RESUMO
In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.
Assuntos
Mucosa Intestinal , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Animais , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Linhagem da Célula , Regeneração , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Camundongos Endogâmicos C57BL , HomeostaseRESUMO
Inflammatory epithelial diseases are spurred by the concomitant dysregulation of immune and epithelial cells. How these two dysregulated cellular compartments simultaneously sustain their heightened metabolic demands is unclear. Single-cell and spatial transcriptomics (ST), along with immunofluorescence, revealed that hypoxia-inducible factor 1α (HIF1α), downstream of IL-17 signaling, drove psoriatic epithelial remodeling. Blocking HIF1α in human psoriatic lesions ex vivo impaired glycolysis and phenocopied anti-IL-17 therapy. In a murine model of skin inflammation, epidermal-specific loss of HIF1α or its target gene, glucose transporter 1, ameliorated epidermal, immune, vascular, and neuronal pathology. Mechanistically, glycolysis autonomously fueled epithelial pathology and enhanced lactate production, which augmented the γδ T17 cell response. RORγt-driven genetic deletion or pharmacological inhibition of either lactate-producing enzymes or lactate transporters attenuated epithelial pathology and IL-17A expression in vivo. Our findings identify a metabolic hierarchy between epithelial and immune compartments and the consequent coordination of metabolic processes that sustain inflammatory disease.
Assuntos
Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Interleucina-17 , Animais , Humanos , Interleucina-17/metabolismo , Interleucina-17/imunologia , Camundongos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pele/imunologia , Pele/patologia , Pele/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Psoríase/imunologia , Psoríase/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Camundongos Knockout , Transdução de Sinais/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Modelos Animais de Doenças , Ácido Láctico/metabolismo , Doença Crônica , Inflamação/imunologia , Camundongos Endogâmicos C57BLRESUMO
A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.
Assuntos
Interleucina-33 , Mastócitos , Proteínas de Ligação a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Animais , Camundongos , Comunicação Celular/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Interleucina-33/metabolismo , Interleucina-33/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/imunologia , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMO
Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.
Assuntos
Acetilcolina , Mucosa Intestinal , Animais , Acetilcolina/metabolismo , Camundongos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Colina O-Acetiltransferase/metabolismo , Interleucina-13/metabolismo , Interleucina-13/imunologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Helmintíase/imunologia , Helmintíase/parasitologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Imunidade Inata , Nematospiroides dubius/imunologia , Células em TufoRESUMO
Glucose is the universal fuel of most mammalian cells, and it is largely replenished through dietary intake. Glucose availability to tissues is paramount for the maintenance of homeostatic energetics and, hence, supply should match demand by the consuming organs. In its journey through the body, glucose encounters cellular barriers for transit at the levels of the absorbing intestinal epithelial wall, the renal epithelium mediating glucose reabsorption, and the tight capillary endothelia (especially in the brain). Glucose transiting through these cellular barriers must escape degradation to ensure optimal glucose delivery to the bloodstream or tissues. The liver, which stores glycogen and generates glucose de novo, must similarly be able to release it intact to the circulation. We present the most up-to-date knowledge on glucose handling by the gut, liver, brain endothelium, and kidney, and discuss underlying molecular mechanisms and open questions. Diseases associated with defects in glucose delivery and homeostasis are also briefly addressed. We propose that the universal problem of sparing glucose from catabolism in favor of translocation across the barriers posed by epithelia and endothelia is resolved through common mechanisms involving glucose transfer to the endoplasmic reticulum, from where glucose exits the cells via unconventional cellular mechanisms.
Assuntos
Encéfalo , Glucose , Animais , Humanos , Glucose/metabolismo , Epitélio/metabolismo , Encéfalo/metabolismo , Transporte Biológico , Intestinos , Mamíferos/metabolismoRESUMO
Cell junctions play key roles in epithelial integrity. During development, when epithelia undergo extensive morphogenesis, these junctions must be remodeled in order to maintain mechanochemical barriers and ensure the cohesion of the tissue. In this Review, we present a comprehensive and integrated description of junctional remodeling mechanisms in epithelial cells during development, from embryonic to adult epithelia. We largely focus on Drosophila, as quantitative analyses in this organism have provided a detailed characterization of the molecular mechanisms governing cell topologies, and discuss the conservation of these mechanisms across metazoans. We consider how changes at the molecular level translate to tissue-scale irreversible deformations, exploring the composition and assembly of cellular interfaces to unveil how junctions are remodeled to preserve tissue homeostasis during cell division, intercalation, invagination, ingression and extrusion.
Assuntos
Drosophila , Junções Intercelulares , Animais , Divisão Celular , Desenvolvimento Embrionário , Células EpiteliaisRESUMO
All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.
Assuntos
Resfriado Comum , Imunidade Inata , Interferons , Mucosa Nasal , SARS-CoV-2 , Transdução de Sinais , Humanos , Mucosa Nasal/virologia , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Interferons/metabolismo , Interferons/imunologia , Resfriado Comum/imunologia , Resfriado Comum/virologia , Transdução de Sinais/imunologia , SARS-CoV-2/imunologia , Replicação Viral , Rhinovirus/imunologia , Coronavirus Humano 229E/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Epiteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavirus Humano NL63/imunologiaRESUMO
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
Assuntos
Mitocôndrias , Oxirredutases , Proteínas de Plantas , Epitélio Pigmentado da Retina , Animais , Mitocôndrias/metabolismo , Camundongos , Oxirredutases/metabolismo , Oxirredutases/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ciona intestinalis/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologiaRESUMO
Epithelial cell death is highly prevalent during development and tissue homeostasis. While we have a rather good understanding of the molecular regulators of programmed cell death, especially for apoptosis, we still fail to predict when, where, how many and which specific cells will die in a tissue. This likely relies on the much more complex picture of apoptosis regulation in a tissular and epithelial context, which entails cell autonomous but also non-cell autonomous factors, diverse feedback and multiple layers of regulation of the commitment to apoptosis. In this review, we illustrate this complexity of epithelial apoptosis regulation by describing these different layers of control, all demonstrating that local cell death probability is a complex emerging feature. We first focus on non-cell autonomous factors that can locally modulate the rate of cell death, including cell competition, mechanical input and geometry as well as systemic effects. We then describe the multiple feedback mechanisms generated by cell death itself. We also outline the multiple layers of regulation of epithelial cell death, including the coordination of extrusion and regulation occurring downstream of effector caspases. Eventually, we propose a roadmap to reach a more predictive understanding of cell death regulation in an epithelial context.
Assuntos
Apoptose , Células Epiteliais , Células Epiteliais/metabolismo , Morte Celular , Apoptose/fisiologiaRESUMO
Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.
Assuntos
Actinas , Peixe-Zebra , Animais , Actinas/metabolismo , Células Fotorreceptoras/metabolismo , Retina , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras de VertebradosRESUMO
Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Junções Intercelulares/metabolismo , Lipoilação , Proteolipídeos/metabolismoRESUMO
Tight junctions (TJs) are specialized regions of contact between cells of epithelial and endothelial tissues that form selective semipermeable paracellular barriers that establish and maintain body compartments with different fluid compositions. As such, the formation of TJs represents a critical step in metazoan evolution, allowing the formation of multicompartmental organisms and true, barrier-forming epithelia and endothelia. In the six decades that have passed since the first observations of TJs by transmission electron microscopy, much progress has been made in understanding the structure, function, molecular composition and regulation of TJs. The goal of this Perspective is to highlight the key concepts that have emerged through this research and the future challenges that lie ahead for the field.
Assuntos
Junções Íntimas , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Humanos , Animais , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Células Epiteliais/citologiaRESUMO
The dentition is critical to animal survival and teeth are present in modern vertebrates including teleost fish, sharks, amphibians, mammals and reptiles. The developmental processes that give rise to teeth are not just preserved through evolution but also share high level of similarity with the embryogenesis of other ectodermal organs. In this review we go beyond the embryonic phase of tooth development to life-long tooth replacement. We will address the origins of successional teeth, the location of putative tissue-resident stem cells, how de novo tooth formation continues throughout life and how teeth are shed in a spatially and temporally controlled manner. We review the evidence that the dental epithelium, which is the earliest recognizable dental structure in the reptilian dentition, serves as a putative niche for tissue-resident epithelial stem cells and recent molecular findings from transcriptomics carried out in reptilian dentitions. We discuss how odontoclasts resorb the primary tooth allowing eruption of the successional tooth. The reptiles, particularly lizards, are emerging as some of the most accessible animals to study tooth replacement which has relevance to evolution of the dentition and human dental disorders.
RESUMO
A delicate balance between photon absorption for vision and the protection of photoreceptors from light damage is pivotal for ocular health. This equilibrium is governed by the light-absorbing 11-cis-retinylidene chromophore of visual pigments, which, upon bleaching, transforms into all-trans-retinal and undergoes regeneration through an enzymatic pathway, named the visual cycle. Chemical side reactions of retinaldehyde during the recycling process can generate by-products that may result in a depletion of retinoids. In our study, we have clarified the crucial roles played by melanin pigmentation and the retinoid transporter STRA6 in preventing this loss and preserving the integrity of the visual cycle. Our experiments initially confirmed that consecutive green and blue light bleaching of isolated bovine rhodopsin produced 9-cis and 13-cis retinal. The same unusual retinoids were found in the retinas of mice exposed to intense light, with elevated concentrations observed in albino mice. Examining the metabolic fate of these visual cycle byproducts revealed that 9-cis-retinal, but not 13-cis-retinal, was recycled back to all-trans-retinal through an intermediate called isorhodopsin. However, investigations in Stra6 knockout mice unveiled that the generation of these visual cycle byproducts correlated with a light-induced loss of ocular retinoids and visual impairment. Collectively, our findings uncover important novel aspects of visual cycle dynamics, with implications for ocular health and photoreceptor integrity.
Assuntos
Proteínas de Membrana , Retinoides , Animais , Bovinos , Camundongos , Diterpenos , Camundongos Knockout , Retina/metabolismo , Retinaldeído/metabolismo , Retinoides/metabolismo , Visão Ocular , Proteínas de Membrana/metabolismoRESUMO
Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.
Assuntos
Actinas , Actomiosina , Humanos , Actinas/metabolismo , Actomiosina/metabolismo , Citocinese , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Junções Íntimas/metabolismo , Células CACO-2 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.
Assuntos
Interferons , Viroses , Humanos , Interferons/farmacologia , Interferons/metabolismo , Células Epiteliais/metabolismo , Linhagem Celular , Viroses/metabolismoRESUMO
MicroRNA (miRNA)-mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA-155 (miR-155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin-7). MiR-155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. MiR-155 localization using in situ hybridization (ISH) in patient biopsies and intra-epithelial compartmentalization of miR-155 show expression predominantly within the basal epithelia. Epithelial miR-155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR-155 stable overexpression induced a functionally deficient epithelial barrier in 3D air-liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR-155 showed notable dilated intra-epithelial spaces. Unbiased RNA-sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin-7. Together, our data reveal a previously unappreciated role for miR-155 in mediating epithelial barrier dysfunction in esophageal inflammation.
Assuntos
Claudinas , Esofagite Eosinofílica , MicroRNAs , Humanos , Claudinas/genética , Esofagite Eosinofílica/genética , Esofagite Eosinofílica/metabolismo , Esofagite Eosinofílica/patologia , Células Epiteliais/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Junções Íntimas/metabolismoRESUMO
Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Catepsina D , Lisossomos , Epitélio Pigmentado da Retina , Doença de Stargardt , Catepsina D/metabolismo , Catepsina D/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Doença de Stargardt/metabolismo , Doença de Stargardt/patologia , Doença de Stargardt/genética , Animais , Humanos , Camundongos , Lisossomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genéticaRESUMO
The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.
Assuntos
Retinopatia Diabética , Ácidos Hidroxâmicos , Edema Macular , Humanos , NF-kappa B/metabolismo , Retinopatia Diabética/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Edema Macular/metabolismo , Transdução de Sinais , Epitélio Pigmentado da Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Junções Íntimas/metabolismo , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Pigmentos da Retina/uso terapêuticoRESUMO
Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.