ABSTRACT
BACKGROUND: Immune cell populations in the intestinal muscularis propria during colitis are poorly resolved. Maintaining homeostasis in this niche is critical, highlighted by the poorer prognosis of inflammatory bowel disease associated with muscularis propria inflammation. METHODS: This study utilizes single-cell RNA sequencing to survey the immune cell populations within the muscularis propria of normal colon and dextran sodium sulfate-induced colitis. Findings are validated by immunohistochemistry, flow cytometry and cell-lineage tracing in vivo, and in vitro assays with muscularis macrophages (MMφ). RESULTS: In naïve conditions, transcriptional duality is observed in MMφs with 2 major subpopulations: conventional resident Cx3cr1+ MMφs and Lyve1+ MMφs. The Lyve1+ population is phagocytic and expresses several known MMφ markers in mouse and human, confirming their identity as a bona fide MMφ subset. Single-cell transcriptomics indicate that resident MMφs are retained during colitis and exhibit plasticity toward an inflammatory profile. Lyve1+ MMφs, which express anti-inflammatory marker CD163, are absent during colitis, as confirmed by flow cytometry. In contrast, lineage tracing finds that resident Cx3cr1+ MMφs remain during colitis and are not completely replaced by the inflammatory infiltrating monocytes. In vitro studies provide biological evidence of the plasticity of resident Cx3cr1+ MMφs in response to lipopolysaccharide (LPS), mirroring transcriptional observations in vivo of their inflammatory plasticity. Potential markers for colitic MMφs, validated in animal models and in individuals with ulcerative colitis, are identified. CONCLUSIONS: Our findings contribute to the understanding of the immune system in the muscularis propria niche during colitis by resolving the heterogeneity and origins of colitic MMφs.
Involvement of the muscularis propria accompanies a poorer prognosis in IBD. This study characterizes muscularis macrophage subpopulations during colitis, highlighting the loss of anti-inflammatory LYVE-1+ macrophages and inflammatory plasticity in resident CX3CR1+ macrophages, providing insights into prognostic and therapeutic targets.
ABSTRACT
The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.
Subject(s)
Bursa of Fabricius , Chickens , Dendritic Cells , Infectious bursal disease virus , Animals , Bursa of Fabricius/virology , Dendritic Cells/physiology , Dendritic Cells/virology , Infectious bursal disease virus/physiology , Birnaviridae Infections/veterinary , Birnaviridae Infections/virology , Poultry Diseases/virology , Poultry Diseases/immunologyABSTRACT
Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAPâ +â cells, whose migration is especially enhanced.
Subject(s)
Agrin , Cell Movement , Neural Stem Cells , Animals , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/transplantation , Mice , Agrin/metabolism , Enteric Nervous System/metabolism , Enteric Nervous System/cytology , Colon/metabolism , Colon/cytology , Neural Crest/metabolism , Neural Crest/cytology , Hirschsprung Disease/metabolism , Hirschsprung Disease/therapy , Stem Cell Transplantation/methodsABSTRACT
Myocardial sleeve around human pulmonary veins plays a critical role in the pathomechanism of atrial fibrillation. Besides the well-known arrhythmogenicity of these veins, there is evidence that myocardial extensions into caval veins and coronary sinus may exhibit similar features. However, studies investigating histologic properties of these structures are limited. We aimed to investigate the immunoreactivity of myocardial sleeves for intermediate filament desmin, which was reported to be more abundant in Purkinje fibers than in ventricular working cardiomyocytes. Sections of 16 human (15 adult and 1 fetal) hearts were investigated. Specimens of atrial and ventricular myocardium, sinoatrial and atrioventricular nodes, pulmonary veins, superior caval vein and coronary sinus were stained with anti-desmin monoclonal antibody. Intensity of desmin immunoreactivity in different areas was quantified by the ImageJ program. Strong desmin labeling was detected at the pacemaker and conduction system as well as in the myocardial sleeves around pulmonary veins, superior caval vein, and coronary sinus of adult hearts irrespective of sex, age, and medical history. In the fetal heart, prominent desmin labeling was observed at the sinoatrial nodal region and in the myocardial extensions around the superior caval vein. Contrarily, atrial and ventricular working myocardium exhibited low desmin immunoreactivity in both adults and fetuses. These differences were confirmed by immunohistochemical quantitative analysis. In conclusion, this study indicates that desmin is abundant in the conduction system and venous myocardial sleeves of human hearts.
Subject(s)
Coronary Sinus , Desmin , Pulmonary Veins , Adult , Humans , Myocardium/pathology , Myocytes, Cardiac , Pulmonary Veins/pathology , Vena Cava, SuperiorABSTRACT
The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000-30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.
Subject(s)
Enteric Nervous System , Neural Stem Cells , Humans , Animals , Swine , Infant , Neurons/metabolism , Intestine, Small , NeurogliaABSTRACT
Previously, the presence of a blood-myenteric plexus barrier and its disruption was reported in experimentally induced colitis via a macrophage-dependent process. The aim of this study is to reveal how myenteric barrier disruption and subsequent neuronal injury affects gut motility in vivo in a murine colitis model. We induced colitis with dextran sulfate sodium (DSS), with the co-administration of liposome-encapsulated clodronate (L-clodronate) to simultaneously deplete blood monocytes contributing to macrophage infiltration in the inflamed muscularis of experimental mice. DSS-treated animals receiving concurrent L-clodronate injection showed significantly decreased blood monocyte numbers and colon muscularis macrophage (MM) density compared to DSS-treated control (DSS-vehicle). DSS-clodronate-treated mice exhibited significantly slower whole gut transit time than DSS-vehicle-treated animals and comparable to that of controls. Experiments with oral gavage-fed Evans-blue dye showed similar whole gut transit times in DSS-clodronate-treated mice as in control animals. Furthermore, qPCR-analysis and immunofluorescence on colon muscularis samples revealed that factors associated with neuroinflammation and neurodegeneration, including Bax1, Hdac4, IL-18, Casp8 and Hif1a are overexpressed after DSS-treatment, but not in the case of concurrent L-clodronate administration. Our findings highlight that MM-infiltration in the muscularis layer is responsible for colitis-associated dysmotility and enteric neuronal dysfunction along with the release of mediators associated with neurodegeneration in a murine experimental model.
Subject(s)
Clodronic Acid , Colitis , Mice , Animals , Clodronic Acid/pharmacology , Colitis/chemically induced , Inflammation , Macrophages , Colon , Dextran Sulfate/toxicity , Mice, Inbred C57BL , Disease Models, AnimalABSTRACT
The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.
Subject(s)
Colorectal Neoplasms , Enteric Nervous System , Humans , Signal Transduction/physiology , Cell Differentiation/physiology , Enteric Nervous System/metabolism , Cell Movement/physiology , Colorectal Neoplasms/metabolism , Neural Crest/metabolism , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolismABSTRACT
There are two types of secretory cells in the chicken bursa of Fabricius (BF): (a) interfollicular epithelial cells (IFE), and (b) bursal secretory dendritic cells (BSDC) in the medulla of bursal follicles. Both cells produce secretory granules, and the cells are highly susceptible to IBDV vaccination and infection. Before and during embryonic follicular bud formation, an electron-dense, scarlet-acid fuchsin positive substance emerges in the bursal lumen, the role of which is unknown. In IFE cells, IBDV infection may induce rapid granular discharge, and in several cells, peculiar granule formation, which suggests that the glycosylation of protein is injured in the Golgi complex. In control birds, the discharged BSDC granules appear in membrane-bound and subsequently solubilized, fine-flocculated forms. The solubilized, fine-flocculated substance is Movat-positive and can be a component of the medullary microenvironment, which prevents the medullary B lymphocytes from nascent apoptosis. Vaccination interferes with the solubilization of the membrane-bound substance, resulting in: (i) aggregation of a secreted substance around the BSDC, and (ii) solid lumps in the depleted medulla. The non-solubilized substance is possibly not "available" for B lymphocytes, resulting in apoptosis and immunosuppression. In IBDV infection, one part of the Movat-positive Mals fuse together to form a medullary, gp-containing "cyst". The other part of Mals migrate into the cortex, recruiting granulocytes and initiating inflammation. During recovery the Movat-positive substance appears as solid, extracellular lumps between the cells of FAE and Mals. Possibly the Mals and Movat-positive extracellular lumps glide into the bursal lumen via FAE to eliminate cell detritus from the medulla.
Subject(s)
Birnaviridae Infections , Infectious bursal disease virus , Poultry Diseases , Vaccines , Animals , Chickens , B-Lymphocytes , Bursa of Fabricius , Glycoproteins , Birnaviridae Infections/veterinaryABSTRACT
The gastrointestinal tract is innervated by an intrinsic neuronal network, known as the enteric nervous system (ENS), and by extrinsic axons arising from peripheral ganglia. The nerve of Remak (NoR) is an avian-specific sacral neural crest-derived ganglionated structure that extends from the cloaca to the proximal midgut and, similar to the pelvic plexus, provides extrinsic innervation to the distal intestine. The molecular mechanisms controlling extrinsic nerve fiber growth into the gut is unknown. In vertebrates, CXCR4, a cell-surface receptor for the CXCL12 chemokine, regulates migration of neural crest cells and axon pathfinding. We have employed chimeric tissue recombinations and organ culture assays to study the role of CXCR4 and CXCL12 molecules in the development of colorectal innervation. CXCR4 is specifically expressed in nerve fibers arising from the NoR and pelvic plexus, while CXCL12 is localized to the hindgut mesenchyme and enteric ganglia. Overexpression of CXCL12 results in significantly enhanced axonal projections to the gut from the NoR, while CXCR4 inhibition disrupts nerve fiber extension, supporting a previously unreported role for CXCR4 and CXCL12 signaling in extrinsic innervation of the colorectum.
Subject(s)
Enteric Nervous System , Gastrointestinal Tract , Animals , Gastrointestinal Tract/innervation , Colon , Neurons/physiology , Signal Transduction , Neural CrestABSTRACT
Enteric nervous system development relies on intestinal colonization by enteric neural crest-derived cells (ENCDCs). This is driven by a population of highly migratory and proliferative ENCDCs at the wavefront, but the molecular characteristics of these cells are unknown. ENCDCs from the wavefront and the trailing region were isolated and subjected to RNA-seq. Wavefront-ENCDCs were transcriptionally distinct from trailing ENCDCs, and temporal modelling confirmed their relative immaturity. This population of ENCDCs exhibited altered expression of ECM and cytoskeletal genes, consistent with a migratory phenotype. Unlike trailing ENCDCs, the wavefront lacked expression of genes related to neuronal or glial maturation. As wavefront ENCDC genes were associated with migration and developmental immaturity, the genes that remain expressed in later progenitor populations may be particularly pertinent to understanding the maintenance of ENCDC progenitor characteristics. Dusp6 expression was specifically upregulated at the wavefront. Inhibiting DUSP6 activity prevented wavefront colonization of the hindgut, and inhibited the migratory ability of post-colonized ENCDCs from midgut and postnatal neurospheres. These effects were reversed by simultaneous inhibition of ERK signaling, indicating that DUSP6-mediated ERK inhibition is required for ENCDC migration in mouse and chick.
Subject(s)
Enteric Nervous System , Mice , Animals , Neural Crest/metabolism , Transcriptome , Cell Movement/physiology , IntestinesABSTRACT
The aim of this study is to follow the gp production in IBDV-vaccinated and challenged birds. The progress of IBDV infection was monitored using anti-VP2 immunocytochemistry, light and transmission electron microscopy. In the medulla of the bursal follicle, the Movat pentachrome staining discovered an extracellular glycoprotein (gp) produced by bursal secretory dendritic cells (BSDCs). The secretory granules of BSDCs either discharge resulting in extracellular gp or fuse together forming intracellular corpuscles. The double fate of granules suggests a dual function of BSDCs: (a.) For the discharged granules, gp contributes to the medullary microenvironment (ME). (b.) The intracellular corpuscles may be the sign of BSDC transformation to a macrophage-like cell (Mal). Infectious bursal disease virus (IBDV) infection accelerates the BSDC transformation to Mal. The decreased number of BSDCs is feedback for the precursor cells of BSDCs lodging in the cortico-medullary epithelial arches (CMEA), where they proliferate. Opening the CMEA, the precursor cells enter the medulla, and differentiate to immature BSDCs. The virus uptake in the corpuscles prevents the granular discharge resulting in the absence of gp and alteration in ME. In vaccine-take birds, the mitotic rate of BSDC precursor cells cannot restore the precursor pool; therefore, in the case of IBDV challenge, the number of newly formed BSDCs is too low for outbreak of clinical disease. The BSDCs, as a primary target of IBDV, may contribute to the long-lasting immunosuppressive status of IBDV-infected chickens.
Subject(s)
Birnaviridae Infections , Infectious bursal disease virus , Poultry Diseases , Viral Vaccines , Animals , Antibodies, Viral , Birnaviridae Infections/veterinary , Bursa of Fabricius , Chickens , Dendritic Cells , GlycoproteinsABSTRACT
[This corrects the article DOI: 10.3389/fnmol.2021.757646.].
ABSTRACT
The bursal secretory dendritic cell (BSDC) was discovered more than 40 yr ago. It is a highly polarized, granulated cell, locating in the medulla of bursal follicle. The cytoplasmic granules either discharge or fuse together forming large, irregular-shaped, dense bodies. Formation of the dense bodies could be the first sign of BSDC transformation to macrophage-like cell (Mal) which is the result of terminal maturation of BSDC. The BSDC is non-phagocytic, unlike Mal. The discharged substance may be attached to the cell membrane (membrane-bound form) and after detaching, appears as a flocculated substance in the extracellular space of medulla. Movat pentachrome staining shows, that this substance is a glycoprotein (gp), which may be contributed to the microenvironment of the medulla. Medullary lymphocytes are floating in the gp. Precursors of the BSDC locate in the corticomedullary epithelial arches, which operate under the effect of Notch/Serrate signaling. The Notch signaling determines the fate of lymphoblast-like precursor cells and inhibits the appearance of immunoglobulin heavy chain. In the arches, the precursor cells proliferate and entering the medulla differentiate. The dense bodies pack the virus particles, which prevents the granular discharge, resulting in disappearance of extracellular gp, but gp emerges inside the virus containing Mal. In infected birds, the Mal contains either apoptotic cells or virus particles. If vaccination or infectious bursal disease virus (IBDV) infection use up the BSDC precursors, the recovery of follicle is critical.
Subject(s)
Birnaviridae Infections , Infectious bursal disease virus , Poultry Diseases , Animals , Birnaviridae Infections/veterinary , Bursa of Fabricius , Chickens , Dendritic Cells , Glycoproteins , MacrophagesABSTRACT
The bursa of Fabricius (BF) plays a central role in the development of B lymphocytes in birds. During embryonic development the BF primordium is colonized by myeloid and lymphoid prebursal stem cells to form the follicle buds, which ultimately develop into lymphoid follicles with a central medullary and an outer cortical region. Lympho-myeloid differentiation within the medulla is fundamental to normal B cell development. In contrast, the complexity of the cellular composition of the follicular cortex and its role in B cell differentiation has only recently begun to be studied. As an effort to characterize the different bursal cells we have produced a large panel of monoclonal antibodies (mAbs) by immunizing mice with a BF cell suspension of guinea fowl (Numida meleagris). One of these antibodies (clone: 7H3) was found to recognize a 80 kDa cell surface antigen expressed first in the yolk sac blood island of 2-day-old guinea fowl and chicken embryos, and later detected in the embryonic circulation and primary lymphoid organs. Double immunofluorescence revealed that chB6+ (Bu-1+) B cells of embryonic BF co-express the 7H3 antigen. 7H3 immunoreactivity of the bursal follicles gradually diminished after hatching and only a subpopulation of cortical B cells expressed the 7H3 antigen. In addition, in post-hatched birds 7H3 mAb recognizes all T lymphocytes of the thymus, peripheral lymphoid organs and blood. Embryonic BF injected with the 7H3 mAb showed a near complete block of lymphoid follicle formation In conclusion, 7H3 mAb labels a new differentiation antigen specific for avian hematopoietic cells, which migrate through the embryonic mesenchyme, colonize the developing BF lymphoid follicles, and differentiate into a subpopulation of cortical B cells. The staining pattern of the 7H3 mAb and the correlation of expression with cell migration suggest that the antigen will serve as valuable immunological marker for studying the ontogeny of avian B cells.
Subject(s)
Bursa of Fabricius , Galliformes , Animals , Antibodies, Monoclonal , B-Lymphocytes , Cell Differentiation , Chick Embryo , Chickens , MiceABSTRACT
Mesenchymal reticular cells (MRCs) form a supporting system in the cortex of the bursal follicle. The stellate-shaped MRCs exhibit a low electron density, which is helpful for their identification. A remarkable feature of MRC is the formation of multiple blebs in the nuclear envelope. The large, irregularly shaped blebs-which are perinuclear spaces-may be detached from the nuclear membrane, creating a sac-like granular endoplasmic reticulum (GER). Inside the bleb, membrane-bound bodies originate from cytoplasmic impressions. The cytoplasm contains a few round mitochondria, in which the internal membranes form either ovoid vesicles or the entire internal structure is indistinct. These mitochondria may be associated with the blebs. The classical Golgi complex with cis and trans faces cannot be recognized, but the accumulation of very small vesicles occurs around two or three stacked flat cisterns. The MRC forms a continuous layer along the corticomedullary basal lamina (CMBL), and during cell migration between the cortex and medulla, it may contribute to the temporary closure of the gap in the CMBL. At the outer surface of the cortex, transitory cells between the MRC and fibrocytes of the interfollicular connective tissue are present, and both cells can produce GER by blebbing. This finding suggests that MRCs and fibrocytes may have a common origin. The other stromal cell is the macrophage (Ma), which may fuse together to form multinucleated giant cells. The definition of histological classification of the third type of stromal cell is questionable, but certain morphological features may be referred to as progenitors of MRCs.
Subject(s)
Bursa of Fabricius , Chickens , Animals , Bursa of Fabricius/anatomy & histology , Cytoplasm , Stromal CellsABSTRACT
BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.
Subject(s)
Induced Pluripotent Stem Cells , Myocytes, Cardiac , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Line , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Myocytes, Cardiac/metabolism , Phenotype , TranscriptomeABSTRACT
The enteric nervous system (ENS), which is derived from enteric neural crest cells (ENCCs), represents the neuronal innervation of the intestine. Compromised ENCC migration can lead to Hirschsprung disease, which is characterized by an aganglionic distal bowel. During the craniocaudal migration of ENCCs along the gut, we find that their proliferation is greatest as the ENCC wavefront passes through the ceca, a pair of pouches at the midgut-hindgut junction in avian intestine. Removal of the ceca leads to hindgut aganglionosis, suggesting that they are required for ENS development. Comparative transcriptome profiling of the cecal buds compared with the interceca region shows that the non-canonical Wnt signaling pathway is preferentially expressed within the ceca. Specifically, WNT11 is highly expressed, as confirmed by RNA in situ hybridization, leading us to hypothesize that cecal expression of WNT11 is important for ENCC colonization of the hindgut. Organ cultures using embryonic day 6 avian intestine show that WNT11 inhibits enteric neuronal differentiation. These results reveal an essential role for the ceca during hindgut ENS formation and highlight an important function for non-canonical Wnt signaling in regulating ENCC differentiation.
Subject(s)
Enteric Nervous System/metabolism , Neural Crest/metabolism , Neurons/metabolism , Wnt Proteins/genetics , Animals , Cell Differentiation/genetics , Cell Movement/genetics , Chick Embryo , Chickens/genetics , Chickens/growth & development , Digestive System/growth & development , Digestive System/metabolism , Enteric Nervous System/growth & development , Hirschsprung Disease/genetics , Hirschsprung Disease/pathology , Humans , Intestines/innervation , Neural Crest/cytology , RNA/genetics , RNA-Seq , Transcriptome/genetics , Wnt Signaling Pathway/geneticsABSTRACT
BACKGROUND & AIMS: Neuroinflammation in the gut is associated with many gastrointestinal (GI) diseases, including inflammatory bowel disease. In the brain, neuroinflammatory conditions are associated with blood-brain barrier (BBB) disruption and subsequent neuronal injury. We sought to determine whether the enteric nervous system is similarly protected by a physical barrier and whether that barrier is disrupted in colitis. METHODS: Confocal and electron microscopy were used to characterize myenteric plexus structure, and FITC-dextran assays were used to assess for presence of a barrier. Colitis was induced with dextran sulfate sodium, with co-administration of liposome-encapsulated clodronate to deplete macrophages. RESULTS: We identified a blood-myenteric barrier (BMB) consisting of extracellular matrix proteins (agrin and collagen-4) and glial end-feet, reminiscent of the BBB, surrounded by a collagen-rich periganglionic space. The BMB is impermeable to the passive movement of 4 kDa FITC-dextran particles. A population of macrophages is present within enteric ganglia (intraganglionic macrophages [IGMs]) and exhibits a distinct morphology from muscularis macrophages, with extensive cytoplasmic vacuolization and mitochondrial swelling but without signs of apoptosis. IGMs can penetrate the BMB in physiological conditions and establish direct contact with neurons and glia. Dextran sulfate sodium-induced colitis leads to BMB disruption, loss of its barrier integrity, and increased numbers of IGMs in a macrophage-dependent process. CONCLUSIONS: In intestinal inflammation, macrophage-mediated degradation of the BMB disrupts its physiological barrier function, eliminates the separation of the intra- and extra-ganglionic compartments, and allows inflammatory stimuli to access the myenteric plexus. This suggests a potential mechanism for the onset of neuroinflammation in colitis and other GI pathologies with acquired enteric neuronal dysfunction.
Subject(s)
Colitis/etiology , Colitis/metabolism , Macrophages/immunology , Macrophages/metabolism , Myenteric Plexus/cytology , Myenteric Plexus/metabolism , Animals , Biomarkers , Colitis/pathology , Disease Models, Animal , Disease Susceptibility , Enteric Nervous System/immunology , Enteric Nervous System/metabolism , Extracellular Matrix , Fluorescent Antibody Technique , Immunohistochemistry , Immunophenotyping , Mice , Myenteric Plexus/ultrastructure , Neuroglia/metabolism , Neuroglia/ultrastructure , Neuroinflammatory Diseases/etiology , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/pathology , Neutrophil InfiltrationABSTRACT
Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb-/- mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb+/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb-/- mice generated functional neurons with similar calcium responses to Ednrb+/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb-/- recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.
Subject(s)
Enteric Nervous System , Hirschsprung Disease , Neural Stem Cells , Animals , Hirschsprung Disease/genetics , Hirschsprung Disease/metabolism , Hirschsprung Disease/therapy , Humans , Intestine, Small/metabolism , Mice , Mice, Inbred C57BL , Neural Crest/metabolism , Neural Stem Cells/metabolismABSTRACT
Primary cilia are ubiquitous microtubule-based organelles that serve as signaling hubs for numerous developmental pathways, most notably the Hedgehog (Hh) pathway. Defects in the structure or function of primary cilia result in a class of diseases called ciliopathies. It is well known that primary cilia participate in transducing a Hh signal, and as such ciliopathies frequently present with phenotypes indicative of aberrant Hh function. Interestingly, the exact mechanisms of cilia-dependent Hh signaling transduction are unclear as some ciliopathic animal models simultaneously present with gain-of-Hh phenotypes in one organ system and loss-of-Hh phenotypes in another. To better understand how Hh signaling is perturbed across different tissues in ciliopathic conditions, we examined four distinct Hh-dependent signaling centers in the naturally occurring avian ciliopathic mutant talpid2 (ta2). In addition to the well-known and previously reported limb and craniofacial malformations, we observed dorsal-ventral patterning defects in the neural tube, and a shortened gastrointestinal tract. Molecular analyses for elements of the Hh pathway revealed that the loss of cilia impact transduction of an Hh signal in a tissue-specific manner at variable levels of the pathway. These studies will provide increased knowledge into how impaired ciliogenesis differentially regulates Hh signaling across tissues and will provide potential avenues for future targeted therapeutic treatments.