RESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Intestinos/citologia , Organoides/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Dibenzazepinas/farmacologia , Células Enteroendócrinas/citologia , Interleucina-4/farmacologia , MacacaRESUMO
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.
Assuntos
Acetilcolina/metabolismo , Diferenciação Celular , Interleucina-4/farmacologia , Intestinos/fisiologia , Organoides/metabolismo , Animais , Perfilação da Expressão Gênica , Interleucina-4/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Macaca fuscata/fisiologia , Macaca mulatta/metabolismo , Macaca mulatta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/fisiologiaRESUMO
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.
Assuntos
Lectinas/biossíntese , Celulas de Paneth/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Interleucinas/farmacologia , Lectinas/genética , Lectinas/metabolismo , Celulas de Paneth/metabolismo , Interleucina 22RESUMO
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.
Assuntos
Duodeno/citologia , Células Enteroendócrinas/metabolismo , Íleo/citologia , Jejuno/citologia , Organoides/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Colecistocinina/genética , Colecistocinina/metabolismo , Polipeptídeo Inibidor Gástrico/genética , Polipeptídeo Inibidor Gástrico/metabolismo , Grelina/genética , Grelina/metabolismo , Glucagon/genética , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Secretina/genética , Secretina/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
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.
Assuntos
Células Enteroendócrinas/metabolismo , Intestinos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células HEK293 , Humanos , Macaca mulatta , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
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.
RESUMO
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.