RESUMEN
Secretory immunoglobulin A (SIgA) can travel to and from the lumen and transport antigen to subepithelial cells. However, IgM can also multimerize into functional secretory component-bound immunoglobulin. While it is already known that both SIgA and SIgM undergo transcytosis to be secreted at the mucosal surface, only SIgA has been shown to perform retrotranscytosis through microfold cells (M cells) of the Peyer's patch. Here, we investigate whether SIgM could also be taken up by M cells via retrotranscytosis. This transport involves FcµR binding at the apical membrane of M cells. We then demonstrate that SIgM can be exploited by SIgM-p24 (HIV-capsid protein) complexes during immunization in the nasal- or gut-associated lymphoid tissue (NALT or GALT), conferring efficient immune responses against p24. Our data demonstrate a mucosal function of SIgM, which could play a role in the regulation of mucosal immunity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Intestinos/fisiología , Proteínas de la Membrana/metabolismo , Transcitosis/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/fisiología , Femenino , Inmunidad Mucosa/fisiología , Inmunoglobulina A Secretora/metabolismo , Inmunoglobulina M/inmunología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ganglios Linfáticos Agregados/citología , Transcitosis/genéticaRESUMEN
Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens, and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, their development and differentiation remain yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development, and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; the absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell-associated markers such as Spi-B and Sox8. In vitro organoid analysis of RankL treated organoid showed an increase of mature marker GP2 expression and other M cell-associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. An increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mice with an increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that PRC2 regulated Atoh8 is one of the factors that regulate the population density of intestinal M cell in the Peyer's patch.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Animales , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Inmunidad Mucosa/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Ratones , Ratones Noqueados , Ganglios Linfáticos Agregados/efectos de los fármacos , Ganglios Linfáticos Agregados/metabolismo , Cultivo Primario de Células , Ligando RANK/farmacología , Receptor Activador del Factor Nuclear kappa-B/farmacología , Linfocitos T/metabolismo , Transcitosis/genéticaRESUMEN
The polyimmunoglobulin receptor (pIgR) transcytoses J chain-containing antibodies through mucosal epithelia. In mammals, two cis-duplicates of PIGR, FCMR, and FCAMR, flank the PIGR gene. A PIGR duplication is first found in amphibians, previously annotated as PIGR2 (herein xlFCAMR), and is expressed by APCs. We demonstrate that xlFcamR is the equivalent of mammalian FcamR. It has been assumed that pIgR is the oldest member of this family, yet our data could not distinguish whether PIGR or FCAMR emerged first; however, FCMR was the last family member to emerge. Interestingly, bony fish "pIgR" is not an orthologue of tetrapod pIgR, and possibly acquired its function via convergent evolution. PIGR/FCAMR/FCMR are members of a larger superfamily, including TREM, CD300, and NKp44, which we name the "double-disulfide Ig superfamily" (ddIgSF). Domains related to each ddIgSF family were identified in cartilaginous fish (sharks, chimeras) and encoded in a single gene cluster syntenic to the human pIgR locus. Thus, the ddIgSF families date back to the earliest antibody-based adaptive immunity, but apparently not before. Finally, our data strongly suggest that the J chain arose in evolution only for Ig multimerization. This study provides a framework for further studies of pIgR and the ddIgSF in vertebrates.
Asunto(s)
Antígenos CD/genética , Inmunidad Mucosa/inmunología , Receptores Fc/genética , Receptores Opioides mu/genética , Receptores de Inmunoglobulina Polimérica/genética , Transcitosis/inmunología , Animales , Antígenos CD/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Humanos , Inmunoglobulinas/metabolismo , Filogenia , Transporte de Proteínas/fisiología , Receptores Fc/inmunología , Receptores Opioides mu/inmunología , Receptores de Inmunoglobulina Polimérica/inmunología , Transcitosis/genética , Xenopus laevisRESUMEN
Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Proteínas Portadoras/genética , Compartimento Celular/genética , Espacio Extracelular/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Microfluídica , Morfogénesis/genética , Transducción de Señal/genética , Transcitosis/genéticaRESUMEN
The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline-rich linker (VP4-P-eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4-P-eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4-P-eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin-mediated endocytosis, the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda.
Asunto(s)
Densovirus/fisiología , Spodoptera/fisiología , Transcitosis/fisiología , Proteínas Virales/fisiología , Animales , Sistema Digestivo/virología , Fenómenos Fisiológicos del Sistema Digestivo , Epitelio/fisiología , Epitelio/virología , Larva/crecimiento & desarrollo , Larva/fisiología , Larva/virología , Spodoptera/crecimiento & desarrollo , Spodoptera/virología , Transcitosis/genéticaRESUMEN
Diabetes is a recognized high-risk factor for the development of atherosclerosis, in which macroautophagy/autophagy is emerging to play essential roles. The retention of low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Here, we identified that high glucose could promote atherosclerosis by stimulating transcytosis of LDL. By inhibiting AMPK-MTOR-PIK3C3 pathway, high glucose suppresses the CAV-CAVIN-LC3B-mediated autophagic degradation of CAV1; therefore, more CAV1 is accumulated in the cytosol and utilized to form more caveolae in the cell membrane and facilitates the LDL transcytosis across endothelial cells. For a proof of concept, higher levels of lipids were accumulated in the subendothelial space of umbilical venous walls from pregnant women with gestational diabetes mellitus (GDM), compared to those of pregnant women without GDM. Our results reveal that high glucose stimulates LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway. ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; AMPK: AMP-activated protein kinase; Bafi: bafilomycin A1; CAV1: caveolin-1; CAVIN1: caveolae associated protein 1; CSD: the CAV1 scaffolding domain; GDM: gestational diabetes mellitus; IMD: intramembrane domain; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule- associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SQSTM1/p62: sequestosome 1.
Asunto(s)
Autofagia/genética , Caveolina 1/metabolismo , Glucosa/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcitosis/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Caveolina 1/genética , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Mutación , Embarazo , Proteínas Quinasas/metabolismo , Estabilidad Proteica , Proteínas de Unión al ARN/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Transcitosis/efectos de los fármacosRESUMEN
FcRn mediates recycling and transcytosis of IgG and albumin in various cell types. The MHC-class-I-like protein of the FcRn heterodimer is encoded by FCGRT. Few determinants of variable FCGRT expression in humans have been identified so far. In this study, we investigated the presence of DNA methylation in regulatory regions of FCGRT in samples of human liver and myocardium tissue, and we examined the impact of FCGRT methylation on FcRn expression in model cell lines. Quantitative DNA methylation analysis of the FCGRT locus revealed differentially methylated regions in DNA from liver and myocardium. Methylation status in individual CpG sites correlated with FCGRT mRNA expression. Data from model cell lines suggest that differential methylation in the -1058 to -587 bp regulatory region of FCGRT contributes to FcRn expression. Chromatin immunoprecipitation assays indicate that CpG site methylation impacts the binding of the methylation sensitive transcription factors Zbtb7a and Sp1. This study provides a foundation to further define the contribution of epigenetic factors during the control of FcRn expression and IgG traffic in human tissues.
Asunto(s)
Proteínas de Unión al ADN/genética , Epigénesis Genética , Hepatocitos/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Miocitos Cardíacos/metabolismo , Receptores Fc/genética , Factor de Transcripción Sp1/genética , Factores de Transcripción/genética , Albúminas/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Hepatocitos/citología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulina G/metabolismo , Hígado/citología , Hígado/metabolismo , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Fc/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Transcitosis/genéticaRESUMEN
Effective delivery of protein therapeutics into the brain remains challenging because of difficulties associated with crossing the blood-brain barrier (BBB). To overcome this problem, many researchers have focused on antibodies binding the transferrin receptor (TfR), which is expressed in endothelial cells, including those of the BBB, and is involved in receptor-mediated transcytosis (RMT). RMT and anti-TfR antibodies provide a useful means of delivering therapeutics into the brain, but the anti-TfR antibody has a short half-life in blood because of its broad expression throughout the body. As a result, anti-TfR antibodies are only maintained at high concentrations in the brain for a short time. To overcome this problem, we developed a different approach which slows down the export of therapeutic antibodies from the brain by binding them to a brain-specific antigen. Here we report a new technology, named AccumuBrain, that achieves both high antibody concentration in the brain and a long half-life in blood by binding to myelin oligodendrocyte glycoprotein (MOG), which is specifically expressed in oligodendrocytes. We report that, using our technology, anti-MOG antibody levels in the brains of mice (Mus musculus) and rats (Rattus norvegicus) were increased several tens of times for a period of one month. The mechanism of this technology is different from that of RMT technologies like TfR and would constitute a breakthrough for central nervous system disease therapeutics.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Barrera Hematoencefálica/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Receptores de Transferrina/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos Antiidiotipos/genética , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Epítopos/inmunología , Humanos , Ratones , Glicoproteína Mielina-Oligodendrócito/genética , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Ratas , Transducción de Señal/inmunología , Transcitosis/genética , Transcitosis/inmunologíaRESUMEN
Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte-like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte-like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte-like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte-like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Cresta Neural/metabolismo , Pericitos/metabolismo , Transcitosis/genética , Animales , Antígenos/genética , Antígenos/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Técnicas de Cocultivo , Células Endoteliales/citología , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/citología , Neuronas/citología , Neuronas/metabolismo , Pericitos/citología , Cultivo Primario de Células , Prosencéfalo/citología , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/metabolismo , Proteoglicanos/genética , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
To test whether genetic variants in PICALM, BIN1, CD2AP, and RIN3-suggested to be involved in blood-brain barrier amyloid-ß transcytosis pathways-associate with Alzheimer's disease, all dementia, suggested vascular dementia, and stroke, and whether such associations are independent of the strong ε4 APOE risk allele. In a prospective cohort study of 74,754 individuals from the general population we genotyped PICALM (rs10792832), BIN1 (rs6733839), CD2AP (rs10948363), and RIN3 (rs10498633), and generated a weighted and a simple allele score. Multifactorially adjusted hazard ratios for the fourth quartile versus the first quartile of the weighted allele score were 1.42 (95% confidence interval 1.22-1.64) for Alzheimer's disease, and 1.33 (1.19-1.48) for all dementia. For suggested vascular dementia and stroke the corresponding estimates were 1.71 (1.18-2.49) and 1.12 (1.04-1.22), respectively. Hazard ratios were similar after APOE adjustment. Genetic variants in PICALM, BIN1, CD2AP, and RIN3 are associated with increased risk of Alzheimer's disease, all dementia, and suggested vascular dementia independent of the strong APOE ε4 allele. These findings may suggest that clathrin-mediated endocytosis in clearance of amyloid-ß across the blood-brain barrier is important for the integrity of both brain tissue and cerebral vessels.
Asunto(s)
Barrera Hematoencefálica , Demencia/genética , Predisposición Genética a la Enfermedad , Accidente Cerebrovascular/genética , Transcitosis/genética , Anciano , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Demencia Vascular/genética , Dinamarca , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Microfold cells (M cells), which are located in the follicle-associated epithelium (FAE) covering mucosal lymphoid follicles, are specialized epithelial cells that initiate mucosal immune responses. These cells take luminal antigens and transport them via transcytosis across the FAE to the antigen-presenting cells underneath. Several intestinal pathogens exploit M cells as their portal for entry to invade the host and cause disease conditions. Recent studies have revealed that the uptake of antigens by M cells is essential for efficient antigen-specific IgA production and that this process likely maintains the homeostasis of mucosal tissues. The present article reviews recent advances in understanding the molecular mechanism of M-cell differentiation and describes the molecules expressed by M cells that are associated with antigen uptake and/or the transcytosis process. Current efforts to augment M-cell-mediated uptake for use in the development of effective mucosal vaccines are also discussed.
Asunto(s)
Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/inmunología , Inmunidad Mucosa , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Mucosa Nasal/citología , Mucosa Nasal/inmunología , Transcitosis/inmunología , Transcitosis/fisiología , Animales , Antígenos/inmunología , Diferenciación Celular/genética , Humanos , Inmunidad Mucosa/genética , Inmunoglobulina A , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , FN-kappa B/fisiología , Ligando RANK/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Transcitosis/genéticaRESUMEN
Primary open-angle glaucoma (POAG) is often caused by elevated intraocular pressure (IOP), which arises due to increased resistance to aqueous humor outflow (AHO). Aqueous humor flows through Schlemm's canal (SC), a lymphatic-like vessel encircling the cornea, and via intercellular spaces of ciliary muscle cells. However, the mechanisms underlying increased AHO resistance are poorly understood. Here, we demonstrate that signaling between angiopoietin (Angpt) and the Angpt receptor Tie2, which is critical for SC formation, is also indispensable for maintaining SC integrity during adulthood. Deletion of Angpt1/Angpt2 or Tie2 in adult mice severely impaired SC integrity and transcytosis, leading to elevated IOP, retinal neuron damage, and impairment of retinal ganglion cell function, all hallmarks of POAG in humans. We found that SC integrity is maintained by interconnected and coordinated functions of Angpt-Tie2 signaling, AHO, and Prox1 activity. These functions diminish in the SC during aging, leading to impaired integrity and transcytosis. Intriguingly, Tie2 reactivation using a Tie2 agonistic antibody rescued the POAG phenotype in Angpt1/Angpt2-deficient mice and rejuvenated the SC in aged mice. These results indicate that the Angpt-Tie2 system is essential for SC integrity. The impairment of this system underlies POAG-associated pathogenesis, supporting the possibility that Tie2 agonists could be a therapeutic option for glaucoma.
Asunto(s)
Angiopoyetina 1/metabolismo , Angiopoyetina 2/metabolismo , Córnea/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Transducción de Señal , Angiopoyetina 1/genética , Angiopoyetina 2/genética , Animales , Córnea/irrigación sanguínea , Córnea/patología , Femenino , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Transcitosis/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The blood-brain barrier (BBB) provides a constant homeostatic brain environment that is essential for proper neural function. An unusually low rate of vesicular transport (transcytosis) has been identified as one of the two unique properties of CNS endothelial cells, relative to peripheral endothelial cells, that maintain the restrictive quality of the BBB. However, it is not known how this low rate of transcytosis is achieved. Here we provide a mechanism whereby the regulation of CNS endothelial cell lipid composition specifically inhibits the caveolae-mediated transcytotic route readily used in the periphery. An unbiased lipidomic analysis reveals significant differences in endothelial cell lipid signatures from the CNS and periphery, which underlie a suppression of caveolae vesicle formation and trafficking in brain endothelial cells. Furthermore, lipids transported by Mfsd2a establish a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Caveolas/metabolismo , Metabolismo de los Lípidos/genética , Proteínas de Transporte de Membrana/genética , Transcitosis/genética , Animales , Barrera Hematoencefálica/ultraestructura , Western Blotting , Caveolas/ultraestructura , Células Endoteliales , Células HEK293 , Humanos , Inmunohistoquímica , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Permeabilidad , SimportadoresRESUMEN
The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.
Asunto(s)
Albúminas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Receptores Fc/metabolismo , Acetaminofén/efectos adversos , Acetaminofén/metabolismo , Animales , Bilis/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Perros , Femenino , Hepatocitos/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Homeostasis , Células de Riñón Canino Madin Darby , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores Fc/genética , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismo , Transcitosis/genéticaRESUMEN
Blood-central nervous system (CNS) barriers partition neural tissues from the blood, providing a homeostatic environment for proper neural function. The endothelial cells that form blood-CNS barriers have specialized tight junctions and low rates of transcytosis to limit the flux of substances between blood and CNS. However, the relative contributions of these properties to CNS barrier permeability are unknown. Here, by studying functional blood-retinal barrier (BRB) formation in mice, we found that immature vessel leakage occurs entirely through transcytosis, as specialized tight junctions are functional as early as vessel entry into the CNS. A functional barrier forms only when transcytosis is gradually suppressed during development. Mutant mice with elevated or reduced levels of transcytosis have delayed or precocious sealing of the BRB, respectively. Therefore, the temporal regulation of transcytosis governs the development of a functional BRB, and suppression of transcytosis is a principal contributor for functional barrier formation.
Asunto(s)
Barrera Hematorretinal/crecimiento & desarrollo , Transcitosis/fisiología , Animales , Barrera Hematorretinal/ultraestructura , Caveolina 1/genética , Caveolina 1/fisiología , Células Endoteliales/fisiología , Femenino , Masculino , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Noqueados , Simportadores , Uniones Estrechas/genética , Uniones Estrechas/fisiología , Transcitosis/genéticaRESUMEN
The blood brain barrier (BBB) presents a challenge for the delivery of brain therapeutics. Trans-BBB delivery methods that use targeting vectors to coopt the vesicle trafficking machinery of BBB endothelial cells have been developed, but these are often hampered by limited flux through the BBB. A solution to this problem lies in the semi-rational engineering of BBB targeting vectors. Leveraging knowledge of intracellular trafficking, researchers have begun to tune selected binding properties of the vector-receptor interaction. Engineered binding affinity, avidity and pH-sensitivity have been shown to affect binding, intracellular sorting and release, ultimately leading to increased brain uptake of the targeting vector and its associated cargo. However, each targeted receptor may exhibit differential responses to engineered binding properties, illustrating the need to better understand vector-receptor interactions and trafficking dynamics.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Ingeniería de Proteínas/métodos , Transcitosis/genética , Animales , Humanos , NanopartículasRESUMEN
INTRODUCTION: Fungal transversal across the brain microvascular endothelial cells (BMECs) is the essential step for the development of cryptococcal meningoencephalitis. Annexin A2 (AnxA2) is an important signaling protein involved in several intracellular processes such as membrane trafficking, endocytosis, and exocytosis. AIM: To investigate the roles and mechanism of AnxA2 during cryptococcal transversal of BMECs. RESULTS: Cryptococcus neoformans infection initiated upregulation of AnxA2 in mouse BMECs. Blockade with anti-AnxA2 antibody led to a reduction in fungal transcytosis activity but no change in its adhesion efficiency. Intriguingly, AnxA2 depletion caused a significant increase in fungal association activity but had no effect on their transcytosis. AnxA2 suppression resulted in marked reduction in its partner protein S100A10, and S100A10 suppression in BMECs significantly reduced the cryptococcal transcytosis efficiency. Furthermore, AnxA2 dephosphorylation at Tyr23 and dephosphorylation of downstream cofilin were required for cryptococcal transversal of BMECs, both of which might be primarily involved in the association of C. neoformans with host cells. CONCLUSIONS: Our work indicated that AnxA2 played complex roles in traversal of C. neoformans across host BMECs, which might be dependent on downstream cofilin to inhibit fungal adhesion but rely on its partner S100A10 to promote cryptococcal transcytosis.
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Anexina A2/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/microbiología , Encéfalo/citología , Cryptococcus neoformans , Células Endoteliales/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Anexina A2/genética , Anexina A2/inmunología , Anticuerpos/farmacología , Barrera Hematoencefálica/patología , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/microbiología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Ratones , Mutación/genética , Fosforilación , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas S100/metabolismo , Factores de Tiempo , Transcitosis/efectos de los fármacos , Transcitosis/genética , Tirosina/metabolismoRESUMEN
Transcytosis is a mechanism for the transcellular transport of biomolecules. Analysis of transcytosis is frequently performed in cells with distinct polarity, such as brain endothelial cells. However, in cells without evident polarity, analysis of transcytosis has not been performed. Here, we describe a method for analyzing transcytosis of a CCN family protein through chondrocytic cells having no apparent polarity.
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Condrocitos/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Transcitosis/genética , Western Blotting , Línea Celular , Humanos , Proteínas Recombinantes de FusiónRESUMEN
M cells reside within the follicle-associated epithelium (FAE) overlying the gut-associated lymphoid tissues. These unique phagocytic epithelial cells enable the mucosal immune system to sample antigens within the lumen of the intestine. The differentiation of M cells from uncommitted precursors in the FAE is dependent on the production of receptor activator of nuclear factor-κB ligand (RANKL) by subepithelial stromal cells. The ligation of a variety of cell surface receptors activates the nuclear factor-κB (NF-κB) family of transcription factors which in-turn induce the transcription of multiple target genes. RANKL-stimulation can stimulate the nuclear translocation of the NF-κB subunit c-Rel. We therefore used c-Rel-deficient mice to determine whether the differentiation and functional maturation of M cells in the Peyer's patches was dependent on c-Rel. Our data show that c-Rel-deficiency does not influence the expression of RANKL or RANK in Peyer's patches, or the induction of M-cell differentiation in the FAE. RANKL-stimulation in the differentiating M cells induces the expression of SpiB which is essential for their subsequent maturation. However, SpiB expression in the FAE was also unaffected in the absence of c-Rel. As a consequence, the functional maturation of M cells was not impaired in the Peyer's patches of c-Rel-deficient mice. Although our data showed that the specific expression of CCL20 and ubiquitin D in the FAE was not impeded in the absence of c-Rel, the expression of ubiquitin D was dramatically reduced in the B cell-follicles of c-Rel-deficient mice. Coincident with this, we also observed that the status of follicular dendritic cells in the B cell-follicles was dramatically reduced in Peyer's patches from c-Rel-deficient mice. Taken together, our data show that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells.
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Diferenciación Celular/genética , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/fisiología , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Biomarcadores , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Fenotipo , Ligando RANK/genética , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transcriptoma , Transcitosis/genética , Transcitosis/inmunologíaRESUMEN
UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.