To Örebro University

MicroRNAs (miRNAs) have been shown to regulate many cellular processes and to play a role in host-pathogen interactions. However, the role of miRNA in urinary tract infection (UTI) remains unclear. The aim of this study was to analyze and compare miRNAs from supernatants of human bladder epithelial cells infected with ESBL-producing (ESBL019) and non-ESBL-producing (CFT073) uropathogenic E. coli (UPEC) strains and to identify miRNA target genes in human cells and uropathogenic bacteria. In total, 402 unique miRNAs were found. The statistical analysis showed differential expression of 30 miRNAs from bladder cells stimulated with ESBL019, while stimulation with CFT073 did not show any significantly expressed miRNAs when compared to unstimulated controls. The 30 differentially expressed miRNAs in ESBL019 stimulated cells showed 747 predicted individual human gene targets. KEGG and REACTOME pathways showed enrichments in pathways mainly connected to immune regulation and stress responses. Of the 30 differentially expressed host miRNAs, nine miRNAs were found to interact with predictive targets of the whole genome from the multi-resistant, ESBL-producing UPEC strain EC958. This study shows that ESBL019-infected bladder epithelial cells release miRNAs with predictive targets in both human and bacterial genes, although their role in UTI cross-species interactions remains to be clarified.

BACKGROUND: 16S rRNA gene sequencing is widely used for bacterial community profiling in both clinical and research contexts. The expanding availability of library preparation protocols and bioinformatic pipelines increases analytical flexibility but may also introduce method-dependent biases that affect inferred microbial composition and relative abundance estimates. The relative impact of library preparation protocol, amplicon region, and bioinformatic pipeline on species-level taxonomic inference and compositional agreement remains insufficiently characterised. We therefore compared the Illumina 16S Metagenomic Sequencing Library Preparation protocol (V3-V4) and the Zymo Quick-16S Plus NGS Library Prep Kit (V1-V2 and V3-V4) in combination with two bioinformatic pipelines, nf-core/ampliseq and TRANA. Performance was assessed using defined microbial community standards and human faecal and colonic biopsy samples.

RESULTS: Pipeline choice was the dominant driver of variation in inferred community composition, exceeding the effects of amplicon regions and library preparation protocols. Genus-level profiles were broadly concordant across methods. Species-level resolution and agreement with expected community composition differed systematically between pipelines, with TRANA demonstrating lower Bray-Curtis dissimilarities to expected compositions than nf-core/ampliseq. Amplicon region had a secondary, pipeline-dependent effect, while protocol differences were minor. In clinical samples, inter-individual biological variation exceeded technical variation.

CONCLUSIONS: Bioinformatic processing substantially influenced species-level inference in short-read 16S sequencing, highlighting the importance of pipeline selection for microbiome study design and cross-study comparability.

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: Pectic rhamnogalacturonan-I (RG-I) is a dietary fiber that modulates the gut-immune axis. This study evaluates a novel variant of RG-I from chicory root (chRG-I).

Methods: In a randomized, double-blind, placebo-controlled trial, 55 healthy adults were stratified by habitual fiber intake and baseline Bifidobacterium levels before receiving 500 mg/day of chRG-I or placebo for four weeks. Primary endpoints included fecal Bifidobacterium counts. Secondary outcomes assessed fecal metabolites, systemic immune cell activation markers, and gastrointestinal symptoms. To provide mechanistic insights, donor-matched fecal samples were used in in vitro fermentation and Caco-2/peripheral blood mononuclear cell co-culture gut barrier models.

Results: Supplementation with chRG-I induced a statistically significant bifidogenic effect, with absolute levels peaking at week three, and lower levels of some fecal short-chain fatty acids (SCFA) compared to placebo. However, donor-matched in vitro fermentations with chRG-I confirmed robust production of SCFA and reduction of branched-chain fatty acids levels (BCFA). Systemically, chRG-I upregulated HLA-DR expression on myeloid dendritic cells. Clinically, chRG-I was well-tolerated and slightly improved stool consistency compared to placebo. In an intestinal barrier challenge model, chRG-I fermentates (a pool of metabolites including SCFA and fragments of chRG-I) protected barrier integrity, modulated the cytokine milieu away from a predominantly pro-inflammatory response, as characterized by increased IL4 and IL22 and reduced IL9, IL17A, and IL21.

Conclusion: Supplementation with a low dose of chRG-I is well-tolerated, beneficially modulates the gut microbiome - which can protect the intestinal barrier, and subtly enhances systemic immune readiness, suggesting that chRG-I may have benefits as a functional food ingredient.

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.