RESUMO
Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.
Assuntos
Imunidade Adaptativa , Colo , Citometria de Fluxo , Imunidade Inata , Animais , Colo/imunologia , Colo/metabolismo , Camundongos , Citometria de Fluxo/métodos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Células Mieloides/metabolismoRESUMO
The enteric nervous system (ENS), known as the intrinsic nervous system of the gastrointestinal tract, is composed of a diverse array of neuronal and glial cell subtypes. Fascinating questions surrounding the generation of cellular diversity in the ENS have captivated ENS biologists for a considerable time, particularly with recent advancements in cell type-specific transcriptomics at both population and single-cell levels. However, the current focus of research in this field is predominantly restricted to the study of enteric neuron subtypes, while the investigation of enteric glia subtypes significantly lags behind. Despite this, enteric glial cells (EGCs) are increasingly recognized as equally important regulators of numerous bowel functions. Moreover, a subset of postnatal EGCs exhibits remarkable plasticity and multipotency, distinguishing them as critical entities in the context of advancing regenerative medicine. In this review, we aim to provide an updated overview of the current knowledge on this subject, while also identifying key questions that necessitate future exploration.
RESUMO
Hirschsprung disease is a congenital malformation where ganglia of the neural crest-derived enteric nervous system are missing over varying lengths of the distal gastrointestinal tract. This complex genetic condition involves both rare and common variants in dozens of genes, many of which have been functionally validated in animal models. Modifier loci present in the genetic background are also believed to influence disease penetrance and severity, but this has not been frequently tested in animal models. Here, we addressed this question using Holstein mice in which aganglionosis is due to excessive deposition of collagen VI around the developing enteric nervous system, thereby allowing us to model trisomy 21-associated Hirschsprung disease. We also asked whether the genetic background might influence the response of Holstein mice to GDNF enemas, which we recently showed to have regenerative properties for the missing enteric nervous system. Compared to Holstein mice in their original FVB/N genetic background, Holstein mice maintained in a C57BL/6N background were found to have a less severe enteric nervous system defect and to be more responsive to GDNF enemas. This change of genetic background had a positive impact on the enteric nervous system only, leaving the neural crest-related pigmentation phenotype of Holstein mice unaffected. Taken together with other similar studies, these results are thus consistent with the notion that the enteric nervous system is more sensitive to genetic background changes than other neural crest derivatives.
Assuntos
Colágeno Tipo VI/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Doença de Hirschsprung/tratamento farmacológico , Doença de Hirschsprung/genética , Animais , Modelos Animais de Doenças , Enema , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medicina Regenerativa , Resultado do TratamentoRESUMO
For a long time, melanocytes were believed to be exclusively derived from neural crest cells migrating from the neural tube toward the developing skin. This notion was then challenged by studies suggesting that melanocytes could also be made from neural crest-derived Schwann cell precursors (SCPs) on peripheral nerves. A SCP origin was inferred from lineage tracing studies in mice using a Plp1 promoter-controlled Cre driver transgene (Plp1-CreERT2) and a fluorescent Rosa26 locus-controlled Cre reporter allele (Rosa26FloxSTOP-YFP ). However, doubts were raised in part because another SCP-directed Cre driver controlled by the Dhh promoter (Dhh-Cre) was apparently unable to label melanocytes when used with a non-fluorescent Rosa26 locus-controlled Cre reporter (Rosa26FloxSTOP-LacZ ). Here, we report that the same Dhh-Cre driver line can efficiently label melanocytes when used in a pure FVB/N background together with the fluorescent instead of the non-fluorescent Rosa26 locus-controlled Cre reporter. Our data further suggest that the vast majority of skin melanocytes are SCP-derived. Interestingly, we also discovered that SCPs contribute inner ear melanocytes in a region-specific manner, extensively contributing to the cochlea but not to the vestibule.
Assuntos
Diferenciação Celular , Cóclea/citologia , Proteínas Hedgehog/metabolismo , Melanócitos/citologia , Células de Schwann/citologia , Pele/citologia , Células-Tronco/citologia , Sistema Vestibular/citologia , Animais , Cóclea/metabolismo , Proteínas Hedgehog/genética , Melanócitos/metabolismo , Camundongos , Camundongos Transgênicos , Células de Schwann/metabolismo , Pele/metabolismo , Células-Tronco/metabolismo , Sistema Vestibular/metabolismoRESUMO
Excess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5'-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.
Assuntos
Amina Oxidase (contendo Cobre)/farmacologia , Histamina/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Colo/metabolismo , Feminino , Peróxido de Hidrogênio/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Músculo Liso/metabolismoRESUMO
Hirschsprung disease (HSCR) is a complex genetic disorder of neural crest development resulting in incomplete formation of the enteric nervous system (ENS). This life-threatening neurocristopathy affects 1/5000 live births, with a currently unexplained male-biased ratio. To address this lack of knowledge, we took advantage of the TashT mutant mouse line, which is the only HSCR model to display a robust male bias. Our prior work revealed that the TashT insertional mutation perturbs a Chr.10 silencer-enriched non-coding region, leading to transcriptional dysregulation of hundreds of genes in neural crest-derived ENS progenitors of both sexes. Here, through sex-stratified transcriptome analyses and targeted overexpression in ENS progenitors, we show that male-biased ENS malformation in TashT embryos is not due to upregulation of Sry-the murine ortholog of a candidate gene for the HSCR male bias in humans-but instead involves upregulation of another Y-linked gene, Ddx3y. This discovery might be clinically relevant since we further found that the DDX3Y protein is also expressed in the ENS of a subset of male HSCR patients. Mechanistically, other data including chromosome conformation captured-based assays and CRISPR/Cas9-mediated deletions suggest that Ddx3y upregulation in male TashT ENS progenitors is due to increased transactivation by p53, which appears especially active in these cells yet without triggering apoptosis. Accordingly, in utero treatment of TashT embryos with the p53 inhibitor pifithrin-α decreased Ddx3y expression and abolished the otherwise more severe ENS defect in TashT males. Our data thus highlight novel pathogenic roles for p53 and DDX3Y during ENS formation in mice, a finding that might help to explain the intriguing male bias of HSCR in humans.
Assuntos
RNA Helicases DEAD-box/genética , Doença de Hirschsprung/genética , Antígenos de Histocompatibilidade Menor/genética , Animais , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Sistema Nervoso Entérico/metabolismo , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Doença de Hirschsprung/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Mutagênese Insercional , Mutação , Crista Neural/metabolismo , Fatores Sexuais , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. METHODS: We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. RESULTS: GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. CONCLUSIONS: GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.
Assuntos
Colo/efeitos dos fármacos , Colo/inervação , Sistema Nervoso Entérico/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Doença de Hirschsprung/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Animais , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Disbiose , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Sistema Nervoso Entérico/fisiopatologia , Microbioma Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/patologia , Doença de Hirschsprung/fisiopatologia , Humanos , Absorção Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Permeabilidade , Recuperação de Função Fisiológica , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Técnicas de Cultura de TecidosRESUMO
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These "enteric" neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret e al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.
RESUMO
Hirschsprung's disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR--named Holstein--that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.
Assuntos
Movimento Celular , Colágeno Tipo VI/biossíntese , Colo/inervação , Doença de Hirschsprung/metabolismo , Crista Neural/metabolismo , Animais , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Colágeno Tipo VI/genética , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Síndrome de Down/complicações , Síndrome de Down/genética , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Doença de Hirschsprung/genética , Doença de Hirschsprung/patologia , Humanos , Lactente , Recém-Nascido , Camundongos , Camundongos Transgênicos , Crista Neural/patologiaRESUMO
BACKGROUND: Intestinal atresia is a rare congenital disorder with an incidence of 3/10,000 birth. About one-third of patients have severe intestinal dysfunction after surgical repair. We examined whether prenatal gastrointestinal obstruction might effect on the myenteric plexus and account for subsequent functional disorders. METHODOLOGY/PRINCIPAL FINDINGS: We studied a rat model of surgically induced antenatal atresia, comparing intestinal samples from both sides of the obstruction and with healthy rat pups controls. Whole-mount preparations of the myenteric plexus were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (nNOS). Quantitative reverse transcription PCR was used to analyze mRNAs for inflammatory markers. Functional motility and permeability analyses were performed in vitro. Phenotypic studies were also performed in 8 newborns with intestinal atresia. In the experimental model, the proportion of nNOS-immunoreactive neurons was similar in proximal and distal segments (6.7±4.6% vs 5.6±4.2%, pâ=â0.25), but proximal segments contained a higher proportion of ChAT-immunoreactive neurons (13.2±6.2% vs 7.5±4.3%, pâ=â0.005). Phenotypic changes were associated with a 100-fold lower concentration-dependent contractile response to carbachol and a 1.6-fold higher EFS-induced contractile response in proximal compared to distal segments. Transcellular (pâ=â0.002) but not paracellular permeability was increased. Comparison with controls showed that modifications involved not only proximal but also distal segments. Phenotypic studies in human atresia confirmed the changes in ChAT expression. CONCLUSION: Experimental atresia in fetal rat induces differential myenteric plexus phenotypical as well as functional changes (motility and permeability) between the two sides of the obstruction. Delineating these changes might help to identify markers predictive of motility dysfunction and to define guidelines for post-surgical care.
Assuntos
Modelos Animais de Doenças , Doenças Fetais/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Atresia Intestinal/fisiopatologia , Plexo Mientérico/patologia , Animais , Área Sob a Curva , Atropina , Colina O-Acetiltransferase/metabolismo , Peroxidase do Rábano Silvestre , Humanos , Recém-Nascido , Manitol , NG-Nitroarginina Metil Éster , Neurônios/citologia , Óxido Nítrico Sintase/metabolismo , Permeabilidade , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Postnatal changes in the enteric nervous system (ENS) are involved in the establishment of colonic motility. In adult rats, butyrate induced neuroplastic changes in the ENS, leading to enhanced colonic motility. Whether butyrate can induce similar changes during the postnatal period remains unknown. Enemas (Na-butyrate) were performed daily in rat pups between postnatal day (PND) 7 and PND 17. Effects of butyrate were evaluated on morphological and histological parameters in the distal colon at PND 21. The neurochemical phenotype of colonic submucosal and myenteric neurons was analyzed using antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS). Colonic motility and neuromuscular transmission was assessed in vivo and ex vivo. Butyrate (2.5 mM) enemas had no impact on pup growth and histological parameters compared with control. Butyrate did not modify the number of Hu-immunoreactive (IR) neurons per ganglia. A significant increase in the proportion (per Hu-IR neurons) of nNOS-IR myenteric and submucosal neurons and ChAT-IR myenteric neurons was observed in the distal colon after butyrate enemas compared with control. In addition, butyrate induced a significant increase in both nitrergic and cholinergic components of the neuromuscular transmission compared with control. Finally, butyrate increased distal colonic transit time compared with control. We concluded that butyrate enemas induced neuroplastic changes in myenteric and submucosal neurons, leading to changes in gastrointestinal functions. Our results support exploration of butyrate as potential therapy for motility disorders in preterm infants with delayed maturation of the ENS.
Assuntos
Butiratos/administração & dosagem , Colo/efeitos dos fármacos , Plexo Mientérico/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Animais Recém-Nascidos , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Colo/inervação , Colo/metabolismo , Enema , Motilidade Gastrointestinal/efeitos dos fármacos , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND & AIMS: Little is known about the environmental and nutritional regulation of the enteric nervous system (ENS), which controls gastrointestinal motility. Short-chain fatty acids (SCFAs) such as butyrate regulate colonic mucosa homeostasis and can modulate neuronal excitability. We investigated their effects on the ENS and colonic motility. METHODS: Effects of butyrate on the ENS were studied in colons of rats given a resistant starch diet (RSD) or intracecal perfusion of SCFAs. Effects of butyrate were also studied in primary cultures of ENS. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) and by quantitative polymerase chain reaction. Signaling pathways involved were analyzed by pharmacologic and molecular biology methods. Colonic motility was assessed in vivo and ex vivo. RESULTS: In vivo and in vitro, RSD and butyrate significantly increased the proportion of ChAT- but not nNOS-immunoreactive myenteric neurons. Acetate and propionate did not reproduce the effects of butyrate. Enteric neurons expressed monocarboxylate transporter 2 (MCT2). Small interfering RNAs silenced MCT2 and prevented the increase in the proportion of ChAT- immunoreactive neurons induced by butyrate. Butyrate and trichostatin A increased histone H3 acetylation in enteric neurons. Effects of butyrate were prevented by inhibitors of the Src signaling pathway. RSD increased colonic transit, and butyrate increased the cholinergic-mediated colonic circular muscle contractile response ex vivo. CONCLUSION: Butyrate or histone deacetylase inhibitors might be used, along with nutritional approaches, to treat various gastrointestinal motility disorders associated with inhibition of colonic transit.