Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

Microbiota-Derived Extracellular Vesicles as a Postbiotic Strategy to Alleviate Diarrhea and Enhance Immunity in Rotavirus-Infected Neonatal Rats.

Rotavirus (RV) infection is a major cause of acute gastroenteritis in children under 5 years old, resulting in elevated mortality rates in low-income countries. The efficacy of anti-RV vaccines is limited in underdeveloped countries, emphasizing the need for novel strategies to boost immunity and alleviate RV-induced diarrhea. This study explores the effectiveness of interventions involving extracellular vesicles (EVs) from probiotic and commensal E. coli in mitigating diarrhea and enhancing immunity in a preclinical model of RV infection in suckling rats. On days 8 and 16 of life, variables related to humoral and cellular immunity and intestinal function/architecture were assessed. Both interventions enhanced humoral (serum immunoglobulins) and cellular (splenic natural killer (NK), cytotoxic T (Tc) and positive T-cell receptor γδ (TCRγδ) cells) immunity against viral infections and downregulated the intestinal serotonin receptor-3 (HTR3). However, certain effects were strain-specific. EcoR12 EVs activated intestinal CD68, TLR2 and IL-12 expression, whereas EcN EVs improved intestinal maturation, barrier properties (goblet cell numbers/mucin 2 expression) and absorptive function (villus length). In conclusion, interventions involving probiotic/microbiota EVs may serve as a safe postbiotic strategy to improve clinical symptoms and immune responses during RV infection in the neonatal period. Furthermore, they could be used as adjuvants to enhance the immunogenicity and efficacy of anti-RV vaccines.

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease.

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

Protocol for the systematic review of the Pneumocystis jirovecii-associated pneumonia in non-HIV immunocompromised patients.

INTRODUCTION: Pneumocystis jirovecii pneumonia (PJP) is a well-known and frequent opportunistic infection in HIV patients. However, there has been an increase in the number of reports of PJP in other immunosuppressed patients with autoimmune inflammatory disorders or because of chemotherapy and high doses of steroids, especially when used in combination as part of immunosuppressive therapy. OBJECTIVE: Despite the increasing importance of PJP in non-HIV patients, there is a lack of comprehensive and updated information on the epidemiology, pathogenesis, diagnosis, microbiology, treatments, and prophylaxis of this infection in this population. Therefore, the objective of this systematic review is to synthesize information on these aspects, from a perspective of evidence-based medicine. METHODS: The protocol is prepared following the preferred reporting items for systematic reviews and meta-analyses (PRISMA-P) guidelines. We will perform a systematic review of literature published between January 2010 and July 2023, using the databases PubMed, Google Scholar, ScienceDirect, and Web of Science. In addition, manual searches will be carried out through related articles, and references to included articles. The main findings and clinical outcomes were extracted from all the eligible studies with a standardized instrument. Two authors will independently screen titles and abstracts, review full texts, and collect data. Disagreements will be resolved by discussion, and a third reviewer will decide if there is no consensus. We will synthesize the results using a narrative or a meta-analytic approach, depending on the heterogeneity of the studies. EXPECTED RESULTS: It is expected that this systematic review will provide a comprehensive and up-to-date overview of the state-of-the-art of PJP in non-HIV patients. Furthermore, the study will highlight possible gaps in knowledge that should be addressed through new research. CONCLUSIONS: Here, we present the protocol for a systematic review which will consider all existing evidence from peer-reviewed publication sources relevant to the primary and secondary outcomes related to diagnosing and managing PJP in non-HIV patients.

Antimicrobial phytoconstituents from Azadirachta indica (neem) with potential inhibitor against FtsZ protein of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a well-known pathogen for its rapid development of multi-drug antibiotic resistance. This pathogen is responsible for numerous human diseases, particularly affecting immunocompromised and elderly patients. Hence, discovering novel therapeutics has become necessary in the fight against antimicrobial resistance. This study is focused on evaluating the potential inhibitory activity of eleven phytocompounds from Azadirachta indica against the nucleotide-binding site of the FtsZ protein of P. aeruginosa through a cheminformatics approach. FtsZ is an indispensable and highly conserved protein in prokaryotic cell division. Docking studies revealed favourable binding energies (ΔG= - 8.3 to - 5.4 kcal/mol) for all selected phytoconstituents. Finally, we selected Nimbiol (CID 11119228), as a lead compound, exhibiting a binding energy (ΔG= -7.8 kcal/mol) for the target. Based on our findings, Nimbiol shows potential as an anti-FtsZ compound, making it a promising candidate for further in vitro and in vivo investigations to assess its antimicrobial activity.

Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 Reduce PepT1 Levels in IL-1ß-Treated Caco-2 Cells via Upregulation of miR-193a-3p.

PepT1, a proton-coupled oligopeptide transporter, is crucial for intestinal homeostasis. It is mainly expressed in small intestine enterocytes, facilitating the absorption of di/tri-peptides from dietary proteins. In the colon, PepT1 expression is minimal to prevent excessive responses to proinflammatory peptides from the gut microbiota. However, increased colonic PepT1 is linked to chronic inflammatory diseases and colitis-associated cancer. Despite promising results from animal studies on the benefits of extracellular vesicles (EVs) from beneficial gut commensals in treating IBD, applying probiotic EVs as a postbiotic strategy in humans requires a thorough understanding of their mechanisms. Here, we investigate the potential of EVs of the probiotic Nissle 1917 (EcN) and the commensal EcoR12 in preventing altered PepT1 expression under inflammatory conditions, using an interleukin (IL)-1-induced inflammation model in Caco-2 cells. The effects are evaluated by analyzing the expression of PepT1 (mRNA and protein) and miR-193a-3p and miR-92b, which regulate, respectively, PepT1 mRNA translation and degradation. The influence of microbiota EVs on PepT1 expression is also analyzed in the presence of bacterial peptides that are natural substrates of colonic PepT1 to clarify how the regulatory mechanisms function under both physiological and pathological conditions. The main finding is that EcN EVs significantly decreases PepT1 protein via upregulation of miR-193a-3p. Importantly, this regulatory effect is strain-specific and only activates in cells exposed to IL-1ß, suggesting that EcN EVs does not control PepT1 expression under basal conditions but can play a pivotal role in response to inflammation as a stressor. By this mechanism, EcN EVs may reduce inflammation in response to microbiota in chronic intestinal disorders by limiting the uptake of bacterial proinflammatory peptides.

Modulation of the Intestinal Barrier Integrity and Repair by Microbiota Extracellular Vesicles through the Differential Regulation of Trefoil Factor 3 in LS174T Goblet Cells.

Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the TFF3 production through TLR2 and downregulated miR7-5-p through PI3K. Consistently, high levels of secreted TFF3 reinforced the tight junctions and stimulated wound healing in the Caco-2 cells. ECOR12 EVs did not cause these effects. TFF3 is a potential therapeutic target in IBD. This study contributes to understanding the molecular players (microbiota EVs) connecting gut microbes to health and may help in designing better nutritional interventions based on microbiota bioactive compounds.