Early-life vitamin A treatment rescues neonatal infection-induced durably impaired tolerogenic properties of celiac lymph nodes.

Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.

Non-professional efferocytosis of Salmonella-infected intestinal epithelial cells in the neonatal host.

The intestinal epithelium is the first line of defense against enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal intestine. Instead, here, we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighboring epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid cocultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis, and identified the involvement of the "eat-me" signals and adaptors phosphatidylserine and C1q as well as the "eat-me" receptors integrin-αv (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighboring epithelial cells may represent a previously unrecognized mechanism of neonatal antimicrobial host defense to maintain barrier integrity.

A physiologically based model of bile acid metabolism in mice.

Bile acid (BA) metabolism is a complex system that includes a wide variety of primary and secondary, as well as conjugated and unconjugated BAs that undergo continuous enterohepatic circulation (EHC). Alterations in both composition and dynamics of BAs have been associated with various diseases. However, a mechanistic understanding of the relationship between altered BA metabolism and related diseases is lacking. Computational modeling may support functional analyses of the physiological processes involved in the EHC of BAs along the gut-liver axis. In this study, we developed a physiologically based model of murine BA metabolism describing synthesis, hepatic and microbial transformations, systemic distribution, excretion, and EHC of BAs at the whole-body level. For model development, BA metabolism of specific pathogen-free (SPF) mice was characterized in vivo by measuring BA levels and composition in various organs, expression of transporters along the gut, and cecal microbiota composition. We found significantly different BA levels between male and female mice that could only be explained by adjusted expression of the hepatic enzymes and transporters in the model. Of note, this finding was in agreement with experimental observations. The model for SPF mice could also describe equivalent experimental data in germ-free mice by specifically switching off microbial activity in the intestine. The here presented model can therefore facilitate and guide functional analyses of BA metabolism in mice, e.g., the effect of pathophysiological alterations on BA metabolism and translation of results from mouse studies to a clinically relevant context through cross-species extrapolation.

Choice of Polymer, but Not Mesh Structure Variation, Reduces the Risk of Bacterial Infection with Staphylococcus aureus In Vivo.

BACKGROUND: Synthetic mesh material is of great importance for surgical incisional hernia repair. The physical and biochemical characteristics of the mesh influence mechanical stability and the foreign body tissue reaction. The influence on bacterial infections, however, remains ill-defined. The aim of the present study was to evaluate the influence of a modified mesh structure with variation in filament linking on the occurrence of bacterial infection that is indicated by the occurrence of CD68+, CD4+, and CD8+ cells in two different materials. METHODS: A total of 56 male Sprague Dawley rats received a surgical mesh implant in a subcutaneous abdominal position. The mesh of two different polymers (polypropylene (PP) and polyvinylidenfluoride (PVDF)) and two different structures (standard structure and bold structure with higher filament linking) were compared. During the implantation, the meshes were infected with Staphylococcus (S.) aureus. After 7 and 21 days, meshes were explanted, and the early and late tissue responses to infection were histologically evaluated. RESULTS: Overall, the inflammatory tissue response was higher at 7 days when compared to 21 days. At 7 days, PP meshes of the standard structure (PP-S) showed the strongest inflammatory tissue response in comparison to all the other groups. At 21 days, no statistically significant difference between different meshes was detected. CD8+ cytotoxic T cells showed a significant difference at 21 days but not at 7 days. PP meshes of both structures showed a higher infiltration of CD8+ T cells than PVDF meshes. CD4+ T helper cells differed at 7 days but not at 21 days, and PVDF meshes in a bold structure showed the highest CD4+ T cell count. The number of CD68+ macrophages was also significantly higher in PP meshes in a standard structure when compared to PVDF meshes at 21 days. CONCLUSION: The inflammatory tissue response to S. aureus infection appears to be highest during the early period after mesh implantation. PP meshes showed a higher inflammatory response than PVDF meshes. The mesh material appears to be more important for the risk of infection than the variation in filament linking.

M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

Gut-liver axis: barriers and functional circuits.

The gut and the liver are characterized by mutual interactions between both organs, the microbiome, diet and other environmental factors. The sum of these interactions is conceptualized as the gut-liver axis. In this Review we discuss the gut-liver axis, concentrating on the barriers formed by the enterohepatic tissues to restrict gut-derived microorganisms, microbial stimuli and dietary constituents. In addition, we discuss the establishment of barriers in the gut and liver during development and their cooperative function in the adult host. We detail the interplay between microbial and dietary metabolites, the intestinal epithelium, vascular endothelium, the immune system and the various host soluble factors, and how this interplay establishes a homeostatic balance in the healthy gut and liver. Finally, we highlight how this balance is disrupted in diseases of the gut and liver, outline the existing therapeutics and describe the cutting-edge discoveries that could lead to the development of novel treatment approaches.

Programmed and environmental determinants driving neonatal mucosal immune development.

The mucosal immune system of neonates goes through successive, non-redundant phases that support the developmental needs of the infant and ultimately establish immune homeostasis. These phases are informed by environmental cues, including dietary and microbial stimuli, but also evolutionary developmental programming that functions independently of external stimuli. The immune response to exogenous stimuli is tightly regulated during early life; thresholds are set within this neonatal "window of opportunity" that govern how the immune system will respond to diet, the microbiota, and pathogenic microorganisms in the future. Thus, changes in early-life exposure, such as breastfeeding or environmental and microbial stimuli, influence immunological and metabolic homeostasis and the risk of developing diseases such as asthma/allergy and obesity.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Blood Endotoxin Levels as Biomarker of Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Growing evidence supports a role of gut-derived metabolites in nonalcoholic fatty liver disease (NAFLD), but the relation of endotoxin levels with gut permeability and NAFLD stage remains unclear. This systematic review with meta-analysis aims to provide further insights. METHODS: PubMed, Embase, and Cochrane Library were searched for studies published until January 2022 assessing blood endotoxins in patients with NAFLD. Meta-analyses and univariate/multivariate meta-regression, as well as correlation analyses, were performed for endotoxin values and potential relationships to disease stage, age, sex, parameters of systemic inflammation, and metabolic syndrome, as well as liver function and histology. RESULTS: Forty-three studies were included, of which 34 were used for meta-analyses. Blood endotoxin levels were higher in patients with simple steatosis vs liver-healthy controls (standardized mean difference, 0.86; 95% confidence interval, 0.62-1.11) as well as in patients with nonalcoholic steatohepatitis vs patients with nonalcoholic fatty liver/non-nonalcoholic steatohepatitis (standardized mean difference, 0.81; 95% confidence interval, 0.27-1.35; P = .0078). Consistently, higher endotoxin levels were observed in patients with more advanced histopathological gradings of liver steatosis and fibrosis. An increase of blood endotoxin levels was partially attributed to a body mass index rise in patients with NAFLD compared with controls. Nevertheless, significant increases of blood endotoxin levels in NAFLD retained after compensation for differences in body mass index, metabolic condition, or liver enzymes. Increases in blood endotoxin levels were associated with increases in C-reactive protein concentrations, and in most cases, paralleled a rise in markers for intestinal permeability. CONCLUSION: Our results support blood endotoxin levels as relevant diagnostic biomarker for NAFLD, both for disease detection as well as staging during disease progression, and might serve as surrogate marker of enhanced intestinal permeability in NAFLD. Registration number in Prospero: CRD42022311166.

The Staphylococcus epidermidis Transcriptional Profile During Carriage.

The virulence factors of the opportunistic human pathogen Staphylococcus epidermidis have been a main subject of research. In contrast, limited information is available on the mechanisms that allow the bacterium to accommodate to the conditions during carriage, a prerequisite for pathogenicity. Here, we tested the hypothesis that the adaptation of S. epidermidis at different anatomical sites is reflected by differential gene regulation. We used qPCR to profile S. epidermidis gene expression in vivo in nose and skin swabs of 11 healthy individuals. Despite some heterogeneity between individuals, significant site-specific differences were detected. For example, expression of the S. epidermidis regulator sarA was found similarly in the nose and on the skin of all individuals. Also, genes encoding colonization and immune evasion factors (sdrG, capC, and dltA), as well as the sphingomyelinase encoding gene sph, were expressed at both anatomical sites. In contrast, expression of the global regulator agr was almost inactive in the nose but readily present on the skin. A similar site-specific expression profile was also identified for the putative chitinase-encoding SE0760. In contrast, expression of the autolysine-encoding gene sceD and the wall teichoic acid (WTA) biosynthesis gene tagB were more pronounced in the nose as compared to the skin. In summary, our analysis identifies site-specific gene expression patterns of S. epidermidis during colonization. In addition, the observed expression signature was significantly different from growth in vitro. Interestingly, the strong transcription of sphingomyelinase together with the low expression of genes encoding the tricarboxylic acid cycle (TCA) suggests very good nutrient supply in both anatomical niches, even on the skin where one might have suspected a rather lower nutrient supply compared to the nose.

Stabilization but No Functional Influence of HIF-1α Expression in the Intestinal Epithelium during Salmonella Typhimurium Infection.

Hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we inferred significant activation of HIF-1 after oral infection of mice with Salmonella enterica serovar Typhimurium. Immunohistochemistry and Western blot analyses confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a-deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced noncanonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact inflammatory gene expression, bacterial spread, or disease outcomes. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro, HIF-1α-deficient macrophages showed overall impaired transcription of mRNA encoding proinflammatory factors; however, the intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

Allulose in human diet: the knowns and the unknowns.

D-Allulose, also referred to as psicose, is a C3-epimer of D-fructose used as a sugar substitute in low energy products. It can be formed naturally during processing of food and drinks containing sucrose and fructose or is prepared by chemical synthesis or via enzymatic treatment with epimerases from fructose. Estimated intakes via Western style diets including sweetened beverages are below 500 mg per d but, when used as a sugar replacement, intake may reach 10 to 30 g per d depending on the food consumed. Due to its structural similarity with fructose, allulose uses the same transport and distribution pathways. But in contrast to fructose, the human genome does not encode for enzymes that are able to metabolise allulose leading to an almost complete renal excretion of the absorbed dose and near-to-zero energetic yield. However, in vitro studies have shown that certain bacteria such as Klebsiella pneumonia are able to utilise allulose as a substrate. This finding has been a subject of concern, since Klebsiella pneumoniae represents an opportunistic human pathogen. It therefore raised the question of whether a high dietary intake of allulose may cause an undesirable growth advantage for potentially harmful bacteria at mucosal sites such as the intestine or at systemic sites following invasive infection. In this brief review, we discuss the current state of science on these issues and define the research needs to better understand the fate of allulose and its metabolic and microbiological effects when ingested as a sugar substitute.

Spatial and temporal key steps in early-life intestinal immune system development and education.

Education of our intestinal immune system early in life strongly influences adult health. This education strongly relies on series of events that must occur in well-defined time windows. From initial colonization by maternal-derived microbiota during delivery to dietary changes from mother's milk to solid foods at weaning, these early-life events have indeed long-standing consequences on our immunity, facilitating tolerance to environmental exposures or, on the contrary, increasing the risk of developing noncommunicable diseases such as allergies, asthma, obesity, and inflammatory bowel diseases. In this review, we provide an outline of the recent advances in our understanding of these events and how they are mechanistically related to intestinal immunity development and education. First, we review the susceptibility of neonates to infections and inflammatory diseases, related to their immune system and microbiota changes. Then, we highlight the maternal factors involved in protection and education of the mucosal immune system of the offspring, the role of the microbiota, and the nature of neonatal immune system until weaning. We also present how the development of some immune responses is intertwined in temporal and spatial windows of opportunity. Finally, we discuss pending questions regarding the neonate particular immune status and the activation of the intestinal immune system at weaning.

Allergic diseases in infancy II-oral tolerance and its failure.

OBJECTIVE: The early window of opportunity describes the timeframe after birth in which essential interactions of the immune system and the newly developing microbiota take place. The infant's immune system has to be reactive to invading pathogens and at the same time tolerant to dietary antigens. If the mechanisms of defense and tolerance induction are disturbed, the risk of infections or allergies is increased. METHOD: This is a narrative review of the recently published information on the topic of neonatal intestinal development and mechanisms of oral tolerance and summarizes the discussions and conclusions from the 8th Human Milk Workshop. RESULTS: The early postnatal period sets the stage for life-long host-microbiome interaction. In this early phase, specific developmental mechanisms ensure physiologic interaction with the developing microbiota. Innate and adaptive immune cells interact in a concerted way to induce and uphold oral tolerance. Factors in human milk can support this induction of tolerance and simultaneously protect against infection and allergy development. CONCLUSION: Understanding the developmental mechanisms in this early phase of immune system development is the first step to develop strategies of pathology prevention. As human milk protects the infant from infections, and aids to develop a tolerogenic immune response, further knowledge on the protective factors in human milk and their effect on the immune system is required.

Allergic diseases in infancy: I - Epidemiology and current interpretation.

Objective: Among non-communicable diseases, the prevalence of allergic diseases has increased significantly in the new millennium. The increase of allergic diseases is linked to the changing environment of infants. Methods: This narrative review summarizes the discussions and conclusions from the 8th Human Milk Workshop. Information from the fields of pediatrics, epidemiology, biology, microbiology, and immunology are summarized to establish a framework describing potential avenues for the prevention of allergic diseases in the future. Results: Several environmental circumstances are linked to the development of allergic diseases. While cesarean section is increasing the risk of allergies, early childhood exposure to a farm environment has a protective effect. From their analysis, nutritive and non-nutritive factors influencing the allergy risk in later life have been identified. The effect of breastfeeding on food allergy development is non-univocal. Human milk components including immunoglobulins, cytokines, and prebiotics have been indicated as important for allergy prevention. Conclusion: Many factors linked to the western lifestyle have been associated with the development of allergic diseases. This suggests several theories that may serve as a basis for new protective interventions. While it is indubitable that mother's milk protects from infectious diseases, its role in the prevention of allergic diseases is to be elucidated.

Adaptation of Staphylococcus aureus to the Human Skin Environment Identified Using an ex vivo Tissue Model.

The healthy human epidermis provides physical protection and is impenetrable for pathogenic microbes. Nevertheless, commensal and pathogen bacteria such as Staphylococcus aureus are able to colonize the skin surface, which may subsequently lead to infection. To identify and characterize regulatory elements facilitating adaptation of S. aureus to the human skin environment we used ex vivo tissue explants and quantified S. aureus gene transcription during co-culture. This analysis provided evidence for a significant downregulation of the global virulence regulator agr upon initial contact with skin, regardless of the growth phase of S. aureus prior to co-culture. In contrast, the alternative sigma factor B (sigB) and the antimicrobial peptide-sensing system (graRS) were expressed during early colonization. Consistently, sigB target genes such as the clumping factor A (clfA) and fibrinogen and fibronectin binding protein A (fnbA) were strongly upregulated upon skin contact. At later timepoints of the adhesion process, wall teichoic acid (WTA) synthesis was induced. Besides the expression of adhesive molecules, transcription of molecules involved in immune evasion were increased during late colonization (staphylococcal complement inhibitor and staphylokinase). Similar to nasal colonization, enzymes involved in cell wall metabolism (sceD and atlA) were highly transcribed. Finally, we detected a strong expression of proteases from all three catalytic classes during the entire colonization process. Taken together, we here present an ex vivo skin colonization model that allows the detailed characterization of the bacterial adaptation to the skin environment.

SPI2 T3SS effectors facilitate enterocyte apical to basolateral transmigration of Salmonella-containing vacuoles in vivo.

Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.

Perinatal development of innate immune topology.

At the transition from intrauterine to postnatal life, drastic alterations are mirrored by changes in cellular immunity. These changes are in part immune cell intrinsic, originate in the replacement of fetal cells, or result from global regulatory mechanisms and adaptation to changes in the tissue microenvironment. Overall, longer developmental trajectories are intersected by events related to mother-infant separation, birth cues, acquisition of microbiota and metabolic factors. Perinatal alterations particularly affect immune niches, where structures with discrete functions meet, the intestinal mucosa, epidermis and lung. Accordingly, the following questions will be addressed in this review.How does the preprogrammed development supported by endogenous cues, steer innate immune cell differentiation, adaptation to tissue structures, and immunity to infection?How does the transition at birth impact on tissue immune make-up including its topology?How do postnatal cues guide innate immune cell differentiation and function at immunological niches?