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Superparamagnetic iron oxide nanoparticles (SPIONs) exhibit unique properties for diverse biomedical applications, including drug delivery and diagnostic imaging. Actively targeted SPIONs enhance delivery to diseased sites, reducing side effects and enhancing treatment efficacy. However, development of reproducible functionalization protocols is challenged by the erratic behavior of nanoparticles in suspensions, such as agglomeration and sedimentation. In this study, we develop and systematically optimize a functionalization method to attach the Fc-region of antibodies onto silica coated SPIONs via click chemistry, ensuring controlled ligand orientation on the particle surface. The synthesis and successive modifications of silica coated SPIONs with organic moieties is presented resulting in the final click conjugation with anti-ICAM1 antibodies. These antibodies target ICAM1, upregulated on epithelial cell surfaces during gastrointestinal inflammation. Thermogravimetric analysis and infrared spectroscopy confirm successful SPION functionalization after each modification step. Cell viability assessment indicates no adverse effects of bioconjugated particles. Quantitative elemental analysis reveals significantly higher iron concentration in inflammation-induced Caco-2 cells exposed to ICAM1-modified particles compared to non-conjugated counterparts. Furthermore, laser scanning confocal microscopy of these cells suggests surface interaction and internalization of bioconjugated SPIONs, underscoring their potential for targeted imaging and therapy in inflammatory diseases.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5 '-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1 beta and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-gamma and TNF-alpha responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

Background: Intestinal Peyer's patches (PPs) form unique niches for bacteria-immune cell interactions that direct host immunity and shape the microbiome. Here we investigate how peroral administration of probiotic bacterium Limosilactobacillus reuteri R2LC affects B lymphocytes and IgA induction in the PPs, as well as the downstream consequences on intestinal microbiota and susceptibility to inflammation.

Results: The B cells of PPs were separated by size to circumvent activation-dependent cell identification biases due to dynamic expression of markers, which resulted in two phenotypically, transcriptionally, and spatially distinct subsets: small IgD(+)/GL7(-)/S1PR1(+)/Bcl6, CCR6-expressing pre-germinal center (GC)-like B cells with innate-like functions located subepithelially, and large GL7(+)/S1PR1(-)/Ki67(+)/Bcl6, CD69-expressing B cells with strong metabolic activity found in the GC. Peroral L. reuteri administration expanded both B cell subsets and enhanced the innate-like properties of pre-GC-like B cells while retaining them in the sub-epithelial compartment by increased sphingosine-1-phosphate/S1PR1 signaling. Furthermore, L. reuteri promoted GC-like B cell differentiation, which involved expansion of the GC area and autocrine TGF beta-1 activation. Consequently, PD-1-T follicular helper cell-dependent IgA induction and production was increased by L. reuteri, which shifted the intestinal microbiome and protected against dextran-sulfate-sodium induced colitis and dysbiosis.

Conclusions: The Peyer's patches sense, enhance and transmit probiotic signals by increasing the numbers and effector functions of distinct B cell subsets, resulting in increased IgA production, altered intestinal microbiota, and protection against inflammation.

Rationale: Ischemic injuries remain a leading cause of mortality and morbidity worldwide, and restoration of functional blood perfusion is vital to limit tissue damage and support healing.

Objective: To reveal a novel role of macrophages in reestablishment of functional tissue perfusion following ischemic injury that can be targeted to improve tissue restoration.

Methods and Results: Using intravital microscopy of ischemic hindlimb muscle in mice, and confocal microscopy of human tissues from amputated legs, we found that macrophages accumulated perivascularly in ischemic muscles, where they expressed high levels of iNOS (inducible nitric oxide [NO] synthase). Genetic depletion of iNOS specifically in macrophages (Cx3cr1-CreERT2;Nos2(fl/fl) or LysM-Cre;Nos2(fl/fl)) did not affect vascular architecture but highly compromised blood flow regulation in ischemic but not healthy muscle, which resulted in aggravated ischemic damage. Thus, the ability to upregulate blood flow was shifted from eNOS (endothelial)-dependence in healthy muscles to completely rely on macrophage-derived iNOS during ischemia. Macrophages in ischemic muscles expressed high levels of CXCR4 (C-X-C chemokine receptor type 4) and CCR2 (C-C chemokine receptor type 2), and local overexpression by DNA plasmids encoding the corresponding chemokines CXCL12 (stromal-derived factor 1) or CCL2 (chemokine [C-C motif] ligand 2) increased macrophage numbers, while CXCL12 but not CCL2 induced their perivascular positioning. As a result, CXCL12-overexpression increased the number of perfused blood vessels in the ischemic muscles, improved functional muscle perfusion in a macrophage-iNOS-dependent manner, and ultimately restored limb function.

Conclusions: This study establishes a new function for macrophages during tissue repair, as they regulate blood flow through the release of iNOS-produced NO. Further, we demonstrate that macrophages can be therapeutically targeted to improve blood flow regulation and functional recovery of ischemic tissues.

Inflammatory bowel disease (IBD) is a chronic condition resulting in impaired intestinal homeostasis. Current practices for diagnosis of IBD are challenged by invasive, demanding procedures. We hypothesized that proteomics analysis could provide a powerful tool for identifying clinical biomarkers for non-invasive IBD diagnosis. Here, the global intestinal proteomes from commonly used in vitro and in vivo models of IBD were analyzed to identify apical and luminal proteins that can be targeted by orally delivered diagnostic agents. Global proteomics analysis revealed upregulated plasma membrane proteins in intestinal segments of proximal- and distal colon from dextran sulfate sodium-treated mice and also in inflamed human intestinal Caco-2 cells pretreated with pro-inflammatory agents. The upregulated colon proteins in mice were compared to the proteome of the healthy ileum, to ensure targeting of diagnostic agents to the inflamed colon. Promising target proteins for future investigations of non-invasive diagnosis of IBD were found in both systems and included Tgm2/TGM2, Icam1/ICAM1, Ceacam1/CEACAM1, and Anxa1/ANXA1. Ultimately, these findings will guide the selection of appropriate antibodies for surface functionalization of imaging agents aimed to target inflammatory biomarkers in situ.

Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo. Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.

IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids—cultured three-dimensional replicas of the mammalian gut—offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.

Obesity is linked to non-alcoholic fatty liver disease and risk factors associated to metabolic syndrome. Bilberry (Vaccinium myrtillus) that contains easily fermentable fiber may strengthen the intestinal barrier function, attenuate inflammation and modulate gut microbiota composition, thereby prevent obesity development. In the current study, liver lipid metabolism, fat depot, cecal and serum short-chain fatty acids (SCFAs) and gut microbiome were evaluated in rats fed bilberries in a high-fat (HFD + BB) or low-fat (LFD + BB) setting for 8 weeks and compared with diets containing equal amount of fiber resistant to fermentation (cellulose, HFD and LFD). HFD fed rats did not obtain an obese phenotype but underwent pre-obesity events including increased liver index, lipid accumulation and increased serum cholesterol levels. This was linked to shifts of cecal bacterial community and reduction of major SCFAs. Bilberry inclusion improved liver metabolism and serum lipid levels. Bilberry inclusion under either LFD or HFD, maintained microbiota homeostasis, stimulated interscapular-brown adipose tissue depot associated with increased mRNA expression of uncoupling protein-1; enhanced SCFAs in the cecum and circulation; and promoted butyric acid and butyrate-producing bacteria. These findings suggest that bilberry may serve as a preventative dietary measure to optimize microbiome and associated lipid metabolism during or prior to HFD.

We developed an experimental set up that enables longitudinal studies of immune cell behavior in situ in the challenged as well as unchallenged kidney of anesthetized mice over several hours. Using highly controlled vacuum to stabilize the kidney, the superficial renal cortex could continuously be visualized with minimal disruption of the local microenvironment. No visible changes in blood flow or neutrophils and macrophages numbers were observed after several hours of visualizing the unchallenged kidney, indicating a stable tissue preparation without apparent tissue damage. Applying this set up to monocyte/macrophage (CX(3)CR1(GFP/+)) reporter mice, we observed the extensive network of stellate-shaped CX(3)CR1 positive cells (previously identified as renal mononuclear phagocytes). The extended dendrites of the CX(3)CR1 positive cells were found to bridge multiple capillaries and tubules and were constantly moving. Light induced sterile tissue injury resulted in rapid neutrophil accumulation to the site of injury. Similarly, microinfusion of uropathogenic Escherichia coli into a single nephron induced a rapid and massive recruitment of neutrophils to the site of infection, in addition to active bacterial clearance by neutrophils. In contrast, the kidney resident mononuclear phagocytes were observed to not increase in numbers or migrate toward the site of injury or infection. In conclusion, this model allows for longitudinal imaging of responses to localized kidney challenges in the mouse.

Scope Emerging evidence suggests that high-fat diet (HFD) is associated with gut microbiome dysbiosis and related disorders. Bilberry is a prebiotic food component with known health benefits. Herein, the dynamics of the colonic mucus layer and microbiome during HFD and bilberry supplementation are addressed. Methods and results The effects on colonic mucus thickness in vivo and gut microbiota composition (Illumina sequencing, quantitative real-time PCR) are investigated in young rats fed a low-fat diet or HFD with or without bilberries for 8 weeks (n = 8). HFD induced significant local colonic effects, despite no observed weight gain or systemic inflammation, as HFD causes epithelial upregulation of inducible nitric oxide synthase, which is counteracted by bilberry. The firmly adherent mucus layer becomes thicker and the mRNA levels of Muc2 and Tff3 are increased by HFD with or without bilberry. In parallel, HFD reduced the colonic abundance of mucolytic bacterial species Akkermansia muciniphila and Bacteroides spp. Finally, bilberry prevents HFD-induced microbiota dysbiosis, including expansion of pathobionts, for example, Enterobacteriaceae. Conclusion HFD expand firmly adherent mucus thickness and reduce mucus-foraging bacteria populations in the colon prior to obesity. Enriching HFD with bilberry protects against intestinal inflammation and marked microbiota encroachment.

Intravital imaging is an invaluable tool for studying the expanding range of immune cell functions. Only in vivo can the complex and dynamic behavior of leukocytes and their interactions with their natural microenvironment be observed and quantified. While the capabilities of high-speed, high-resolution confocal and multiphoton microscopes are well-documented and steadily improving, other crucial hardware required for intravital imaging is often developed in-house and less commonly published in detail. In this report, we describe a low-cost, multipurpose, and tissue-stabilizing in vivo imaging platform that enables sensing and regulation of local tissue temperature. The effect of tissue temperature on local blood flow and leukocyte migration is demonstrated in muscle and skin. Two different models of vacuum windows are described in this report, however, the design of the vacuum window can easily be adapted to fit different organs and tissues.