Dynamic changes in extracellular vesicle-associated miRNAs elicited by ultrasound in inflammatory bowel disease patients.

Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to molecular shifts of EV contents, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the inflammation-specific molecular shifts in EVs like altered EV-miRNA expression, which in turn can be detected in the peripheral blood. 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 min after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing. Candidate inducible EV-miRNAs were functionally assessed in vitro by transfection of miRNA mimics and qPCR of predicted target genes. Serum EV-miRNA concentration at baseline correlated with disease severity, as determined by clinical activity scores and sonographic findings. Three miRNAs (miR-942-5p, mir-5588, mir-3195) were significantly induced by sonography. Among the significantly regulated EV-miRNAs, miR-942-5p was strongly induced in higher grade intestinal inflammation and correlated with clinical activity in Crohn's disease. Prediction of target regulation and transfection of miRNA mimics inferred a role of this EV-miRNA in regulating barrier function in inflammation. Induction of mir-5588 and mir-3195 did not correlate with inflammation grade. This proof-of-concept trial highlights the principle of induced molecular shifts in EVs from inflamed tissue through transabdominal ultrasound. These inducible EVs and their molecular cargo like miRNA could become novel biomarkers for intestinal inflammation in IBD.

Vulnerability to climate change of United States marine mammal stocks in the western North Atlantic, Gulf of Mexico, and Caribbean.

Climate change and climate variability are affecting marine mammal species and these impacts are projected to continue in the coming decades. Vulnerability assessments provide a framework for evaluating climate impacts over a broad range of species using currently available information. We conducted a trait-based climate vulnerability assessment using expert elicitation for 108 marine mammal stocks and stock groups in the western North Atlantic, Gulf of Mexico, and Caribbean Sea. Our approach combined the exposure (projected change in environmental conditions) and sensitivity (ability to tolerate and adapt to changing conditions) of marine mammal stocks to estimate vulnerability to climate change, and categorize stocks with a vulnerability index. The climate vulnerability score was very high for 44% (n = 47) of these stocks, high for 29% (n = 31), moderate for 20% (n = 22), and low for 7% (n = 8). The majority of stocks (n = 78; 72%) scored very high exposure, whereas 24% (n = 26) scored high, and 4% (n = 4) scored moderate. The sensitivity score was very high for 33% (n = 36) of these stocks, high for 18% (n = 19), moderate for 34% (n = 37), and low for 15% (n = 16). Vulnerability results were summarized for stocks in five taxonomic groups: pinnipeds (n = 4; 25% high, 75% moderate), mysticetes (n = 7; 29% very high, 57% high, 14% moderate), ziphiids (n = 8; 13% very high, 50% high, 38% moderate), delphinids (n = 84; 52% very high, 23% high, 15% moderate, 10% low), and other odontocetes (n = 5; 60% high, 40% moderate). Factors including temperature, ocean pH, and dissolved oxygen were the primary drivers of high climate exposure, with effects mediated through prey and habitat parameters. We quantified sources of uncertainty by bootstrapping vulnerability scores, conducting leave-one-out analyses of individual attributes and individual scorers, and through scoring data quality for each attribute. These results provide information for researchers, managers, and the public on marine mammal responses to climate change to enhance the development of more effective marine mammal management, restoration, and conservation activities that address current and future environmental variation and biological responses due to climate change.

Pomegranate Extract Affects Gut Biofilm Forming Bacteria and Promotes Intestinal Mucosal Healing Regulating the Crosstalk between Epithelial Cells and Intestinal Fibroblasts.

Background: Pomegranate (Punica granatum) can be used to prepare a bioactive extract exerting anti-inflammatory activities. Clinical studies demonstrated an improvement in clinical response in inflammatory bowel disease (IBD) patients when pomegranate extract (PG) was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. This study investigates the effect of PG on bacterial biofilm formation and the promotion of mucosal wound healing. Methods: The acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) by oral gavage for 11 days. Colitis was scored daily by evaluating body weight loss, bleeding, and stool consistency. In vivo intestinal permeability was evaluated by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation was assessed by fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity were monitored by immunostaining for JAM-A and MUC-2 markers. Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 h. Primary fibroblasts were isolated from healthy and inflamed areas of 8 IBD patients, and Caco-2 cells were stimulated with or without PG (5 µg/mL). Inflammatory mediators were measured at the mRNA and protein level by RT-PCR, WB, or Bio-plex multiplex immunoassay, respectively. Results: In vivo, PG boosted the recovery phase of colitis, promoting a complete restoration of the intestinal barrier with the regeneration of the mucus layer, as also demonstrated by the absence of bacterial spread into the mucosa and the enrichment of crypt-associated fibroblasts. Microfluidic experiments did not highlight a specific effect of PG on Enterobacterales biofilm formation, even though Citrobacter freundii biofilm was slightly impaired in the presence of PG. In vitro, inflamed fibroblasts responded to PG by downregulating the release of metalloproteinases, IL-6, and IL-8 and upregulating the levels of HGF. Caco-2 cells cultured in a medium supplemented with PG increased the expression of SOX-9 and CD44, whereas in the presence of HGF or plated with a fibroblast-conditioned medium, they displayed a decrease in SOX-9 and CD44 expression and an increase in AXIN2, a negative regulator of Wnt signaling. Conclusions: These data provide new insight into the manifold effects of PG on promoting mucosal homeostasis in IBD by affecting pathogen biofilm formation and favoring the regeneration of the intestinal barrier through the regulation of the crosstalk between epithelial and stromal cells.

Dysfunctional Extracellular Matrix Remodeling Supports Perianal Fistulizing Crohn's Disease by a Mechanoregulated Activation of the Epithelial-to-Mesenchymal Transition.

BACKGROUND AND AIMS: Perianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation. METHODS: Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed. RESULTS: A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli. CONCLUSIONS: By mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.