Protective Role of Flavonoids against Intestinal Pro-Inflammatory Effects of Silver Nanoparticles

Silver nanoparticles (AgNP) have been increasingly incorporated into food-related and hygiene products for their unique antimicrobial and preservative properties. The consequent oral exposure may then result in unpredicted harmful effects in the gastrointestinal tract (GIT), which should be considered in the risk assessment and risk management of these materials. In the present study, the toxic effects of polyethyleneimine (PEI)-coated AgNP (4 and 19 nm) were evaluated in GIT-relevant cells (Caco-2 cell line as a model of human intestinal cells, and neutrophils as a model of the intestinal inflammatory response). This study also evaluated the putative protective action of dietary flavonoids against such harmful effects. The obtained results showed that AgNP of 4 and 19 nm effectively induced Caco-2 cell death by apoptosis with concomitant production of nitric oxide, irrespective of the size. It was also observed that AgNP induced human neutrophil oxidative burst. Interestingly, some flavonoids, namely quercetin and quercetagetin, prevented the deleterious effects of AgNP in both cell types. Overall, the data of the present study provide a first insight into the promising protective role of flavonoids against the potentially toxic effects of AgNP at the intestinal level.

Cadmium affects autophagy in the human intestinal cells Caco-2 through ROS-mediated ERK activation

Cadmium is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium has the capacity to accumulate high levels of this metal. We have previously shown that Cd induces ERK1/2 activation in differentiated but not proliferative human enterocytic-like Caco-2 cells. As autophagy is a dynamic process that plays a critical role in intestinal mucosa, we aimed the present study 1) to investigate the role of p-ERK1/2 in constitutive autophagy in proliferative Caco-2 cells and 2) to investigate whether Cd-induced activation of ERK1/2 modifies autophagic activity in postconfluent Caco-2 cell monolayers. Western blot analyses of ERK1/2 and autophagic markers (LC3, SQSTM1), and cellular staining with acridine orange showed that ERK1/2 and autophagic activities both decreased with time in culture. GFP-LC3 fluorescence was also associated with proliferative cells and the presence of a constitutive ERK1/2-dependent autophagic flux was demonstrated in proliferative but not in postconfluent cells. In the latter condition, serum and glucose deprivation triggered autophagy via a transient phosphorylation of ERK1/2, whereas Cd-modified autophagy via a ROS-dependent sustained activation of ERK1/2. Basal autophagy flux in proliferative cells and Cd-induced increases in autophagic markers in postconfluent cells both involved p-ERK1/2. Whether Cd blocks autophagic flux in older cell cultures remains to be clarified but our data suggest dual effects. Our results prompt further studies investigating the consequences that Cd-induced ERK1/2 activation and the related effect on autophagy may have on the intestinal cells, which may accumulate and trap high levels of Cd under some nutritional conditions. Graphical abstract

Development of Chrysin Loaded Oil-in-Water Nanoemulsion for Improving Bioaccessibility

Chrysin (5,7-dihydroxyflavone) is a remarkable flavonoid exhibiting many health-promoting activities, such as antioxidant, anti-inflammatory, and anti-Alzheimer’s disease (AD). Nevertheless, chrysin has been addressed regarding its limited applications, due to low bioaccessibility. Therefore, to improve chrysin bioaccessibility, a colloidal delivery system involving nanoemulsion was developed as chrysin nanoemulsion (chrysin-NE) using an oil-in-water system. Our results show that chrysin can be loaded by approximately 174.21 µg/g nanoemulsion (100.29 ± 0.53% w/w) when medium chain triglyceride (MCT) oil was used as an oil phase. The nanocolloidal size, polydispersity index, and surface charge of chrysin-NE were approximately 161 nm, 0.21, and −32 mV, respectively. These properties were stable for at least five weeks at room temperature. Furthermore, in vitro chrysin bioactivities regarding antioxidant and anti-AD were maintained as pure chrysin, suggesting that multistep formulation could not affect chrysin properties. Interestingly, the developed chrysin-NE was more tolerant of gastrointestinal digestion and significantly absorbed by the human intestinal cells (Caco-2) than pure chrysin. These findings demonstrate that the encapsulation of chrysin using oil-in-water nanoemulsion could enhance the bioaccessibility of chrysin, which might be subsequently applied to food and nutraceutical industries.

New Variants of Squash Mosaic Viruses Detected in Human Fecal Samples

Squash mosaic virus (SqMV) is a phytovirus that infects great diversity of plants worldwide. In Brazil, the SqMV has been identified in the states of Ceará, Maranhão, Piauí, Rio Grande do Norte, and Tocantins. The presence of non-pathogenic viruses in animals, such as phytoviruses, may not be completely risk-free. Similarities in gene repertories between these viruses and viruses that affect animal species have been reported. The present study describes the fully sequenced genomes of SqMV found in human feces, collected in Tocantins, and analyzes the viral profile by metagenomics in the context of diarrhea symptomatology. The complete SqMV genome was obtained in 39 of 253 analyzed samples (15.5%); 97.4% of them belonged to children under 5 years old. There was no evidence that the observed symptoms were related to the presence of SqMV. Of the different virus species detected in these fecal samples, at least 4 (rotavirus, sapovirus, norovirus, parechovirus) are widely known to cause gastrointestinal symptoms. The presence of SqMV nucleic acid in fecal samples is likely due to recent dietary consumption and it is not evidence of viral replication in the human intestinal cells. Identifying the presence of SqMV in human feces and characterization of its genome is a relevant precursor to determining whether and how plant viruses interact with host cells or microorganisms in the human gastrointestinal tract.

A LCMS Metabolomic Workflow to Investigate Metabolic Patterns in Human Intestinal Cells Exposed to Hydrolyzed Crab Waste Materials

We have developed a LCMS metabolomic workflow to investigate metabolic patterns from human intestinal cells treated with simulated gastrointestinal-digested hydrolyzed crab waste materials. This workflow facilitates smart and reproducible comparisons of cell cultures exposed to different treatments. In this case the variable was the hydrolysis methods, also accounting for the GI digestion giving an output of direct correlation between cellular metabolic patterns caused by the treatments. In addition, we used the output from this workflow to select treatments for further evaluation of the Caco-2 cell response in terms of tentative anti-inflammatory activity in the hopes to find value in the crab waste materials to be used for food products. As hypothesized, the treatment identified to change the cellular metabolomic pattern most readily, was also found to cause the greatest effect in the cells, although the response was pro-inflammatory rather than anti-inflammatory, it proves that changes in cellular metabolic patterns are useful predictors of bioactivity. We conclude that the developed workflow allows for cost effective, rapid sample preparation as well as accurate and repeatable LCMS analysis and introduces a data pipeline specifically for probe the novel metabolite patterns created as a means to assess the performing treatments.

M2R: a Python add-on to cobrapy for modifying human genome-scale metabolic reconstruction using the gut microbiota models

Motivation The gut microbiota is the human body’s largest population of microorganisms that interact with human intestinal cells. They use ingested nutrients for fundamental biological processes and have important impacts on human physiology, immunity and metabolome in the gastrointestinal tract. Results Here, we present M2R, a Python add-on to cobrapy that allows incorporating information about the gut microbiota metabolism models to human genome-scale metabolic models (GEMs) like RECON3D. The idea behind the software is to modify the lower bounds of the exchange reactions in the model using aggregated in- and out-fluxes from selected microbes. M2R enables users to quickly and easily modify the pool of the metabolites that enter and leave the GEM, which is particularly important for those looking into an analysis of the metabolic interaction between the gut microbiota and human cells and its dysregulation. Availability and implementation M2R is freely available under an MIT License at https://github.com/e-weglarz-tomczak/m2r. Supplementary information Supplementary data are available at Bioinformatics online.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

CADHERIN-11, AN ESSENTIAL REGULATOR OF CELL-CELL ADHESION UPREGULATED IN IBD PATIENTS, PLAYS ESSENTIAL ROLES IN PROMOTING FIBROBLAST ACTIVATION AND DEVELOPMENT OF INTESTINAL INFLAMMATION AND FIBROSIS

Background Intestinal fibrosis is a severe complication of inflammatory bowel diseases (IBD) leading to intestinal strictures and need for surgery. No effective anti-fibrotic therapy is available. Cadherin-11 (Cad-11) is an adherens junction protein, which is upregulated in rheumatoid arthritis (RA), idiopathic pulmonary fibrosis (IPF) and skin fibrosis. Inhibition of cadherin-11 has shown beneficial effects in RA and IPF animal models. A phase II clinical trial of cadherin-11 inhibition in RA has shown a good safety profile. Our aim was to evaluate the expression levels and function of Cad-11 in IBD patients using intestinal tissues, primary human intestinal cells, and the murine dextran sulfate sodium (DSS)-induced chronic colitis model. Methods IBD (Crohn’s disease (CD) n=20; Ulcerative colitis (UC) (n=10) and control (n=10) full thickness resected intestinal tissues were procured from adults in accordance with IRB approval. Protein and mRNA were extracted for western blot (WB) and quantitative polymerase chain reaction (qPCR). Distribution of Cad-11 was evaluated by immunofluorescence (IF) and RNA hybridization in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections, respectively. Primary human intestinal myofibroblasts (HIMF) were used in functional experiments. Recombinant human Fc and Cad-11 extracellular domain (hCAD-11-Fc) was used as activator and siRNA as inhibitor of Cad-11 in HIMF. Murine chronic colitis was induced in wildtype BALB/c mice and cadherin-11 knockout mice by DSS. Anti-Cad-11 monoclonal antibody (H1M1) was used for the treatment of BALB/c mouse colitis. Results Increased gene and protein expression levels of Cad-11 were found in intestinal full thickness IBD tissue compared to controls (45-fold, p<0.01). Cad-11 colocalized with alpha smooth muscle actin (α-SMA) (Figure 1), indicating that Cad-11 is selectively expressed in intestinal myofibroblasts and smooth muscle cells. Among all the primary human intestinal cells, Cad-11 was expressed exclusively in HIMF and HIMC cells. Level of Cad-11 was increased in IBD HIMFs compared to non-IBD controls, and increased upon stimulation with TNF-α, IL-1β, b-FGF and TGF-β (all p<0.01). Knocking down Cad-11 with siRNA decreased FN expression, while hCAD-11-Fc increased the expression FN in a dose- and time-dependent manner as well as the proliferation of HIMF. Upon treatment with H1M1 antibody, DSS-treated mice showed lower clinical scores and weight loss compared to control mice (p<0.001. Figure 2), as well as less FN expression (p<0.001). Cadherin-11 knockout mice also showed lower clinical scores and weight loss compared to wild type mice (p<0.001). Conclusions Cad-11 expression is increased in CD stricture tissues and its blockade reduces profibrotic effects in HIMF in vitro. Inhibition of Cad-11 in vivo reduces clinical severity and fibrosis of experimental colitis.