RESUMEN
Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.
Asunto(s)
COVID-19 , Mucosa Intestinal , SARS-CoV-2 , Uniones Estrechas , Replicación Viral , Humanos , SARS-CoV-2/patogenicidad , Células CACO-2 , COVID-19/virología , COVID-19/patología , Mucosa Intestinal/virología , Mucosa Intestinal/patología , Uniones Estrechas/virología , Alanina/análogos & derivados , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Antivirales/farmacología , Células HT29 , Ocludina/metabolismo , Ocludina/genética , Adenosina Monofosfato/análogos & derivadosRESUMEN
Most nanomaterial-based medicines are intravenously applied since oral administration comprises challenging-related biological obstacles, such as interactions with distinct digestive fluids and their transport through the intestinal barrier. Moreover, there is a lack of nanoparticle-based studies that faithfully consider the above-cited obstacles and boost oral-administered nanomedicines' rational design. In this study, the physicochemical stability of fluorescent model silica nanoparticles (f-SiO2NPs) passing through all simulated gastrointestinal fluids (salivary, gastric, and intestinal) and their absorption and transport across a model human intestinal epithelium barrier are investigated. An aggregation/disaggregation f-SiO2NPs process is identified, although these particles remain chemically and physically stable after exposure to digestive fluids. Further, fine imaging of f-SiO2NPs through the absorption and transport across the human intestinal epithelium indicates that nanoparticle transport is time-dependent. The above-presented protocol shows tremendous potential for deciphering fundamental gastrointestinal nanoparticles' evolution and can contribute to rational oral administration-based nanomedicine design.
Asunto(s)
Líquidos Corporales , Nanopartículas , Humanos , Mucosa Intestinal , Tracto Gastrointestinal , Administración OralRESUMEN
In vitro intestinal epithelium models have drawn great attention to investigating intestinal biology in recent years. However, the difficulty to maintain the normal physiological status of primary intestinal epithelium in vitro limits the applications. Here, we designed patterned electrospun polylactic acid (PLA) nanofibrous membranes with crypt-like topography and mimic ECM fibrous network to support crypt culture and construct in vitro intestinal epithelium models. The patterned electrospun PLA nanofibrous membranes modified with Matrigels at 0 °C showed high biocompatibility and promoted cell growth and proliferation. The constructed duodenum epithelium models and colon epithelium models on the patterned electrospun PLA nanofibrous membranes expressed the typical differentiation markers of intestinal epithelia and the gene expression levels were close to the original tissues, especially with the help of probiotics. The constructed intestinal epithelium models could be used to assess probiotic adhesion and colonization, which were verified to show significant differences with the Caco-2 cell models due to the different cell types. These findings provide new insights and a better understanding of the roles of biophysical, biochemical, and biological signals in the construction of in vitro intestinal epithelium models as well as the potential applications of these models in the study of host-gut microbes interactions. KEY POINTS: ⢠Patterned electrospun scaffold has crypt-like topography and ECM nanofibrous network. ⢠Matrigels at 0°C modify scaffolds more effectively than at 37°C. ⢠Synergy of biomimic scaffold and probiotics makes in vitro model close to tissue.
Asunto(s)
Nanofibras , Andamios del Tejido , Humanos , Ingeniería de Tejidos , Células CACO-2 , Diferenciación Celular , Mucosa Intestinal/metabolismo , Poliésteres/metabolismoRESUMEN
Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.