To Örebro University

Intestinal luminal microbial metabolites affect tryptophan and serotonin metabolism, and cross or modify the blood-brain barrier (BBB). Understanding those mechanisms further necessitates integrated gut-brain axis model systems. Using an ex vivo-in vitro approach, H2O2-stressed or non-stressed human dermal fibroblasts - representing the BBB - are cultured with serosal fluids of healthy or irritable bowel syndrome human colonic biopsies collected from Ussing chamber experiments, after participant's colon was exposed to butyrate in vivo, fecal fiber fermentation or control supernatant ex vivo. Culturing fibroblasts with serosal fluids does not compromise viability or have cytotoxic effects. Serosal fluids alone do not alter expression of tryptophan-related large amino acid membrane transporter genes and proteins, nor their activity (i.e., tryptophan uptake). However, adding serosal fluids to fibroblasts prior to oxidative stress indicate a protective role. This new model allows investigation of direct effects of serosal content on BBB-representing fibroblasts and is highly promising for more personalized applications.

Background: Rhamnogalacturonan-I (RG-I) is a pectic polysaccharide with emerging prebiotic and immunomodulatory potential. This randomised, double-blind, placebo-controlled trial (ID: NCT06081972) evaluated the effects of carrot-derived RG-I (cRG-I) supplementation, compared to placebo (maltodextrin), on gut microbiota composition and immune cell activation in healthy adults.

Methods: A total of 54 participants (18-70 years old) were randomised in a double-blind manner to receive either 500 mg/day of cRG-I or placebo for four weeks. Pre-screening ensured balanced randomisation based on habitual fibre intake and faecal Bifidobacterium counts. Questionnaires assessed potential gut health and well-being effects, while in vitro and ex vivo models were used to evaluate effects on intestinal permeability.

Results: cRG-I was well tolerated with excellent compliance. Faecal Bifidobacterium counts increased significantly, peaking at week 3. Isobutyric acid levels rose, though no other SCFAs differed. Immunologically, cRG-I enhanced the percentage of circulating myeloid dendritic cells expressing activation markers (CD86, HLA-DR) on. Stool consistency improved slightly. Preclinical models further showed that cRG-I and its fermentation products protected intestinal barrier integrity under stress.

Conclusion: These results support cRG-I as a safe, low-dose dietary intervention capable of beneficially modulating gut microbiota, immune responses, and barrier function in healthy adults within a short supplementation period.

Background: This study investigates the impact of fermentation supernatants (FSs) from Pleurotus eryngii whole mushrooms (PEWS), as well as its subcomponents, digested (PEWSD) and extracted (PEWSE) forms, on intestinal barrier function and immune modulation in lipopolysaccharide (LPS) -stimulated Caco-2 cells.

Methods: Gene expression of tight junction (TJs) genes, cytokines, and key immune/metabolic receptors was assessed via qRT-PCR, while cytokine protein levels were measured using ELISA to explore post-transcriptional regulation.

Results: LPS challenge significantly downregulated TJs zonula occludens-1 (ZO-1,) occludin, and claudin-1, compromising epithelial integrity. Treatment with FS-PEWS notably restored ZO-1 and occludin expression, outperforming FS-PEWSD and FS-PEWSE, which only partially mitigated the LPS-induced damage. FS-PEWS further demonstrated potent immunomodulatory effects, upregulating anti-inflammatory IL-10 and pro-inflammatory cytokines such as IL-8 and TNF-α. The activation of key receptors like TLR-2 and mTOR suggests that FS-PEWS modulates critical immune and metabolic pathways, such as NF-kB signaling, to maintain immune homeostasis. Although mRNA expression of pro-inflammatory cytokines was altered, no corresponding protein release was detected, suggesting potential post-transcriptional regulation.

Conclusions: FS-PEWS preserves intestinal barrier integrity and modulates immune responses, particularly in low-grade inflammation, highlighting the whole food matrix's role in enhancing its bioactivity and functional food potential.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infection (UTI). While the role of UPEC in triggering host immune responses is well studied, less is known about how host-derived cytokines influence UPEC virulence. The aim of this study was to investigate how the pro-inflammatory cytokine IL-1β affects UPEC virulence, metabolism, and host-pathogen interactions. Using the CFT073 strain, we found that IL-1β exposure induced a rapid metabolic shift characterized by decreased oxygen consumption rate (OCR) and increased extracellular acidification rate (ECAR), indicating a transition from respiration to fermentative metabolism. Microarray analysis confirmed the upregulation of fermentative (hyc) and antioxidant (katG, ahpF, grxA) genes, along with increased purine biosynthesis, supporting a metabolic state favouring stress resistance and proliferation. IL-1β also increased fimH and papC gene expression, leading to increased adhesion and invasion of bladder epithelial cells. Furthermore, IL-1β-stimulated CFT073 suppressed the gene expression of several innate immune related genes in a C. elegans infection model. Taken together, our findings suggest that IL-1β induces a virulence-enhancing metabolic change in UPEC, which could enhance its persistence and colonization of the urinary tract. This cross-kingdom signaling may have implications for infection dynamics during a UTI.

Pleurotus eryngii (PE), an edible mushroom rich in bioactive compounds, has been shown to exert immunomodulatory, anti-inflammatory, antioxidant, anti-carcinogenic, anti-microbial, antihepatotoxic and hypolipidemic activities, all important for the well-being of the ageing population. This study assessed in vitro the prebiotic-like effects of multiple forms of this mushroom. An in vitro static batch fermentation was performed for 24 h with faecal inocula from five apparently healthy older adults in the presence of the following PE forms: whole food matrix (PEWS), in vitro digested (PEWSD) and rich in beta-glucans extract (PEWSE). The changes in bacterial communities upon fermentation at family, genera and species level were detected via 16S rRNA Next Generation Sequencing and Quantitative real-time PCR. Short-chain fatty acids (SCFAs) were quantified using gas chromatography (GC), whereas other metabolites were analysed through ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). DEseq2 analysis indicated that PEWS presence exhibited the largest impact on faecal microbial families' and genera's abundance compared to negative (noncarbon source) and positive (inulin) controls. Only PEWS significantly increased Bifidobacterium spp. and F. prausnitzii populations, while all three forms robustly increased Bacteroides spp. levels and levels of butyrate, acetate and propionate acids. Statistical significance was set at p < 0.05. Overall, the findings highlight the beneficial effect of PE on intestinal health of older adults supporting its potential incorporation into innovative functional foods. However, additional in vivo studies are required to substantiate these findings before translating them into dietary guidelines or clinical applications.

INTRODUCTION: Studies suggest that immune checkpoints play a role in accelerating the formation of atherosclerosis. We aimed to assess the expression of soluble and membrane-bound immune checkpoints in patients with peripheral artery disease (PAD).

METHODS: The levels of 14 soluble immune checkpoints were assessed in blood plasma of PAD patients (n= 37) and healthy controls (HCs, n=39) by Multiplex protein assay. The surface expression of immune checkpoints on peripheral blood immune cells was determined by flow cytometry. Cytokine production capacity was measured by flow cytometry in TIM-3+ T cells to determine immune exhaustion.

RESULTS: Soluble levels of PD-L2 were decreased in female PAD patients, whereas soluble levels of TIM-3 showed a trend towards an increased concentration in female PAD patients. PD-L2+ frequencies were higher within all monocyte subsets in PAD patients. CD4+ T cells from PAD patients had increased frequencies of TIM-3+ cells, showing little overlap with other immune exhaustion markers. TIM-3+ CD4+ T cells from both PAD patients and HCs, had a low capacity to produce pro-inflammatory cytokines, but a higher capacity to produce IL-10 compared to TIM-3- CD4+ T cells.

CONCLUSION: PAD patients show differences in the expression of membrane-bound and soluble immune checkpoints. Some of these differences might be caused by prolonged immune activation, although immune exhaustion markers did not always overlap.

Background: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation.

Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated.

Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1.

Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions.

Aim: The cell matrix of plant foods has received little attention in prebiotic fiber research. We aimed to understand the impact of the plant cell matrix in dried chicory root on its breakdown in the human gut to explain its reported beneficial effects on gut and metabolic health.

Methods: We applied in vitro digestion and fermentation models together with an ex vivo gut barrier integrity model. Plant cell matrix intactness in the upper gastrointestinal tract was investigated by scanning electron microscopy. Colonic breakdown of inulin, and chicory root cubes and powder was assessed by gut microbiota analysis using 16S rRNA gene amplicon sequencing and determining the kinetics of changes in pH, gas, and short- chain fatty acid (SCFA) production. Finally, effects on gut barrier integrity were explored by exposing colonic biopsies to fermentation supernatants in an Ussing chamber model.

Results: The plant cell matrix of dried chicory root cubes remained intact throughout upper gastrointestinal transit. Dried chicory root fermentation resulted in higher final relative abundances of pectin-degrading Monoglobus and butyrate-producing Roseburia spp. compared to inulin and a seven-fold increase in Bifidobacterium spp. in donors where these species were present. Dried chicory root cubes yielded similar total SCFAs but higher final butyrate levels than chicory root powder or isolated inulin with less gas produced. No uniform but donor-specific effects of fermentation supernatants on the maintenance of gut barrier integrity were detected.

Conclusion: The intact plant cell matrix of dried chicory root affected its colonic breakdown kinetics and microbiota, underpinning its beneficial effect in vivo.

The appropriateness of the fecal microbiota to adequately reflect the gut microbiota composition from more difficult to access luminal content at different colonic locations has been debated. Here, in a healthy population, luminal samples were collected from terminal ileum to rectum using an unique sampling technique without the need of prior bowel cleansing/preparation. Rectal swabs were collected immediately prior colonoscopy by an experienced physician, and fecal samples were collected at home by the participants themselves. Microbiota composition was evaluated as relative abundance, α-diversity and Bray-Curtis dissimilarities. Our data suggest that fecal samples and rectal swabs present noninvasive, easily accessible, low-cost sampling tools that are accurate proxies to characterize luminal large intestinal microbiota composition.