Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Branched-chain amino acids regulate hyaluronan synthesis and PPARα expression in the skin.

We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in branched-chain amino acid catabolism, on hyaluronan synthesis in mice. The skin levels of hyaluronan and the gene expression levels of hyaluronan synthase (Has)2, Has3, and peroxisome proliferator-activated receptor-α were significantly lower in the BDK-knockout group than in the wild-type group.

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

Interleukin-4 Promotes Tuft Cell Differentiation and Acetylcholine Production in Intestinal Organoids of Non-Human Primate.

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.

Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids.

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1ß, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00487-y.

Up-regulation of gasdermin C in mouse small intestine is associated with lytic cell death in enterocytes in worm-induced type 2 immunity.

"Taste-like" tuft cells in the intestine trigger type 2 immunity in response to worm infection. The secretion of interleukin-13 (IL-13) from type 2 innate lymphoid cells (ILC2) represents a key step in the tuft cell-ILC2 cell-intestinal epithelial cell circuit that drives the clearance of worms from the gut via type 2 immune responses. Hallmark features of type 2 responses include tissue remodeling, such as tuft and goblet cell expansion, and villus atrophy, yet it remains unclear if additional molecular changes in the gut epithelium facilitate the clearance of worms from the gut. Using gut organoids, we demonstrated that IL-4 and IL-13, two type 2 cytokines with similar functions, not only induced the classical type 2 responses (e.g., tuft cell expansion) but also drastically up-regulated the expression of gasdermin C genes (Gsdmcs). Using an in vivo worm-induced type 2 immunity model, we confirmed the up-regulation of Gsdmcs in Nippostrongylus brasiliensis-infected wild-type C57BL/6 mice. Consistent with gasdermin family members being principal effectors of pyroptosis, overexpression of Gsdmc2 in human embryonic kidney 293 (HEK293) cells triggered pyroptosis and lytic cell death. Moreover, in intestinal organoids treated with IL-4 or IL-13, or in wild-type mice infected with N. brasiliensis, lytic cell death increased, which may account for villus atrophy observed in worm-infected mice. Thus, we propose that the up-regulated Gsdmc family may be major effectors for type 2 responses in the gut and that Gsdmc-mediated pyroptosis may provide a conduit for the release of antiparasitic factors from enterocytes to facilitate the clearance of worms.

Expression of TAS2R14 in the intestinal endocrine cells of non-human primates.

BACKGROUND: Recent studies have demonstrated that genes related to bitter taste receptors (TAS2Rs) on various chromosomes are expressed in extra-oral organs of various animals. The bitter taste receptor TAS2R14 is conserved among primate species and shows broad ligand sensitivity. Mice have a number of orthologues to primate TAS2R14 located in tandem on chromosome 16; however, their expression patterns are not unique. OBJECTIVE: We characterized the expression of TAS2R14 in various cell types in the intestines of the rhesus macaque and evaluated its role in hormone production in the gut. METHODS: TAS2R14 expression was examined in the intestines of rhesus macaques, a common non-human primate model, by RT-qPCR and immunohistochemical staining. RESULTS: Mean expression levels of TAS2R14 in the duodenum, ileum, and colon were similar to each other and were lower than those in circumvallate papillae. An immunohistochemical analysis revealed TAS2R14 immunoreactivity in enteroendocrine cells positive for cholecystokinin, serotonin, and the G protein GNAT3. CONCLUSION: These results suggest that primate TAS2R14 is broadly expressed in the intestine, mainly in enteroendocrine cells, and promotes gut hormone secretion in response to bitter stimuli.

Generation of intestinal chemosensory cells from nonhuman primate organoids.

Several gastrointestinal epithelial cells are involved in taste signal transduction. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans. The 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal and human tissues. Here, we report the generation of intestinal chemosensory cells from nonhuman primates, macaques, using an organoid culture system. We were able to maintain macaque intestinal organoids in the proliferation medium for more than six months. Upon switching to differentiation medium, we observed a drastic change in organoid morphology and chemosensory cell marker protein expression. This switch from proliferation to differentiation was confirmed by transcriptome analysis of the duodenum, jejunum, and ileum organoids. We further observed that the supplementation of culture media with interleukin (IL)-4 or the Notch inhibitor dibenzazepine (DBZ) accelerated terminal cell differentiation into chemosensory cells. Overall, we generated monkey intestinal organoids for the first time. These organoids are suitable for studying the function of primate chemosensory cells.

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.

Interleukin-4 suppresses the proliferation and alters the gene expression in enteroids.

Interleukin (IL)-4 is known as a cytokine mainly involved in allergy and inflammation, but recent studies have suggested that IL-4 plays a part in the differentiation process of various cells. Since the effect of IL-4 on intestinal epithelial cells, particularly cryptic cells including stem cells, is poorly understood, we investigated IL-4-induced changes in intestinal epithelial cells using mouse jejunal organoids called enteroids. IL-4 treatment decreased cell proliferation, the expression of the stem cell markers leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4), and Lgr5-positive cells in enteroids. Among the differentiation markers, IL-4 significantly decreased the gene expression levels of the Paneth cell markers lysozyme 1 (Lyz1) and regenerating islet-derived protein 3 gamma (Reg3γ). A fluorescent immunostaining showed that IL-4 attenuated the emission and fluorescence intensity derived from lysozyme, which is enriched in Paneth cells. These results suggest that functional changes in Paneth cells caused by IL-4 may contribute to the reduction in Lgr5-positive cells and proliferative activity. IL-4 may affects gut function by altering the proliferation and the gene expression in enteroids.

Negative effects of a low-quality protein diet on wound healing via modulation of the MMP2 activity in rats.

Protein malnutrition is largely associated with a delay or failure of the healing process. However, the effect of dietary protein quality on wound healing is largely unknown. This study aimed to reveal the effect of dietary protein quality on wound healing and elucidate the regulatory mechanisms in a rat model of full-thickness cutaneous wounds. Rats were fed a normal diet for a week, and then they were divided into three groups that were fed the following diet for the experimental period: casein diet, gluten diet and gluten + lysine diet. The gluten diet significantly decreased body weight and wound healing compared with the casein diet, but this effect was reversed by supplementation with lysine. The numbers of leukocytes were significantly higher in the skin of the gluten group than those in the casein group. The wounded skin tissues of the gluten group showed lower amounts of collagen deposition compared with that in the casein group. Our results also showed that both matrix metalloproteinase (MMP) 2 activity and MMP14 mRNA levels were significantly increased in the skin of the gluten group, compared with the casein group. In summary, this study suggests low-quality protein diets have negative effects on wound healing via modulation of MMP2 activity in rats.

Maternal gut microbiota in pregnancy influences offspring metabolic phenotype in mice.

Antibiotics and dietary habits can affect the gut microbial community, thus influencing disease susceptibility. Although the effect of microbiota on the postnatal environment has been well documented, much less is known regarding the impact of gut microbiota at the embryonic stage. Here we show that maternal microbiota shapes the metabolic system of offspring in mice. During pregnancy, short-chain fatty acids produced by the maternal microbiota dictate the differentiation of neural, intestinal, and pancreatic cells through embryonic GPR41 and GPR43. This developmental process helps maintain postnatal energy homeostasis, as evidenced by the fact that offspring from germ-free mothers are highly susceptible to metabolic syndrome, even when reared under conventional conditions. Thus, our findings elaborate on a link between the maternal gut environment and the developmental origin of metabolic syndrome.

Expression of Bitter Taste Receptors in the Intestinal Cells of Non-Human Primates.

(1) Background: Recent studies have investigated the expression of taste-related genes in the organs of various animals, including humans; however, data for additional taxa are needed to facilitate comparative analyses within and among species. (2) Methods: We investigated the expression of taste-related genes in the intestines of rhesus macaques, the non-human primates most commonly used in experimental models. (3) Results: Based on RNAseq and qRT-PCR, genes encoding bitter taste receptors and the G-protein gustducin were expressed in the gut of rhesus macaques. RNAscope analysis showed that one of the bitter receptors, TAS2R38, was expressed in some cells in the small intestine, and immunohistochemical analysis revealed the presence of T2R38-positive cells in the villi of the intestines. (4) Conclusions: These results suggest that bitter receptors are expressed in the gut of rhesus macaques, supporting the use of macaques as a model for studies of human taste, including gut analyses.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Identification of Reg3ß-producing cells using IL-22-stimulated enteroids.

Reg3ß, a lectin, displays antibacterial activity. This study investigated Reg3ß-expressing cells using IL-22-stimulated enteroids. IL-22 stimulation elevated the mRNA and protein levels of Reg3ß. IL-22 also increased the mRNA levels of CD133 (a transit-amplifying cell marker) and lysozyme (a Paneth cell marker). Immunohistochemistry showed partial colocalization of Reg3ß- and lysozyme-positive cells, suggesting that Paneth cells are one of Reg3ß-producing cells.

Generation and characterization of Suncus murinus intestinal organoid: a useful tool for studying motilin secretion.

Motilin, a 22-amino-acid peptide produced in the upper small intestine, induces strong gastric contraction in fasted state. In many rodents, motilin and its cognate receptors exist as pseudogenes, which has delayed motilin research in the past decades. Recently, the house musk shrew (Suncus murinus) was developed as a useful model for studying motilin and gastrointestinal motility. However, due to a lack of motilin-producing cell lines and difficulties in culturing small intestinal cells, the regulatory mechanisms of motilin secretion and its messenger RNA (mRNA) transcription have remained largely unclear. In this study, we generated small intestinal organoids from S. murinus for the first time. Using methods similar to mouse organoid generation, we found crypt-like budding structures 3 days after isolating intestinal tissues. The organoids grew gradually with time. In addition, the generated organoids were able to be passaged and maintained for 6 months or longer. Motilin messenger RNA (mRNA) and immunopositive cells were observed in both S. murinus intestinal organoids and primary tissues. This is the first report of intestinal organoids in S. murinus, and our results suggest that S. murinus intestinal organoids could be useful for analyzing motilin secretion and transcription.

Transient receptor potential vanilloid 4 mediates sour taste sensing via type III taste cell differentiation.

Taste buds are comprised of taste cells, which are classified into types I to IV. Transient receptor potential (TRP) channels play a significant role in taste perception. TRP vanilloid 4 (TRPV4) is a non-selective cation channel that responds to mechanical, thermal, and chemical stimuli. The present study aimed to define the function and expression of TRPV4 in taste buds using Trpv4-deficient mice. In circumvallate papillae, TRPV4 colocalized with a type IV cell and epithelial cell marker but not type I, II, or III markers. Behavioural studies showed that Trpv4 deficiency reduced sensitivity to sourness but not to sweet, umami, salty, and bitter tastes. Trpv4 deficiency significantly reduced the expression of type III cells compared with that in wild type (WT) mice in vivo and in taste bud organoid experiments. Trpv4 deficiency also significantly reduced Ki67-positive cells and ß-catenin expression compared with those in WT circumvallate papillae. Together, the present results suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice.

Wound fluid of rats fed protein-free diets delays wound healing through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Adequate nutrition is required to maintain healthy skin integrity, and malnourished patients with poor protein diet often experience delayed wound healing. Understanding the cellular mechanisms of protein malnutrition will justify the importance of optimal protein diets in health and disease defense. Therefore in the present study, we examined the effects of changes in wound fluid composition and its function caused by protein malnutrition on wound healing. Rats were fed a control (CO; 20% protein) diet or a protein-free (PF) diet for 2 weeks; we then created full-thickness wounds on the dorsolateral skin. On day 5 after wounding, frozen sections of the wounds were created to investigate the state of granulation tissues, and wound fluid obtained from the rats was collected to examine variations in cytokine levels and its function. Wound closure was significantly delayed from day 4 until total wound closure in rats fed a PF diet. Thickness of granulation tissue, which is composed of mainly dermal fibroblasts, and Ki67 immunohistochemical staining were significantly decreased in rats fed PF diets. PF diets decreased insulin-like growth factor (IGF)-I, which promotes wound healing, and increased IGF-binding protein-1, which inhibits IGF-I bioavailability, in wound fluid. Wound fluid obtained from rats fed a PF diet suppressed dermal fibroblast proliferation. Furthermore, the wound fluid remarkably decreased the phosphorylation level of IGF-I receptor ß (IGF-IR) and extracellular signal-regulated kinase (ERK)(1/2) in dermal fibroblasts. These results show that wound fluid of rats fed PF diets delays wound healing by inhibiting granulation tissue formation through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Starvation reduces hyaluronan synthesis by suppressing TGF-ß1/IGF-I signaling in rat skin.

Although starvation has been reported to influence the functions of various tissues, its effects on the skin are not well understood. In this study, we investigated the effect of starvation on hyaluronan synthesis in rat skin. Starvation reduced hyaluronan synthesis in the skin. Starvation also decreased the skin mRNA expression of transforming growth factor (TGF)-ß1, which enhances the gene expression of rhas2 and rhas3. The serum levels of insulin-like growth factor (IGF)-I, which enhances rhas2, rhas3, and TGF-ß1 mRNA expression, in the starvation group were considerably lower than those in the control (CO) group. IGF-IR phosphorylation was substantially lower in the starvation group compared with the CO group. These findings suggest that starvation reduces hyaluronan synthesis in the skin by suppressing TGF-ß1/IGF-I signaling. Abbreviations: HAS: hyaluronan synthase; IGF-I: insulin-like growth factor-I; IGFBP-1: insulin-like growth factor binding protein-1; TGF-ß1: transforming growth factor-ß1; TBST: tris buffered saline containing 0.5% (v/v) Tween 20; HABP: hyaluronic acid binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.