that supplementing human bile or the primary bile acid chenodeoxycholic acid inhibited HEV replication in organoids via the farnesoid X receptor (FXR) signaling pathway. The effects of the secondary bile acid, ursodeoxycholic acid, were opposite and promoted viral replication. In conclusion, this model provides a novel approach to study the gut-liver axis in HEV transmission and the impact of bile acids in modulating HEV infection.

The historical lack of models recapitulating the complexity of the human intestinal epithelium has hindered studies into many aspects of human enteric virus biology. Immortalized and transformed cell lines are typically limited by the presence of only one cell type, whereas susceptibility in animal models often requires infection routes that differ from humans or necessitates modification of immune system components [1, 2]. In addition, comparing animals from different species remains a confounding factor when trying to infer how findings may apply to human health. Thus, the development of human gastrointestinal organoids to study virus–host interactions marks a significant advance. They provide a physiologically relevant ex vivo platform in which human enteric microorganisms can be studied interacting with the human intestinal epithelium. Furthermore, their intermediate complexity, falling between cell lines and animal models, adds an additional tool to better understand these viruses. Here, we highlight how gastrointestinal organoids have provided new insights into the biology of human enteric viruses and the potential of this technology for advancing the field. The different flavors of gastrointestinal organoids Gastrointestinal organoids are three-dimensional (3D) structures derived from primary tissues (i.e., patient biopsy) containing intestinal stem/progenitor cells or from human pluripotent stem cells (hPSCs) [3]. They contain multiple intestinal epithelial cell types that perform critical functions that are also observed in the human intestine (e.g., absorption, barrier function, differentiation). Induced pluripotent stem cell–derived human intestinal organoids (HIOs) most closely resemble the human fetal intestine [4] and may encompass an epithelium alone [5] or also contain a mesenchyme [6]. However, most infectious disease laboratories work with human intestinal enteroids (HIEs), patient-derived, 3D epithelium-only structures that can be transitioned to a 2D monolayer on plates or transwells. HIEs maintain the physiological and genetic characteristics of their sources for long periods [7–10]. They can be differentiated from the crypt-like state into villus-like state by withdrawing growth factors required to maintain stem cells (i.e., WNT3A) from the culture media [11–13]. Cellular differentiation of specific cell lineages can be further achieved by pharmacologic or genetic means. For example, secretory cells are enriched following dibenzazepine (DBZ) treatment to block NOTCH signaling [12] and enteroendocrine cells by overexpression of NEUROGENIN-3 [14]. In addition, receptor activator of NF-κB ligand (RANKL) treatment of HIE with and without tumor necrosis factor alpha (TNF-α) addition has been used to drive microfold cell development [15, 16]. However, HIE and HIO do not completely mimic the intestinal epithelium in vivo. For example, they do not entirely reproduce the cellularity observed in vivo as few Paneth cells and no Tuft cells are present [17]. Furthermore, the lack of villi and genetic or pharmacologic skewing of differentiation may also affect the proportionality of individual cell lineages.

Inflammatory bowel disease (IBD) is a chronic remitting disorder with increasing incidence worldwide. The intestinal epithelial barrier plays a major role in IBD, contributing to its pathogenesis, evolution, and perpetuation over time. Until recently, studies focused on exploring the role of the intestinal epithelium in IBD were hampered by the lack of techniques for the long-term culturing of human primary epithelial cells ex vivo. Recently, however, a methodology for generating stable human 3D epithelial cultures directly from adult intestinal stem cells was established. These long-term cultures, called organoids, mimic the tissue of origin and can be generated from small-size intestinal tissue samples, making it a promising tool for modeling the course of IBD.In this review, we provide an overview of the versatility of human organoid cultures in IBD modeling. We discuss recent advances and current limitations in the application of this tool for modeling the contribution of the intestinal epithelium alone and in combination with other key cellular and molecular players in the context of IBD pathophysiology. Finally, we outline the pressing need for technically standardizing the laboratory manipulation of human epithelial organoids for their broader implementation in clinically oriented IBD studies.

BackgroundTimely resolution of innate immune responses activated by surgical intervention is crucial for patient recovery. While cytokines and innate immune cells are critical in inflammation resolution, the specific role of IL-18 in these processes remains controversial and underexplored.MethodsWe investigate determinants of successful recovery using peripheral blood samples from orthopedic surgery (ORT) patients (n = 33) at T0 (before surgery), T1 (24 h after surgery) and T2 (3 days after surgery). Monocytes from ORT patients underwent immunophenotyping together with bulk transcriptomic analysis. We found that IL-18 strongly defines the recovery immune signature. These results were further validated in vitro by comparing IL-18 and TNF-α effects on monocytes, and in 3D human intestine organoids together with single cell (sc)-RNAseq analysis.ResultsTranscriptomics of ORT monocytes revealed upregulation of ITG family integrins, namely ITGB3 and ITGB5, CXCL family chemokines, notably CXCL1-3, CXCL5, and SCL/TAL1 factor controlling differentiation and migration, but not pro-inflammatory genes. Similar changes were observed in IL-18 stimulated healthy donor monocytes in vitro, including an increase in CD11b, CD64, and CD86 levels, accompanied by increased phosphorylation of Akt but not NFκB. These changes were attenuated in the presence of TNF-α, thus showing a unique role of IL-18 when acting alone without its most frequent paired cytokine TNF-α. We further confirmed that IL-18 induces monocyte-macrophage transition and migration using human intestinal organoids. Finally, TNF-α/IL-18 ratio showed a high predictive value of clinical severity in septic patients.ConclusionsWe propose a novel role of IL-18 on monocyte migration and macrophage transition characterizing successful orthopedic surgery recovery, as well as the ratio of IL-18/TNF-α as a novel marker of inflammation resolution, with potential implications for patient monitoring and therapeutic strategies.

We previously demonstrated that neurotensin, a 13-amino-acid gut hormone peptide, enhances small intestinal epithelial cell fatty acid uptake through inhibition of AMPK. Here, utilizing Drosophila and mouse models in vivo, as well as mouse and human small intestinal epithelial organoids or monolayers ex vivo, we determine the targets of neurotensin and AMPK associated with obesity and aging. High-fat diet and aging decreased AMPK and insulin signaling, which was prevented by neurotensin deficiency. High-fat diet feeding increased FABP1 protein expression in wild-type mice; this effect was attenuated in neurotensin-deficient mice. AICAR and metformin increased AMPK phosphorylation in young but not in aged small intestinal epithelial cells. By contrast, AICAR and metformin inhibited FABP1 mRNA and protein expression. Moreover, cytosolic colocalization of AMPKα1 and FABP1 was noted in IEC-6 cells. AMPK phosphorylation and FABP1 expression was decreased in aged wild-type small intestinal epithelial cells; however, this effect was reversed in neurotensin-deficient cells. Results from human duodenal organoids confirm the effects of neurotensin, palmitic acid and metformin on AMPK phosphorylation and FABP1. Finally, overexpressing neurotensin in enteroendocrine cells reduced the lifespan of Drosophila; neurotensin deficiency extended the lifespan of mice fed a high-fat diet. Our findings indicate that neurotensin inhibits AMPK and increases FABP1 in small intestinal epithelial cells under conditions of obesity. Neurotensin deficiency preserves AMPK and FABP1 levels, thus attenuating some of the negative effects of obesity and aging.

Immunomodulatory Microbial Metabolites Identified in Allo-SCT Patients Protect from GvHD By Sting Dependent Modulation of Intestinal Tissue Homeostasis

Organoid vs In Vivo Mouse Model: Which is Better Research Tool to Understand the Biologic Mechanisms of Intestinal Epithelium?

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

A feeding trial was designed to assess the effects of dietary protein and lipid content on growth, feed utilization efficiency, and muscle proximate composition of juvenile mandarin fish, Siniperca scherzeri. Six experimental diets were formulated with a combination of three protein (35, 45, and 55%) and two dietary lipid levels (7 and 14%). Each diet was fed to triplicate groups of fish (8.3 ± 0.1 g) to apparent satiation for 8 weeks. The results showed that growth performance in terms of weight gain (WG) and specific growth rate (SGR) increased with increasing dietary protein level from 35 to 55% at the same dietary lipid level. At the same dietary lipid content, WG and SGR obtained with diets containing 55% protein was significantly higher than those obtained with diets containing 45 and 35% protein. No significant effect on growth rate was found when the dietary level of lipid was increased from 7 to 14%. While the levels of protein and lipid in the diets had no significant effect on feed intake, other nutrient utilization efficiency parameters including daily protein intake (DPI), feed efficiency (FE), and protein efficiency ratio (PER) showed a similar trend to that of growth rates, with the highest values obtained with diets containing 55% protein. Muscle chemical composition was not significantly affected by the different dietary treatments for each dietary lipid or protein level tested. These findings may suggest that a practical diet containing 55% protein and 7% lipid provides sufficient nutrient and energy to support the acceptable growth rates and nutrient utilization of mandarin fish juveniles.

BackgroundPregnant women are considered a high-risk population for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as the virus can infect the placenta and embryos. Recently, SARS-CoV-2 has been widely reported to cause retinal pathological changes and to infect the embryonic retina. The infection of host cells by SARS-CoV-2 is primarily mediated through spike (S) protein, which also plays a crucial role in the pathogenesis of SARS-CoV-2. However, it remains poorly understood how the S protein of SARS-CoV-2 affects retinal development, and the underlying mechanism has not yet been clarified.MethodsWe used human embryonic stem cell-derived retinal organoids (hEROs) as a model to study the effect of S protein exposure at different stages of retinal development. hEROs were treated with 2 μg/mL of S protein on days 90 and 280. Immunofluorescence staining, RNA sequencing, and RT-PCR were performed to assess the influence of S protein exposure on retinal development at both early and late stages.ResultsThe results showed that ACE2 and TMPRSS2, the receptors facilitating SARS-CoV-2 entry into host cells, were expressed in hEROs. Exposure to the S protein induced an inflammatory response in both the early and late stages of retinal development in the hEROs. Additionally, RNA sequencing indicated that early exposure of the S protein to hEROs affected nuclear components and lipid metabolism, while late-stages exposure resulted in changes to cell membrane components and the extracellular matrix.ConclusionThis work highlights the differential effects of SARS-CoV-2 S protein exposure on retinal development at both early and late stages, providing insights into the cellular and molecular mechanisms underlying SARS-CoV-2-induced developmental impairments in the human retina.

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB−/− and MTTP−/− organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

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GENERAL COMMENTARY article Front. Genet., 25 January 2013Sec. RNA Volume 4 - 2013 | https://doi.org/10.3389/fgene.2013.00005