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1.
Gastroenterology ; 159(1): 214-226.e1, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32247021

RESUMEN

BACKGROUND & AIMS: Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. METHODS: We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. RESULTS: A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. CONCLUSIONS: We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.


Asunto(s)
Diferenciación Celular/inmunología , Linfotoxina-alfa/metabolismo , Ganglios Linfáticos Agregados/inmunología , Transducción de Señal/inmunología , Tretinoina/metabolismo , Animales , Presentación de Antígeno/inmunología , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Humanos , Íleon/citología , Íleon/inmunología , Inmunidad Mucosa , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Ratones , FN-kappa B/metabolismo , Organoides , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/metabolismo , Cultivo Primario de Células , Proteínas Recombinantes/metabolismo
2.
J Virol ; 91(18)2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28679762

RESUMEN

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

3.
Dev Biol ; 384(2): 166-80, 2013 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-24140541

RESUMEN

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left-right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer's vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left-right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left-right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.


Asunto(s)
Tipificación del Cuerpo/fisiología , Corazón/embriología , Proteínas de Choque Térmico Pequeñas/fisiología , Pez Cebra/embriología , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Choque Térmico Pequeñas/genética , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa
4.
Front Immunol ; 10: 1970, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31481960

RESUMEN

The role of intestinal epithelial cells (IECs) in mucosal tolerance and immunity remains poorly understood. We present a method for inducing MHC class II (MHC-II) in human enteroids, "mini-guts" derived from small intestinal crypt stem cells, and show that the intracellular MHC-II peptide-pathway is intact and functional in IECs. Our approach enables human enteroids to be used for novel in vitro studies into IEC MHC-II regulation and function during health and disease.


Asunto(s)
Antígenos de Histocompatibilidad Clase II , Mucosa Intestinal/inmunología , Organoides/inmunología , Humanos
5.
Front Immunol ; 9: 2144, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30319613

RESUMEN

As the primary barrier between an organism and its environment, epithelial cells are well-positioned to regulate tolerance while preserving immunity against pathogens. Class II major histocompatibility complex molecules (MHC class II) are highly expressed on the surface of epithelial cells (ECs) in both the lung and intestine, although the functional consequences of this expression are not fully understood. Here, we summarize current information regarding the interactions that regulate the expression of EC MHC class II in health and disease. We then evaluate the potential role of EC as non-professional antigen presenting cells. Finally, we explore future areas of study and the potential contribution of epithelial surfaces to gut-lung crosstalk.


Asunto(s)
Presentación de Antígeno , Células Epiteliales/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Mucosa Intestinal/inmunología , Mucosa Respiratoria/inmunología , Microbioma Gastrointestinal/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Mucosa Intestinal/citología , Pulmón/citología , Pulmón/inmunología , Mucosa Respiratoria/citología
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